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WO2019243624A1 - Production of benzylisoquinoline alkaloids in recombinant hosts - Google Patents

Production of benzylisoquinoline alkaloids in recombinant hosts Download PDF

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Publication number
WO2019243624A1
WO2019243624A1 PCT/EP2019/066561 EP2019066561W WO2019243624A1 WO 2019243624 A1 WO2019243624 A1 WO 2019243624A1 EP 2019066561 W EP2019066561 W EP 2019066561W WO 2019243624 A1 WO2019243624 A1 WO 2019243624A1
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seq
set forth
polypeptide
amino acid
acid sequence
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French (fr)
Inventor
Jens Houghton-Larsen
Esben Halkjaer Hansen
Vincent J.J. Martin
Michael E. Pyne
Lauren NARCROSS
Kaspar KEVVAI
Leanne BOURGEOIS
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Valorbec LP
River Stone Biotech ApS
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Valorbec LP
River Stone Biotech ApS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring

Definitions

  • This disclosure relates to recombinant production of benzylisoquinoline alkaloids in recombinant hosts.
  • this disclosure relates to production of (S)-norcoclaurine and derivatives thereof in Saccharomyces cerevisiae.
  • (S)-norcoclaurine synthase is an enzyme involved in committing and rate- limiting steps of benzylisoquinoline (BIA) biosynthesis.
  • BSA benzylisoquinoline
  • 4-hydroxyphenylacetaldehyde (4-HPAA) and dopamine are condensed in a Pictet- Spengler reaction to form (S)-norcoclaurine. See Figure 1.
  • (S)-norcoclaurine synthase is a catalytically inefficient enzyme, having K m values in the double-digit millimolar range.
  • the molar concentration of produced norcoclaurine is about 100 times lower than the concentration of the precursor dopamine. Narcross et al., Trends Biotechnol. 34:228-41 (2016); DeLoache et al., Nat. Chem. Biol. 11 :465-71 (2015).
  • the Ehrlich pathway which involves catabolism of branched-chain amino acids including leucine, valine, and isoleucine; aromatic amino acids including phenylalanine, tyrosine, and tryptophan; and the sulfur-containing amino acid (methionine), leads to the formation of fusel alcohols and fusel acids.
  • S. cerevisiae several dehydrogenase polypeptides and reductase polypeptide are involved.
  • the Ehrlich pathway includes conversion of 4-HPAA to tyrosol.
  • Intracellular availability of 4-HPAA and dopamine in yeast is also highly dependent on the aromatic amino acid biosynthesis pathways, particularly the shikimake pathway, because both precursors are tyrosine derivatives. See Figure 1.
  • benzylisoquinoline alkaloids are of great medical interest because of their pharmacological activity, such as, for example, the antibiotic sanguinarine, the muscle relaxants papaverine and tubocurarine, and the analgesics codeine and morphine. Galanie et al., Science
  • the invention provides a recombinant S. cerevisiae host cell capable of producing one or more benzylisoquinoline alkaloids, comprising a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine and having reduced expression of:
  • the one or more endogenous transporter genes encode a polypeptide having at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:70 or 74, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 76, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72.
  • the one or more endogenous transcription factor genes encode a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 184 or 186.
  • the invention further provides a recombinant host cell comprising reduced expression of the one or more transporter genes encoding the one or more polypeptides having at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:70 or 74, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 76, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72; and the one or more endogenous transporter genes encoding one or more polypeptides having at least 90% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 86 or 88, or at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 102, 112, or at least 70% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 78, 80, 98, 114, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 92, 94, 106, or 1
  • the invention further provides a recombinant host cell comprising reduced expression of the transporter gene encoding a polypeptide having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:70 and further comprising:
  • the invention further provides a recombinant host cell comprising reduced expression of the endogenous gene encoding the one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:154, 152, or 150.
  • the recombinant host cell disclosed herein further comprises reduced expression of:
  • benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:146.
  • the invention further provides a recombinant S. cerevisiae host cell capable of producing one or more benzylisoquinoline alkaloids, comprising a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine, wherein:
  • the gene has a copy number of 2 or more and the host cell further
  • the one or more polypeptides comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 154, 152, or 150; or
  • the host cell has reduced expression of one or more endogenous genes encoding one or more NCS-compatible substrate pathway polypeptide or one or more transcription factor genes that regulate expression of the one or more endogenous genes.
  • the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine has a copy number of 2 or more.
  • the polypeptide capable of synthesizing (S)-norcoclaurine comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, or 198.
  • the benzylisoquinoline alkaloid precursor is 4-hydroxyphenylacetaldehyde (4-HPAA) or 3,4- dihydroxyphenylacetaldehyde (3,4-DHPAA).
  • the one or more endogenous genes encode the one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:154, 152, or 150.
  • the NCS-compatible substrate pathway is a tryptophan biosynthesis pathway or a phenylalanine biosynthesis pathway.
  • the endogenous gene encodes an NCS-compatible pathway polypeptide having at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 166 or 168, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 164.
  • the recombinant host cell disclosed herein further comprises:
  • the invention further provides a recombinant host cell, comprising:
  • the recombinant host cell disclosed herein further comprises:
  • polypeptide capable of synthesizing phenylpyruvate from prephenate comprising a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:164; and/or at least one endogenous gene encoding a polypeptide capable of synthesizing anthranilate from chorismate comprising a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO: 166 or 168.
  • the invention further provides a recombinant host cell, comprising:
  • the one or more benzylisoquinoline alkaloids is (S)-norcoclaurine or (S)-norlaudanosoline.
  • the host cell is further capable of producing a natural or a non-natural benzylisoquinoline alkaloid derivative of (S)- norcoclaurine.
  • the derivative is (S)- reticuline, (R)-reticuline, salutaridinol, thebaine neopinone, and codeinone.
  • the invention further provides a method of producing one or more benzylisoquinoline alkaloids in a cell culture, comprising culturing the host cell disclosed herein in the cell culture, under conditions in which the genes are expressed; wherein the one or more benzylisoquinoline alkaloids is produced by the host cell.
  • the genes are constitutively expressed.
  • the host cell further produces a natural or a non-natural benzylisoquinoline alkaloid derivative of (S)-norcoclaurine,
  • the derivative is (S)-reticuline, (R)- reticuline, berberine, papaverine, morphine, sanguinarine, noscapine, codeine, thebaine, northebaine, oripavine, nororipavine, neopinone, codeinone, oxycodone, or buprenorphine.
  • the host cell is cultured in a fermentor with a feed profile and at a temperature for a period of time, wherein the feed profile, the temperature, and the period of time facilitate reduced formation of an acetaldehyde.
  • the cell culture comprises a non- fermentable carbon source, and wherein the cell culture has a total level of acetaldehyde that is lower than the total level of acetaldehyde in a corresponding cell culture including a glucose carbon source and/or a sucrose carbon source.
  • the non-fermentable carbon source is an acetate carbon source or a glycerol carbon source.
  • the method further comprises isolating one or more benzylisoquinoline alkaloids produced by the recombinant host cell.
  • the method further comprises enzymatically or chemically converting one or more benzylisoquinoline alkaloids produced by the recombinant host cell to provide a benzylisoquinoline alkaloid derivative.
  • the benzylisoquinoline alkaloid derivative is berberine, papaverine, morphine, sanguinarine, noscapine, neomorphine, hydrocodone, codeine, oxycodone, oxymorphone, dihydromorphine, or buprenorphine.
  • the host cell is cultured in a fermentor with a feed profile and at a temperature for a period of time, wherein the feed profile, the temperature, and the period of time facilitate production of the one or more benzylisoquinoline alkaloids.
  • the one or more benzylisoquinoline alkaloids is (S)-norcoclaurine.
  • the invention further provides a cell culture broth, comprising the host cell disclosed herein and further comprising:
  • supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), nucleobases, and/or amino acids; and optionally,
  • the one or more benzylisoquinoline alkaloids are present at a concentration of at least 100 mg/liter of the cell culture.
  • the cell culture comprises less than 10 g/L of ethanol.
  • the invention further provides a cell lysate from the host cell disclosed herein grown in the cell culture, comprising:
  • supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), nucleobases, and/or amino acids; and optionally,
  • Figure 1 shows a biochemical pathway for producing (S)-norcoclaurine
  • a DAHP synthase polypeptide (Aro4), a pentafunctional AROM polypeptide (Aro1 ), a bifunctional chorismate synthase / flavin reductase polypeptide (Aro2), a chorismate mutase polypeptide (Aro7), a prephenate dehydrogenase polypeptide (Tyr1 ), an aromatic aminotransferase I polypeptide (Aro8), an aromatic aminotransferase II polypeptide (Aro9), a phenylpyruvate decarboxylase polypeptide (Aro10), a tyrosine hydroxylase polypeptide (TyrH), an L-DOPA decarboxylase polypeptide (DODC), an (S)-norcoclaurine synthase polypeptide (NCS), a pre
  • Figure 2 shows representative benzylisoquinoline alkaloids obtainable from (S)- norcoclaurine.
  • Figure 3 shows production of dopamine (left) and (S)-norcoclaurine (right) from a control S. cerevisiae strain (Strain C) and a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in Example 4, below).
  • Figure 4 shows visible- and fluorescence-microscopy images (left) and specific fluorescence measurements of S. cerevisiae strains expressing GFP-tagged polypeptides capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in
  • Figure 5 shows production of dopamine and (S)-norcoclaurine (from left to right for each strain) from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 6 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 7 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 8 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 9 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 10 shows off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in Example 5, below.
  • Figure 1 1 shows off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in Example 5, below.
  • Figure 12 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 13 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 14 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 15 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 16 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 17 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 18 shows production of certain off-target condensation products (products 2, 3, and 4; from bottom to top for each strain) of a polypeptide capable of synthesizing (S)- norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 19 shows accumulation of biomass (OD 6 oo) and ethanol during the fed-batch phases of cultivations of S. cerevisiae strains as described in Example 6, below.
  • Figure 20 shows accumulation of norcoclaurine during the fed-batch phases of cultivations of S. cerevisiae strains as described in Example 6, below.
  • Figure 21 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more transporter polypeptides, as described in Example 9, below.
  • Figure 22 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 23 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 24 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 25 shows production of dopamine, tyrosol, 4-HPAC, and (S)-norcoclaurine (from left to right for each strain) from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a
  • nucleic acid means one or more nucleic acids.
  • the term“substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation.
  • the term“substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
  • Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (PCR) techniques. See, for example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989, CURRENT
  • nucleic acid can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof, in either single-stranded or double-stranded embodiments depending on context as understood by the skilled worker.
  • the terms“microorganism,”“microorganism host,”“microorganism host cell,”“recombinant host,” and“recombinant host cell” can be used interchangeably.
  • the term“recombinant host” is intended to refer to a Saccharomyces cerevisiae ( S . cerevisiae) host, the genome of which has been augmented by at least one DNA sequence.
  • DNA sequences include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein (“expressed”), and other genes or DNA sequences which one desires to introduce into a host.
  • introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene.
  • the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis.
  • the term“cell culture” refers to a culture medium comprising one or more recombinant hosts.
  • a cell culture may comprise a single strain of recombinant host, or may comprise two or more distinct host strains.
  • the culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; yeast extract; aminoglycoside antibiotics such as G418 and hygromycin B; etc.
  • recombinant gene refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. “Introduced,” or“augmented” in this context, is known in the art to mean introduced or augmented by the hand of man.
  • a recombinant gene can be a DNA sequence from another species or can be a DNA sequence that originated from or is present in the same species but has been incorporated into a host by recombinant methods to form a recombinant host.
  • a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA.
  • said recombinant genes are encoded by cDNA.
  • recombinant genes are synthetic and/or codon-optimized for expression in S. cerevisiae.
  • the term“engineered biosynthetic pathway” refers to a biosynthetic pathway that occurs in a recombinant host, as described herein. In some aspects, one or more steps of the biosynthetic pathway do not naturally occur in an unmodified host. In some embodiments, a heterologous version of a gene is introduced into a host that comprises an endogenous version of the gene.
  • the term“endogenous” gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell. In some
  • an endogenous S. cerevisiae gene is overexpressed.
  • the term “overexpress” is used to refer to the expression of a gene in an organism at levels higher than the level of gene expression in a wild type organism, i.e., increased expression of a recombinant gene in a host cell relative to a corresponding host cell that does not contain the recombinant gene (see, e.g., Prelich, 2012, Genetics 190:841-54).
  • an endogenous yeast gene is deleted. See, e.g., Giaever & Nislow, 2014, Genetics 197(2):451-65.
  • the terms“deletion,”“deleted,”“knockout,” and“knocked out” can be used
  • heterologous sequence and“heterologous coding sequence” are used to describe a sequence derived from a species other than the recombinant host.
  • a heterologous coding sequence for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant S. cerevisiae host expressing the heterologous sequence.
  • a coding sequence is a sequence that is native to the host.
  • A“selectable marker” can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a detectable change, e.g. a color change.
  • Linearized DNA fragments of the gene replacement vector then are introduced into the cells using methods well known in the art ( see below). Integration of the linear fragments into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, polymerase chain reaction (PCR) or Southern blot analysis. Subsequent to its use in selection, a selectable marker can be removed from the genome of the host cell by, e.g., Cre-LoxP systems (see, e.g., Gossen et al., 2002, Ann. Rev.
  • a gene replacement vector can be constructed in such a way as to include a portion of the gene to be disrupted, where the portion is devoid of any endogenous gene promoter sequence and encodes none, or an inactive fragment of, the coding sequence of the gene.
  • the terms“variant” and“mutant” are used to describe a protein sequence that has been modified, by man or nature, at one or more amino acids, compared to the wild-type sequence of a particular protein.
  • the term“inactive fragment” is a fragment of the gene that encodes a protein having, e.g., less than about 10% ⁇ e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 %, or 0%) of the activity of the protein produced from the full-length coding sequence of the gene.
  • Such a portion of a gene is inserted in a vector in such a way that no known promoter sequence is operably linked to the gene sequence, but that a stop codon and a transcription termination sequence are operably linked to the portion of the gene sequence.
  • This vector can be subsequently linearized in the portion of the gene sequence and transformed into a cell. By way of single homologous recombination, this linearized vector is then integrated in the endogenous counterpart of the gene with resulting inactivation thereof.
  • “alkaloid” refers to any of the group of naturally occurring chemical compounds that mostly contain basic nitrogen atoms.
  • “alkaloids” include“true alkaloids” (i.e., amino-acid-derived compounds containing nitrogen in the heterocycle), for example, atropine, nicotine, morphine, ergotamine, coniine, and coniceine; “protoalkaloids” (i.e., amino-acid-derived compounds containing nitrogen), for example, mescaline, adrenaline, and ephedrine;“polyamine alkaloids” (i.e., derivatives of putrescine, spermidine, and spermine);“peptide and cyclopeptide alkaloids”; and“pseudoalkaloids” (i.e., alkaloid-like compounds that do not originate from amino acids), for example, terpene-like alkaloids, steroid-like alkaloids, and purine-like alkaloids (
  • alkaloid also includes non-naturally occurring compounds derived from or otherwise resembling any naturally occurring alkaloid. See M. Hesse,“Alkaloids. Nature’s Curse or Blessing?” (2002); T. Aniszewski,“Alkaloids - Secrets of Life” (2007). Alkaloids can contain asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. Each chiral center can be defined in terms of absolute stereochemistry as (R)- or (S)-. As used herein, the term“alkaloid” includes all such possible isomers, as well as their racemic and optically pure forms.
  • alkaloids can be classified by skeleton (e.g., pyrrolidine, quinoline, benzylisoquinoline, indole, and terpenoid).
  • skeleton e.g., pyrrolidine, quinoline, benzylisoquinoline, indole, and terpenoid.
  • the term“benzylisoquinoline alkaloid” refers to alkaloids having a benzylisoquinoline skeleton, including, for example, (S)-norcoclaurine, coclaurine, berberine, papaverine, morphine, sanguinarine, hydrastine, and noscapine. See Figure 2; see also, Singla et al., “BIAdb: A curated database of benzylisoquinoline alkaloids,” BMC Pharmacol. 10:4 (March 2010)).
  • benzylisoquinoline alkaloid precursor compound are used to refer to intermediate compounds in the benzylisoquinoline alkaloid biosynthetic pathway.
  • Benzylisoquinoline alkaloid precursors include, but are not limited to, phosphoenolpyruvate (PEP), erythrose 4-phosphate (E4P), 3- deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), 5-enolpyruvateshikimate 3-phosphate (EPSP), chorismate, prephenate, L-tyrosine, L-3,4-dihydroxyphenylalaine (L-DOPA), 4-(2- aminoethyl)benzene-1 ,2-diol (dopamine), 4-hydroxyphenylpyruvate (4-HPP), and 4- hydroxyphenylacetaldehyde (4-HPAA). See Figure 1.
  • PEP phosphoenolpyruvate
  • E4P erythrose 4-phosphate
  • DAHP
  • benzylisoquinoline alkaloid precursors are themselves benzylisoquinoline alkaloids.
  • (S)-norcoclaurine is a benzylisoquinoline alkaloid precursor of morphine. See Figure 2.
  • Benzylisoquinoline alkaloids and/or precursors thereof can be produced in vivo, (i.e., in a recombinant host), in vitro (i.e., enzymatically or chemically), or by whole cell bioconversion.
  • the terms“produce” and“accumulate” can be used interchangeably to describe synthesis of benzylisoquinoline alkaloids and precursors thereof in vivo, in vitro, or by whole cell conversion.
  • a culture broth can comprise a carbon source such as dextrose, sucrose, fructose, glycerol, xylose, ethanol, glucose, or acetate; a phosphate source; vitamins; trace elements; salts; yeast nitrogen base (YNB); amino acids; nucleobases; and a nitrogen source.
  • the nitrogen source can include, for example, malt extract, corn steep liquor, casein hydrosylate, yeast extract, urea, amino acids, ammonia and/or ammonium salts, etc.
  • a culture broth can comprise one or more benzylisoquinoline alkaloids added to the medium or produced de novo by a recombinant host, as described herein.
  • a non-fermentable carbon source e.g ., an acetate carbon source or a glycerol carbon source
  • reduced intracellular acetaldehyde limits or eliminates spontaneous condensation of acetaldehyde and dopamine.
  • benzylisoquinoline alkaloids and/or precursors thereof are produced in vivo through expression of one or more enzymes involved in the benzylisoquinoline alkaloid biosynthetic pathway in a recombinant S. cerevisiae host.
  • a recombinant S. cerevisiae host For example, a recombinant S.
  • DAHP 3-deoxy- D-arabinoheptulosonate-7-phosphate
  • E4P erythrose 4-phosphate
  • DAHP synthesis polypeptide e.g., SEQ ID NO:2 or SEQ ID NO:4
  • EBP erythrose 4-phosphate
  • a gene encoding a polypeptide capable of synthesizing 5-enolpyruvateshikimate 3-phosphate (EPSP) from DAHP ⁇ e.g., a pentafunctional AROM polypeptide; e.g., SEQ ID NO:6)
  • a gene encoding a polypeptide capable of synthesizing chorismate from EPSP ⁇ e.g., bifunctional chorismate synthase / flavin reductase polypeptide; e.g., SEQ ID NO:8)
  • a gene encoding a polypeptide capable of synthesizing chorismate from EPSP ⁇ e.g., bifunctional chorismate synth
  • production of benzylisoquinoline alkaloids can be accomplished by differential copy numbers of the benzylisoquinoline alkaloid
  • biosynthesis pathway genes in the recombinant cell, differential promoter strengths, and/or by utilizing mutants with increased specificity/activity towards the product of interest.
  • additional copies of a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in recombinant cells as otherwise described herein can increase the production of (S)-norcoclaurine.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing DAHP from E4P and PEP.
  • the gene encoding a polypeptide capable of synthesizing DAHP from E4P and PEP is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • a terminator such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing DAHP from E4P and PEP results in a total expression level of genes encoding a polypeptide capable of synthesizing DAHP from E4P and PEP that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing DAHP from E4P and PEP, i.e., an overexpression of a polypeptide capable of synthesizing DAHP from E4P and PEP.
  • the polypeptide capable of synthesizing DAHP from E4P and PEP is a feedback-resistant (FBR) polypeptide.
  • a feedback resistant version of and/or overexpression of a benzylisoquinoline alkaloid biosynthesis polypeptide e.g., a polypeptide capable of synthesizing DAHP from E4P and PEP, e.g., a DAHP synthase polypeptide; e.g., a polypeptide capable of synthesizing prephenate from chorismate, e.g., a chorismate mutase polypeptide
  • pathway regulation e.g., by reducing or eliminating endogenous transcriptional regulation of expression.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing prephenate from chorismate.
  • the gene encoding a polypeptide capable of synthesizing prephenate from chorismate is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPH (SEQ ID NO:179), or pCCW12 (SEQ ID NQ:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPH (SEQ ID NO:179), or pCCW12 (SEQ ID NQ:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • a terminator such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing prephenate from chorismate results in a total expression level of genes encoding a polypeptide capable of synthesizing prephenate from chorismate that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing prephenate from chorismate, i.e., an overexpression of a polypeptide capable of synthesizing prephenate from chorismate.
  • the polypeptide capable of synthesizing prephenate from chorismate is a feedback-resistant (FBR) polypeptide.
  • IAA indole-3-acetaldehyde
  • 4-HPAA 4-HPAA for condensation with dopamine
  • dopamine catalyzed, e.g., by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine.
  • overexpression of a polypeptide capable of synthesizing prephenate from chorismate can increase the benzylisoquinoline alkaloid biosynthesis flux relative to competing pathways from chorismate towards IAA, and accordingly increase the availability of intracellular dopamine for condensation with 4-HPAA.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate.
  • the gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NQ:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NQ:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • a terminator such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate results in a total expression level of genes encoding a polypeptide capable of synthesizing 4-HPP from prephenate that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing 4-HPP from prephenate, i.e., an overexpression of a polypeptide capable of synthesizing 4-HPP from prephenate.
  • PAA phenylacetaldehyde
  • S synthesizing
  • overexpression of a polypeptide capable of synthesizing 4-HPP from prephenate can increase benzylisoquinoline alkaloid biosynthesis flux relative to competing pathways from prephenate towards PAA, and accordingly increase the availability of intracellular dopamine for condensation with 4-HPAA.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine.
  • the gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO:177), tADH1 (SEQ ID NO: 178), tTDH 1 (SEQ ID NO:181 ), or tPGK1 (SEQ ID NO: 182).
  • a terminator such as, for example, for example, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO:177), tADH1 (SEQ ID NO: 178), tTDH 1 (SEQ ID NO:181 ), or tPGK1 (SEQ ID NO: 182).
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine results in a total expression level of genes encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine, i.e., an overexpression of a polypeptide capable of synthesizing L-DOPA from L-tyrosine.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA.
  • the gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), P ⁇ H 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • a terminator such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), P ⁇ H 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA results in a total expression level of genes encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing dopamine from L- DOPA, i.e., an overexpression of a polypeptide capable of synthesizing dopamine from L-
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP.
  • the gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO: 169), pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGH (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • a terminator such as, for example, tPGH (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • synthesizing 4-HPAA from 4-HPP results in a total expression level of genes encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP, i.e., an overexpression of a polypeptide capable of synthesizing 4-HPAA from 4-HPP.
  • overexpression of a polypeptide capable of synthesizing 4-HPAA from 4-HPP can increase benzylisoquinoline alkaloid biosynthesis flux relative to competing pathways from 4-HPP.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA.
  • the gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from (S)- norcoclaurine from dopamine and 4-HPAA is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO: 170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO: 170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO: 174), tCYC1 (SEQ ID NO: 175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 174), tPGM (SEQ ID NO: 174), tCYC1 (SEQ ID NO: 175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA results in a total expression level of genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4- HPAA that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA, i.e., an overexpression of a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA.
  • the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA is a truncated polypeptide such as, for example, a truncated NCS polypeptide lacking an N-terminal signal peptide (e.g ., SEQ ID NO:42 or SEQ ID NO:44), a truncated NCS polypeptide lacking a C-terminal transmembrane domain (e.g., SEQ ID NO:52 or SEQ ID NO:54), or a truncated NCS polypeptide lacking an N-terminal signal peptide and a C- terminal transmembrane domain.
  • a truncated NCS polypeptide such as, for example, a truncated NCS polypeptide lacking an N-terminal signal peptide (e.g ., SEQ ID NO:42 or SEQ ID NO:44), a truncated NCS polypeptide lacking a C-terminal transmembr
  • a polypeptide lacking a signal peptide and/or a transmembrane domain, expressed in a recombinant host may have increased solubility in the cytosol relative to a corresponding polypeptide comprising the signal peptide and/or transmembrane domain.
  • the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA has a copy number of two (i.e., is present in the recombinant host in two copies).
  • the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA e.g ., an NCS polypeptide
  • the polypeptide capable of synthesizing DAHP from PEP and E4P comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 1 ) or SEQ ID NO:4 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:3).
  • the polypeptide capable of synthesizing EPSP from DAHP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:6 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:5).
  • the polypeptide capable of synthesizing chorismate from EPSP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:8 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:7).
  • the polypeptide capable of synthesizing prephenate from chorismate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:10 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:9) or SEQ ID NO:12 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:1 1 ).
  • the polypeptide capable of synthesizing 4-HPP from prephenate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:14 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 13).
  • the polypeptide capable of synthesizing L-tyrosine from 4-HPP and/or the polypeptide capable of synthesizing 4-HPP from L-tyrosine comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:16 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 15) or SEQ ID NO:18 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:17).
  • the polypeptide capable of synthesizing 4-HPAA from 4-HPP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:20 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 19).
  • the polypeptide capable of synthesizing L-DOPA from L-tyrosine comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:22 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:21 ) or SEQ ID NO:24 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:23).
  • the polypeptide capable of synthesizing dopamine from L-DOPA comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:26 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:25) or SEQ ID NO:28 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:27).
  • the polypeptide capable of synthesizing (S)-norcoclaurine from 4- HPAA and dopamine comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:32 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:31 ), or SEQ ID NO:34 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:33), or SEQ ID NO:36 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:35), or SEQ ID NO:38 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:37), or SEQ ID NO:40 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:39), or SEQ ID NO:42 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:41 ), or SEQ ID NO:44 (which can be encoded by the nucleo
  • benzylisoquinoline alkaloids and/or precursors thereof are produced in vivo through expression of one or more enzymes involves in the benzylisoquinoline alkaloid biosynthetic pathway.
  • a recombinant S for example, a recombinant S.
  • cerevisiae host comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P (e.g ., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephenate from chorismate (e.g., a recombinant gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 12); a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate (e.g., a
  • the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA has a copy number of two, three, four, five, six, seven, eight, or more.
  • (S)-Norcoclaurine can be produced in vivo (i.e., in a recombinant host), in vitro (i.e., enzymatically), or by whole cell bioconversion.
  • the terms“produce” and “accumulate” can be used interchangeably to describe synthesis of (S)-norcoclaurine in vivo, in vitro, or by whole cell bioconversion.
  • a cell is permeabilized to take up a substrate to be modified or to excrete a modified product.
  • a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out.
  • the cells are permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween.
  • the cells are permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide (CTAB).
  • CTAB cetyltrimethylammonium bromide
  • the cells are permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock.
  • a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant.
  • the resulting supernatant can then be applied to a chromatography column, e.g., a C18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol.
  • the compound(s) can then be further purified by preparative HPLC.
  • (S)-norcoclaurine can be produced by co-culturing of two or more hosts.
  • one or more hosts each expressing one or more enzymes involved in the (S)-norcoclaurine biosynthetic pathway, produce (S)-norcoclaurine.
  • a host expressing a gene encoding a polypeptide capable of polypeptide capable of synthesizing (S)-norcoclaurine; a gene encoding a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:2 and 4; a gene encoding a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:6; a gene encoding a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:8; a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:10 and 12; a gene encoding a polypeptide having at least 55% sequence identity to the amino acid sequence set forth
  • the benzylisoquinoline alkaloid comprises, for example, (S)- norcoclaurine, (S)-reticuline, (R)-reticuline, salutaridinol, thebaine, neopinone, and codeinone.
  • a recombinant S. cerevisiae host as otherwise described herein, further comprising a gene encoding a polypeptide capable of synthesizing (S)-coclaurine from (S)- norcoclaurine (e.g ., a 6-O-methyltransferase (6-OMT) polypeptide; e.g., a polypeptide having at least 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO:201 ), a gene encoding a polypeptide capable of synthesizing 3’-hydroxy-coclaurine from (S)-coclaurine or 3’- hydroxy-A/-methyl-coclaurine from /V-methyl-coclaurine (e.g ., an N-methylcoclaurine 3’- monooxygenase (NMCH) polypeptide; e.g., a polypeptide having at least 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO:202
  • 6-OMT 6-O
  • a recombinant S. cerevisiae host capable of producing (S)-reticuline in vivo (e.g., as described herein), further comprising a gene encoding a polypeptide capable of synthesizing (R)-reticuline from (S)-reticuline (e.g., a 1 ,2- dehydroreticuline synthase-1 , 2-dehydroreticuline reductase (DRS-DRR) polypeptide; e.g., a polypeptide having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO:205), a gene encoding a polypeptide capable of synthesizing salutaridine from (R)- reticuline (e.g., a salutaridine synthase (SAS) polypeptide; e.g., a polypeptide having at least 55% sequence identity to an amino acid sequence set forth in SEQ ID NO:206), a gene encoding a polypeptide capable
  • a recombinant S. cerevisiae host capable of producing thebaine in vivo e.g as described herein), further comprising a gene encoding a polypeptide capable of synthesizing neopinone from thebaine ⁇ e.g., a morphinone reductase polypeptide) and a gene encoding a polypeptide capable of synthesizing hydrocodone from codeinone ⁇ e.g., a thebaine 6-O-demethylase polypeptide), can produce hydrocodone in vivo.
  • benzylisoquinoline alkaloids precursors produced in vivo ⁇ e.g., (S)-norcoclaurine, (S)-reticuline, (R)-reticuline, salutaridinol, thebaine, neopinone, codeinone
  • can be converted in vitro e.g., chemically or enzymatically to derivatives including berberine, papaverine, morphine, sanguinarine, noscapine, neomorphine, hydrocodone, codeine, oxycodone, oxymorphone, dihydromorphine, and buprenorphine.
  • codeinone produced by a recombinant host described herein is enzymatically converted to hydrocodone by contacting the codeinone with a polypeptide capable of synthesizing hydrocodone from codeinone ⁇ e.g., a thebaine 6-O-demethylase polypeptide) in vitro.
  • thebaine produced by a recombinant host described herein is chemically converted to buprenorphine in vitro ⁇ see, e.g., WO 2018/21 1331 ; Machara et al., Adv. Synth. Catal. 354(4):613-26 (2012); Werner et al., J. Org.
  • thebaine produced by a recombinant host described herein is chemically or enzymatically converted to morphine, neomorphine, hydrocodone, codeine, oxycodone, oxymorphone, dihydromorphine, or etorphine.
  • the terms“detectable amount,”“detectable concentration,” “measurable amount,” and“measurable concentration” refer to a level of benzylisoquinoline alkaloids or precursors thereof measured in AUC, pM/OD 6 oo, mg/L, mM, or mM.
  • Benzylisoquinoline alkaloid production i.e., total, supernatant, and/or intracellular
  • benzylisoquinoline alkaloid levels can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high-performance liquid
  • HPLC ultraviolet-visible spectroscopy/ spectrophotometry
  • MS mass spectrometry
  • NMR nuclear magnetic resonance spectroscopy
  • the term“undetectable concentration” refers to a level of a compound that is too low to be measured and/or analyzed by techniques such as TLC, HPLC, UV-Vis, MS, or NMR.
  • a compound of an“undetectable concentration” is not present in a composition of benzylisoquinoline alkaloids and/or precursors thereof.
  • one or more benzylisoquinoline alkaloids e.g ., (S)-norcoclaurine
  • precursors thereof are produced in vivo by culturing a recombinant S.
  • cerevisiae host comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephenate from chorismate (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:12); a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate (e.g., a
  • the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA has a copy number of two, three, four, five, six, seven, eight, or more.
  • benzylisoquinoline alkaloids e.g., (S)-norcoclaurine
  • the methods of the present invention can optionally comprise one or more additional steps. These steps can for example be directed toward isolation and/or purification of one or more benzylisoquinoline alkaloids from the fermentation broth or host cells.
  • the isolating steps may comprise: (a) contacting the cell culture comprising the benzylisoquinoline alkaloids with: (i) one or more adsorbent resins in a packed column in order to bind at least a portion of the
  • benzylisoquinoline alkaloids to the resin, thereby isolating the benzylisoquinoline alkaloid compounds; or (ii) one or more ion exchange or reversed-phase chromatography columns in order to bind at least a portion of the benzylisoquinoline alkaloid in the column, thereby isolating the benzylisoquinoline alkaloid; or (b) crystallizing and/or organic solvent extracting the benzylisoquinoline alkaloids from the cell culture, thereby isolating the benzylisoquinoline alkaloids;(i) contacting the cell culture with an organic solvent immiscible with water and separating the organic phase enriched in benzylisoquinoline alkaloids (c) separating the cell culture into a solid phase and a liquid phase, wherein the liquid phase comprises of the benzylisoquinoline alkaloids; and (i) contacting the liquid phase with one or more adsorbent resins in order to
  • the isolating step can comprise separating the solid phase from the liquid phase using a process comprising tangential flow filtration with diafiltration membranes to generate a permeate stream comprising the benzylisoquinoline alkaloids, wherein the membranes used in the tangential flow filtration are ultrafiltration or nanofiltration membranes.
  • the permeate stream is extracted by an organic solvent which phase-separates from the aqueous phase to generate an extracted benzylisoquinoline alkaloids in the organic solvent.
  • the permeate stream containing the benzylisoquinoline alkaloids product could be concentrated by some evaporation to produce a crystallized benzylisoquinoline alkaloid.
  • the aqueous permeate or the concentrate can be extracted by an organic solvent which phase-separates from the aqueous phase.
  • the solvent extraction could be performed in a counter-current extraction centrifuge such as a Podbelniak extractor, or in a counter-current extraction column such as a Karr or Scheibel column. This yields the benzylisoquinoline alkaloids products in an organic solvent suitable for subsequent purification processing.
  • the isolating step can also comprise separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced one or more benzylisoquinoline alkaloids, and (a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced one or more
  • organic solvent extraction can be replaced with a series of process operations which yield a similar organic solution of benzylisoquinoline alkaloids.
  • the series of process operations would include (a) precipitation of benzylisoquinoline alkaloids from the aqueous concentrate produced by addition of acid until acidic pH; (b) filtration and optionally water-washing of the resulting solids; and (c) dissolution of the filtered benzylisoquinoline alkaloids containing solids into an organic solvent suitable for further purification.
  • the organic extract can be contacted with carbon to adsorb impurities and color bodies.
  • the carbon contacting can be done by mixing carbon in the organic extract and filtering the carbon out of the resulting suspension, or by feeding the organic extract to a column or filter containing a fixed bed of carbon and collecting a purified effluent stream.
  • the organic extract can be crystallized by concentrating the solution evaporatively.
  • the resulting benzylisoquinoline alkaloids products crystals can be filtered, washed, and dried to yield a high-purity
  • the reaction mixture can be filtered in order to remove the solid in the media (cell debris etc.).
  • the resulting aqueous solution can be extracted repeatedly with an organic solvent not miscible with water (this can be chloroform, toluene, dichloromethane, ethyl acetate, or the like).
  • the resulting organic phase can be concentrated into small quantity (resulting into a syrup).
  • the aqueous phase can be discarded.
  • the resulting residue benzylisoquinoline alkaloid crude material can be then crystallized from any short chain alcohol, such as methanol or it can be purified with other suitable purification technique such as chromatography or other standard techniques.
  • Another possible procedure to extract the alkaloids from the fermentation broth or cells can be a caustic wash of the broth/cells followed by a filtration in order to remove the biological material and other solids.
  • the alkaloids can then be precipitated from the basic solution as salt after adjusting the pH to acidic with addition of acid (for example, sulphuric acid or hydrochloric acid, etc.).
  • acid for example, sulphuric acid or hydrochloric acid, etc.
  • the benzylisoquinoline alkaloids can be extracted from the cells/broth trough percolation via an organic solvent.
  • the resulting organics can be concentrated into small quantities.
  • the resulting residue can be purified with other suitable purification technique such as crystallization and/or chromatography or other standard techniques.
  • the terms“or” and“and/or” are used to describe multiple components in combination or exclusive of one another.
  • “x, y, and/or z” can refer to“x” alone,“y” alone,“z” alone,“x, y, and z,”“(x and y) or z,”“x or (y and z),” or“x or y or z.”
  • “and/or” is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group.
  • “and/or” is used to refer to production of benzylisoquinoline alkaloids and/or benzylisoquinoline alkaloid precursors.
  • “and/or” is used to refer to production of benzylisoquinoline alkaloids, wherein one or more benzylisoquinoline alkaloids are produced.
  • “and/or” is used to refer to production of benzylisoquinoline alkaloids, wherein one or more benzylisoquinoline alkaloids are produced through the following steps: culturing a recombinant S. cerevisiae host, synthesizing one or more benzylisoquinoline alkaloids in a recombinant S. cerevisiae host, and/or isolating one or more benzylisoquinoline alkaloids.
  • Functional homologs of the polypeptide described above are also suitable for use in producing benzylisoquinoline alkaloids and/or precursors thereof in a recombinant S. cerevisiae host.
  • a functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide.
  • a functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs.
  • Variants of a naturally occurring functional homolog can themselves be functional homologs.
  • Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally occurring polypeptides (“domain swapping”).
  • Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs.
  • the term“functional homolog” is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
  • Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of benzylisoquinoline alkaloid biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using, for example, the amino acid sequence of an NCS polypeptide as the reference sequence. An amino acid sequence is, in some instances, deduced from a corresponding nucleotide sequence.
  • nucleic acids and polypeptides are identified from transcriptome data based on expression levels rather than by using BLAST analysis.
  • conserveed regions can be identified by locating a region within the primary amino acid sequence of a benzylisoquinoline alkaloid biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer et al., Nucl.
  • conserveed regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.
  • polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions.
  • conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g ., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity).
  • a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
  • polypeptides suitable for producing norcoclaurine in a recombinant S. cerevisiae host include NCS polypeptides and functional homologs thereof.
  • a candidate sequence typically has a length that is from 80% to 250% of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250% of the length of the reference sequence.
  • a functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, or 120% of the length of the reference sequence, or any range between.
  • a % sequence identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows.
  • a reference sequence e.g., a nucleic acid sequence or an amino acid sequence described herein
  • Clustal Omega version 1.2.1 , default parameters
  • Clustal Omega calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments.
  • word size 2; window size: 4; scoring method: %age; number of top diagonals: 4; and gap penalty: 5.
  • gap opening penalty 10.0; gap extension penalty: 5.0; and weight transitions: yes.
  • word size 1 ; window size: 5; scoring
  • the Clustal Omega output is a sequence alignment that reflects the relationship between sequences.
  • Clustal Omega can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European
  • % sequence identity of a candidate nucleic acid or amino acid sequence to a reference sequence
  • the sequences are aligned using Clustal Omega, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100.
  • the% sequence identity value can be rounded to the nearest tenth. For example, 78.1 1 , 78.12, 78.13, and 78.14 are rounded down to 78.1 , while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
  • a nucleic acid sequence encoding a benzylisoquinoline alkaloid biosynthesis polypeptide can include a tag sequence that encodes a“tag” designed to facilitate subsequent manipulation (e.g ., to facilitate purification or detection), solubility, secretion, or localization of the encoded polypeptide.
  • Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide.
  • Non-limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), disulfide oxidoreductase (DsbA), maltose binding protein (MBP), N-utilization substance (NusA), small ubiquitin-like modifier (SUMO), and FlagTM tag (Kodak, New Haven, CT).
  • Other examples of tags include a chloroplast signal peptide, a mitochondrial signal peptide, an amyloplast peptide, signal peptide, or a secretion tag.
  • a benzylisoquinoline alkaloid biosynthesis polypeptide is a protein altered by domain swapping.
  • domain swapping is used to describe the process of replacing a domain of a first protein with a domain of a second protein.
  • the domain of the first protein and the domain of the second protein are functionally identical or functionally similar.
  • the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein.
  • an NCS polypeptide e.g., a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA is altered by domain swapping.
  • benzylisoquinoline alkaloid biosynthesis polypeptides can include additional amino acids that are not involved in the enzymatic activities carried out by the polypeptides.
  • benzylisoquinoline alkaloid biosynthesis polypeptides are fusion proteins.
  • the terms“chimera,”“fusion polypeptide,” “fusion protein,”“fusion enzyme,”“fusion construct,”“chimeric protein,” “chimeric polypeptide,” “chimeric construct,” and“chimeric enzyme” can be used interchangeably herein to refer to proteins engineered through the joining of two or more genes that code for different proteins.
  • a protein is altered by circular permutation, which involves covalent attachment of the ends of a protein that would be opened elsewhere afterwards.
  • circular permutation involves covalent attachment of the ends of a protein that would be opened elsewhere afterwards.
  • the order of the sequence is altered without causing changes in the amino acids of the protein.
  • a targeted circular permutation can be produced, for example but not limited to, by designing a spacer to join the ends of the original protein. Once the spacer has been defined, there are several possibilities to generate permutations through generally accepted molecular biology techniques, for example but not limited to, by producing
  • concatemers by means of PCR and subsequent amplification of specific permutations inside the concatemer or by amplifying discrete fragments of the protein to exchange to join them in a different order.
  • the step of generating permutations can be followed by creating a circular gene by binding the fragment ends and cutting back at random, thus forming collections of permutations from a unique construct.
  • Modification of transport systems in a recombinant S. cerevisiae host that are involved in transport of benzylisoquinoline alkaloids and/or precursors thereof can allow for more effective production of benzylisoquinoline alkaloids in recombinant hosts.
  • the terms“transport of a benzylisoquinoline alkaloid (precursor),” “benzylisoquinoline alkaloid (precursor) transport,”“excretion of a benzylisoquinoline alkaloid (precursor),” and“benzylisoquinoline alkaloid (precursor) excretion” can be used
  • transporter also referred to as a membrane transport protein refers to a membrane protein involved in the movement of small molecules, macromolecules (such as carbohydrates), and ions across a biological membrane.
  • Transporters span the membrane in which they are localized and across which they transport substances. Transporter proteins can assist in the movement (i.e., transport or excretion) of a substance from the intracellular space to the culture medium, or from a vacuolar space to the intracellular space. Transporters are known to function as passive transport systems, carrying molecules down their concentration gradient, or as active transport systems, using energy to carry molecules uphill against their concentration gradient. Active transport is mediated by carriers which couple transport directly to the use of energy derived from hydrolysis of an ATP molecule or by carriers which make use of a pre-established electrochemical ion gradient to drive co-transport of the nutrient molecule and a co-transported ion. The latter category comprises symporters and antiporters, which carry the ion in the same or opposite direction, respectively, as the transported substrate.
  • Transport proteins have been classified according to various criteria at the
  • Transporter Classification Database (on the world-wide web at tcdb.org). See Saier Jr. et al., Nucl. Acids Res., 42(1 ):D251-258 (2014).
  • Non-limiting examples thereof include, among others, the family of Multiple Drug Resistance (MDR) plasma membrane transporters that is thought to be ubiquitous among living organisms.
  • MDR transporter superfamily can be further subdivided according to the mode of operation by which the substrate is transported from one side of the membrane to the other. Transporters can operate to move substances across membranes in response to chemiosmotic ion gradients or by active transport.
  • ABC transporters are transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to carry out translocation of various substrates across membranes. They can transport a wide variety of substrates across the plasma membrane and intracellular membranes, including metabolic products, lipids and sterols, and drugs.
  • endogenous ABC transporter genes include PDR5, PDR10, PDR12, PDR15, PDR18, SNQ2, YDR061W, YOR1 , YOL075C, MDL2,
  • ABC transporters transport benzylisoquinoline alkaloid precursors and/or benzylisoquinoline alkaloids.
  • MFS transporters are monomeric polypeptides that can transport small solutes in response to proton gradients.
  • the MFS transporter family is sometimes referred to as the uniporter-symporter-antiporter family.
  • MFS transporters function in, inter alia, in sugar uptake and drug efflux systems.
  • MFS transporters typically comprise conserved MFS-specific motifs.
  • Non-limiting examples of endogenous MFS transporter genes include TP01 , TP02, TP03, TP04, QDR1 , QDR2, QDR3, FLR1 , DTR1 , YHK8, SE01 , YBR241 C, VBA3, FEN2, SNF3, STL1 , HXT10, AZR1 , MPH3, VBA5, GEX2, SNQ1 , AQR1 , MCH1 , MCH5, ATG22, HXT15, MPH2, ITR1 , SIT 1 , VPS73, HXT5, SOA1 , HXT9, YMR279C, YIL166C, HOL1 , and ENB1 (or a functional homolog thereof).
  • MFS transporters transport benzylisoquinoline alkaloid precursors and/or benzylisoquinoline alkaloids.
  • cerevisiae host that involve one or more benzylisoquinoline alkaloids precursors can allow for more effective production of benzylisoquinoline alkaloids in recombinant hosts.
  • oxidoreductases in yeast can reduce the level of reduction of 4-HPAA to tyrosol and thereby improve norcoclaurine or norlaudanosoline yields in recombinant strains expressing a norcoclaurine synthase (NCS) enzyme, with ARM (YGL157W) being the most prominent, with clear indication in the presented data from that ADH6 disruption also has an effect.
  • NCS norcoclaurine synthase
  • ARM YGL157W
  • WO 2018/029282 Kristy Hawkins,“Metabolic engineering of Saccharomyces cerevisiae for the production of benzylisoquinoline alkaloids” 1 January 2009. Caltech Thesis, XP55361294, pp. 1-154.
  • the fold-increase yield demonstrated by these approaches are still not high enough to make the process commercially viable since much 4-HPAA are still lost to Tyrosol and yields of norcoclaurine still are in pg/L or low mg/L scale
  • S. cerevisiae comprises a number of endogenous genes encoding dehydrogenases and reductases that act on substrates similar to one or more benzylisoquinoline alkaloid precursors such as, for example, 4-HPAA.
  • S. cerevisiae comprises five aldehyde dehydrogenase genes (ALD2, ALD3, ALD4, ALD5, and ALD6) and seven primary alcohol dehydrogenase genes (ADH1 , ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7).
  • S. cerevisiae further includes a formaldehyde dehydrogenase gene (SFA1 ), and an aldehyde reductase gene (ARM ).
  • Other endogenous genes similar to ARI1 include YGL039W, YDR541C, YPR1 , GCY1 , GRE2, AAD3, AAD4, and AAD14.
  • tyrosine e.g ., by expressing one or more pathway polypeptides such as Aro10, Aro7, and/or Tyr1 from a constitutive, non-native promoter, and/or by expression one or more feedback-resistant pathway polypeptides such as Aro7-FBR (SEQ ID NO: 12) or Aro4-FBR (SEQ ID NO:4) to remove feedback inhibition from tyrosine results in a downregulated, reinforced tyrosine pathway.
  • pathway polypeptides such as Aro10, Aro7, and/or Tyr1
  • a feedback-resistant pathway polypeptides such as Aro7-FBR (SEQ ID NO: 12) or Aro4-FBR (SEQ ID NO:4)
  • S. cerevisiae comprises an endogenous biosynthetic pathway towards
  • phenylalanine of which the intermediate PAA can, in certain embodiments, be condensed with dopamine by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine.
  • the phenylalanine pathway involves endogenous gene PHA2.
  • S. cerevisiae also comprises an endogenous biosynthetic pathway towards tryptophan, on which the intermediate IAA can, in certain embodiments, be condensed with dopamine by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine.
  • the tryptophan pathway involves endogenous genes TRP2 and TRP3.
  • acetaldehyde (AA), 2-methylbutanal (2-MB) 3-methylbutanal (3-MB) (collectively, methylbutanal (MB)), acetoin, and pyruvate can, in certain embodiments, be condensed with dopamine by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine.
  • AA, IAA, PAA, 2-MB, 3-MB, acetoin, and pyruvate are intermediates in various biosynthetic pathways (e.g., an endogenous biosynthetic pathway towards tyrosine, leucine, isoleucine, valine, or methionine).
  • NCS-compatible substrate includes any aldehyde other than 4-HPAA that can be condensed with dopamine by a polypeptide also capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine (e.g., an (S)-norcoclaurine synthase (NCS) polypeptide; e.g., SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:54) such as, for example, AA, IAA, PAA, 2-MB, 3-MB, acetoin, and pyruvate.
  • S S-norcoclaurine synthase
  • NCS-compatible substrate pathway refers to any biosynthetic pathway that includes an NCS- compatible substrate as an intermediate or final product, such as, for example, the aromatic acid biosynthesis pathways endogenous to S. cerevisiae.
  • recombinant S. cerevisiae host cells capable of producing one or more benzylisoquinoline alkaloids and/or precursors thereof comprise one or more inactivated endogenous genes.
  • An endogenous gene is typically inactivated by disrupting expression of the gene or introducing a mutation to reduce or even completely eliminate endogenous gene activity in a host comprising the mutation.
  • a disruption in one or more endogenous transporter genes reduces or deletes transport expression or activity for the transporter encoded by the disrupted gene(s).
  • reduced expression refers to any level of expression that is less than that of a corresponding gene not having reduced expression, including“repressed expression,”“lowered expression,”“no expression,” and“deletion.”
  • reduced expression can be produced in a host cell by disrupting or deleting the gene locus of the one or more endogenous genes.
  • recombinant S. cerevisiae host cells comprising reduced expression of at least one endogenous transporter gene or a transcription factor that regulates expression of at least one endogenous transporter gene are capable of producing at least one benzylisoquinoline alkaloid such as, for example, (S)-norcoclaurine.
  • reducing endogenous transporter activity of a recombinant S. cerevisiae host cell increases the intracellular accumulation of one or more benzylisoquinoline alkaloid precursors (e.g ., dopamine) by the recombinant host, which can subsequently increase production of one or more benzylisoquinoline alkaloids derived therefrom.
  • benzylisoquinoline alkaloid precursors e.g ., dopamine
  • Recombinant S. cerevisiae hosts comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene, as disclosed herein, can include one or more
  • benzylisoquinoline alkaloid biosynthesis genes as disclosed herein, such as a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P; a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP; a gene encoding a polypeptide capable of synthesizing prephenate from chorismate; a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate; a gene encoding a polypeptide capable of synthesizing L-tyrosine from 4- HPP; a gene encoding a polypeptide capable of synthesizing 4-HPP from L-tyrosine; a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP; a gene encoding a polypeptide capable of synthesizing L-DOPA from L-
  • endogenous genes encoding one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor e.g., 4-HPAA
  • a disruption in one or more endogenous genes encoding one or more polypeptides capable of synthesizing 4-hydroxyphenylethanol (tyrosol) or 4- hydroxyphenylacetate (4-HPAC) from 4-HPAA reduces expression or activity for the one or more polypeptides encoded by the disrupted gene(s).
  • recombinant S As set forth herein, recombinant S.
  • cerevisiae host cells comprising reduced expression (i.e., repressed expression or deletion) of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing 4-HPAA, as disclosed herein, are capable of producing at least one benzylisoquinoline alkaloid such as, for example, (S)-norcoclaurine.
  • cerevisiae host cell increases the intracellular accumulation of one or more benzylisoquinoline alkaloid precursors (e.g., 4-HPAA) by the recombinant host, which can subsequently increase production of one or more benzylisoquinoline alkaloids derived therefrom.
  • benzylisoquinoline alkaloid precursors e.g., 4-HPAA
  • Recombinant S. cerevisiae hosts comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., 4-HPAA) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., 4-HPAA), as disclosed herein, can include one or more benzylisoquinoline alkaloid biosynthesis genes as disclosed herein, such as a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P; a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP; a gene encoding a polypeptide capable of synthesizing prephenate from
  • endogenous genes encoding one or more NCS-compatible substrate pathway polypeptides reduces or deletes expression or activity for the one or more polypeptides encoded by the disrupted gene(s).
  • a disruption in one or more endogenous genes encoding one or more polypeptides involved in tyrosine reduces or deletes expression or activity for the one or more polypeptides encoded by the disrupted gene(s).
  • phenylalanine, tryptophan, leucine, isoleucine, valine, or methionine biosynthetic reduces expression or activity for the one or more polypeptides encoded by the disrupted gene(s).
  • recombinant S. cerevisiae host cells comprising reduced expression (i.e., repressed expression or deletion) of at least one NCS-compatible substrate pathway
  • polypeptides disclosed herein are capable of producing at least one benzylisoquinoline alkaloid such as, for example, (S)-norcoclaurine.
  • reducing activity of one or more NCS-compatible substrate pathway polypeptides of a recombinant S. cerevisiae host cell decreases the intracellular accumulation of NCS-compatible substrates (e.g., AA, PAA, IA, 2- MB, 3-MP, acetoin, pyruvate) by the recombinant host, decreasing competition with 4-HPAA, and increasing the relative amount of intracellular dopamine available for condensation with 4- HPAA by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine.
  • NCS-compatible substrates e.g., AA, PAA, IA, 2- MB, 3-MP, acetoin, pyruvate
  • Recombinant S. cerevisiae hosts comprising reduced expression of at least one endogenous gene encoding one or more NCS-compatible substrate pathway polypeptides or a transcription factor gene that regulates expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide, as disclosed herein, can include one or more benzylisoquinoline alkaloid biosynthesis genes such as a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P; a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP; a gene encoding a polypeptide capable of synthesizing prephenate from chorismate; a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate; a gene encoding a polypeptide capable of synthesizing L-tyrosine from
  • Endogenous genes can be reduced, or endogenous genes can be deleted by mutations that disrupt the gene.
  • a gene replacement vector can be constructed in such a way to include a selectable marker gene flanked at both the 5' and 3' ends by portions of the gene of sufficient length to mediate homologous recombination.
  • the selectable marker can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change.
  • Linearized DNA fragments of the gene replacement vector, containing no plasmid DNA or an element, are then introduced into cells using known methods. Integration of the linear fragment into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, Southern blot analysis.
  • the resulting cells contain an inactivated mutant gene, due to insertion of the selectable marker at the locus for the polypeptide.
  • a deletion-disruption gene replacement vector can be constructed in a similar way using known techniques and, by way of homologous recombinant, integrated in the endogenous gene, thereby inactivating it.
  • the selectable marker can be removed from the genome of the host cell after determining that the desired disruption mutation has been introduced. See, e.g., Gossen et al. (2002) Ann. Rev. Genetics 36:153-173.
  • endogenous genes can also be reduced, or endogenous genes can be deleted by utilizing CRISPR systems (see, e.g., Adli,“The CRISPR tool kit for genome editing and beyond,” Nature Communications 9:1911 (2016)), transcription activator-like effector nucleases (TALENs) (see, e.g., Joung & Sander,“TALENs: a widely applicable technology for targeted genome editing,” Nat. Rev. Mol. Cell. Biol. 14(1 ):49-55 (2013)), or modified zinc finger nucleases (see, e.g., Carroll,“Genome Engineering With Zinc-Finger Nucleases,” Genetics 188(4):773-82 (2011 ) to introduce desired insertion or deletion mutations.
  • CRISPR systems see, e.g., Adli,“The CRISPR tool kit for genome editing and beyond,” Nature Communications 9:1911 (2016)
  • TALENs transcription activator-like effector nucleases
  • Joung & Sander “TALENs:
  • an endogenous gene is inactivated by introducing a mutation that results in insertions of nucleotides, deletions of nucleotides, or transition or transversion point mutations in the wild-type gene sequence.
  • Other types of mutations that may be introduced in a gene include duplications and inversions in the wild-type sequence. Mutations can be made in the coding sequence at a locus for the polypeptide, as well as in noncoding sequences such as regulatory regions, introns, and other untranslated sequences. Mutations in the coding sequence can result in insertions of one or more amino acids, deletions of one or more amino acids, and/or non-conservative amino acid substitutions in the corresponding gene product. In some cases, the sequence of a gene comprises more than one mutation or more than one type of mutation. Insertion or deletion of amino acids in a coding sequence can, for example, disrupt the conformation of a substrate-binding pocket of the resulting gene product.
  • Amino acid insertions or deletions can also disrupt catalytic sites important for gene product activity. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids.
  • Non-conservative substitutions can make a substantial change in the charge or hydrophobicity of the gene product.
  • Non-conservative amino acid substitutions can also make a substantial change in the bulk of the residue side chain, e.g., substituting an alanine residue for an isoleucine residue. Examples of non conservative substitutions include a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid.
  • a mutation in a gene may result in no amino acid changes but, although not affecting the amino acid sequence of the encoded polypeptide, may alter transcriptional levels (e.g., increasing or decreasing transcription), decrease translational levels, alter secondary structure of DNA or mRNA, alter binding sites for transcriptional or translational machinery, or decrease tRNA binding efficiency.
  • Mutations in loci for polypeptides can be generated by site-directed mutagenesis of the transporter gene sequence in vitro, followed by homologous recombination to introduce the mutation into the host genome as described above.
  • mutations can also be generated by inducing mutagenesis in cells of the host, using a mutagenic agent to induce genetic mutations within a population of cells. Mutagenesis is particularly useful for those species or strains for which in vitro mutagenesis and homologous recombination is less well established or is inconvenient.
  • the dosage of the mutagenic chemical or radiation for a particular species or strain is determined experimentally such that a mutation frequency is obtained that is below a threshold level characterized by lethality or reproductive sterility.
  • Modification of transcription factor expression can also be used to reduce or eliminate gene expression.
  • the yeast transcriptions factors PDR1 and/or PDR3 regulate expression of the genes encoding ABC transporters PDR5, SNQ2 and YOR1.
  • a benzylisoquinoline alkaloid-producing host comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene.
  • the endogenous transporter gene is PDR5, PDR12, PDR15, and/or SNQ2.
  • the endogenous transporter genes are PDR5 and one or more of PDR12, PDR15 and SNQ2.
  • the endogenous transporter genes are one or more of PDR5, PDR12, PDR15, and/or SNQ2 (e.g., PDR5 and PDR12), and one or more of AUS1 , PDR10, YOR1 , TP01 , TP02, TP03, TP04, QDR1 , QDR2, QDR3, FLR1 , YOL075C, PDR18, DTR1 , YHK8, NFT1 , STE6, YCF1 , YBT1 , BPT1 , and VMR1.
  • PDR5 and PDR12 e.g., PDR5 and PDR12
  • a benzylisoquinoline alkaloid-producing host comprises reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor, or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor.
  • the at least one endogenous gene encodes a polypeptide capable of synthesizing tyrosol or 4-HPAC from 4-HPAA, such as, for example, ARM , ALD4, ADH6, YPR1 , or YDR541 C.
  • the endogenous gene is ARM , ALD4, ADH6, YPR1 , YDR541 C, YGL039W,
  • a benzylisoquinoline alkaloid-producing host comprises reduced expression of at least one endogenous gene encoding one or more NCS-compatible substrate pathway polypeptides.
  • the NCS-compatible substrate pathway is an aromatic amino acid biosynthesis pathway, such as, for example, tryptophan (e.g., involving TRP2 and TRP3) or phenylalanine (e.g., involving PHA2) biosynthesis.
  • the NCS-compatible substrate pathway polypeptide is capable of synthesizing phenylpyruvate from prephenate (e.g., PHA2), capable of synthesizing anthranilate from chorismate (e.g., TRP2, and/or TRP3).
  • prephenate e.g., PHA2
  • chorismate e.g., TRP2, and/or TRP3
  • the PDR5 gene which can have the nucleotide sequence set forth in SEQ ID NO:69, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:70.
  • the PDR12 gene which can have the nucleotide sequence set forth in SEQ ID NO:71 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:72.
  • the PDR15 gene which can have the nucleotide sequence set forth in SEQ ID NO:73) encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:74.
  • the SNQ2 gene which can have the nucleotide sequence set forth in SEQ ID NO:75 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:76.
  • the PDR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:183, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 184.
  • the PDR3 gene which can have the nucleotide sequence set forth in SEQ ID NO: 185, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:186.
  • the AUS1 gene which can have the nucleotide sequence set forth in SEQ ID NO:77 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:78.
  • the PDR10 gene which can have the nucleotide sequence set forth in SEQ ID NO:79, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:80.
  • the YOR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:81 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:82.
  • the TP01 gene which can have the nucleotide sequence set forth in SEQ ID NO:83 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:84.
  • the TP02 gene which can have the nucleotide sequence set forth in SEQ ID NO:85 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:86.
  • the TP03 gene which can have the nucleotide sequence set forth in SEQ ID NO:87, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:88.
  • the TP04 gene which can have the nucleotide sequence set forth in SEQ ID NO:89, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:90.
  • the QDR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:91 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:92.
  • the QDR2 gene which can have the nucleotide sequence set forth in SEQ ID NO:93, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:94.
  • the QDR3 gene which can have the nucleotide sequence set forth in SEQ ID NO:95, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:96.
  • the FLR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:97, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:98.
  • the YOL075C gene which can have the nucleotide sequence set forth in SEQ ID NO:99, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:100.
  • the PDR18 gene which can have the nucleotide sequence set forth in SEQ ID NO:101 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:102.
  • the DTR1 which can have the nucleotide sequence set forth in SEQ ID NO: 103, gene encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:104.
  • the YHK8 gene which can have the nucleotide sequence set forth in SEQ ID NO:105, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:106.
  • the NFT 1 gene which can have the nucleotide sequence set forth in SEQ ID NO:107, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 108.
  • the STE6 gene which can have the nucleotide sequence set forth in SEQ ID NO:109, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:1 10.
  • the YCF1 gene which can have the nucleotide sequence set forth in SEQ ID NO:1 1 1 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:1 12.
  • the YBT 1 gene which can have the nucleotide sequence set forth in SEQ ID NO: 113 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1 14.
  • the BPT2 gene which can have the nucleotide sequence set forth in SEQ ID NO:1 15 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:1 16.
  • the VMR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:1 17 encodes a polypeptide having an amino acid sequence set as forth in SEQ ID NO:1 18.
  • the ARI1 gene which can have the nucleotide sequence set forth in SEQ ID NO:145, encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO: 146.
  • the ALD4 gene which can have the nucleotide sequence set forth in SEQ ID NO:123, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:124.
  • the ADH6 gene which can have the nucleotide sequence set forth in SEQ ID NO: 139, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 140.
  • the YPR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:153, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 154.
  • the YDR541 C gene which can have the nucleotide sequence set forth in SEQ ID NO:149 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 150.
  • the PHA2 gene which can have the nucleotide sequence set forth in SEQ ID NO: 163 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 164.
  • the TRP2 gene which can have the nucleotide sequence set forth in SEQ ID NO:165 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 166.
  • the TRP3 gene which can have the nucleotide sequence set forth in SEQ ID NO:167, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:168.
  • the AAD3 gene which can have the nucleotide sequence set forth in SEQ ID NO:159, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:160.
  • the GRE2 gene which can have the nucleotide sequence set forth in SEQ ID NO: 151 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 152.
  • benzylisoquinoline alkaloids and/or precursors thereof are produced in vivo through expression of one or more recombinant genes encoding one or more benzylisoquinoline biosynthesis polypeptides in a recombinant host comprising reduced expression of one or more endogenous genes.
  • a recombinant S for example, a recombinant S.
  • cerevisiae host comprising reduced expression of at least one endogenous transporter gene (e.g ., PDR5, PDR12, PDR15, and/or SNQ2) or a transcription factor that regulates expression of at least one endogenous transporter gene (e.g., PDR1 and/or PDR3), and further comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephen
  • the recombinant S. cerevisiae host comprises reduced expression of one, or two, or three, or each of PDR5 (SEQ ID NO:69, SEQ ID NO:70), PDR12 (SEQ ID NO:71 , SEQ ID NO:72), PDR15 (SEQ ID NO:73, SEQ ID NO:74), and SNQ2 (SEQ ID NO:75, SEQ ID NO:76).
  • the polypeptide capable of synthesizing (S)- norcoclaurine from 4-HPAA and dopamine has the amino acid sequence set forth in any of SEQ ID NO:42 or 30.
  • a recombinant S. cerevisiae host comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., ARM , ALD4, ADH6, YPR1 , AAD3,
  • GRE2 GRE2, and/or YDR541 C
  • a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor, and further comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephenate from chorismate (e.g., a recombinant gene encoding
  • the recombinant S. cerevisiae host comprises reduced expression of one, or two, or three, or four, or five, or six, or each of ARM (SEQ ID NO:145, SEQ ID NO:146), ALD4 (SEQ ID NO:123, SEQ ID NO:124), ADH6 (SEQ ID NO:139, SEQ ID NO:140), YPR1 (SEQ ID NO:153, SEQ ID NO:154), AAD3 (SEQ ID NO:159, SEQ ID NO: 160), GRE2 (SEQ ID NO: 151 , SEQ ID NO:152), and YDR541 C (SEQ ID NO:149, SEQ ID NO:150).
  • ARM SEQ ID NO:145, SEQ ID NO:146
  • ALD4 SEQ ID NO:123, SEQ ID NO:124
  • ADH6 SEQ ID NO:139, SEQ ID NO:140
  • YPR1 SEQ ID NO:153, SEQ ID NO:154
  • AAD3 SEQ ID NO:159,
  • the recombinant S. cerevisiae host comprises reduced expression of one, or two, or three, or four, or five, or six, or each of ARM (SEQ ID NO:145, SEQ ID NO: 146), ALD4 (SEQ ID NO:123, SEQ ID NO:124), ADH6 (SEQ ID NO:139, SEQ ID NO:140), YPR1 (SEQ ID NO:153, SEQ ID NO:154), AAD3 (SEQ ID NO: 159, SEQ ID NO:160), GRE2 (SEQ ID NO: 151 , SEQ ID NO:152), and YDR541C (SEQ ID NO: 149, SEQ ID NO:150), and further comprises reduced expression of one, or two, or each of TRP2 (SEQ ID NO:165, SEQ ID NO: 166), TRP3 (SEQ ID NO: 167, SEQ ID NO: 168), and PHA2 (SEQ ID NO: 163, SEQ ID NO:164).
  • ARM SEQ ID NO:
  • the polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine is a truncated polypeptide (e.g., lacking an N-terminal signal peptide; e.g. having an amino acid sequence as set forth in SEQ ID NO:42) .
  • the polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine is a polypeptide having an amino acid sequence as set forth in SEQ ID NO:30.
  • a recombinant S. cerevisiae host comprising reduced expression of at least one endogenous transporter gene (e.g ., PDR5, PDR12, PDR15, and/or SNQ2) or a transcription factor that regulates expression of at least one endogenous transporter gene, further comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., ARM , ALD4, ADH6, YPR1 , AAD3, GRE2, and/or YDR541 C) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor and/or reduced expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide (e.g., PHA2, TRP2, and/or T
  • expression of one or more recombinant genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA e.g., a polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, and 198
  • S synthesizing
  • cerevisiae host comprising reduced expression of one or more endogenous transporter genes (e.g., PDR5, PDR12, PDR15, and/or SNQ2) or a transcription factor that regulates expression of at least one endogenous transporter gene (e.g., PDR1 and/or PDR3) increases the amount of (S)- norcoclaurine produced by the host cell by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%.
  • endogenous transporter genes e.g., PDR5, PDR12, PDR15, and/or SNQ2
  • a transcription factor that regulates expression of at least one endogenous transporter gene e.g., PDR1 and/or
  • expression of one or more recombinant genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA e.g ., a polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, and 198
  • S synthesizing
  • cerevisiae host comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., ARM , ALD4, ADH6, YPR1 , AAD3, GRE2, and/or YDR541 C) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor and/or reduced expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide (e.g., PHA2, TRP2, and/or TRP3) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide, increases the amount of (S)-norcoclaurine produced by the host cell by at least about 50%, or at least about 100%, or at least about 200%, or at least about
  • a recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired.
  • a coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence.
  • the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.
  • the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid.
  • the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism.
  • a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct.
  • stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found.
  • Regulatory region refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product.
  • Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5 ' and 3 ' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof.
  • a regulatory region typically comprises at least a core (basal) promoter.
  • a regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • a regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence.
  • the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter.
  • a regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
  • regulatory regions The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
  • One or more genes can be combined in a recombinant nucleic acid construct in “modules” useful for a discrete aspect of production of benzylisoquinoline alkaloids and/or precursors thereof. Combining a plurality of genes in a module, particularly a polycistronic module, facilitates the use of the module in a variety of species.
  • a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species.
  • nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid.
  • codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host ⁇ e.g.,
  • these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
  • Recombinant S. cerevisiae hosts can be used to express polypeptides as otherwise described herein for producing benzylisoquinoline alkaloids.
  • the recombinant host is grown in a fermenter at a temperature for a period of time, wherein the temperature and period of time facilitate production of one or more benzylisoquinoline alkaloids.
  • the constructed and genetically engineered S. cerevisiae hosts provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, semi-continuous fermentations such as draw and fill, continuous perfusion fermentation, and continuous perfusion cell culture.
  • Levels of substrates and intermediates e.g., E4P, DAHP, EPSP, chorismate, prephenate, 4-HPP, 4-HPAA, L-tyrosine, L-DOPA, and dopamine, can be determined by extracting samples from culture media for analysis according to published methods.
  • Carbon sources of use in the instant method include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the benzylisoquinoline alkaloids.
  • suitable carbon sources include sucrose, fructose, xylose, ethanol, glycerol, and glucose.
  • the carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during a fed-batch phase.
  • the present invention further relates to a fermentation process, including but not limited to small-scale or batch and/or fed-batch fermentation process and a large-scale fermentation process, for the production of (S)-norcoclaurine, as described herein, whereby the cultivation regime of the process comprises at least one production stage fermentation phase.
  • the fermentation process is a batch cultivation.
  • the fermentation process is a fed-batch process.
  • a base medium supports initial growth and production, and a continuously or periodically supplied feeding medium prevents depletion of nutrients and sustains the production stage.
  • the media can be selected to accommodate the distinct metabolic requirements during different cultivation phases.
  • Process parameters including feeding strategy and control parameters define the chemical and physical environments suitable for cell growth and/or (S)-norcoclaurine production.
  • Benzylisoquinoline alkaloids and precursors thereof can be isolated from a cell culture using a method described herein. For example, following fermentation, cultures were diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose. After 72-96 h growth, metabolites were extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice- cold 100% acetonitrile (ACN; 80% final concentration). Cells were incubated for 5 minutes and subsequently diluted with 417 mI of 0.16% formic acid (FA) to give a final concentration of 30% ACN and 0.1 % FA. The resulting extract was utilized for LC-MS analysis. Cell broth was diluted 13.34-fold using this method.
  • Fluorescence levels were measured after cultivating strains overnight in SC with 2% glucose. Overnight cultures were diluted 10* into fresh media and incubated for an additional 4 h to obtain log phase cells. Fluorescence was measured from cell suspension using a microplate reader and normalized against OD 6 oo for three biological replicates. Fluorescence was detected by the TECAN M200 plate reader using an excitation wavelength of 485 nm and an emission wavelength of 525 nm. Gain was adjusted for each sample. A background strain lacking GFP was used to correct for autofluorescence generated by cells and media.
  • Dopamine-producing S. cerevisiae strains comprising and expressing a recombinant gene encoding a feedback-resistant variant of DAHP synthase polypeptide (SEQ ID NO:3, SEQ ID NO:4), a recombinant gene encoding a tyrosine hydroxylase polypeptide (SEQ ID NO:21 , SEQ ID NO:22), and a recombinant gene encoding an L-DOPA decarboxylase polypeptide
  • a dopamine-producing S. cerevisiae strain as described in Example 3 was transformed with a vector comprising a codon-optimized nucleotide sequence encoding an NCS polypeptide, optionally N-terminal truncated to remove a signal peptide, C-terminal truncated to remove a transmembrane domain, or C-terminal domain-swapped, and/or optionally green fluorescent protein (GFP)-tagged, operably linked to a pTEF1 promoter (SEQ ID NO:173 and a tPGM terminator (SEQ ID NO:174), as summarized in Table 1 , below.
  • GFP green fluorescent protein
  • Dopamine-producing S. cerevisiae cells were cultivated in YPD medium (10 g/L yeast extract, 20 g/L tryptone, 20 g/L dextrose; Thermo Fisher Scientific) for routine strain growth and maintenance.
  • YPD was supplemented, if necessary, with 200 pg/ml G418 or 200 pg/nnl hygromycin B. G418 concentration was increased to 400 pg/ml for selection on agar- solidified YPD.
  • E. coli DH5a was utilized for routine plasmid maintenance and propagation and was cultivated in Lysogeny Broth (LB) (Sigma Aldrich). Ampicillin or kanamycin at
  • Colonies were picked in triplicate and inoculated into 500 pi of 2* synthetic complete (SC) medium containing 4% glucose or sucrose in 96-well two ml deep well plates.
  • the present inventors have determined that sucrose, in place of glucose, increases production of metabolites derived from the shikimate pathway by 50%.
  • cultures were diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose.
  • metabolites were extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice-cold 100% acetonitrile (ACN; 80% final concentration).
  • a dopamine-producing S. cerevisiae strain as described in Example 3, further engineered to express at least one (S)-norcoclaurine synthase as described in Example 4 (SEQ ID NO:31 , SEQ ID NO:32; SEQ ID NO:41 , SEQ ID NO:42) was engineered to downregulate expression of one or more native genes encoding dehydrogenase and reductase polypeptides having potential activity on one or more benzylisoquinoline alkaloid precursors, and optionally to overexpress one or more benzylisoquinoline alkaloid biosynthesis polypeptides, as summarized in Table 4, below.
  • Colonies were picked in triplicate and inoculated into 500 pi of 2* synthetic complete (SC) medium containing 4% glucose or sucrose in 96-well two ml deep well plates.
  • the present inventors have determined that sucrose, in place of glucose, increases production of metabolites derived from the shikimate pathway by 50%.
  • cultures were diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose.
  • metabolites were extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice-cold 100% acetonitrile (ACN; 80% final concentration).
  • S. cerevisiae genes with potential activity for 4-HPAA were targeted for deletion (strains 20-40) including all 5 aldehyde dehydrogenase genes (ALD2, ALD3, ALD4, ALD5, and ALD6), all 7 primary alcohol dehydrogenase genes (ADH1-ADH7), the gene encoding formaldehyde dehydrogenase (SFA1 ), and 7 genes similar to ARI1 (YGL039W, YDR541C, YPR1 , GCY1 , GRE2, AAD3, and AAD14).
  • S formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenas
  • Strain 32 showed a 3.5-fold increase in (S)-norcoclaurine production.
  • Strains 21 and 23 demonstrated increased dopamine formation; strain 21 (lacking ALD6) showed a 2.8-fold increase.
  • Strain 40 produced no dopamine or (S)-norcoclaurine.
  • strains further overexpressing N-terminal truncated NdNCS and lacking the combination of ARM , ALD4, ADH6, YPR1 showed no additional improvement over strain 66, similarly overexpressing N-terminal truncated NdNCS and lacking the combination of ARM , ALD4, ADH6, YPR1 , and YDR541 C.
  • strain 66 demonstrated a 121-fold increase of (S)-norcoclaurine production. See Figure 9.
  • LC-MS spectra were examined for peaks indicating off-target NCS condensation products by first searching for 35 unique m/z values derived from condensation of dopamine with 63 possible endogenous aldehydes and ketones, including aliphatic, aromatic, and cyclic species. From this analysis, six putative Pictet-Spengler-competent carbonyl species were identified. See Figure 10. A major peak consistent with salsolinol (1 ), derived from condensation of dopamine with acetaldehyde, was observed and was larger than the (S)-norcoclaurine peak in all LC-MS spectra.
  • PAA and IAA aromatic Ehrlich pathway aldehydes
  • deletion of ALD4 enhanced formation of all condensation products by a factor of 2.2-10, likely due to increased levels of dopamine and presumably higher flux through the aromatic amino acid (AAA) pathway.
  • strain 87 a strain having inactivated phenylalanine and tryptophan biosynthesis polypeptides was further engineered to express eight copies of the recombinant gene encoding N-terminal truncated NdNCS of Example 4 (strain 87). Further overexpression of a chorismate synthase polypeptide in strain 88 led to an increase in production of (S)-norcoclaurine relative to strain 87. Of strains 89-93, further engineered to delete, respectively, ADH5, AAD14, ADH3, YGL029W, or AAD3, strain 93 showed the greatest increase in production of (S)-norcoclaurine relative to strain 88. See Figures 22-23.
  • Strains overexpressing N-terminal truncated NdNCS, Tyr1 , Aro7, Aro10, and Aro2, and lacking ARM , ALD4, ADH6, YPR1 , YDR541C, PHA2, TRP3, and AAD3, were further engineered to delete AAD4, ADH7, SFA1 , YGL039, AAD14, GRE2, GCY1 , or ALD2 and ALD3 to provide, respectively, strains 94-101.
  • strain 99 demonstrated an 82% decrease in tyrosol production and a 97% increase in (S)-norcoclaurine production relative to strain 93. See Figures 24-25.
  • aldo-keto reductases e.g., ARM , ADH6, YPR1 , YDR541C, AAD3, GRE2
  • tyrosol formation i.e., from 4-HPAA
  • deletion of genes not directly related tyrosol formation ⁇ e.g., ALD4, PHA2, TRP3
  • ALD4, PHA2, TRP3 can further increase carbon flux to L-tyrosine.
  • Strains 67 and 68 of Example 5 were each cultivated in a fed-batch bioreactor. Controlled fed-batch cultivations were carried out in a 3 L BioBundle fermenter (Applikon) equipped with d0 2 , pH, and temperature sensors. Cultivation was controlled using an EZ- Control bio-controller and BioXpert cultivation control software (Applikon). A variable speed pump (Watson-Marlow) was used to supply the feeding medium. Culture pH was maintained at 4.5 by titration with 3 M NaOH and 3 N H 2 S0 4 . Dissolved oxygen was maintained at 10 % of air saturation by automatically adjusting the stirring rate (aeration rate 0.5 L/min). Cultivation temperature was maintained at 30 °C.
  • the batch medium contained per L: 2.5 g KH 2 P0 4 , 5 g NH CI, 1 g MgS0 4 -7H 2 0, 5 ml vitamin stock, 5 ml microelement stock, 1.6 g yeast synthetic drop-out medium without leucine, 380 mg L-leucine, and (i) 40 g sucrose or (ii) 30 g glycerol.
  • Vitamin stock contained per L: 5 mg biotin, 100 mg calcium pantothenate, 100 mg nicotinic acid, 2,500 mg myo-inositol, 100 mg thiamine hydrochloride, 100 mg pyridoxine, and 20 mg p-aminobenzoic acid.
  • Microelement stock contained per L: 15 g disodium EDTA, 2.9 g CaCI 2 , 9.2 g ZnS0 4 -7H 2 0, 5.1 g FeS0 4 -7H 2 0, 0.5 g CuS0 4 , 0.43 g MnS0 4 H 2 0, 0.47 g CoCI 2 , and 0.48 g Na 2 Mo0 4 .
  • the culture was grown in batch mode for 16 to 96 hours.
  • the fed-batch phase was carried out with a constant feeding rate of 3.5 mL/h.
  • the feeding medium contained per L: 10 g KH 2 P0 4 , 40 g NH 4 CI, 4 g
  • MgS0 -7H 2 0, 10 ml vitamin stock, 10 mL microelement stock, 400 mg yeast synthetic drop-out medium without leucine, 2.5 g L-leucine, 810 mg L-histidine, 570 mg L-methionine, 800 mg uracil, and 240 g sucrose.
  • the total cultivation time was seven to ten days and samples (5 mL) were collected daily.
  • Cell densities were determined by dilution of fermentation broth with water followed by measurement of absorption at 600 nm (OD 6 oo). Dry cell weight (g/L) was calculated using a conversion factor of 0.59 g/L per OD 6 oo (determined gravimetrically). Culture broths were analyzed for metabolite production according to Example 1.
  • Results are shown in Figures 19-20.
  • the batch medium for strain 67 contained sucrose as a carbon source.
  • the cells were unable to metabolize the ethanol produced during the batch phase of the cultivation, ultimately leading to growth arrest. See Figure 19. Under these conditions, this strain produced 215 mg/L (S)-norcoclaurine. See Figure 20.
  • glycerol was used as a carbon source in the batch medium of strain 68. Ethanol levels remained low during the fed-batch phase, and the strain produced 383 mg/L (S)- norcoclaurine. See Figure 20.
  • An (S)-norcoclaurine stock solution was prepared in DMSO at a concentration of 10 mM. Standard solutions were prepared at concentrations of 4 mM, 2 mM, 1 mM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, and 10 nM from the stock solution. Samples were injected into an Agilent 1290 UPLC coupled to an Ultivo Triple Quadrupole. The LC-MS method was as follows: Mobile Phase A. H 2 0 + 0.1 % Formic acid; Mobile Phase B: Acetonitrile + 0.1 % Formic acid; Column: Phenomenex Kinetex 1.7 pm XB-C18 100A, 2.1x100mm.
  • Table 5 shows the elution gradient and the LC-MS conditions are given in Table 6.
  • Table 7 shows the mass spectrometer source and detector parameters and Table 8 shows the target compound, retention time, parent ion, transition ions (MRM) as well as dwell time, fragmentor voltage and collision energy used.
  • MRM transition ions
  • (S)-norcoclaurine-producing S. cerevisiae strains comprising and expressing a recombinant gene encoding a DAHP synthase polypeptide (SEQ ID NO:3, SEQ ID NO:4), operably linked to a pPDC1 promoter (SEQ ID NO:169) and a tCYC1 terminator (SEQ ID NO:175); a recombinant gene encoding a tyrosine hydroxylase polypeptide (SEQ ID NO:SEQ ID NO:23, SEQ ID NO:24), operably linked to a pTEF2 promoter (SEQ ID NO:170) and a tFBA1 terminator (SEQ ID NO: 176); a recombinant gene encoding an L-DOPA decarboxylase polypeptide (SEQ ID NO:SEQ ID NO:27, SEQ ID NO:28), operably linked to a pTDH3 promoter (SEQ ID NO
  • Yeast transformants were grown in 96-deep well plates in 500 pL liquid synthetic complete media for 3 days at 30 °C with shaking at 230 rpm in a Kuhner Climo-Shaker ISF1-X.
  • Culture samples for LC-MS were prepared by extraction as follows: 96% ethanol and culture sample were mixed 1 :1 and incubated on a heating block at 80 °C for 10 minutes. After heating, cells were pelleted in an Eppendorff tabletop centrifuge and the supernatant was then transferred to a new tube and diluted 1 :10 in water.
  • (S)-norcoclaurine-producing S. cerevisiae strains comprising and expressing a recombinant gene encoding a DAHP synthase polypeptide (SEQ ID NO:3, SEQ ID NO:4), a recombinant gene encoding a tyrosine hydroxylase polypeptide (SEQ ID NO:21 , SEQ ID NO:22; SEQ ID NO:23, SEQ ID NO:24), a recombinant gene encoding an L-DOPA decarboxylase polypeptide (SEQ ID NO:25, SEQ ID NO:26; SEQ ID NO:27, SEQ ID NO:28), a recombinant gene encoding a prephenate dehydrogenase (SEQ ID NO:13, SEQ ID NO:14), a recombinant gene encoding a phenylpyruvate decarboxylase (SEQ ID NO:19, SEQ ID NO:20), and/or
  • strains are further engineered to comprise reduced expression of one or more endogenous transporter genes PDR5 (SEQ ID NO:69, SEQ ID NO:70), PDR12 (SEQ ID NO:71 , SEQ ID NO:72), PDR15 (SEQ ID NO:73, SEQ ID NO:74), SNQ2 (SEQ ID NO:75, SEQ ID NO:76), and/or one or more endogenous transporter genes AUS1 (SEQ ID NO:77, SEQ ID NO:78), PDR10 (SEQ ID NO:79, SEQ ID NO:80), YOR1 (SEQ ID NO:81 , SEQ ID NO:82),
  • TP01 (SEQ ID NO:83, SEQ ID NO:84), TP02 (SEQ ID NO:85, SEQ ID NO:86), TP03 (SEQ ID NO:87, SEQ ID NO:88), TP04 (SEQ ID NO:89, SEQ ID NO:90), QDR1 (SEQ ID NO:91 , SEQ ID NO:92), QDR2 (SEQ ID NO:93, SEQ ID NO:94), QDR3 (SEQ ID NO:95, SEQ ID NO:96), FLR1 (SEQ ID NO:97, SEQ ID NO:98), YOL075C (SEQ ID NO:99, SEQ ID NO:100), PDR18 (SEQ ID NO: 101 , SEQ NO:102), DTR1 (SEQ ID NO:103, SEQ ID NO:104), YHK8 (SEQ ID NO:105, SEQ ID NQ:106), NFT1 (SEQ ID NQ:107, SEQ ID NQ: 108), STE6 (SEQ ID NQ:109
  • BPT1 SEQ ID NO:1 15, SEQ ID NO:1 16
  • VMR1 SEQ ID NO:1 17, SEQ ID NO: 1 18
  • the strains are further engineered to comprise reduced expression of one or more endogenous genes ARI1 (SEQ ID NO:145, SEQ ID NO:146), ALD4 (SEQ ID NO:123, SEQ ID NO:124), ADH6 (SEQ ID NO:139, SEQ ID NO: 140), YPR1 (SEQ ID NO:153, SEQ ID NO:154), YDR541C (SEQ ID NO: 149, SEQ ID NO:150), PHA2 (SEQ ID NO:163, SEQ ID NO: 164), TRP2 (SEQ ID NO: 165, SEQ ID NO:166), and TRP3 (SEQ ID NO: 167, SEQ ID NO:168).
  • ARI1 SEQ ID NO:145, SEQ ID NO:146
  • ALD4 SEQ ID NO:123, SEQ ID NO:124
  • ADH6 SEQ ID NO:139, SEQ ID NO: 140
  • YPR1 SEQ ID NO:153, SEQ ID NO:154
  • YDR541C S
  • Colonies of the (S)-norcoclaurine-producing strains as described in Example 10 are picked in triplicate and inoculated into 500 pi of 2* synthetic complete (SC) medium containing 4% glucose or sucrose in 96-well two ml deep well plates.
  • SC synthetic complete
  • the present inventors have determined that sucrose, in place of glucose, increases production of metabolites derived from the shikimate pathway by 50%.
  • cultures are diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose.
  • metabolites are extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice- cold 100% acetonitrile (ACN; 80% final concentration). Cells are incubated for 5 minutes and subsequently diluted with 417 pi of 0.16% formic acid (FA) to give a final concentration of 30% ACN and 0.1 % FA. The resulting extract is utilized for LC-MS analysis according to Example 1. Cell broth is diluted 13.34-fold using this method.
  • Strains as described in Example 10 are each cultivated in a fed-batch bioreactor. Controlled fed-batch cultivations are carried out in a 3 L BioBundle fermenter (Applikon) equipped with d0 2 , pH, and temperature sensors. Cultivation is controlled using an EZ-Control bio-controller and BioXpert cultivation control software (Applikon). A variable speed pump (Watson-Marlow) s used to supply the feeding medium. Culture pH is maintained at 4.5 by titration with 3 M NaOH and 3 N H 2 SO 4 . Dissolved oxygen is maintained at 10 % of air saturation by automatically adjusting the stirring rate (aeration rate 0.5 L/min). Cultivation temperature is maintained at 30 °C.
  • the batch medium contains per L: 2.5 g KH 2 PO 4 , 5 g NH 4 CI, 1 g MgS0 4 -7H 2 0, 5 ml vitamin stock, 5 ml microelement stock, 1.6 g yeast synthetic drop-out medium without leucine, 380 mg L-leucine, and (i) 40 g sucrose or (ii) 30 g glycerol.
  • Vitamin stock contains per L: 5 mg biotin, 100 mg calcium pantothenate,
  • Microelement stock contains per L: 15 g disodium EDTA, 2.9 g CaCh, 9.2 g ZnS0 4 -7H 2 0, 5.1 g FeS0 4 -7H 2 0, 0.5 g CuS0 4 , 0.43 g MnS0 4 H 2 0, 0.47 g C0CI 2 , and 0.48 g Na 2 Mo0 4 .
  • the culture is grown in batch mode for 16 to 96 hours.
  • the fed-batch phase is carried out with a constant feeding rate of 3.5 mL/h.
  • the feeding medium contains per L: 10 g KH 2 PO 4 , 40 g NH 4 CI, 4 g MgS0 4 -7H 2 0, 10 ml vitamin stock, 10 mL microelement stock, 400 mg yeast synthetic drop-out medium without leucine, 2.5 g L-leucine, 810 mg L-histidine, 570 mg L-methionine, 800 mg uracil, and 240 g sucrose.
  • the total cultivation time is seven to ten days and samples (5 mL) are collected daily. Cell densities are determined by dilution of fermentation broth with water followed by measurement of absorption at 600 nm (OD 6 oo). Dry cell weight (g/L) is calculated using a conversion factor of 0.59 g/L per OD 6 OO (determined gravimetrically). Culture broths are analyzed for metabolite production according to Example 1.
  • NCS enzymes inherently are promiscuous and can perform the Pictet-Spengler reaction with aldehydes other than 4-HPAA, resulting in unwanted side products and carbon loss.
  • GLINDPDVNN TFNINKGLQS ARQLFVNLTN IGLP IGSEML DTISPQYLAD LVSFGAIGAR 180
  • MAPVTIEKFV NQEERHLVSN RSATIPFGEY IFKRLLSIDT KSVFGVPGDF NLSLLEYLYS 60 PSVESAGLRW VGTCNELNAA YAADGYSRYS NKIGCLITTY GVGELSALNG IAGSFAENVK 120 VLHIVGVAKS IDSRSSNFSD RNLHHLVPQL HDSNFKGPNH KVYHDMVKDR VACSVAYLED 180 IETACDQVDN VIRDIYKYSK PGYIFVPADF ADMSVTCDNL VNVPRISQQD CIVYPSENQL 240 SDI INKITSW IYSSKTPAIL GDVLTDRYGV SNFLNKLICK TGIWNFSTVM GKSVIDESNP 300 TYMGQYNGKE GLKQVYEHFE LCDLVLHFGV DINEINNGHY TFTYKPNAKI IQFHPNYIRL 360 VDTRQGNEQM FKGINFAP IL KELYKRIDV
  • MDHATLAMIL AILFISFHFI KLLFSQQTTK LLPPGPKPLP I IGNILEVGK KPHRSFANLA 60 KIHGPLISLR LGSVTTIVVS SADVAKEMFL KKDHPLSNRT IPNSVTAGDH HKLTMSWLPV 120 SPKWRNFRKI TAVHLLSPQR LDACQTFRHA KVQQLYEYVQ ECAQKGQAVD IGKAAFTTSL 180 NLLSKLFFSV ELAHHKSHTS QEFKELIWNI MEDIGKPNYA DYFP ILGCVD PSGIRRRLAC 240 SFDKLIAVFQ GIICERLAPD SSTTTTTTTD DVLDVLLQLF KQNELTMGEI NHLLVDIFDA 300 GTDTTSSTLE WVMTELIRNP EMMEKAQEEI KQVLGKDKQI QESDIINLPY LQAI IKETLR 360 LHPPTVFLLP RKADTDVELY GYIVPKDAQI LVNLWAIG
  • MDHATLAMIL AILFISFHFI KLLFSQQTTK LLPPGPKPLP I IGNILEVGK KPHRSFANLA 60 KIHGPLISLR LGSVTTIVVS SADVAKEMFL KKDHPLSNRT IPNSVTAGDH HKLTMSWLPV 120 SPKWRNFRKI TAVHLLSPQR LDACQTFRHA KVQQLYEYVQ ECAQKGQAVD IGKAAFTTSL 180 NLLSKLFFSV ELAHHKSHTS QEFKELIWNI MEDIGKPNYA DYFP ILGCVD PSGIRRRLAC 240 SFDKLIAVFQ GIICERLAPD SSTTTTTTTD DVLDVLLQLF KQNELTMGEI NHLLVDIFDA 300 GTDTTSSTLE WVMTELIRNP EMMEKAQEEI KQVLGKDKQI QESDIINLPY LQAI IKETLR 360 LHPPTVFLLP RKADTDVELY GYIVPKDAQI LVNLWAIG
  • KIVLENKAKH KGFIEIGMSK GEELFTGWP ILVELDGDVN GHKFSVSGEG EGDATYGKLT 240
  • Atgtcatcag atatcagaga cgtagaggaa cgaaattcgc ggagctcgag ctcaagctcg 60 agctcgaact ctgccgccca atccattgga cagcatccat accgcggttt cgacagcgaa 120 gccgcggaaa gggtgcatga gttggctaga acactcacat cgcagagttt actatacact 180 gctaactcaa acaatagctc ttccagcaac cataatgcgc acaatgcgga ctcgagatcc 240 gtattttcta cggacatgga aggtgtgaac ccggtgttca ctaacccgga caccccggga 300 tacaatccca a
  • MSSDIRDVEE RNSRSSSSSS SSNSAAQSIG QHPYRGFDSE AAERVHELAR TLTSQSLLYT 60 ANSNNSSSSN HNAHNADSRS VFSTDMEGVN PVFTNPDTPG YNPKLDPNSD QFSSTAWVQN 120 MANICTSDPD FYKPYSLGCV WKNLSASGDS ADVSYQSTFA NIVPKLLTKG LRLLKPSKEE 180 DTFQILKPMD GCLNPGELLV VLGRPGSGCT TLLKSISSNS HGFKIAKDSI VSYNGLSSSD 240 IRKHYRGEW YNAESDIHLP HLTVYQTLFT VARMKTPQNR IKGVDREAYA NHVTEVAMAT 300 YGLSHTRDTK VGNDLVRGVS GGERKRVSIA EVAICGARFQ CWDNATRGLD SATALEFIRA 360 LKTQAD IGKT AATVAIYQCS QDAYDLFDKV CVLDDGYQLY F
  • MSQQENGDVA TELIENRLSF SRIPRISLHV RDLSIVASKT NTTLVNTFSM DLPSGSVMAV 60 MGGSGSGKTT LLNVLASKIS GGLTHNGSIR YVLEDTGSEP NETEPKRAHL DGQDHPIQKH 120 VIMAYLPQQD VLSPRLTCRE TLKFAADLKL NSSERTKKLM VEQLIEELGL KDCADTLVGD 180 NSHRGLSGGE KRRLSIGTQM ISNPSIMFLD EPTTGLDAYS AFLVIKTLKK LAKEDGRTFI 240 MSIHQPRSDI LFLLDQVCIL SKGNWYCDK MDNTIPYFES IGYHVPQLVN PADYFIDLSS 300 VDSRSDKEEA ATQSRLNSLI DHWHDYERTH LQLQAESYIS NATEIQIQNM TTRLPFWKQV 360 TVLTRRNFKL NFSDYVTLIS TFAEPLI IGT VCGWIYYKPD KSS
  • GVEMANSSEF GLGSGIETES LSTGLKVAKM LKAGTVWINT YNDFDSRVPF
  • HGVINVSVSE AAIEASTRYV RANGTTVLVG MPAGAKCCSD VFNQWKSIS IVGSYVGNRA 300
  • HGI INVSVSE AAIEASTRYC RANGTWLVG LPAGAKCSSD VFNHWKSIS IVGSYVGNRA 300
  • PAWWSAANY YATSYGKTPF SIYQGKWNVL NRDFERDIIP MARHFGMALA PWDVMGGGRF 240
  • HGVINVSVSE AAIEASTRYV RANGTTVLVG MPAGAKCCSD VFNQWKSIS IVGSYVGNRA 300

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Abstract

The invention relates to recombinant microorganisms and methods for producing benzylisoquinoline alkaloids and precursors thereof.

Description

PRODUCTION OF BENZYLISOQUINOLINE ALKALOIDS IN RECOMBINANT HOSTS
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] This disclosure relates to recombinant production of benzylisoquinoline alkaloids in recombinant hosts. In particular, this disclosure relates to production of (S)-norcoclaurine and derivatives thereof in Saccharomyces cerevisiae.
Description of Related Art
[0002] (S)-norcoclaurine synthase (NCS) is an enzyme involved in committing and rate- limiting steps of benzylisoquinoline (BIA) biosynthesis. During biosynthesis, two precursor molecules, 4-hydroxyphenylacetaldehyde (4-HPAA) and dopamine are condensed in a Pictet- Spengler reaction to form (S)-norcoclaurine. See Figure 1.
[0003] Generally, (S)-norcoclaurine synthase is a catalytically inefficient enzyme, having Km values in the double-digit millimolar range. In yeast strains producing norcoclaurine, the molar concentration of produced norcoclaurine is about 100 times lower than the concentration of the precursor dopamine. Narcross et al., Trends Biotechnol. 34:228-41 (2016); DeLoache et al., Nat. Chem. Biol. 11 :465-71 (2015). The low affinity of (S)-norcoclaurine synthase for 4-HPAA and dopamine necessitate relatively high intracellular accumulation of 4-HPAA and dopamine; but dopamine is actively secreted by yeast, and 4-HPAA is highly reactive and subject to the Ehrlich pathway.
[0004] The Ehrlich pathway, which involves catabolism of branched-chain amino acids including leucine, valine, and isoleucine; aromatic amino acids including phenylalanine, tyrosine, and tryptophan; and the sulfur-containing amino acid (methionine), leads to the formation of fusel alcohols and fusel acids. In S. cerevisiae, several dehydrogenase polypeptides and reductase polypeptide are involved. The Ehrlich pathway includes conversion of 4-HPAA to tyrosol. [0005] Intracellular availability of 4-HPAA and dopamine in yeast is also highly dependent on the aromatic amino acid biosynthesis pathways, particularly the shikimake pathway, because both precursors are tyrosine derivatives. See Figure 1.
[0006] These circumstances create significant obstacles to efficient production of (S)- norcoclaurine in yeast. This situation has important implications for producing any
benzylisoquinoline alkaloids in yeast, because (S)-norcoclaurine is the precursor to nearly all natural and non-natural benzylisoquinoline alkaloids. See, e.g., Figure 2. Many
benzylisoquinoline alkaloids are of great medical interest because of their pharmacological activity, such as, for example, the antibiotic sanguinarine, the muscle relaxants papaverine and tubocurarine, and the analgesics codeine and morphine. Galanie et al., Science
349(6252): 1095-100 (August 2015) disclosed a complete biosynthesis of opioids through enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast, thereby enabling production of thebaine, oripavine, neopinone, codeinone, hydrocodone, morphine, oxycodone and codeine, albeit not in commercially useful amounts.. Furthermore, DeLoache et al., Nat. Chem. Biol. 1 1 (7):465-71 (May 2015) described full reconstitution of the seven-enzyme pathway from L-tyrosine to (S)-reticuline, thereby showing that production of noscapine, berberine, sanguinarine, tubocurarine and papaverine could be achieved in the laboratory.
[0007] Currently, the amount of (S)-norcoclaurine that can be produced in yeast is too low to drive a commercially useful benzylisoquinoline alkaloid biosynthetic pathway. Accordingly, there remains a need for a yeast expression system involving (S)-norcoclaurine synthase capable of increased production of (S)-norcoclaurine sufficient to support commercial production of (a S)-norcoclaurine and useful benzylisoquinoline alkaloids that can be produced therefrom SUMMARY OF THE INVENTIONS
[0008] It is against the above background that the present invention provides certain advantages and advancements over the prior art.
[0009] Although the invention as described herein is not limited to specific advantages or functionalities, such as, for example, the ability to scale up production of one or more benzylisoquinoline alkaloids, the invention provides a recombinant S. cerevisiae host cell capable of producing one or more benzylisoquinoline alkaloids, comprising a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine and having reduced expression of:
(a) one or more endogenous transporter genes or one or more endogenous transcription factor genes that regulates expression of the one or more endogenous transporter genes;
(b) one or more endogenous genes encoding one or more polypeptides
capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor or one or more transcription factor genes that regulate expression of the one or more endogenous genes; and/or
(c) one or more endogenous genes encoding one or more NCS-compatible substrate pathway polypeptides or one or more transcription factor genes that regulate expression of the one or more endogenous genes.
[0010] In one aspect of the recombinant host cell disclosed herein, the one or more endogenous transporter genes encode a polypeptide having at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:70 or 74, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 76, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72.
[0011] In one aspect of the recombinant host cell disclosed herein, the one or more endogenous transcription factor genes encode a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 184 or 186.
[0012] The invention further provides a recombinant host cell comprising reduced expression of the one or more transporter genes encoding the one or more polypeptides having at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:70 or 74, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 76, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72; and the one or more endogenous transporter genes encoding one or more polypeptides having at least 90% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 86 or 88, or at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 102, 112, or at least 70% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 78, 80, 98, 114, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 92, 94, 106, or 1 16, or at least 60% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 100, 104, or 110, or at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 18, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:82, 84, 90, 96, or 108.
[0013] The invention further provides a recombinant host cell comprising reduced expression of the transporter gene encoding a polypeptide having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:70 and further comprising:
(a) reduced expression of the transporter gene encoding a polypeptide
having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72; (b) reduced expression of the transporter gene encoding a polypeptide
having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:74;
(c) reduced expression of the transporter gene encoding a polypeptide
having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:76;
(d) reduced expression of the transporter gene encoding a polypeptide
having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:74 and reduced expression of the transporter gene encoding a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:76; or
(e) reduced expression of the transporter gene encoding the polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72, reduced expression of the transporter gene encoding the polypeptide having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:74, and reduced expression of the transporter gene encoding the polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:76.
[0014] The invention further provides a recombinant host cell comprising reduced expression of the endogenous gene encoding the one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:154, 152, or 150. [0015] In one aspect, the recombinant host cell disclosed herein further comprises reduced expression of:
(a) the one or more transporter genes encoding the one or more polypeptides having at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:70 or 74, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 76, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72; and
(b) the one or more polypeptides capable of reducing or oxidizing a
benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:146.
[0016] The invention further provides a recombinant S. cerevisiae host cell capable of producing one or more benzylisoquinoline alkaloids, comprising a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine, wherein:
(a) the gene has a copy number of 2 or more and the host cell further
comprises reduced expression of one or more endogenous genes encoding one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor or one or more transcription factor genes that regulate expression of the one or more endogenous gene; wherein the one or more polypeptides comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 154, 152, or 150; or
(b) the host cell has reduced expression of one or more endogenous genes encoding one or more NCS-compatible substrate pathway polypeptide or one or more transcription factor genes that regulate expression of the one or more endogenous genes.
[0017] In one aspect of the recombinant host cell disclosed herein, the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine has a copy number of 2 or more.
[0018] In one aspect of the recombinant host cell disclosed herein, the polypeptide capable of synthesizing (S)-norcoclaurine comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, or 198.
[0019] In one aspect of the recombinant host cell disclosed herein, the benzylisoquinoline alkaloid precursor is 4-hydroxyphenylacetaldehyde (4-HPAA) or 3,4- dihydroxyphenylacetaldehyde (3,4-DHPAA).
[0020] In one aspect of the recombinant host cell disclosed herein, the one or more endogenous genes encode the one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:154, 152, or 150.
[0021] In one aspect of the recombinant host cell disclosed herein, the NCS-compatible substrate pathway is a tryptophan biosynthesis pathway or a phenylalanine biosynthesis pathway. [0022] In one aspect of the recombinant host cell disclosed herein, the endogenous gene encodes an NCS-compatible pathway polypeptide having at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 166 or 168, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 164.
[0023] In one aspect, the recombinant host cell disclosed herein further comprises:
(a) a gene encoding a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:2 or 4;
(b) a gene encoding a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:6;
(c) a gene encoding a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:8;
(d) a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:10 or 12;
(e) a gene encoding a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14;
(f) a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:16 or 18; and
(g) a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:20;
wherein at least one of the genes is overexpressed.
[0024] The invention further provides a recombinant host cell, comprising:
(a) a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, the polypeptide lacking an N-terminal signal peptide; and
(b) reduced expression of one or more endogenous genes encoding one or more polypeptides capable of synthesizing tyrosol or 4-HPAC from 4- HPAA, each comprising a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ I D NOs: 124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:154, 152, or 150.
[0025] In one aspect, the recombinant host cell disclosed herein further comprises:
(c) reduced expression of at least one endogenous gene encoding a
polypeptide capable of synthesizing phenylpyruvate from prephenate comprising a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:164; and/or at least one endogenous gene encoding a polypeptide capable of synthesizing anthranilate from chorismate comprising a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO: 166 or 168.
[0026] The invention further provides a recombinant host cell, comprising:
(a) a polypeptide having the amino acid sequence set forth in SEQ ID NO:4;
(b) a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:24; and
(c) a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:28.
[0027] In one aspect of the recombinant host cell disclosed herein, the one or more benzylisoquinoline alkaloids is (S)-norcoclaurine or (S)-norlaudanosoline. [0028] In one aspect of the recombinant host cell disclosed herein, the host cell is further capable of producing a natural or a non-natural benzylisoquinoline alkaloid derivative of (S)- norcoclaurine.
[0029] In one aspect of the recombinant host cell disclosed herein, the derivative is (S)- reticuline, (R)-reticuline, salutaridinol, thebaine neopinone, and codeinone.
[0030] The invention further provides a method of producing one or more benzylisoquinoline alkaloids in a cell culture, comprising culturing the host cell disclosed herein in the cell culture, under conditions in which the genes are expressed; wherein the one or more benzylisoquinoline alkaloids is produced by the host cell.
[0031] In one aspect of the method disclosed herein, the genes are constitutively expressed.
[0032] In one aspect of the method disclosed herein, expression of the genes is induced.
[0033] In one aspect of the method disclosed herein, the host cell further produces a natural or a non-natural benzylisoquinoline alkaloid derivative of (S)-norcoclaurine,
[0034] In one aspect of the method disclosed herein, the derivative is (S)-reticuline, (R)- reticuline, berberine, papaverine, morphine, sanguinarine, noscapine, codeine, thebaine, northebaine, oripavine, nororipavine, neopinone, codeinone, oxycodone, or buprenorphine.
[0035] In one aspect of the method disclosed herein, the host cell is cultured in a fermentor with a feed profile and at a temperature for a period of time, wherein the feed profile, the temperature, and the period of time facilitate reduced formation of an acetaldehyde.
[0036] In one aspect of the method disclosed herein, formation of the acetaldehyde is reduced compared to a corresponding host cell cultured under conditions that do not facilitate reduced formation of an acetaldehyde. [0037] In one aspect of the method disclosed herein, the cell culture comprises a non- fermentable carbon source, and wherein the cell culture has a total level of acetaldehyde that is lower than the total level of acetaldehyde in a corresponding cell culture including a glucose carbon source and/or a sucrose carbon source.
[0038] In one aspect of the method disclosed herein, the non-fermentable carbon source is an acetate carbon source or a glycerol carbon source.
[0039] In one aspect of the method disclosed herein, the method further comprises isolating one or more benzylisoquinoline alkaloids produced by the recombinant host cell.
[0040] In one aspect of the method disclosed herein, the method further comprises enzymatically or chemically converting one or more benzylisoquinoline alkaloids produced by the recombinant host cell to provide a benzylisoquinoline alkaloid derivative.
[0041] In one aspect of the method disclosure herein, the benzylisoquinoline alkaloid derivative is berberine, papaverine, morphine, sanguinarine, noscapine, neomorphine, hydrocodone, codeine, oxycodone, oxymorphone, dihydromorphine, or buprenorphine.
[0042] In one aspect of the method disclosed herein, the host cell is cultured in a fermentor with a feed profile and at a temperature for a period of time, wherein the feed profile, the temperature, and the period of time facilitate production of the one or more benzylisoquinoline alkaloids.
[0043] In one aspect of the method disclosed herein, the one or more benzylisoquinoline alkaloids is (S)-norcoclaurine.
[0044] The invention further provides a cell culture broth, comprising the host cell disclosed herein and further comprising:
(a) one or more benzylisoquinoline alkaloids produced by the recombinant host cell; (b) dextrose, sucrose, fructose, glycerol, xylose, ethanol, glucose, and/or acetate; and
(c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), nucleobases, and/or amino acids; and optionally,
(d) malt extract, corn steep liquor, casein hydrosylate, yeast extract, urea, amino acids, ammonia and/or ammonium salts;
wherein the one or more benzylisoquinoline alkaloids are present at a concentration of at least 100 mg/liter of the cell culture.
[0045] In one aspect of the cell culture broth described herein, the cell culture comprises less than 10 g/L of ethanol.
[0046] The invention further provides a cell lysate from the host cell disclosed herein grown in the cell culture, comprising:
(a) one or more benzylisoquinoline alkaloids produced by the recombinant host cell;
(b) dextrose, sucrose, fructose, glycerol, xylose, ethanol, glucose, and/or acetate; and
(c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), nucleobases, and/or amino acids; and optionally,
(d) malt extract, corn steep liquor, casein hydrosylate, yeast extract, urea, amino acids, ammonia and/or ammonium salts;
wherein the one or more benzylisoquinoline alkaloids are present at a concentration of at least 1 mg/liter of the cell culture. [0047] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:
[0049] Figure 1 shows a biochemical pathway for producing (S)-norcoclaurine,
phenylpyruvate, and anthranilate from benzylisoquinoline precursors using a DAHP synthase polypeptide (Aro4), a pentafunctional AROM polypeptide (Aro1 ), a bifunctional chorismate synthase / flavin reductase polypeptide (Aro2), a chorismate mutase polypeptide (Aro7), a prephenate dehydrogenase polypeptide (Tyr1 ), an aromatic aminotransferase I polypeptide (Aro8), an aromatic aminotransferase II polypeptide (Aro9), a phenylpyruvate decarboxylase polypeptide (Aro10), a tyrosine hydroxylase polypeptide (TyrH), an L-DOPA decarboxylase polypeptide (DODC), an (S)-norcoclaurine synthase polypeptide (NCS), a prephenate dehydratase polypeptide (PHA2), an anthranilate synthase polypeptide (TRP2), and a multifunctional tryptophan biosynthesis protein (TRP3).
[0050] Figure 2 shows representative benzylisoquinoline alkaloids obtainable from (S)- norcoclaurine.
[0051] Figure 3 shows production of dopamine (left) and (S)-norcoclaurine (right) from a control S. cerevisiae strain (Strain C) and a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in Example 4, below). [0052] Figure 4 shows visible- and fluorescence-microscopy images (left) and specific fluorescence measurements of S. cerevisiae strains expressing GFP-tagged polypeptides capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in
Example 4, below.
[0053] Figure 5 shows production of dopamine and (S)-norcoclaurine (from left to right for each strain) from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
[0054] Figure 6 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
[0055] Figure 7 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
[0056] Figure 8 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
[0057] Figure 9 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
[0058] Figure 10 shows off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in Example 5, below.
[0059] Figure 1 1 shows off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in Example 5, below.
[0060] Figure 12 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
[0061] Figure 13 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
[0062] Figure 14 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
[0063] Figure 15 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
[0064] Figure 16 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
[0065] Figure 17 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below. [0066] Figure 18 shows production of certain off-target condensation products (products 2, 3, and 4; from bottom to top for each strain) of a polypeptide capable of synthesizing (S)- norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
[0067] Figure 19 shows accumulation of biomass (OD6oo) and ethanol during the fed-batch phases of cultivations of S. cerevisiae strains as described in Example 6, below.
[0068] Figure 20 shows accumulation of norcoclaurine during the fed-batch phases of cultivations of S. cerevisiae strains as described in Example 6, below.
[0069] Figure 21 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more transporter polypeptides, as described in Example 9, below.
[0070] Figure 22 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
[0071] Figure 23 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
[0072] Figure 24 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
[0073] Figure 25 shows production of dopamine, tyrosol, 4-HPAC, and (S)-norcoclaurine (from left to right for each strain) from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a
benzylisoquinoline alkaloid precursor, as described in Example 5, below.
[0074] Skilled artisans will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the Figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0075] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.
[0076] Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. For example, reference to a“nucleic acid” means one or more nucleic acids.
[0077] It is noted that terms like“preferably,”“commonly,” and“typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
[0078] For the purposes of describing and defining the present invention it is noted that the term“substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term“substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
[0079] Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (PCR) techniques. See, for example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley
Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, CA).
[0080] As used herein, the terms“polynucleotide”,“nucleotide”,“oligonucleotide”, and “nucleic acid” can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof, in either single-stranded or double-stranded embodiments depending on context as understood by the skilled worker.
[0081] As used herein, the terms“microorganism,”“microorganism host,”“microorganism host cell,”“recombinant host,” and“recombinant host cell” can be used interchangeably. As used herein, the term“recombinant host” is intended to refer to a Saccharomyces cerevisiae ( S . cerevisiae) host, the genome of which has been augmented by at least one DNA sequence. Such DNA sequences include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein (“expressed”), and other genes or DNA sequences which one desires to introduce into a host. It will be appreciated that typically the genome of a recombinant host described herein is augmented through stable introduction of one or more recombinant genes. Generally, introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene. In some instances, the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis.
[0082] As used herein, the term“cell culture” refers to a culture medium comprising one or more recombinant hosts. A cell culture may comprise a single strain of recombinant host, or may comprise two or more distinct host strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; yeast extract; aminoglycoside antibiotics such as G418 and hygromycin B; etc.
[0083] As used herein, the term“recombinant gene” refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. “Introduced,” or“augmented” in this context, is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene can be a DNA sequence from another species or can be a DNA sequence that originated from or is present in the same species but has been incorporated into a host by recombinant methods to form a recombinant host. It will be appreciated that a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA. In some aspects, said recombinant genes are encoded by cDNA. In other embodiments, recombinant genes are synthetic and/or codon-optimized for expression in S. cerevisiae.
[0084] As used herein, the term“engineered biosynthetic pathway” refers to a biosynthetic pathway that occurs in a recombinant host, as described herein. In some aspects, one or more steps of the biosynthetic pathway do not naturally occur in an unmodified host. In some embodiments, a heterologous version of a gene is introduced into a host that comprises an endogenous version of the gene.
[0085] As used herein, the term“endogenous” gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell. In some
embodiments, an endogenous S. cerevisiae gene is overexpressed. As used herein, the term “overexpress” is used to refer to the expression of a gene in an organism at levels higher than the level of gene expression in a wild type organism, i.e., increased expression of a recombinant gene in a host cell relative to a corresponding host cell that does not contain the recombinant gene (see, e.g., Prelich, 2012, Genetics 190:841-54). In some embodiments, an endogenous yeast gene is deleted. See, e.g., Giaever & Nislow, 2014, Genetics 197(2):451-65. As used herein, the terms“deletion,”“deleted,”“knockout,” and“knocked out” can be used
interchangeably to refer to an endogenous gene that has been manipulated to no longer be expressed in an organism.
[0086] As used herein, the terms“heterologous sequence” and“heterologous coding sequence” are used to describe a sequence derived from a species other than the recombinant host. A heterologous coding sequence, for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant S. cerevisiae host expressing the heterologous sequence. In some embodiments, a coding sequence is a sequence that is native to the host.
[0087] A“selectable marker” can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a detectable change, e.g. a color change. Linearized DNA fragments of the gene replacement vector then are introduced into the cells using methods well known in the art ( see below). Integration of the linear fragments into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, polymerase chain reaction (PCR) or Southern blot analysis. Subsequent to its use in selection, a selectable marker can be removed from the genome of the host cell by, e.g., Cre-LoxP systems (see, e.g., Gossen et al., 2002, Ann. Rev. Genetics 36:153- 173 and U.S. 2006/0014264). Alternatively, a gene replacement vector can be constructed in such a way as to include a portion of the gene to be disrupted, where the portion is devoid of any endogenous gene promoter sequence and encodes none, or an inactive fragment of, the coding sequence of the gene.
[0088] As used herein, the terms“variant” and“mutant” are used to describe a protein sequence that has been modified, by man or nature, at one or more amino acids, compared to the wild-type sequence of a particular protein.
[0089] As used herein, the term“inactive fragment” is a fragment of the gene that encodes a protein having, e.g., less than about 10% {e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 %, or 0%) of the activity of the protein produced from the full-length coding sequence of the gene. Such a portion of a gene is inserted in a vector in such a way that no known promoter sequence is operably linked to the gene sequence, but that a stop codon and a transcription termination sequence are operably linked to the portion of the gene sequence. This vector can be subsequently linearized in the portion of the gene sequence and transformed into a cell. By way of single homologous recombination, this linearized vector is then integrated in the endogenous counterpart of the gene with resulting inactivation thereof.
[0090] As used herein, the term“alkaloid” refers to any of the group of naturally occurring chemical compounds that mostly contain basic nitrogen atoms. As used herein,“alkaloids” include“true alkaloids” (i.e., amino-acid-derived compounds containing nitrogen in the heterocycle), for example, atropine, nicotine, morphine, ergotamine, coniine, and coniceine; “protoalkaloids” (i.e., amino-acid-derived compounds containing nitrogen), for example, mescaline, adrenaline, and ephedrine;“polyamine alkaloids” (i.e., derivatives of putrescine, spermidine, and spermine);“peptide and cyclopeptide alkaloids”; and“pseudoalkaloids” (i.e., alkaloid-like compounds that do not originate from amino acids), for example, terpene-like alkaloids, steroid-like alkaloids, and purine-like alkaloids (e.g., caffeine, theobromine, the acrine, theophylline). As used herein, the term“alkaloid” also includes non-naturally occurring compounds derived from or otherwise resembling any naturally occurring alkaloid. See M. Hesse,“Alkaloids. Nature’s Curse or Blessing?” (2002); T. Aniszewski,“Alkaloids - Secrets of Life” (2007). Alkaloids can contain asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. Each chiral center can be defined in terms of absolute stereochemistry as (R)- or (S)-. As used herein, the term“alkaloid” includes all such possible isomers, as well as their racemic and optically pure forms.
[0091] The person of ordinary skill in the art will appreciate that alkaloids can be classified by skeleton (e.g., pyrrolidine, quinoline, benzylisoquinoline, indole, and terpenoid). As used herein, the term“benzylisoquinoline alkaloid” refers to alkaloids having a benzylisoquinoline skeleton, including, for example, (S)-norcoclaurine, coclaurine, berberine, papaverine, morphine, sanguinarine, hydrastine, and noscapine. See Figure 2; see also, Singla et al., “BIAdb: A curated database of benzylisoquinoline alkaloids,” BMC Pharmacol. 10:4 (March 2010)).
[0092] As used herein, the terms“benzylisoquinoline alkaloid precursor” and
“benzylisoquinoline alkaloid precursor compound” are used to refer to intermediate compounds in the benzylisoquinoline alkaloid biosynthetic pathway. Benzylisoquinoline alkaloid precursors include, but are not limited to, phosphoenolpyruvate (PEP), erythrose 4-phosphate (E4P), 3- deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), 5-enolpyruvateshikimate 3-phosphate (EPSP), chorismate, prephenate, L-tyrosine, L-3,4-dihydroxyphenylalaine (L-DOPA), 4-(2- aminoethyl)benzene-1 ,2-diol (dopamine), 4-hydroxyphenylpyruvate (4-HPP), and 4- hydroxyphenylacetaldehyde (4-HPAA). See Figure 1. In some embodiments,
benzylisoquinoline alkaloid precursors are themselves benzylisoquinoline alkaloids. For example, (S)-norcoclaurine is a benzylisoquinoline alkaloid precursor of morphine. See Figure 2.
[0093] Benzylisoquinoline alkaloids and/or precursors thereof can be produced in vivo, (i.e., in a recombinant host), in vitro (i.e., enzymatically or chemically), or by whole cell bioconversion. As used herein, the terms“produce” and“accumulate” can be used interchangeably to describe synthesis of benzylisoquinoline alkaloids and precursors thereof in vivo, in vitro, or by whole cell conversion.
[0094] As used herein, the terms“culture broth,”“culture medium,” and“growth medium” can be used interchangeably to refer to a liquid that supports growth of a cell. A culture broth can comprise a carbon source such as dextrose, sucrose, fructose, glycerol, xylose, ethanol, glucose, or acetate; a phosphate source; vitamins; trace elements; salts; yeast nitrogen base (YNB); amino acids; nucleobases; and a nitrogen source. The nitrogen source can include, for example, malt extract, corn steep liquor, casein hydrosylate, yeast extract, urea, amino acids, ammonia and/or ammonium salts, etc. Additionally, a culture broth can comprise one or more benzylisoquinoline alkaloids added to the medium or produced de novo by a recombinant host, as described herein. In certain embodiments, a non-fermentable carbon source ( e.g ., an acetate carbon source or a glycerol carbon source) prevents buildup of acetaldehyde and/or ethanol. In such embodiments, reduced intracellular acetaldehyde limits or eliminates spontaneous condensation of acetaldehyde and dopamine.
[0095] In some embodiments, benzylisoquinoline alkaloids and/or precursors thereof are produced in vivo through expression of one or more enzymes involved in the benzylisoquinoline alkaloid biosynthetic pathway in a recombinant S. cerevisiae host. For example, a recombinant S. cerevisiae host comprising a gene encoding a polypeptide capable of synthesizing 3-deoxy- D-arabinoheptulosonate-7-phosphate (DAHP) from phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) {e.g., DAHP synthesis polypeptide; e.g., SEQ ID NO:2 or SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing 5-enolpyruvateshikimate 3-phosphate (EPSP) from DAHP {e.g., a pentafunctional AROM polypeptide; e.g., SEQ ID NO:6); a gene encoding a polypeptide capable of synthesizing chorismate from EPSP {e.g., bifunctional chorismate synthase / flavin reductase polypeptide; e.g., SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephenate from chorismate {e.g., a chorismate mutase; e.g., SEQ ID NO:10 or SEQ ID NO: 12); a gene encoding a polypeptide capable of synthesizing 4-hydroxyphenylpyruvate (4-HPP) from prephenate {e.g., a prephenate dehydrogenase; e.g., SEQ ID NO:14); a gene encoding a polypeptide capable of synthesizing L-tyrosine from 4-HPP {e.g., an aromatic aminotransferase polypeptide; e.g., SEQ ID NO:16 or SEQ ID NO:18); a gene encoding a polypeptide capable of synthesizing 4-HPP from L-tyrosine {e.g., an aromatic aminotransferase polypeptide; e.g., SEQ ID NO:16 or SEQ ID NO:18); a gene encoding a polypeptide capable of synthesizing 4-hydroxyphenylacetaldehyde (4-HPAA) from 4-HPP {e.g., a phenylpyruvate decarboxylase polypeptide; e.g., SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing L-3,4-dihydroxyphenylalanine (L-DOPA) from L-tyrosine ( e.g ., a tyrosine hydroxylase polypeptide; e.g., SEQ ID NO:22 or SEQ ID NO:24); a gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA (e.g., an L-DOPA decarboxylase polypeptide; e.g., SEQ ID NO:26 or SEQ ID NO:28); and/or a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine (e.g., an (S)- norcoclaurine synthase (NCS) polypeptide; e.g., SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:54) can produce one or more benzylisoquinoline alkaloids (e.g., (S)- norcoclaurine) and/or precursors thereof in vivo. See, e.g., Figures 1-2. The person of ordinary skill in the art will appreciate that one or more of these genes can be endogenous to the S. cerevisiae host provided that at least one of these genes is a recombinant gene introduced into the recombinant host.
[0096] In some aspects, production of benzylisoquinoline alkaloids (e.g., (S)-norcoclaurine) can be accomplished by differential copy numbers of the benzylisoquinoline alkaloid
biosynthesis pathway genes (see Figure 1 ) in the recombinant cell, differential promoter strengths, and/or by utilizing mutants with increased specificity/activity towards the product of interest. For example, additional copies of a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in recombinant cells as otherwise described herein can increase the production of (S)-norcoclaurine.
[0097] In some embodiments, a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing DAHP from E4P and PEP. In some aspects, the gene encoding a polypeptide capable of synthesizing DAHP from E4P and PEP is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
In some aspects, the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180). In some aspects, the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182). In some aspects, expression of a recombinant gene encoding a polypeptide capable of synthesizing DAHP from E4P and PEP results in a total expression level of genes encoding a polypeptide capable of synthesizing DAHP from E4P and PEP that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing DAHP from E4P and PEP, i.e., an overexpression of a polypeptide capable of synthesizing DAHP from E4P and PEP. In some aspects, the polypeptide capable of synthesizing DAHP from E4P and PEP is a feedback-resistant (FBR) polypeptide. The present inventors have discovered that,
advantageously, expression of a feedback resistant version of and/or overexpression of a benzylisoquinoline alkaloid biosynthesis polypeptide (e.g., a polypeptide capable of synthesizing DAHP from E4P and PEP, e.g., a DAHP synthase polypeptide; e.g., a polypeptide capable of synthesizing prephenate from chorismate, e.g., a chorismate mutase polypeptide) can reduce or eliminate pathway regulation (e.g., by reducing or eliminating endogenous transcriptional regulation of expression).
[0098] In some embodiments, a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing prephenate from chorismate. In some aspects, the gene encoding a polypeptide capable of synthesizing prephenate from chorismate is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
In some aspects, the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPH (SEQ ID NO:179), or pCCW12 (SEQ ID NQ:180). In some aspects, the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182). In some aspects, expression of a recombinant gene encoding a polypeptide capable of synthesizing prephenate from chorismate results in a total expression level of genes encoding a polypeptide capable of synthesizing prephenate from chorismate that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing prephenate from chorismate, i.e., an overexpression of a polypeptide capable of synthesizing prephenate from chorismate. In some aspects, the polypeptide capable of synthesizing prephenate from chorismate is a feedback-resistant (FBR) polypeptide.
[0099] The present inventors have discovered that indole-3-acetaldehyde (IAA) can compete with 4-HPAA for condensation with dopamine (catalyzed, e.g., by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine). In certain
embodiments, overexpression of a polypeptide capable of synthesizing prephenate from chorismate can increase the benzylisoquinoline alkaloid biosynthesis flux relative to competing pathways from chorismate towards IAA, and accordingly increase the availability of intracellular dopamine for condensation with 4-HPAA.
[00100] In some embodiments, a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate. In some aspects, the gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
In some aspects, the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NQ:180). In some aspects, the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182). In some aspects, expression of a recombinant gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate results in a total expression level of genes encoding a polypeptide capable of synthesizing 4-HPP from prephenate that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing 4-HPP from prephenate, i.e., an overexpression of a polypeptide capable of synthesizing 4-HPP from prephenate.
[00101] The present inventors have further discovered that phenylacetaldehyde (PAA) can compete with 4-HPAA for condensation with dopamine (catalyzed, e.g., by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine). In certain
embodiments, overexpression of a polypeptide capable of synthesizing 4-HPP from prephenate can increase benzylisoquinoline alkaloid biosynthesis flux relative to competing pathways from prephenate towards PAA, and accordingly increase the availability of intracellular dopamine for condensation with 4-HPAA.
[00102] In some embodiments, a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine. In some aspects, the gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence. In some aspects, the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180). In some aspects, the recombinant gene is operably linked to a terminator, such as, for example, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO:177), tADH1 (SEQ ID NO: 178), tTDH 1 (SEQ ID NO:181 ), or tPGK1 (SEQ ID NO: 182). In some aspects, expression of a recombinant gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine results in a total expression level of genes encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine, i.e., an overexpression of a polypeptide capable of synthesizing L-DOPA from L-tyrosine.
[00103] In some embodiments, a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA. In some aspects, the gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence. In some aspects, the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180). In some aspects, the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), PΌH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182). In some aspects, expression of a recombinant gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA results in a total expression level of genes encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing dopamine from L- DOPA, i.e., an overexpression of a polypeptide capable of synthesizing dopamine from L-
DOPA. [00104] In some embodiments, a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP. In some aspects, the gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence. In some aspects, the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID
NO:172), pTEF1 (SEQ ID NO:173), pTPH (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180). In some aspects, the recombinant gene is operably linked to a terminator, such as, for example, tPGH (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182). In some aspects, expression of a recombinant gene encoding a polypeptide capable of
synthesizing 4-HPAA from 4-HPP results in a total expression level of genes encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP, i.e., an overexpression of a polypeptide capable of synthesizing 4-HPAA from 4-HPP. In certain embodiments, overexpression of a polypeptide capable of synthesizing 4-HPAA from 4-HPP can increase benzylisoquinoline alkaloid biosynthesis flux relative to competing pathways from 4-HPP.
[00105] In some embodiments, a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA. In some aspects, the gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from (S)- norcoclaurine from dopamine and 4-HPAA is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence. In some aspects, the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO: 170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180). In some aspects, the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO: 174), tCYC1 (SEQ ID NO: 175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID
NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182). In some aspects, expression of a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA results in a total expression level of genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4- HPAA that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA, i.e., an overexpression of a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA.
[00106] In some embodiments, the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA is a truncated polypeptide such as, for example, a truncated NCS polypeptide lacking an N-terminal signal peptide ( e.g ., SEQ ID NO:42 or SEQ ID NO:44), a truncated NCS polypeptide lacking a C-terminal transmembrane domain (e.g., SEQ ID NO:52 or SEQ ID NO:54), or a truncated NCS polypeptide lacking an N-terminal signal peptide and a C- terminal transmembrane domain. The person of ordinary skill in the art will appreciate that a polypeptide lacking a signal peptide and/or a transmembrane domain, expressed in a recombinant host, may have increased solubility in the cytosol relative to a corresponding polypeptide comprising the signal peptide and/or transmembrane domain.
[00107] In some embodiments, the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA (e.g., an NCS polypeptide) has a copy number of two (i.e., is present in the recombinant host in two copies). For example, in some embodiments, the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA ( e.g ., an NCS polypeptide) has a copy number of two, three, four, five, six, seven, eight, or more. See Example 5.
[00108] In some aspects, the polypeptide capable of synthesizing DAHP from PEP and E4P comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 1 ) or SEQ ID NO:4 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:3).
[00109] In some aspects, the polypeptide capable of synthesizing EPSP from DAHP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:6 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:5).
[00110] In some aspects, the polypeptide capable of synthesizing chorismate from EPSP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:8 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:7).
[00111] In some aspects, the polypeptide capable of synthesizing prephenate from chorismate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:10 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:9) or SEQ ID NO:12 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:1 1 ).
[00112] In some aspects, the polypeptide capable of synthesizing 4-HPP from prephenate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:14 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 13).
[00113] In some aspects, the polypeptide capable of synthesizing L-tyrosine from 4-HPP and/or the polypeptide capable of synthesizing 4-HPP from L-tyrosine comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:16 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 15) or SEQ ID NO:18 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:17). [00114] In some aspects, the polypeptide capable of synthesizing 4-HPAA from 4-HPP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:20 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 19).
[00115] In some aspects, the polypeptide capable of synthesizing L-DOPA from L-tyrosine comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:22 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:21 ) or SEQ ID NO:24 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:23).
[00116] In some aspects, the polypeptide capable of synthesizing dopamine from L-DOPA comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:26 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:25) or SEQ ID NO:28 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:27).
[00117] In some aspects, the polypeptide capable of synthesizing (S)-norcoclaurine from 4- HPAA and dopamine comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:32 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:31 ), or SEQ ID NO:34 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:33), or SEQ ID NO:36 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:35), or SEQ ID NO:38 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:37), or SEQ ID NO:40 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:39), or SEQ ID NO:42 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:41 ), or SEQ ID NO:44 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:43), or SEQ ID NO:52 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:51 ), or SEQ ID NO:54 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:53), or SEQ ID NO: or SEQ ID NO:30 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:29), or SEQ ID NO:188 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:187), or SEQ ID NO:190 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:189), or SEQ ID NO: 192 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:191 ), or SEQ ID NO:194 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:193), or SEQ ID NO:196 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:195), or SEQ ID NO:198 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:197.
[00118] In some embodiments, benzylisoquinoline alkaloids and/or precursors thereof are produced in vivo through expression of one or more enzymes involves in the benzylisoquinoline alkaloid biosynthetic pathway. For example, a recombinant S. cerevisiae host comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P ( e.g ., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephenate from chorismate (e.g., a recombinant gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 12); a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 14); a gene encoding a polypeptide capable of synthesizing L-tyrosine from 4-HPP and/or 4-HPP from L-tyrosine; a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:22 or SEQ ID NO:24); a gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:26 or SEQ ID NO:28); and/or a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine ( e.g ., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in any of SEQ ID NOs:30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190,
192, 194, 196, and 198) can produce one or more benzylisoquinoline alkaloids (e.g., (S)- norcoclaurine) and/or precursors thereof in vivo. In certain such embodiments, the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA (e.g., an NCS polypeptide) has a copy number of two, three, four, five, six, seven, eight, or more.
[00119] (S)-Norcoclaurine can be produced in vivo (i.e., in a recombinant host), in vitro (i.e., enzymatically), or by whole cell bioconversion. As used herein, the terms“produce” and “accumulate” can be used interchangeably to describe synthesis of (S)-norcoclaurine in vivo, in vitro, or by whole cell bioconversion.
[00120] In some embodiments, a cell is permeabilized to take up a substrate to be modified or to excrete a modified product. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. In some embodiments, the cells are permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween. In some embodiments, the cells are permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide (CTAB). In some embodiments, the cells are permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock. For example, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant. The resulting supernatant can then be applied to a chromatography column, e.g., a C18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol. The compound(s) can then be further purified by preparative HPLC. [00121] In some embodiments, (S)-norcoclaurine can be produced by co-culturing of two or more hosts. In some embodiments, one or more hosts, each expressing one or more enzymes involved in the (S)-norcoclaurine biosynthetic pathway, produce (S)-norcoclaurine. For example, a host expressing a gene encoding a polypeptide capable of polypeptide capable of synthesizing (S)-norcoclaurine; a gene encoding a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:2 and 4; a gene encoding a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:6; a gene encoding a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:8; a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:10 and 12; a gene encoding a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14; a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 16 and 18; a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:20; a gene encoding a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:22 and 24; and/or a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:26 and 28 can produce (S)-norcoclaurine.
[00122] In some embodiments, the benzylisoquinoline alkaloid comprises, for example, (S)- norcoclaurine, (S)-reticuline, (R)-reticuline, salutaridinol, thebaine, neopinone, and codeinone.
[00123] For example, a recombinant S. cerevisiae host as otherwise described herein, further comprising a gene encoding a polypeptide capable of synthesizing (S)-coclaurine from (S)- norcoclaurine ( e.g ., a 6-O-methyltransferase (6-OMT) polypeptide; e.g., a polypeptide having at least 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO:201 ), a gene encoding a polypeptide capable of synthesizing 3’-hydroxy-coclaurine from (S)-coclaurine or 3’- hydroxy-A/-methyl-coclaurine from /V-methyl-coclaurine ( e.g ., an N-methylcoclaurine 3’- monooxygenase (NMCH) polypeptide; e.g., a polypeptide having at least 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO:202), a gene encoding a polypeptide capable of synthesizing /V-methyl-coclaurine from (S)-coclaurine or 3’-hydroxy-A/-methyl- coclaurine from 3’-hydroxy-coclaurine (e.g., a coclaurine-A/-methyltransferase (CNMT) polypeptide; e.g., a polypeptide having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO:203), and a gene encoding a polypeptide capable of synthesizing (S)-reticuline from 3’-hydroxy-/V-methyl-coclaurine (e.g., a 3’-hydroxy-/V-methyl-(S)- coclaurine 4’-0-methyltransferase (4’-OMT) polypeptide; e.g., a polypeptide having at least 55% sequence identity to an amino acid sequence as set forth in SEQ ID NO:204), can produce (S)- reticuline in vivo. In another example, a recombinant S. cerevisiae host capable of producing (S)-reticuline in vivo (e.g., as described herein), further comprising a gene encoding a polypeptide capable of synthesizing (R)-reticuline from (S)-reticuline (e.g., a 1 ,2- dehydroreticuline synthase-1 , 2-dehydroreticuline reductase (DRS-DRR) polypeptide; e.g., a polypeptide having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO:205), a gene encoding a polypeptide capable of synthesizing salutaridine from (R)- reticuline (e.g., a salutaridine synthase (SAS) polypeptide; e.g., a polypeptide having at least 55% sequence identity to an amino acid sequence set forth in SEQ ID NO:206), a gene encoding a polypeptide capable of synthesizing salutaridinol from salutaridine (e.g., a salutaridine reductase (SAR) polypeptide; e.g., a polypeptide having at least 65% sequence identity to an amino acid sequence set forth in SEQ ID NO:207), and a gene encoding a polypeptide capable of synthesizing thebaine from salutaridinol (e.g., a salutaridinol 7-0- acetyltransferase (SAT) polypeptide and a thebaine synthase (THS) polypeptide; e.g., a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:208 and a polypeptide having at least 65% sequence identity to an amino acid sequence set forth in SEQ ID NQ:209), can produce thebaine in vivo. In another example, a recombinant S. cerevisiae host capable of producing thebaine in vivo e.g as described herein), further comprising a gene encoding a polypeptide capable of synthesizing neopinone from thebaine {e.g., a morphinone reductase polypeptide) and a gene encoding a polypeptide capable of synthesizing hydrocodone from codeinone {e.g., a thebaine 6-O-demethylase polypeptide), can produce hydrocodone in vivo. See, e.g., Galanie et al.,“Complete biosynthesis of opioids in yeast,” Science 349(6252): 1095-100 (August 2015); Chen et al.,“A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis,” Nat. Chem. Biol. 14(7):738-43 (May 2018); WO 2016/183023; WO 2016179296; WO 2018005553.
[00124] In some embodiments, benzylisoquinoline alkaloids precursors produced in vivo {e.g., (S)-norcoclaurine, (S)-reticuline, (R)-reticuline, salutaridinol, thebaine, neopinone, codeinone) can be converted in vitro (e.g., chemically or enzymatically) to derivatives including berberine, papaverine, morphine, sanguinarine, noscapine, neomorphine, hydrocodone, codeine, oxycodone, oxymorphone, dihydromorphine, and buprenorphine. For example, in certain such embodiments, codeinone produced by a recombinant host described herein is enzymatically converted to hydrocodone by contacting the codeinone with a polypeptide capable of synthesizing hydrocodone from codeinone {e.g., a thebaine 6-O-demethylase polypeptide) in vitro. In another example, in certain such embodiments, thebaine produced by a recombinant host described herein is chemically converted to buprenorphine in vitro {see, e.g., WO 2018/21 1331 ; Machara et al., Adv. Synth. Catal. 354(4):613-26 (2012); Werner et al., J. Org. Chem. 76(1 1 ):4628-34 (201 1 )). In another example, in certain such embodiments, thebaine produced by a recombinant host described herein is chemically or enzymatically converted to morphine, neomorphine, hydrocodone, codeine, oxycodone, oxymorphone, dihydromorphine, or etorphine.
[00125] As used herein, the terms“detectable amount,”“detectable concentration,” “measurable amount,” and“measurable concentration” refer to a level of benzylisoquinoline alkaloids or precursors thereof measured in AUC, pM/OD6oo, mg/L, mM, or mM.
Benzylisoquinoline alkaloid production (i.e., total, supernatant, and/or intracellular
benzylisoquinoline alkaloid levels) can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high-performance liquid
chromatography (HPLC), ultraviolet-visible spectroscopy/ spectrophotometry (UV-Vis), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR).
[00126] As used herein, the term“undetectable concentration” refers to a level of a compound that is too low to be measured and/or analyzed by techniques such as TLC, HPLC, UV-Vis, MS, or NMR. In some embodiments, a compound of an“undetectable concentration” is not present in a composition of benzylisoquinoline alkaloids and/or precursors thereof.
[00127] In some aspects, one or more benzylisoquinoline alkaloids ( e.g ., (S)-norcoclaurine) and/or precursors thereof are produced in vivo by culturing a recombinant S. cerevisiae host comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephenate from chorismate (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:12); a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:14); a gene encoding a polypeptide capable of synthesizing L- tyrosine from 4-HPP and/or 4-HPP from L-tyrosine; a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine ( e.g ., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:22 or SEQ ID NO:24); a gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:26 or SEQ ID NO:28); and/or a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in any of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, and 198) in a cell culture under conditions in which the genes are expressed, under conditions sufficient to facilitate production of the one or more
benzylisoquinoline alkaloids (e.g., (S)-norcoclaurine) and/or precursors thereof. In certain such embodiments, the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA (e.g., an NCS polypeptide) has a copy number of two, three, four, five, six, seven, eight, or more.
[00128] After the recombinant microorganism has been grown in culture for a period of time, one or more produced benzylisoquinoline alkaloids (e.g., (S)-norcoclaurine) can then be recovered from the culture. The methods of the present invention can optionally comprise one or more additional steps. These steps can for example be directed toward isolation and/or purification of one or more benzylisoquinoline alkaloids from the fermentation broth or host cells.
[00129] A number of different methods can be used to isolate and purify benzylisoquinoline alkaloids produced by the methods disclosed herein. For example, the isolating steps may comprise: (a) contacting the cell culture comprising the benzylisoquinoline alkaloids with: (i) one or more adsorbent resins in a packed column in order to bind at least a portion of the
benzylisoquinoline alkaloids to the resin, thereby isolating the benzylisoquinoline alkaloid compounds; or (ii) one or more ion exchange or reversed-phase chromatography columns in order to bind at least a portion of the benzylisoquinoline alkaloid in the column, thereby isolating the benzylisoquinoline alkaloid; or (b) crystallizing and/or organic solvent extracting the benzylisoquinoline alkaloids from the cell culture, thereby isolating the benzylisoquinoline alkaloids;(i) contacting the cell culture with an organic solvent immiscible with water and separating the organic phase enriched in benzylisoquinoline alkaloids (c) separating the cell culture into a solid phase and a liquid phase, wherein the liquid phase comprises of the benzylisoquinoline alkaloids; and (i) contacting the liquid phase with one or more adsorbent resins in order to bind at least a portion of the benzylisoquinoline alkaloids to the resin, thereby isolating the products; (ii) contacting the liquid phase with one or more ion exchange or reversed-phase chromatography columns in order to bind at least a portion of the
benzylisoquinoline alkaloids in the column, thereby isolating the pure products; or (iii) crystallizing and/or extracting the benzylisoquinoline alkaloids from the liquid phase, thereby isolating the benzylisoquinoline alkaloids.
[00130] The isolating step can comprise separating the solid phase from the liquid phase using a process comprising tangential flow filtration with diafiltration membranes to generate a permeate stream comprising the benzylisoquinoline alkaloids, wherein the membranes used in the tangential flow filtration are ultrafiltration or nanofiltration membranes. In an embodiment, the permeate stream is extracted by an organic solvent which phase-separates from the aqueous phase to generate an extracted benzylisoquinoline alkaloids in the organic solvent. Optionally the permeate stream containing the benzylisoquinoline alkaloids product could be concentrated by some evaporation to produce a crystallized benzylisoquinoline alkaloid. The aqueous permeate or the concentrate can be extracted by an organic solvent which phase-separates from the aqueous phase. The solvent extraction could be performed in a counter-current extraction centrifuge such as a Podbelniak extractor, or in a counter-current extraction column such as a Karr or Scheibel column. This yields the benzylisoquinoline alkaloids products in an organic solvent suitable for subsequent purification processing.
[00131] The isolating step can also comprise separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced one or more benzylisoquinoline alkaloids, and (a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced one or more
benzylisoquinoline alkaloids; or (b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the produced one or more benzylisoquinoline alkaloids; or (c) crystallizing or extracting the produced one or more benzylisoquinoline alkaloids; thereby isolating the produced one or more
benzylisoquinoline alkaloids.
[00132] It will be understood that organic solvent extraction can be replaced with a series of process operations which yield a similar organic solution of benzylisoquinoline alkaloids. The series of process operations would include (a) precipitation of benzylisoquinoline alkaloids from the aqueous concentrate produced by addition of acid until acidic pH; (b) filtration and optionally water-washing of the resulting solids; and (c) dissolution of the filtered benzylisoquinoline alkaloids containing solids into an organic solvent suitable for further purification. Optionally the organic extract can be contacted with carbon to adsorb impurities and color bodies. Optionally the carbon contacting can be done by mixing carbon in the organic extract and filtering the carbon out of the resulting suspension, or by feeding the organic extract to a column or filter containing a fixed bed of carbon and collecting a purified effluent stream. The organic extract can be crystallized by concentrating the solution evaporatively. The resulting benzylisoquinoline alkaloids products crystals can be filtered, washed, and dried to yield a high-purity
benzylisoquinoline alkaloid product. The reaction mixture can be filtered in order to remove the solid in the media (cell debris etc.). The resulting aqueous solution can be extracted repeatedly with an organic solvent not miscible with water (this can be chloroform, toluene, dichloromethane, ethyl acetate, or the like). The resulting organic phase can be concentrated into small quantity (resulting into a syrup). The aqueous phase can be discarded. The resulting residue benzylisoquinoline alkaloid crude material) can be then crystallized from any short chain alcohol, such as methanol or it can be purified with other suitable purification technique such as chromatography or other standard techniques. Another possible procedure to extract the alkaloids from the fermentation broth or cells can be a caustic wash of the broth/cells followed by a filtration in order to remove the biological material and other solids. The alkaloids can then be precipitated from the basic solution as salt after adjusting the pH to acidic with addition of acid (for example, sulphuric acid or hydrochloric acid, etc.). The benzylisoquinoline alkaloids can be extracted from the cells/broth trough percolation via an organic solvent. The resulting organics can be concentrated into small quantities. The resulting residue can be purified with other suitable purification technique such as crystallization and/or chromatography or other standard techniques.
[00133] As used herein, the terms“or” and“and/or” are used to describe multiple components in combination or exclusive of one another. For example,“x, y, and/or z” can refer to“x” alone,“y” alone,“z” alone,“x, y, and z,”“(x and y) or z,”“x or (y and z),” or“x or y or z.” In some embodiments,“and/or” is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group. In some embodiments,“and/or” is used to refer to production of benzylisoquinoline alkaloids and/or benzylisoquinoline alkaloid precursors. In some
embodiments,“and/or” is used to refer to production of benzylisoquinoline alkaloids, wherein one or more benzylisoquinoline alkaloids are produced. In some embodiments,“and/or” is used to refer to production of benzylisoquinoline alkaloids, wherein one or more benzylisoquinoline alkaloids are produced through the following steps: culturing a recombinant S. cerevisiae host, synthesizing one or more benzylisoquinoline alkaloids in a recombinant S. cerevisiae host, and/or isolating one or more benzylisoquinoline alkaloids.
Functional Homologs
[00134] Functional homologs of the polypeptide described above are also suitable for use in producing benzylisoquinoline alkaloids and/or precursors thereof in a recombinant S. cerevisiae host. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide. A functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, can themselves be functional homologs. Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally occurring polypeptides (“domain swapping”). Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term“functional homolog” is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
[00135] Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of benzylisoquinoline alkaloid biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using, for example, the amino acid sequence of an NCS polypeptide as the reference sequence. An amino acid sequence is, in some instances, deduced from a corresponding nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a benzylisoquinoline alkaloid biosynthesis polypeptide. An amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in benzylisoquinoline alkaloid biosynthesis polypeptides, e.g., conserved functional domains. In some embodiments, nucleic acids and polypeptides are identified from transcriptome data based on expression levels rather than by using BLAST analysis.
[00136] Conserved regions can be identified by locating a region within the primary amino acid sequence of a benzylisoquinoline alkaloid biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer et al., Nucl. Acids Res., 26:320- 322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman et al., Nucl. Acids Res., 27:260-262 (1999). Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.
[00137] Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity ( e.g ., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
[00138] For example, polypeptides suitable for producing norcoclaurine in a recombinant S. cerevisiae host include NCS polypeptides and functional homologs thereof.
[00139] Methods to modify the substrate specificity of, for example, a benzylisoquinoline alkaloid biosynthesis polypeptide, are known to those skilled in the art, and include without limitations site-directed/rational mutagenesis approaches, random directed evolution
approaches, and combinations in which random mutagenesis/saturation techniques are performed near the active site of the enzyme. See, e.g., Osmani et al., 2009, Phytochemistry 70: 325-347.
[00140] A candidate sequence typically has a length that is from 80% to 250% of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250% of the length of the reference sequence. A functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, or 120% of the length of the reference sequence, or any range between. A % sequence identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows. A reference sequence (e.g., a nucleic acid sequence or an amino acid sequence described herein) is aligned to one or more candidate sequences using the computer program Clustal Omega (version 1.2.1 , default parameters), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et al ., 2003, Nucleic Acids Res. 31 (13):3497-500.
[00141] Clustal Omega calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: %age; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1 ; window size: 5; scoring
method:%age; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gin, Glu, Arg, and Lys; residue-specific gap penalties: on. The Clustal Omega output is a sequence alignment that reflects the relationship between sequences. Clustal Omega can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European
Bioinformatics Institute site at http://www.ebi. ac.uk/Tools/msa/clustalo/.
[00142] To determine a % sequence identity of a candidate nucleic acid or amino acid sequence to a reference sequence, the sequences are aligned using Clustal Omega, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the% sequence identity value can be rounded to the nearest tenth. For example, 78.1 1 , 78.12, 78.13, and 78.14 are rounded down to 78.1 , while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. [00143] In some embodiments, a nucleic acid sequence encoding a benzylisoquinoline alkaloid biosynthesis polypeptide can include a tag sequence that encodes a“tag” designed to facilitate subsequent manipulation ( e.g ., to facilitate purification or detection), solubility, secretion, or localization of the encoded polypeptide. Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), disulfide oxidoreductase (DsbA), maltose binding protein (MBP), N-utilization substance (NusA), small ubiquitin-like modifier (SUMO), and Flag™ tag (Kodak, New Haven, CT). Other examples of tags include a chloroplast signal peptide, a mitochondrial signal peptide, an amyloplast peptide, signal peptide, or a secretion tag.
[00144] In some embodiments, a benzylisoquinoline alkaloid biosynthesis polypeptide is a protein altered by domain swapping. As used herein, the term“domain swapping” is used to describe the process of replacing a domain of a first protein with a domain of a second protein. In some embodiments, the domain of the first protein and the domain of the second protein are functionally identical or functionally similar. In some embodiments, the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein. In some embodiments, an NCS polypeptide (e.g., a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA) is altered by domain swapping.
[00145] It will be appreciated that benzylisoquinoline alkaloid biosynthesis polypeptides can include additional amino acids that are not involved in the enzymatic activities carried out by the polypeptides. In some embodiments, benzylisoquinoline alkaloid biosynthesis polypeptides (e.g., an NCS polypeptide) are fusion proteins. The terms“chimera,”“fusion polypeptide,” “fusion protein,”“fusion enzyme,”“fusion construct,”“chimeric protein,” "chimeric polypeptide," “chimeric construct,” and“chimeric enzyme” can be used interchangeably herein to refer to proteins engineered through the joining of two or more genes that code for different proteins.
[00146] In some embodiments, a protein is altered by circular permutation, which involves covalent attachment of the ends of a protein that would be opened elsewhere afterwards. Thus, the order of the sequence is altered without causing changes in the amino acids of the protein.
In some embodiments, a targeted circular permutation can be produced, for example but not limited to, by designing a spacer to join the ends of the original protein. Once the spacer has been defined, there are several possibilities to generate permutations through generally accepted molecular biology techniques, for example but not limited to, by producing
concatemers by means of PCR and subsequent amplification of specific permutations inside the concatemer or by amplifying discrete fragments of the protein to exchange to join them in a different order. The step of generating permutations can be followed by creating a circular gene by binding the fragment ends and cutting back at random, thus forming collections of permutations from a unique construct.
Endogenous Transporters
[00147] Modification of transport systems in a recombinant S. cerevisiae host that are involved in transport of benzylisoquinoline alkaloids and/or precursors thereof can allow for more effective production of benzylisoquinoline alkaloids in recombinant hosts.
[00148] As used herein, the terms“transport of a benzylisoquinoline alkaloid (precursor),” “benzylisoquinoline alkaloid (precursor) transport,”“excretion of a benzylisoquinoline alkaloid (precursor),” and“benzylisoquinoline alkaloid (precursor) excretion” can be used
interchangeably.
[00149] As used herein, the term“transporter” (also referred to as a membrane transport protein) refers to a membrane protein involved in the movement of small molecules, macromolecules (such as carbohydrates), and ions across a biological membrane.
Transporters span the membrane in which they are localized and across which they transport substances. Transporter proteins can assist in the movement (i.e., transport or excretion) of a substance from the intracellular space to the culture medium, or from a vacuolar space to the intracellular space. Transporters are known to function as passive transport systems, carrying molecules down their concentration gradient, or as active transport systems, using energy to carry molecules uphill against their concentration gradient. Active transport is mediated by carriers which couple transport directly to the use of energy derived from hydrolysis of an ATP molecule or by carriers which make use of a pre-established electrochemical ion gradient to drive co-transport of the nutrient molecule and a co-transported ion. The latter category comprises symporters and antiporters, which carry the ion in the same or opposite direction, respectively, as the transported substrate.
[00150] Transport proteins have been classified according to various criteria at the
Transporter Classification Database (on the world-wide web at tcdb.org). See Saier Jr. et al., Nucl. Acids Res., 42(1 ):D251-258 (2014). Non-limiting examples thereof include, among others, the family of Multiple Drug Resistance (MDR) plasma membrane transporters that is thought to be ubiquitous among living organisms. The MDR transporter superfamily can be further subdivided according to the mode of operation by which the substrate is transported from one side of the membrane to the other. Transporters can operate to move substances across membranes in response to chemiosmotic ion gradients or by active transport. ATP-binding cassette transporter polypeptides (ABC transporters) are transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to carry out translocation of various substrates across membranes. They can transport a wide variety of substrates across the plasma membrane and intracellular membranes, including metabolic products, lipids and sterols, and drugs. Particular non-limiting examples of endogenous ABC transporter genes include PDR5, PDR10, PDR12, PDR15, PDR18, SNQ2, YDR061W, YOR1 , YOL075C, MDL2,
ADP1 , CAF16, VMR1 , AUS1 , NFT1 , STE6, YCF1 , YBT1 , BPT1 , and STE6 (or a functional homolog thereof). In some aspects, ABC transporters transport benzylisoquinoline alkaloid precursors and/or benzylisoquinoline alkaloids.
[00151] Another transporter family, the Major Facilitator Superfamily (MFS) transporters are monomeric polypeptides that can transport small solutes in response to proton gradients. The MFS transporter family is sometimes referred to as the uniporter-symporter-antiporter family. MFS transporters function in, inter alia, in sugar uptake and drug efflux systems. MFS transporters typically comprise conserved MFS-specific motifs. Non-limiting examples of endogenous MFS transporter genes include TP01 , TP02, TP03, TP04, QDR1 , QDR2, QDR3, FLR1 , DTR1 , YHK8, SE01 , YBR241 C, VBA3, FEN2, SNF3, STL1 , HXT10, AZR1 , MPH3, VBA5, GEX2, SNQ1 , AQR1 , MCH1 , MCH5, ATG22, HXT15, MPH2, ITR1 , SIT 1 , VPS73, HXT5, SOA1 , HXT9, YMR279C, YIL166C, HOL1 , and ENB1 (or a functional homolog thereof). In some aspects, MFS transporters transport benzylisoquinoline alkaloid precursors and/or benzylisoquinoline alkaloids.
Endogenous Dehydrogenases and Reductases
[00152] Modification of endogenous biosynthetic pathway genes in a recombinant S.
cerevisiae host that involve one or more benzylisoquinoline alkaloids precursors can allow for more effective production of benzylisoquinoline alkaloids in recombinant hosts.
[00153] For example, it has been shown that disruption of certain endogenous
oxidoreductases in yeast can reduce the level of reduction of 4-HPAA to tyrosol and thereby improve norcoclaurine or norlaudanosoline yields in recombinant strains expressing a norcoclaurine synthase (NCS) enzyme, with ARM (YGL157W) being the most prominent, with clear indication in the presented data from that ADH6 disruption also has an effect. See, e.g., WO 2018/029282; Kristy Hawkins,“Metabolic engineering of Saccharomyces cerevisiae for the production of benzylisoquinoline alkaloids” 1 January 2009. Caltech Thesis, XP55361294, pp. 1-154. However, the fold-increase yield demonstrated by these approaches are still not high enough to make the process commercially viable since much 4-HPAA are still lost to Tyrosol and yields of norcoclaurine still are in pg/L or low mg/L scale.
[00154] S. cerevisiae comprises a number of endogenous genes encoding dehydrogenases and reductases that act on substrates similar to one or more benzylisoquinoline alkaloid precursors such as, for example, 4-HPAA. For example, S. cerevisiae comprises five aldehyde dehydrogenase genes (ALD2, ALD3, ALD4, ALD5, and ALD6) and seven primary alcohol dehydrogenase genes (ADH1 , ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7). S. cerevisiae further includes a formaldehyde dehydrogenase gene (SFA1 ), and an aldehyde reductase gene (ARM ). Other endogenous genes similar to ARI1 include YGL039W, YDR541C, YPR1 , GCY1 , GRE2, AAD3, AAD4, and AAD14.
[00155] Notably, several endogenous dehydrogenases and reductases are involved in NADPH cofactor supply and regeneration, an important cofactor in certain Ehrlich pathway steps. Modification of such genes can provide secondary effects on endogenous biosynthesis pathways. For example, modification of an ALD4 gene can affect NADPH regeneration, which NADPH is involved in reduction of 4-HPAA to tyrosol (i.e., away from (S)-norcoclaurine).
Endogenous Amino Acid Biosynthesis Polypeptides
[00156] Modification of endogenous biosynthetic pathway genes in a recombinant S.
cerevisiae host that produce compounds that can act as substrates for benzylisoquinoline alkaloid biosynthesis polypeptides can allow for more effective production of benzylisoquinoline alkaloids in recombinant hosts. [00157] For example, removal of native transcriptional control of an endogenous biosynthetic pathway forwards tyrosine ( e.g ., by expressing one or more pathway polypeptides such as Aro10, Aro7, and/or Tyr1 from a constitutive, non-native promoter, and/or by expression one or more feedback-resistant pathway polypeptides such as Aro7-FBR (SEQ ID NO: 12) or Aro4-FBR (SEQ ID NO:4) to remove feedback inhibition from tyrosine) results in a downregulated, reinforced tyrosine pathway.
[00158] S. cerevisiae comprises an endogenous biosynthetic pathway towards
phenylalanine, of which the intermediate PAA can, in certain embodiments, be condensed with dopamine by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine. The phenylalanine pathway involves endogenous gene PHA2. S. cerevisiae also comprises an endogenous biosynthetic pathway towards tryptophan, on which the intermediate IAA can, in certain embodiments, be condensed with dopamine by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine. The tryptophan pathway involves endogenous genes TRP2 and TRP3.
[00159] In addition to IAA and PAA, a number of aldehyde products such as, for example, acetaldehyde (AA), 2-methylbutanal (2-MB) 3-methylbutanal (3-MB) (collectively, methylbutanal (MB)), acetoin, and pyruvate can, in certain embodiments, be condensed with dopamine by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine. The person of skill in the art will appreciate that AA, IAA, PAA, 2-MB, 3-MB, acetoin, and pyruvate are intermediates in various biosynthetic pathways (e.g., an endogenous biosynthetic pathway towards tyrosine, leucine, isoleucine, valine, or methionine).
[00160] As used herein, the term“NCS-compatible substrate” includes any aldehyde other than 4-HPAA that can be condensed with dopamine by a polypeptide also capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine (e.g., an (S)-norcoclaurine synthase (NCS) polypeptide; e.g., SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:54) such as, for example, AA, IAA, PAA, 2-MB, 3-MB, acetoin, and pyruvate. As used herein, the term “NCS-compatible substrate pathway” refers to any biosynthetic pathway that includes an NCS- compatible substrate as an intermediate or final product, such as, for example, the aromatic acid biosynthesis pathways endogenous to S. cerevisiae.
Endogenous Gene Inactivation
[00161] In some aspects, recombinant S. cerevisiae host cells capable of producing one or more benzylisoquinoline alkaloids and/or precursors thereof comprise one or more inactivated endogenous genes. An endogenous gene is typically inactivated by disrupting expression of the gene or introducing a mutation to reduce or even completely eliminate endogenous gene activity in a host comprising the mutation. For example, in certain embodiments, a disruption in one or more endogenous transporter genes reduces or deletes transport expression or activity for the transporter encoded by the disrupted gene(s). As used herein, the term“reduced expression” refers to any level of expression that is less than that of a corresponding gene not having reduced expression, including“repressed expression,”“lowered expression,”“no expression,” and“deletion.” For example, reduced expression can be produced in a host cell by disrupting or deleting the gene locus of the one or more endogenous genes. As set forth herein, recombinant S. cerevisiae host cells comprising reduced expression of at least one endogenous transporter gene or a transcription factor that regulates expression of at least one endogenous transporter gene are capable of producing at least one benzylisoquinoline alkaloid such as, for example, (S)-norcoclaurine. In certain embodiments, reducing endogenous transporter activity of a recombinant S. cerevisiae host cell increases the intracellular accumulation of one or more benzylisoquinoline alkaloid precursors ( e.g ., dopamine) by the recombinant host, which can subsequently increase production of one or more benzylisoquinoline alkaloids derived therefrom.
[00162] Recombinant S. cerevisiae hosts comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene, as disclosed herein, can include one or more
benzylisoquinoline alkaloid biosynthesis genes as disclosed herein, such as a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P; a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP; a gene encoding a polypeptide capable of synthesizing prephenate from chorismate; a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate; a gene encoding a polypeptide capable of synthesizing L-tyrosine from 4- HPP; a gene encoding a polypeptide capable of synthesizing 4-HPP from L-tyrosine; a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP; a gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine; a gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA; and/or a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine; wherein expression of these genes results in production of one or more benzylisoquinoline alkaloids ( e.g ., (S)-norcoclaurine).
[00163] In another example, in certain embodiments, a disruption in one or more
endogenous genes encoding one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., 4-HPAA), as disclosed herein, reduces or deletes expression or activity for the one or more polypeptides encoded by the disrupted gene(s). For example, in certain embodiments, a disruption in one or more endogenous genes encoding one or more polypeptides capable of synthesizing 4-hydroxyphenylethanol (tyrosol) or 4- hydroxyphenylacetate (4-HPAC) from 4-HPAA, as disclosed herein, reduces expression or activity for the one or more polypeptides encoded by the disrupted gene(s). As set forth herein, recombinant S. cerevisiae host cells comprising reduced expression (i.e., repressed expression or deletion) of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing 4-HPAA, as disclosed herein, are capable of producing at least one benzylisoquinoline alkaloid such as, for example, (S)-norcoclaurine. In certain embodiments, reducing activity of one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor ( e.g ., 4-HPAA), as disclosed herein, of a recombinant S. cerevisiae host cell increases the intracellular accumulation of one or more benzylisoquinoline alkaloid precursors (e.g., 4-HPAA) by the recombinant host, which can subsequently increase production of one or more benzylisoquinoline alkaloids derived therefrom.
[00164] Recombinant S. cerevisiae hosts comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., 4-HPAA) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., 4-HPAA), as disclosed herein, can include one or more benzylisoquinoline alkaloid biosynthesis genes as disclosed herein, such as a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P; a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP; a gene encoding a polypeptide capable of synthesizing prephenate from chorismate; a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate; a gene encoding a polypeptide capable of synthesizing L-tyrosine from 4- HPP; a gene encoding a polypeptide capable of synthesizing 4-HPP from L-tyrosine; a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP; a gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine; a gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA; and/or a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine; wherein expression of these genes results in production of one or more benzylisoquinoline alkaloids ( e.g ., (S)-norcoclaurine).
[00165] In another example, in certain embodiments, a disruption in one or more
endogenous genes encoding one or more NCS-compatible substrate pathway polypeptides, as disclosed herein, reduces or deletes expression or activity for the one or more polypeptides encoded by the disrupted gene(s). For example, in certain embodiments, a disruption in one or more endogenous genes encoding one or more polypeptides involved in tyrosine,
phenylalanine, tryptophan, leucine, isoleucine, valine, or methionine biosynthetic reduces expression or activity for the one or more polypeptides encoded by the disrupted gene(s). As set forth herein, recombinant S. cerevisiae host cells comprising reduced expression (i.e., repressed expression or deletion) of at least one NCS-compatible substrate pathway
polypeptides disclosed herein are capable of producing at least one benzylisoquinoline alkaloid such as, for example, (S)-norcoclaurine. In certain embodiments, reducing activity of one or more NCS-compatible substrate pathway polypeptides of a recombinant S. cerevisiae host cell decreases the intracellular accumulation of NCS-compatible substrates (e.g., AA, PAA, IA, 2- MB, 3-MP, acetoin, pyruvate) by the recombinant host, decreasing competition with 4-HPAA, and increasing the relative amount of intracellular dopamine available for condensation with 4- HPAA by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine.
[00166] Recombinant S. cerevisiae hosts comprising reduced expression of at least one endogenous gene encoding one or more NCS-compatible substrate pathway polypeptides or a transcription factor gene that regulates expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide, as disclosed herein, can include one or more benzylisoquinoline alkaloid biosynthesis genes such as a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P; a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP; a gene encoding a polypeptide capable of synthesizing prephenate from chorismate; a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate; a gene encoding a polypeptide capable of synthesizing L-tyrosine from 4-HPP; a gene encoding a polypeptide capable of synthesizing 4-HPP from L-tyrosine; a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP; a gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine; a gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA; and/or a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine; wherein expression of these genes results in production of one or more benzylisoquinoline alkaloids ( e.g ., (S)-norcoclaurine).
[00167] Expression of endogenous genes can be reduced, or endogenous genes can be deleted by mutations that disrupt the gene. For example, reduced expression of an
endogenous gene can be produced in a host cell by disrupting or deleting the gene locus. For example, a gene replacement vector can be constructed in such a way to include a selectable marker gene flanked at both the 5' and 3' ends by portions of the gene of sufficient length to mediate homologous recombination. The selectable marker can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change. Linearized DNA fragments of the gene replacement vector, containing no plasmid DNA or an element, are then introduced into cells using known methods. Integration of the linear fragment into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, Southern blot analysis. The resulting cells contain an inactivated mutant gene, due to insertion of the selectable marker at the locus for the polypeptide. A deletion-disruption gene replacement vector can be constructed in a similar way using known techniques and, by way of homologous recombinant, integrated in the endogenous gene, thereby inactivating it. In some embodiments, the selectable marker can be removed from the genome of the host cell after determining that the desired disruption mutation has been introduced. See, e.g., Gossen et al. (2002) Ann. Rev. Genetics 36:153-173.
[00168] Expression of endogenous genes can also be reduced, or endogenous genes can be deleted by utilizing CRISPR systems (see, e.g., Adli,“The CRISPR tool kit for genome editing and beyond,” Nature Communications 9:1911 (2018)), transcription activator-like effector nucleases (TALENs) (see, e.g., Joung & Sander,“TALENs: a widely applicable technology for targeted genome editing,” Nat. Rev. Mol. Cell. Biol. 14(1 ):49-55 (2013)), or modified zinc finger nucleases (see, e.g., Carroll,“Genome Engineering With Zinc-Finger Nucleases,” Genetics 188(4):773-82 (2011 ) to introduce desired insertion or deletion mutations. In some
embodiments, an endogenous gene is inactivated by introducing a mutation that results in insertions of nucleotides, deletions of nucleotides, or transition or transversion point mutations in the wild-type gene sequence. Other types of mutations that may be introduced in a gene include duplications and inversions in the wild-type sequence. Mutations can be made in the coding sequence at a locus for the polypeptide, as well as in noncoding sequences such as regulatory regions, introns, and other untranslated sequences. Mutations in the coding sequence can result in insertions of one or more amino acids, deletions of one or more amino acids, and/or non-conservative amino acid substitutions in the corresponding gene product. In some cases, the sequence of a gene comprises more than one mutation or more than one type of mutation. Insertion or deletion of amino acids in a coding sequence can, for example, disrupt the conformation of a substrate-binding pocket of the resulting gene product.
[00169] Amino acid insertions or deletions can also disrupt catalytic sites important for gene product activity. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids. Non-conservative substitutions can make a substantial change in the charge or hydrophobicity of the gene product. Non-conservative amino acid substitutions can also make a substantial change in the bulk of the residue side chain, e.g., substituting an alanine residue for an isoleucine residue. Examples of non conservative substitutions include a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid.
[00170] In some embodiments, a mutation in a gene may result in no amino acid changes but, although not affecting the amino acid sequence of the encoded polypeptide, may alter transcriptional levels (e.g., increasing or decreasing transcription), decrease translational levels, alter secondary structure of DNA or mRNA, alter binding sites for transcriptional or translational machinery, or decrease tRNA binding efficiency.
[00171] Mutations in loci for polypeptides can be generated by site-directed mutagenesis of the transporter gene sequence in vitro, followed by homologous recombination to introduce the mutation into the host genome as described above. However, mutations can also be generated by inducing mutagenesis in cells of the host, using a mutagenic agent to induce genetic mutations within a population of cells. Mutagenesis is particularly useful for those species or strains for which in vitro mutagenesis and homologous recombination is less well established or is inconvenient. The dosage of the mutagenic chemical or radiation for a particular species or strain is determined experimentally such that a mutation frequency is obtained that is below a threshold level characterized by lethality or reproductive sterility.
[00172] Modification of transcription factor expression can also be used to reduce or eliminate gene expression. For example, the yeast transcriptions factors PDR1 and/or PDR3 regulate expression of the genes encoding ABC transporters PDR5, SNQ2 and YOR1.
[00173] In some embodiments, a benzylisoquinoline alkaloid-producing host comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene. In some embodiments, the endogenous transporter gene is PDR5, PDR12, PDR15, and/or SNQ2. In some embodiments, the endogenous transporter genes are PDR5 and one or more of PDR12, PDR15 and SNQ2. In some embodiments the endogenous transporter genes are one or more of PDR5, PDR12, PDR15, and/or SNQ2 (e.g., PDR5 and PDR12), and one or more of AUS1 , PDR10, YOR1 , TP01 , TP02, TP03, TP04, QDR1 , QDR2, QDR3, FLR1 , YOL075C, PDR18, DTR1 , YHK8, NFT1 , STE6, YCF1 , YBT1 , BPT1 , and VMR1.
[00174] In some embodiments, a benzylisoquinoline alkaloid-producing host comprises reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor, or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor. In some embodiments, the at least one endogenous gene encodes a polypeptide capable of synthesizing tyrosol or 4-HPAC from 4-HPAA, such as, for example, ARM , ALD4, ADH6, YPR1 , or YDR541 C. In some embodiments, the endogenous gene is ARM , ALD4, ADH6, YPR1 , YDR541 C, YGL039W,
AAD3, and/or GRE2.
[00175] In some embodiments, a benzylisoquinoline alkaloid-producing host comprises reduced expression of at least one endogenous gene encoding one or more NCS-compatible substrate pathway polypeptides. In some embodiments, the NCS-compatible substrate pathway is an aromatic amino acid biosynthesis pathway, such as, for example, tryptophan (e.g., involving TRP2 and TRP3) or phenylalanine (e.g., involving PHA2) biosynthesis. In some embodiments, the NCS-compatible substrate pathway polypeptide is capable of synthesizing phenylpyruvate from prephenate (e.g., PHA2), capable of synthesizing anthranilate from chorismate (e.g., TRP2, and/or TRP3).
[00176] In some embodiments, the PDR5 gene, which can have the nucleotide sequence set forth in SEQ ID NO:69, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:70. In some embodiments, the PDR12 gene, which can have the nucleotide sequence set forth in SEQ ID NO:71 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:72. In some embodiments, the PDR15 gene, which can have the nucleotide sequence set forth in SEQ ID NO:73) encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:74. In some embodiments, the SNQ2 gene, which can have the nucleotide sequence set forth in SEQ ID NO:75 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:76. In some embodiments, the PDR1 gene, which can have the nucleotide sequence set forth in SEQ ID NO:183, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 184. In some embodiments, the PDR3 gene, which can have the nucleotide sequence set forth in SEQ ID NO: 185, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:186.
[00177] In some embodiments, the AUS1 gene, which can have the nucleotide sequence set forth in SEQ ID NO:77 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:78. In some embodiments, the PDR10 gene, which can have the nucleotide sequence set forth in SEQ ID NO:79, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:80. In some embodiments, the YOR1 gene, which can have the nucleotide sequence set forth in SEQ ID NO:81 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:82. In some embodiments the TP01 gene, which can have the nucleotide sequence set forth in SEQ ID NO:83 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:84. In some embodiments the TP02 gene, which can have the nucleotide sequence set forth in SEQ ID NO:85 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:86. In some embodiments the TP03 gene, which can have the nucleotide sequence set forth in SEQ ID NO:87, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:88. In some
embodiments the TP04 gene, which can have the nucleotide sequence set forth in SEQ ID NO:89, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:90. In some embodiments the QDR1 gene, which can have the nucleotide sequence set forth in SEQ ID NO:91 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:92. In some embodiments the QDR2 gene, which can have the nucleotide sequence set forth in SEQ ID NO:93, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:94. In some embodiments the QDR3 gene, which can have the nucleotide sequence set forth in SEQ ID NO:95, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:96. In some embodiments the FLR1 gene, which can have the nucleotide sequence set forth in SEQ ID NO:97, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:98. In some embodiments the YOL075C gene, which can have the nucleotide sequence set forth in SEQ ID NO:99, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:100. In some embodiments the PDR18 gene, which can have the nucleotide sequence set forth in SEQ ID NO:101 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:102. In some embodiments the DTR1 , which can have the nucleotide sequence set forth in SEQ ID NO: 103, gene encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:104. In some embodiments the YHK8 gene, which can have the nucleotide sequence set forth in SEQ ID NO:105, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:106. In some embodiments the NFT 1 gene, which can have the nucleotide sequence set forth in SEQ ID NO:107, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 108. In some embodiments the STE6 gene, which can have the nucleotide sequence set forth in SEQ ID NO:109, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:1 10. In some embodiments the YCF1 gene, which can have the nucleotide sequence set forth in SEQ ID NO:1 1 1 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:1 12. In some embodiments the YBT 1 gene, which can have the nucleotide sequence set forth in SEQ ID NO: 113 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1 14. In some embodiments the BPT2 gene, which can have the nucleotide sequence set forth in SEQ ID NO:1 15 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:1 16. In some embodiments the VMR1 gene, which can have the nucleotide sequence set forth in SEQ ID NO:1 17 encodes a polypeptide having an amino acid sequence set as forth in SEQ ID NO:1 18.
[00178] In some embodiments, the ARI1 gene, which can have the nucleotide sequence set forth in SEQ ID NO:145, encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO: 146. In some embodiments, the ALD4 gene, which can have the nucleotide sequence set forth in SEQ ID NO:123, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:124. In some embodiments, the ADH6 gene, which can have the nucleotide sequence set forth in SEQ ID NO: 139, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 140. In some embodiments, the YPR1 gene, which can have the nucleotide sequence set forth in SEQ ID NO:153, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 154. In some embodiments, the YDR541 C gene, which can have the nucleotide sequence set forth in SEQ ID NO:149 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 150. In some embodiments, the PHA2 gene, which can have the nucleotide sequence set forth in SEQ ID NO: 163 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 164. In some embodiments, the TRP2 gene, which can have the nucleotide sequence set forth in SEQ ID NO:165 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 166. In some embodiments, the TRP3 gene, which can have the nucleotide sequence set forth in SEQ ID NO:167, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:168. In some embodiments, the AAD3 gene, which can have the nucleotide sequence set forth in SEQ ID NO:159, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:160. In some embodiments, the GRE2 gene, which can have the nucleotide sequence set forth in SEQ ID NO: 151 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 152.
[00179] In some embodiments, benzylisoquinoline alkaloids and/or precursors thereof are produced in vivo through expression of one or more recombinant genes encoding one or more benzylisoquinoline biosynthesis polypeptides in a recombinant host comprising reduced expression of one or more endogenous genes. For example, a recombinant S. cerevisiae host comprising reduced expression of at least one endogenous transporter gene ( e.g ., PDR5, PDR12, PDR15, and/or SNQ2) or a transcription factor that regulates expression of at least one endogenous transporter gene (e.g., PDR1 and/or PDR3), and further comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephenate from chorismate (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:12); a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 14); a gene encoding a polypeptide capable of synthesizing L-tyrosine from 4-HPP and/or 4-HPP from L-tyrosine; a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:22 or SEQ ID NO:24); a gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:26 and SEQ ID NO:28); and/or a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine ( e.g ., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in any of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, and 198) can produce (S)-norcoclaurine in vivo. In certain embodiments, the recombinant S. cerevisiae host comprises reduced expression of one, or two, or three, or each of PDR5 (SEQ ID NO:69, SEQ ID NO:70), PDR12 (SEQ ID NO:71 , SEQ ID NO:72), PDR15 (SEQ ID NO:73, SEQ ID NO:74), and SNQ2 (SEQ ID NO:75, SEQ ID NO:76). In certain such embodiments, the polypeptide capable of synthesizing (S)- norcoclaurine from 4-HPAA and dopamine has the amino acid sequence set forth in any of SEQ ID NO:42 or 30.
[00180] In another example, a recombinant S. cerevisiae host comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., ARM , ALD4, ADH6, YPR1 , AAD3,
GRE2, and/or YDR541 C) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor, and further comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephenate from chorismate (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 12); a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 14); a gene encoding a polypeptide capable of synthesizing L-tyrosine from 4-HPP and/or 4-HPP from L-tyrosine; a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP ( e.g ., a recombinant gene having an amino acid sequence as set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:22 or SEQ ID NO:24); a gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:26 or SEQ ID NO:28); and/or a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in any of SEQ ID NOs:30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188,
190, 192, 194, 196, and 198) can produce (S)-norcoclaurine in vivo. In certain embodiments, the recombinant S. cerevisiae host comprises reduced expression of one, or two, or three, or four, or five, or six, or each of ARM (SEQ ID NO:145, SEQ ID NO:146), ALD4 (SEQ ID NO:123, SEQ ID NO:124), ADH6 (SEQ ID NO:139, SEQ ID NO:140), YPR1 (SEQ ID NO:153, SEQ ID NO:154), AAD3 (SEQ ID NO:159, SEQ ID NO: 160), GRE2 (SEQ ID NO: 151 , SEQ ID NO:152), and YDR541 C (SEQ ID NO:149, SEQ ID NO:150). In certain embodiments, the recombinant S. cerevisiae host comprises reduced expression of one, or two, or three, or four, or five, or six, or each of ARM (SEQ ID NO:145, SEQ ID NO: 146), ALD4 (SEQ ID NO:123, SEQ ID NO:124), ADH6 (SEQ ID NO:139, SEQ ID NO:140), YPR1 (SEQ ID NO:153, SEQ ID NO:154), AAD3 (SEQ ID NO: 159, SEQ ID NO:160), GRE2 (SEQ ID NO: 151 , SEQ ID NO:152), and YDR541C (SEQ ID NO: 149, SEQ ID NO:150), and further comprises reduced expression of one, or two, or each of TRP2 (SEQ ID NO:165, SEQ ID NO: 166), TRP3 (SEQ ID NO: 167, SEQ ID NO: 168), and PHA2 (SEQ ID NO: 163, SEQ ID NO:164). In certain such embodiments, the polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine is a truncated polypeptide (e.g., lacking an N-terminal signal peptide; e.g. having an amino acid sequence as set forth in SEQ ID NO:42) . In certain such embodiments, the polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine is a polypeptide having an amino acid sequence as set forth in SEQ ID NO:30.
[00181] In another example, a recombinant S. cerevisiae host comprising reduced expression of at least one endogenous transporter gene ( e.g ., PDR5, PDR12, PDR15, and/or SNQ2) or a transcription factor that regulates expression of at least one endogenous transporter gene, further comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., ARM , ALD4, ADH6, YPR1 , AAD3, GRE2, and/or YDR541 C) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor and/or reduced expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide (e.g., PHA2, TRP2, and/or TRP3) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide, and also comprising one or more recombinant genes encoding one or more benzylisoquinoline biosynthesis polypeptides can produce one or more benzylisoquinoline alkaloids (e.g., (S)- norcoclaurine) in vivo.
[00182] In some embodiments, expression of one or more recombinant genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA (e.g., a polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, and 198) in a recombinant S. cerevisiae host comprising reduced expression of one or more endogenous transporter genes (e.g., PDR5, PDR12, PDR15, and/or SNQ2) or a transcription factor that regulates expression of at least one endogenous transporter gene (e.g., PDR1 and/or PDR3) increases the amount of (S)- norcoclaurine produced by the host cell by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%.
[00183] In some embodiments, expression of one or more recombinant genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA ( e.g ., a polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, and 198) in a recombinant S. cerevisiae host comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., ARM , ALD4, ADH6, YPR1 , AAD3, GRE2, and/or YDR541 C) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor and/or reduced expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide (e.g., PHA2, TRP2, and/or TRP3) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide, increases the amount of (S)-norcoclaurine produced by the host cell by at least about 50%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%, or at least about 1 ,000%, or at least about 1 ,500%, or at least about 2,000%, or at least about 5,000%, or at least about 7,500%, or at least about 10,000%.
Benzylisoquinoline Alkaloid Biosynthesis Nucleic Acids
[00184] A recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired. A coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence. Typically, the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.
[00185] In many cases, the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid. Thus, if the recombinant host is a microorganism, the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism.
A native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. “Regulatory region” refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. A regulatory region typically comprises at least a core (basal) promoter. A regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). A regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence. For example, to operably link a coding sequence and a promoter sequence, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter. A regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
[00186] The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
[00187] One or more genes can be combined in a recombinant nucleic acid construct in “modules” useful for a discrete aspect of production of benzylisoquinoline alkaloids and/or precursors thereof. Combining a plurality of genes in a module, particularly a polycistronic module, facilitates the use of the module in a variety of species. In additional to genes useful for benzylisoquinoline alkaloid production, a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species.
[00188] It will be appreciated that because of the degeneracy of the genetic code, a number of nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. Thus, codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host {e.g.,
microorganism). As isolated nucleic acids, these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
Host Microorganisms
[00189] Recombinant S. cerevisiae hosts can be used to express polypeptides as otherwise described herein for producing benzylisoquinoline alkaloids. Typically, the recombinant host is grown in a fermenter at a temperature for a period of time, wherein the temperature and period of time facilitate production of one or more benzylisoquinoline alkaloids. The constructed and genetically engineered S. cerevisiae hosts provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, semi-continuous fermentations such as draw and fill, continuous perfusion fermentation, and continuous perfusion cell culture. Levels of substrates and intermediates, e.g., E4P, DAHP, EPSP, chorismate, prephenate, 4-HPP, 4-HPAA, L-tyrosine, L-DOPA, and dopamine, can be determined by extracting samples from culture media for analysis according to published methods.
[00190] Carbon sources of use in the instant method include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the benzylisoquinoline alkaloids. Examples of suitable carbon sources include sucrose, fructose, xylose, ethanol, glycerol, and glucose. The carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during a fed-batch phase.
[00191] The present invention further relates to a fermentation process, including but not limited to small-scale or batch and/or fed-batch fermentation process and a large-scale fermentation process, for the production of (S)-norcoclaurine, as described herein, whereby the cultivation regime of the process comprises at least one production stage fermentation phase. In certain embodiments, the fermentation process is a batch cultivation. In other embodiments, the fermentation process is a fed-batch process. In such a fed-batch process, a base medium supports initial growth and production, and a continuously or periodically supplied feeding medium prevents depletion of nutrients and sustains the production stage. The media can be selected to accommodate the distinct metabolic requirements during different cultivation phases. Process parameters including feeding strategy and control parameters define the chemical and physical environments suitable for cell growth and/or (S)-norcoclaurine production.
[00192] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
[00193] The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
Example 1 : LC-MS and LC-UV Analytical Procedures
[00194] Metabolites were separated by reverse-phase HPLC (Perkin Elmer SERIES 200 Micropump, Perkin Elmer, Norfolk, CT, USA) using an Agilent Zorbax Rapid Resolution HT C18 2.1 *30 mm, 1.8 micron column. Samples were separated using a linear gradient: 0-5 min 98% A (0.1 % formic acid) / 2% B (100% acetonitrile), 5-7 min 90% A / 10% B, 7-7.1 min 15% A / 85% B at a flow rate of 0.3 ml/min, followed by a 3 min equilibration at 100% A at a flow rate of 0.4 ml/min. Ten microliters of broth extract was loaded on the HPLC column run at a flow rate of 0.3 ml/min. Following LC separation, metabolites were injected into the LTQ-FT-MS (ESI source in positive ion mode). Identification of alkaloids was done using retention time and exact mass (<10 ppm) of the monoisotopic mass of the protonated molecular ion. LC-FT-MS data were processed using Xcalibur (Thermo Scientific, Bremen, Germany).
[00195] Benzylisoquinoline alkaloids and precursors thereof can be isolated from a cell culture using a method described herein. For example, following fermentation, cultures were diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose. After 72-96 h growth, metabolites were extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice- cold 100% acetonitrile (ACN; 80% final concentration). Cells were incubated for 5 minutes and subsequently diluted with 417 mI of 0.16% formic acid (FA) to give a final concentration of 30% ACN and 0.1 % FA. The resulting extract was utilized for LC-MS analysis. Cell broth was diluted 13.34-fold using this method.
Example 2: Fluorescence Detection Procedures
[00196] Fluorescence levels were measured after cultivating strains overnight in SC with 2% glucose. Overnight cultures were diluted 10* into fresh media and incubated for an additional 4 h to obtain log phase cells. Fluorescence was measured from cell suspension using a microplate reader and normalized against OD6oo for three biological replicates. Fluorescence was detected by the TECAN M200 plate reader using an excitation wavelength of 485 nm and an emission wavelength of 525 nm. Gain was adjusted for each sample. A background strain lacking GFP was used to correct for autofluorescence generated by cells and media.
Example 3: Strain Engineering
[00197] Dopamine-producing S. cerevisiae strains comprising and expressing a recombinant gene encoding a feedback-resistant variant of DAHP synthase polypeptide (SEQ ID NO:3, SEQ ID NO:4), a recombinant gene encoding a tyrosine hydroxylase polypeptide (SEQ ID NO:21 , SEQ ID NO:22), and a recombinant gene encoding an L-DOPA decarboxylase polypeptide
(SEQ ID NO:25, SEQ ID NO:26) were engineered.
Example 4: NCS Polypeptide Expression in S. cerevisiae
[00198] A dopamine-producing S. cerevisiae strain as described in Example 3 was transformed with a vector comprising a codon-optimized nucleotide sequence encoding an NCS polypeptide, optionally N-terminal truncated to remove a signal peptide, C-terminal truncated to remove a transmembrane domain, or C-terminal domain-swapped, and/or optionally green fluorescent protein (GFP)-tagged, operably linked to a pTEF1 promoter (SEQ ID NO:173 and a tPGM terminator (SEQ ID NO:174), as summarized in Table 1 , below.
Table 1. NCS Polypeptides Expressed
Figure imgf000077_0001
Figure imgf000078_0001
[00199] Dopamine-producing S. cerevisiae cells were cultivated in YPD medium (10 g/L yeast extract, 20 g/L tryptone, 20 g/L dextrose; Thermo Fisher Scientific) for routine strain growth and maintenance. YPD was supplemented, if necessary, with 200 pg/ml G418 or 200 pg/nnl hygromycin B. G418 concentration was increased to 400 pg/ml for selection on agar- solidified YPD. E. coli DH5a was utilized for routine plasmid maintenance and propagation and was cultivated in Lysogeny Broth (LB) (Sigma Aldrich). Ampicillin or kanamycin at
concentrations of 125 and 30 pg/ml, respectively, were used for plasmid selection in E. coli. Selected constructs were cloned using the CloneJet PCR cloning kit (Thermo Fisher Scientific). The gfp expression cassette was amplified from pGREG503 (Jansen). Oligonucleotides were purchased from Life Technologies and high-fidelity PCR was performed using Phusion DNA Polymerase (Thermo Fisher Scientific). Yeast colony PCR was performed using the Phire Plant Direct PCR kit.
[00200] Colonies were picked in triplicate and inoculated into 500 pi of 2* synthetic complete (SC) medium containing 4% glucose or sucrose in 96-well two ml deep well plates. The present inventors have determined that sucrose, in place of glucose, increases production of metabolites derived from the shikimate pathway by 50%. Following growth at 30 °C and 350 rpm for 18-24 h, cultures were diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose. After 72-96 h growth, metabolites were extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice-cold 100% acetonitrile (ACN; 80% final concentration). Cells were incubated for 5 minutes and subsequently diluted with 417 pi of 0.16% formic acid (FA) to give a final concentration of 30% ACN and 0.1 % FA. The resulting extract was utilized for LC- MS analysis according to Example 1. Cell broth was diluted 13.34-fold using this method. [00201] Results are shown in Figure 3. Strains 1-5, and particularly strains 1 and 3
(expressing NdNCS and ScNCS, respectively) showed increased levels of (S)-norcoclaurine. After 96 hours, strains 1 and 3 produced 0.63 and 0.65 mg/L (S)-norcoclaurine, respectively. N- terminal truncation of the 20-amino-acid signal peptide of NdNCS and ScNCS led to a 1.4-fold increase in (S)-norcoclaurine production; strains 6 and 7 produced 0.94 and 0.92 mg/L (S)- norcoclaurine, respectively. Conversely, N-terminal truncation of the 14-amino-acid signal peptide of PsNCS3, SdNCS, and TfNCS abolished (S)-norcoclaurine production in strains 8-10.
[00202] C-terminal truncation of the 22-amino-acid transmembrane domain of SdNCS and TfNCS led to increased (S)-norcoclaurine production; strains 1 1 and 12, expressing C-terminal truncated SdNCS and TfNCS, respectively, produced 74% and 44% more (S)-norcoclaurine than strains 4 and 5, but overall less than strains 6 and 7. C-terminal domain swapped SdNCS and TfNCS (with the corresponding C-terminal domain of PsNCS3) significantly decreased (S)- norcoclaurine production in strains 13 and 14.
Table 2. (S)- Norcoclaurine Production
Figure imgf000079_0001
aRelative to strain 1 ; bRelative to strain 3; cRelative to strain 4; dRelative to strain 5 (see Table 1 ).
[00203] The effect of polypeptide localization within recombinant S. cerevisiae was studied by comparing GFP-tagged NdNCS and ScNCS with GFP-tagged, N-terminal truncated NdNCS and ScNCS. Fluorescence intensity measurements were performed according to Example 2, above. Strains 15-18 were visualized using high resolution confocal microscopy; results are shown in Figure 4. Whereas control strain 19 displayed diffuse fluorescence throughout the cytoplasm, GFP-tagged NdNCS (strain 15) and ScNCS (strain 17) produced punctate foci often associated with the vacuole. This pattern is consistent with inclusion bodies caused by aggregation of poorly soluble NdNCS and ScNCS proteins. Conversely, cells harboring GFP- tagged, N-truncated NdNCS and ScNCS proteins in strains 16 and 18, respectively, showed a reduced occurrence of punctate foci and substantially higher levels of fluorescence distributed throughout the cytoplasm. The results indicated that the enhanced NCS activity of the truncated NdNCS and ScNCS polypeptides was due to increased solubility following removal of heterologous N-terminal signal peptides. This conclusion was corroborated by fluorescence intensity measurements of single cells, which showed a 1.5- to 3-fold increase in specific GFP fluorescence was in strains 16 and 18, relative to strains 15 and 17.
Table 3. Fluorescence
Figure imgf000080_0001
Example 5: Off-Target Endogenous Gene Inactivation in S. cerevisiae
[00204] A dopamine-producing S. cerevisiae strain as described in Example 3, further engineered to express at least one (S)-norcoclaurine synthase as described in Example 4 (SEQ ID NO:31 , SEQ ID NO:32; SEQ ID NO:41 , SEQ ID NO:42) was engineered to downregulate expression of one or more native genes encoding dehydrogenase and reductase polypeptides having potential activity on one or more benzylisoquinoline alkaloid precursors, and optionally to overexpress one or more benzylisoquinoline alkaloid biosynthesis polypeptides, as summarized in Table 4, below.
Table 4. Endogenous Genes Deletion
Figure imgf000080_0002
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
[00205] Colonies were picked in triplicate and inoculated into 500 pi of 2* synthetic complete (SC) medium containing 4% glucose or sucrose in 96-well two ml deep well plates. The present inventors have determined that sucrose, in place of glucose, increases production of metabolites derived from the shikimate pathway by 50%. Following growth at 30 °C and 350 rpm for 18-24 h, cultures were diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose. After 72-96 h growth, metabolites were extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice-cold 100% acetonitrile (ACN; 80% final concentration). Cells were incubated for 5 minutes and subsequently diluted with 417 mI of 0.16% formic acid (FA) to give a final concentration of 30% ACN and 0.1 % FA. The resulting extract was utilized for LC- MS analysis according to Example 1. Cell broth was diluted 13.34-fold using this method.
[00206] Twenty S. cerevisiae genes with potential activity for 4-HPAA were targeted for deletion (strains 20-40) including all 5 aldehyde dehydrogenase genes (ALD2, ALD3, ALD4, ALD5, and ALD6), all 7 primary alcohol dehydrogenase genes (ADH1-ADH7), the gene encoding formaldehyde dehydrogenase (SFA1 ), and 7 genes similar to ARI1 (YGL039W, YDR541C, YPR1 , GCY1 , GRE2, AAD3, and AAD14). (S)-norcoclaurine production is shown in Figure 5. Strains 24, 35, and 37 exhibited a significant growth defect. The growth rate of an ADH1 -deleted S. cerevisiae host cell could be enhanced by further engineering the strain to express a gene encoding Lactococcal NADH oxidase (LlnoxE) (strain 40).
[00207] While most of deletion strains 20-40 demonstrated (S)-norcoclaurine production similar to or less than that of strain 1 (expressing NdNCS), strains 32, 21 , and 23, lacking ARM , ALD4, and ALD6, respectively, demonstrated increased (S)-norcoclaurine production. Strain 32 showed a 3.5-fold increase in (S)-norcoclaurine production. Strains 21 and 23 demonstrated increased dopamine formation; strain 21 (lacking ALD6) showed a 2.8-fold increase. Strain 40 produced no dopamine or (S)-norcoclaurine.
[00208] In order to stabilize a high intracellular concentration of 4-HPAA in (S)- norcoclaurine-producing S. cerevisiae, combinations of one or more of the targets of strains 20- 40 were targeted for deletion (strains 41-66 and 75-82). (S)-norcoclaurine production is shown in Figures 6-9. Strains lacking the combination of ARM , ALD4, and ADH6 ( e.g ., strains 47, 55-56) showed increased (S)-norcoclaurine performance; additional deletions failed to further enhance performance (strains 50-54). The adversely impacted growth of strain 50 (lacking YPR1 ) likely indicated a toxic buildup of aldehyde, due to an inability of NCS to efficiently transfer the pool of available 4-HPAA.
[00209] Certain deletion strains (55-66 and 75-82) were engineered to express one or more copies of the recombinant gene encoding N-terminal truncated NdNCS of Example 4. (S)- norcoclaurine production is shown in Figures 7-9. Strain 57, overexpressing N-terminal truncated NdNCS and lacking the combination of ARM , ALD4, ADH6, and YPR1 demonstrated increased production of (S)-norcoclaurine without an associated decline in culture growth.
Additional deletion of YDR541C and ADH3 in strains 58 and 59, respectively, further increased (S)-norcoclaurine production, but deletion of both YDR541 C and ADH3 in combination with ARM , ALD4, ADH6, and YPR1 (strains 73 and 74) showed no improvement over deletion of just one of YDR541 C and ADH3 in the same combination. Additional deletion of ADH3, ADH5, ADH7, SFA1 , YGL039W, AAD3, and AAD14 in strains further overexpressing N-terminal truncated NdNCS and lacking the combination of ARM , ALD4, ADH6, YPR1 (strains 75-82) showed no additional improvement over strain 66, similarly overexpressing N-terminal truncated NdNCS and lacking the combination of ARM , ALD4, ADH6, YPR1 , and YDR541 C. Relative to strain 1 (expressing NdNCS), strain 66 demonstrated a 121-fold increase of (S)-norcoclaurine production. See Figure 9. To probe NCS promiscuity in the engineered strains, LC-MS spectra were examined for peaks indicating off-target NCS condensation products by first searching for 35 unique m/z values derived from condensation of dopamine with 63 possible endogenous aldehydes and ketones, including aliphatic, aromatic, and cyclic species. From this analysis, six putative Pictet-Spengler-competent carbonyl species were identified. See Figure 10. A major peak consistent with salsolinol (1 ), derived from condensation of dopamine with acetaldehyde, was observed and was larger than the (S)-norcoclaurine peak in all LC-MS spectra. Three putative condensation products were consistent with condensation of dopamine and endogenous Ehrlich pathway aldehydes, namely phenylacetaldehyde (PAA) from phenylalanine (product 2), indole acetaldehyde (IAA) from tryptophan (product 3), and the isomers 2- methylbutanal (2MB) or 3-methylbutanal (3MB) from isoleucine and leucine, respectively (products 4a and 4b, respectively).
[00210] Peaks corresponding to condensation of dopamine and Ehrlich pathway aldehydes derived from valine or methionine were not identified. The two remaining putative dopamine condensation products were consistent with the ketone moieties of acetoin (product 5) or pyruvate (product 6). To assess promiscuity of NdNCS for the amine substrate, LC-MS data was queried using the seven Pictet-Spengler-competent carbonyl species (acetaldehyde, PAA, IAA, 2MB/3MB, acetoin, pyruvate, and 4-HPAA) and a series of 12 amine metabolites. Amines included in this analysis were tyramine, tryptamine, and phenethylamine derived through decarboxylation of the aromatic amino acids, as well as the dopamine derivatives
hydroxydopamine and dopamine quinone. Of these 84 m/z values, an additional four putative promiscuous products were revealed. All four of these putative peaks were derived from acetaldehyde via condensation with tyramine (7), hydroxydopamine (8), dopamine quinone (9), or tryptamine (10). An additional two BIA-like products were identified corresponding to predicted molecular formulas of C17H19NO4 (11 ) and C18H19NO4 (12). See Figure 1 1.
[00211] The levels of compounds 1-12 were compared across wild-type S. cerevisiae and strains C, 1 , and 66. Results are shown in Figures 12-14. Whereas none of the promiscuous products were identified in supernatants derived from the wild-type strain, several products, particularly those derived from acetaldehyde (compounds 1 and 7-10), were formed
spontaneously in the strain C. Although compounds 7 and 10 are not derived from dopamine, the dopamine pathway is required to observe their formation through promiscuous activity of the L-DOPA decarboxylase enzyme on tyrosine and tryptophan, yielding tyramine and tryptamine, respectively. With the exception of compounds 7-9, the abundance of all promiscuous NCS- catalyzed products increased as (S)-norcoclaurine titer was improved throughout the course of the gene deletion experiments. The order of condensation products from greatest peak area to smallest in strain 66 was 1 , (S)-norcoclaurine, 2, 7, 8, 3, 4, 11 , 5, 12, 6, 9, 10. See Figure 15.
An NCS polypeptide was required to detect products derived from Ehrlich pathway aldehydes (compounds 2-4), including (S)-norcoclaurine. Interestingly, compounds 3-5 were not identified in strain 1 , yet yielded substantial peaks in strain 57. While synthesis of (S)-norcoclaurine was improved by a factor of 121 , levels of compound 2 were also increased 100-fold. These data indicate that several mutations shown to enhance (S)-norcoclaurine output had a comparable effect on the synthesis of other NCS-catalyzed products. For example, levels of compounds 2 and 3 increased through deletion of ARM , deletion of YPR1 and ADH6 improved formation of compounds 3 and 4, respectively, and deletion of YDR541 C enhanced levels of all Ehrlich pathway products (compounds 2-4). This in turn implies that certain enzymes acting on 4-HPAA are also involved in the reduction of the other aromatic Ehrlich pathway aldehydes (PAA and IAA), in addition to certain aliphatic Ehrlich pathway analogues (2- or 3-methylbutanal). With the exception of compounds 7 and 9, deletion of ALD4 enhanced formation of all condensation products by a factor of 2.2-10, likely due to increased levels of dopamine and presumably higher flux through the aromatic amino acid (AAA) pathway.
[00212] In order to block formation of compounds 2 and 3, which are derived from
phenylalanine and tryptophan within the AAA pathway, expression of benzylisoquinoline alkaloid biosynthesis polypeptides with activity towards (S)-norcoclaurine was increased. Results are shown in Figures 15-18. Prephenate dehydrogenase (Tyr1 ) was overexpressed in strain 67, which demonstrated reduced formation of compound 2 by 44%, and increased production of (S)-norcoclaurine by 120%. Because levels of dopamine-derived compounds 1 , 3-6, and 8-9 were also enhanced in strain 67, a feedback-resistant variant of chorismate mutase (Aro7) was further expressed in strain 68, which demonstrated further increased production of (S)- norcoclaurine by 26%, and reduced formation of compound 3 by 76%. See Figures 16-18. Further overexpressing phenylpyruvate decarboxylase (Aro10) in strain 69 increased production of (S)-norcoclaurine by 24%, and reduced residual dopamine by 46%, indicating enhanced flux to 4-HPAA. See Figure 18.
[00213] To further impede formation of compounds 2 and 3, phenylalanine and tryptophan biosynthesis polypeptides were inactivated in strains 70-72. Deletion of PHA2 in strain 70 caused a 61 % reduction in production of compound 2, and a 74% increase in production of (S)- norcoclaurine. Further deletion of TRP2 (strain 71 ) or TRP3 (strain 72) led to 13% or 42% decreases in formation of compound 3, respectively. See Figures 17-18.
[00214] To further improve (S)-norcoclaurine production, a strain having inactivated phenylalanine and tryptophan biosynthesis polypeptides was further engineered to express eight copies of the recombinant gene encoding N-terminal truncated NdNCS of Example 4 (strain 87). Further overexpression of a chorismate synthase polypeptide in strain 88 led to an increase in production of (S)-norcoclaurine relative to strain 87. Of strains 89-93, further engineered to delete, respectively, ADH5, AAD14, ADH3, YGL029W, or AAD3, strain 93 showed the greatest increase in production of (S)-norcoclaurine relative to strain 88. See Figures 22-23.
[00215] Strains overexpressing N-terminal truncated NdNCS, Tyr1 , Aro7, Aro10, and Aro2, and lacking ARM , ALD4, ADH6, YPR1 , YDR541C, PHA2, TRP3, and AAD3, were further engineered to delete AAD4, ADH7, SFA1 , YGL039, AAD14, GRE2, GCY1 , or ALD2 and ALD3 to provide, respectively, strains 94-101. Notably, strain 99 demonstrated an 82% decrease in tyrosol production and a 97% increase in (S)-norcoclaurine production relative to strain 93. See Figures 24-25. Without being bound by theory, the results indicate that deletion of genes encoding aldo-keto reductases (e.g., ARM , ADH6, YPR1 , YDR541C, AAD3, GRE2) can significantly limit, or even nearly abolish tyrosol formation (i.e., from 4-HPAA), and deletion of genes not directly related tyrosol formation {e.g., ALD4, PHA2, TRP3) can further increase carbon flux to L-tyrosine. The results moreover show that combination of such deletions with overexpression of benzylisoquinoline alkaloid biosynthesis pathway genes (e.g., Aro2, Tyr1 , Aro10, Aro7FBR, Aro4FBR) can even further enhance overall flux to L-tyrosine and/or 4-HPAA.
Example 6: Fed-Batch Fermentation
[00216] Strains 67 and 68 of Example 5 were each cultivated in a fed-batch bioreactor. Controlled fed-batch cultivations were carried out in a 3 L BioBundle fermenter (Applikon) equipped with d02, pH, and temperature sensors. Cultivation was controlled using an EZ- Control bio-controller and BioXpert cultivation control software (Applikon). A variable speed pump (Watson-Marlow) was used to supply the feeding medium. Culture pH was maintained at 4.5 by titration with 3 M NaOH and 3 N H2S04. Dissolved oxygen was maintained at 10 % of air saturation by automatically adjusting the stirring rate (aeration rate 0.5 L/min). Cultivation temperature was maintained at 30 °C.
[00217] Bioreactor inoculum was generated in four 125 ml. shake flasks containing 25 ml. YPD medium and incubated at 30 °C for 24 h. The cells from each of the flasks were centrifuged, washed once with 50 ml. 0.9 % w/v NaCI, centrifuged again, pooled in 50 ml. 0.9 % NaCI, and used to inoculate 950 ml. (starting OD6oo = 0.4) of batch medium. The batch medium contained per L: 2.5 g KH2P04, 5 g NH CI, 1 g MgS04-7H20, 5 ml vitamin stock, 5 ml microelement stock, 1.6 g yeast synthetic drop-out medium without leucine, 380 mg L-leucine, and (i) 40 g sucrose or (ii) 30 g glycerol. Vitamin stock contained per L: 5 mg biotin, 100 mg calcium pantothenate, 100 mg nicotinic acid, 2,500 mg myo-inositol, 100 mg thiamine hydrochloride, 100 mg pyridoxine, and 20 mg p-aminobenzoic acid. Microelement stock contained per L: 15 g disodium EDTA, 2.9 g CaCI2, 9.2 g ZnS04-7H20, 5.1 g FeS04-7H20, 0.5 g CuS04, 0.43 g MnS04 H20, 0.47 g CoCI2, and 0.48 g Na2Mo04. The culture was grown in batch mode for 16 to 96 hours. The fed-batch phase was carried out with a constant feeding rate of 3.5 mL/h. The feeding medium contained per L: 10 g KH2P04, 40 g NH4CI, 4 g
MgS0 -7H20, 10 ml vitamin stock, 10 mL microelement stock, 400 mg yeast synthetic drop-out medium without leucine, 2.5 g L-leucine, 810 mg L-histidine, 570 mg L-methionine, 800 mg uracil, and 240 g sucrose. The total cultivation time was seven to ten days and samples (5 mL) were collected daily. Cell densities were determined by dilution of fermentation broth with water followed by measurement of absorption at 600 nm (OD6oo). Dry cell weight (g/L) was calculated using a conversion factor of 0.59 g/L per OD6oo (determined gravimetrically). Culture broths were analyzed for metabolite production according to Example 1.
[00218] Results are shown in Figures 19-20. The batch medium for strain 67 contained sucrose as a carbon source. The cells were unable to metabolize the ethanol produced during the batch phase of the cultivation, ultimately leading to growth arrest. See Figure 19. Under these conditions, this strain produced 215 mg/L (S)-norcoclaurine. See Figure 20. In order to limit ethanol production, glycerol was used as a carbon source in the batch medium of strain 68. Ethanol levels remained low during the fed-batch phase, and the strain produced 383 mg/L (S)- norcoclaurine. See Figure 20.
Example 7: LC-MS and LC-UV Analytical Procedures
[00219] An (S)-norcoclaurine stock solution was prepared in DMSO at a concentration of 10 mM. Standard solutions were prepared at concentrations of 4 mM, 2 mM, 1 mM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, and 10 nM from the stock solution. Samples were injected into an Agilent 1290 UPLC coupled to an Ultivo Triple Quadrupole. The LC-MS method was as follows: Mobile Phase A. H20 + 0.1 % Formic acid; Mobile Phase B: Acetonitrile + 0.1 % Formic acid; Column: Phenomenex Kinetex 1.7 pm XB-C18 100A, 2.1x100mm. The elution gradient is shown in Table 5 and the LC-MS conditions are given in Table 6. Table 7 shows the mass spectrometer source and detector parameters and Table 8 shows the target compound, retention time, parent ion, transition ions (MRM) as well as dwell time, fragmentor voltage and collision energy used.
Table 5. LC-MS Gradient
Figure imgf000096_0001
Table 6. LC-MS Conditions
Figure imgf000096_0002
Table 7. Source and Detector Parameters
Figure imgf000097_0001
Table 8. LC-MS Gradient
Figure imgf000097_0002
Example 8: Strain Engineering
[00220] (S)-norcoclaurine-producing S. cerevisiae strains comprising and expressing a recombinant gene encoding a DAHP synthase polypeptide (SEQ ID NO:3, SEQ ID NO:4), operably linked to a pPDC1 promoter (SEQ ID NO:169) and a tCYC1 terminator (SEQ ID NO:175); a recombinant gene encoding a tyrosine hydroxylase polypeptide (SEQ ID NO:SEQ ID NO:23, SEQ ID NO:24), operably linked to a pTEF2 promoter (SEQ ID NO:170) and a tFBA1 terminator (SEQ ID NO: 176); a recombinant gene encoding an L-DOPA decarboxylase polypeptide (SEQ ID NO:SEQ ID NO:27, SEQ ID NO:28), operably linked to a pTDH3 promoter (SEQ ID NO: 171 ) and a tEN02 terminator (SEQ ID NO: 177); and a recombinant gene encoding an (S)-norcoclaurine synthase (SEQ ID NO:29, SEQ ID NO:30), operably linked to a pPGK1 promoter (SEQ ID NO:172) and a tADH1 terminator (SEQ ID NO:178), and further comprising downregulated expression of the endogenous ARM gene, were engineered.
Example 9: Endogenous Transporter Gene Inactivation in S. cerevisiae
[00221] An (S)-norcoclaurine-producing S. cere visiae strain as described in Example 8 was engineered to downregulate or delete expression of one or more native genes encoding an ABC transporter polypeptide, as summarized in Table 9, below.
Table 9. Transporter Genes Deleted
Figure imgf000098_0001
[00222] Yeast transformants were grown in 96-deep well plates in 500 pL liquid synthetic complete media for 3 days at 30 °C with shaking at 230 rpm in a Kuhner Climo-Shaker ISF1-X. Culture samples for LC-MS were prepared by extraction as follows: 96% ethanol and culture sample were mixed 1 :1 and incubated on a heating block at 80 °C for 10 minutes. After heating, cells were pelleted in an Eppendorff tabletop centrifuge and the supernatant was then transferred to a new tube and diluted 1 :10 in water.
[00223] LC-MS analysis was performed according to Example 6. Results are shown in Figure 21. Strain 84 showed a dramatic increase in (S)-norcoclaurine production, and strain 83 shows a modest but consistent increase in (S)-norcoclaurine production. Without being bound by theory, the results indicate that preventing excretion of benzylisoquinoline precursors such as L-DOPA or dopamine by downregulating expression of one or more ABC transporters capable of transporting those precursors increases production of (S)-norcoclaurine. Example 10: Strain Engineering
[00224] (S)-norcoclaurine-producing S. cerevisiae strains comprising and expressing a recombinant gene encoding a DAHP synthase polypeptide (SEQ ID NO:3, SEQ ID NO:4), a recombinant gene encoding a tyrosine hydroxylase polypeptide (SEQ ID NO:21 , SEQ ID NO:22; SEQ ID NO:23, SEQ ID NO:24), a recombinant gene encoding an L-DOPA decarboxylase polypeptide (SEQ ID NO:25, SEQ ID NO:26; SEQ ID NO:27, SEQ ID NO:28), a recombinant gene encoding a prephenate dehydrogenase (SEQ ID NO:13, SEQ ID NO:14), a recombinant gene encoding a phenylpyruvate decarboxylase (SEQ ID NO:19, SEQ ID NO:20), and/or a recombinant gene encoding an (S)-norcoclaurine synthase (SEQ ID NO:29, SEQ ID NO:30; SEQ ID NO:31 , SEQ ID NO:32; SEQ ID NO:33, SEQ ID NO:34; SEQ ID NO:35, SEQ ID NO:36; SEQ ID NO:37, SEQ ID NO:38; SEQ ID NO:39, SEQ ID NO:40; SEQ ID NO:41 , SEQ ID NO:42; SEQ ID NO:43, SE ID NO:44; SEQ ID NO:51 , SEQ ID NO:52; SEQ ID NO:53, SEQ ID NO:54) are engineered.
[00225] The strains are further engineered to comprise reduced expression of one or more endogenous transporter genes PDR5 (SEQ ID NO:69, SEQ ID NO:70), PDR12 (SEQ ID NO:71 , SEQ ID NO:72), PDR15 (SEQ ID NO:73, SEQ ID NO:74), SNQ2 (SEQ ID NO:75, SEQ ID NO:76), and/or one or more endogenous transporter genes AUS1 (SEQ ID NO:77, SEQ ID NO:78), PDR10 (SEQ ID NO:79, SEQ ID NO:80), YOR1 (SEQ ID NO:81 , SEQ ID NO:82),
TP01 (SEQ ID NO:83, SEQ ID NO:84), TP02 (SEQ ID NO:85, SEQ ID NO:86), TP03 (SEQ ID NO:87, SEQ ID NO:88), TP04 (SEQ ID NO:89, SEQ ID NO:90), QDR1 (SEQ ID NO:91 , SEQ ID NO:92), QDR2 (SEQ ID NO:93, SEQ ID NO:94), QDR3 (SEQ ID NO:95, SEQ ID NO:96), FLR1 (SEQ ID NO:97, SEQ ID NO:98), YOL075C (SEQ ID NO:99, SEQ ID NO:100), PDR18 (SEQ ID NO: 101 , SEQ NO:102), DTR1 (SEQ ID NO:103, SEQ ID NO:104), YHK8 (SEQ ID NO:105, SEQ ID NQ:106), NFT1 (SEQ ID NQ:107, SEQ ID NQ: 108), STE6 (SEQ ID NQ:109, SEQ ID NO:1 10), YCF1 (SEQ ID NO:1 11 , SEQ ID NO: 112), YBT1 (SEQ ID NO:1 13, SEQ ID
NO:1 14), BPT1 (SEQ ID NO:1 15, SEQ ID NO:1 16), and VMR1 (SEQ ID NO:1 17, SEQ ID NO: 1 18).
[00226] The strains are further engineered to comprise reduced expression of one or more endogenous genes ARI1 (SEQ ID NO:145, SEQ ID NO:146), ALD4 (SEQ ID NO:123, SEQ ID NO:124), ADH6 (SEQ ID NO:139, SEQ ID NO: 140), YPR1 (SEQ ID NO:153, SEQ ID NO:154), YDR541C (SEQ ID NO: 149, SEQ ID NO:150), PHA2 (SEQ ID NO:163, SEQ ID NO: 164), TRP2 (SEQ ID NO: 165, SEQ ID NO:166), and TRP3 (SEQ ID NO: 167, SEQ ID NO:168).
Example 11 : Benzylisoquinoline alkaloid biosynthesis
[00227] Colonies of the (S)-norcoclaurine-producing strains as described in Example 10 are picked in triplicate and inoculated into 500 pi of 2* synthetic complete (SC) medium containing 4% glucose or sucrose in 96-well two ml deep well plates. The present inventors have determined that sucrose, in place of glucose, increases production of metabolites derived from the shikimate pathway by 50%. Following growth at 30 °C and 350 rpm for 18-24 h, cultures are diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose. After 72-96 h growth, metabolites are extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice- cold 100% acetonitrile (ACN; 80% final concentration). Cells are incubated for 5 minutes and subsequently diluted with 417 pi of 0.16% formic acid (FA) to give a final concentration of 30% ACN and 0.1 % FA. The resulting extract is utilized for LC-MS analysis according to Example 1. Cell broth is diluted 13.34-fold using this method.
Example 12: Fed-Batch Fermentation
[00228] Strains as described in Example 10 are each cultivated in a fed-batch bioreactor. Controlled fed-batch cultivations are carried out in a 3 L BioBundle fermenter (Applikon) equipped with d02, pH, and temperature sensors. Cultivation is controlled using an EZ-Control bio-controller and BioXpert cultivation control software (Applikon). A variable speed pump (Watson-Marlow) s used to supply the feeding medium. Culture pH is maintained at 4.5 by titration with 3 M NaOH and 3 N H2SO4. Dissolved oxygen is maintained at 10 % of air saturation by automatically adjusting the stirring rate (aeration rate 0.5 L/min). Cultivation temperature is maintained at 30 °C.
[00229] Bioreactor inoculum is generated in four 125 ml. shake flasks containing 25 ml. YPD medium and incubated at 30 °C for 24 h. The cells from each of the flasks are centrifuged, washed once with 50 ml. 0.9 % w/v NaCI, centrifuged again, pooled in 50 ml. 0.9 % NaCI, and used to inoculate 950 ml. (starting OD6oo = 0.4) of batch medium. The batch medium contains per L: 2.5 g KH2PO4, 5 g NH4CI, 1 g MgS04-7H20, 5 ml vitamin stock, 5 ml microelement stock, 1.6 g yeast synthetic drop-out medium without leucine, 380 mg L-leucine, and (i) 40 g sucrose or (ii) 30 g glycerol. Vitamin stock contains per L: 5 mg biotin, 100 mg calcium pantothenate,
100 mg nicotinic acid, 2,500 mg myo-inositol, 100 mg thiamine hydrochloride, 100 mg pyridoxine, and 20 mg p-aminobenzoic acid. Microelement stock contains per L: 15 g disodium EDTA, 2.9 g CaCh, 9.2 g ZnS04-7H20, 5.1 g FeS04-7H20, 0.5 g CuS04, 0.43 g MnS04 H20, 0.47 g C0CI2, and 0.48 g Na2Mo04. The culture is grown in batch mode for 16 to 96 hours. The fed-batch phase is carried out with a constant feeding rate of 3.5 mL/h. The feeding medium contains per L: 10 g KH2PO4, 40 g NH4CI, 4 g MgS04-7H20, 10 ml vitamin stock, 10 mL microelement stock, 400 mg yeast synthetic drop-out medium without leucine, 2.5 g L-leucine, 810 mg L-histidine, 570 mg L-methionine, 800 mg uracil, and 240 g sucrose. The total cultivation time is seven to ten days and samples (5 mL) are collected daily. Cell densities are determined by dilution of fermentation broth with water followed by measurement of absorption at 600 nm (OD6oo). Dry cell weight (g/L) is calculated using a conversion factor of 0.59 g/L per OD6OO (determined gravimetrically). Culture broths are analyzed for metabolite production according to Example 1.
[00230] The above Examples demonstrate a substantial increase in norcoclaurine yield, more than an order of magnitude higher than the prior art.
[00231] The remarkable effect of the combined disruption of oxidoreductases disclosed herein is highly surprising. Several of the oxidoreductases disrupted have been tested in the prior art using a similar setup, with no effect. But by combinatorially disrupting them, further in combination with augmented NCS activity, these disruptions suddenly have a large impact is achieved.
[00232] Additionally surprising is that reducing the formation of other types of aldehydes in yeast further augments the norcoclaurine yield. The present inventors have determined that NCS enzymes inherently are promiscuous and can perform the Pictet-Spengler reaction with aldehydes other than 4-HPAA, resulting in unwanted side products and carbon loss. These reductions of side products formation are in the present work demonstrated both by gene deletions to reduce formation of PAA and IAA as well as optimization of fermentation conditions to reduce the formation of acetaldehyde.
Table 9. Sequences disclosed herein
SEQ ID NO:1
Saccharomyces cerevisiae
atgagtgaat ctccaatgtt cgctgccaac ggcatgccaa aggtaaatca aggtgctgaa 60 gaagatgtca gaattttagg ttacgaccca ttagcttctc cagctctcct tcaagtgcaa 120 atcccagcca caccaacttc tttggaaact gccaagagag gtagaagaga agctatagat 180 attattaccg gtaaagacga cagagttctt gtcattgtcg gtccttgttc catccatgat 240 ctagaagccg ctcaagaata cgctttgaga ttaaagaaat tgtcagatga attaaaaggt 300 gatttatcca tcattatgag agcatacttg gagaagccaa gaacaaccgt cggctggaaa 360 ggtctaatta atgaccctga tgttaacaac actttcaaca tcaacaaggg tttgcaatcc 420 gctagacaat tgtttgtcaa cttgacaaat atcggtttgc caattggttc tgaaatgctt 480 gataccattt ctcctcaata cttggctgat ttggtctcct tcggtgccat tggtgccaga 540 accaccgaat ctcaactgca cagagaattg gcctccggtt tgtctttccc agttggtttc 600 aagaacggta ccgatggtac cttaaatgtt gctgtggatg cttgtcaagc cgctgctcat 660 tctcaccatt tcatgggtgt tactaagcat ggtgttgctg ctatcaccac tactaagggt 720 aacgaacact gcttcgttat tctaagaggt ggtaaaaagg gtaccaacta cgacgctaag 780 tccgttgcag aagctaaggc tcaattgcct gccggttcca acggtctaat gattgactac 840 tctcacggta actccaataa ggatttcaga aaccaaccaa aggtcaatga cgttgtttgt 900 gagcaaatcg ctaacggtga aaacgccatt accggtgtca tgattgaatc aaacatcaac 960 gaaggtaacc aaggcatccc agccgaaggt aaagccggct tgaaatatgg tgtttccatc 1020 actgatgctt gtataggttg ggaaactact gaagacgtct tgaggaaatt ggctgctgct 1080 gtcagacaaa gaagagaagt taacaagaaa tag 1113
SEQ ID NO:2
Saccharomyces cerevisiae
MSESPMFAAN GMPKVNQGAE EDVRILGYDP LASPALLQVQ IPATPTSLET AKRGRREAID 60
I ITGKDDRVL VIVGPCSIHD LEAAQEYALR LKKLSDELKG DLSI IMRAYL EKPRTTVGWK 120
GLINDPDVNN TFNINKGLQS ARQLFVNLTN IGLP IGSEML DTISPQYLAD LVSFGAIGAR 180
TTESQLHREL ASGLSFPVGF KNGTDGTLNV AVDACQAAAH SHHFMGVTKH GVAAITTTKG 240
NEHCFVILRG GKKGTNYDAK SVAEAKAQLP AGSNGLMIDY SHGNSNKDFR NQPKVNDWC 300
EQIANGENAI TGVMIESNIN EGNQGIPAEG KAGLKYGVS I TDACIGWETT EDVLRKLAAA 360
VRQRREVNKK 370
SEQ ID NO:3
atgagtgaat ctccaatgtt cgctgccaac ggcatgccaa aggtaaatca aggtgctgaa 60 gaagatgtca gaattttagg ttacgaccca ttagcttctc cagctctcct tcaagtgcaa 120 atcccagcca caccaacttc tttggaaact gccaagagag gtagaagaga agctatagat 180 attattaccg gtaaagacga cagagttctt gtcattgtcg gtccttgttc catccatgat 240 ctagaagccg ctcaagaata cgctttgaga ttaaagaaat tgtcagatga attaaaaggt 300 gatttatcca tcattatgag agcatacttg gagaagccaa gaacaaccgt cggctggaaa 360 ggtctaatta atgaccctga tgttaacaac actttcaaca tcaacaaggg tttgcaatcc 420 gctagacaat tgtttgtcaa cttgacaaat atcggtttgc caattggttc tgaaatgctt 480 gataccattt ctcctcaata cttggctgat ttggtctcct tcggtgccat tggtgccaga 540 accaccgaat ctcaactgca cagagaattg gcctccggtt tgtctttccc agttggtttc 600 aagaacggta ccgatggtac cttaaatgtt gctgtggatg cttgtcaagc cgctgctcat 660 tctcaccatt tcatgggtgt tactttgcat ggtgttgctg ctatcaccac tactaagggt 720 aacgaacact gcttcgttat tctaagaggt ggtaaaaagg gtaccaacta cgacgctaag 780 tccgttgcag aagctaaggc tcaattgcct gccggttcca acggtctaat gattgactac 840 tctcacggta actccaataa ggatttcaga aaccaaccaa aggtcaatga cgttgtttgt 900 gagcaaatcg ctaacggtga aaacgccatt accggtgtca tgattgaatc aaacatcaac 960 gaaggtaacc aaggcatccc agccgaaggt aaagccggct tgaaatatgg tgtttccatc 1020 actgatgctt gtataggttg ggaaactact gaagacgtct tgaggaaatt ggctgctgct 1080 gtcagacaaa gaagagaagt taacaagaaa tag 1113 SEQ ID NO:4
MSESPMFAAN GMPKVNQGAE EDVRILGYDP LASPALLQVQ IPATPTSLET AKRGRREAID 60 I ITGKDDRVL VIVGPCSIHD LEAAQEYALR LKKLSDELKG DLSI IMRAYL EKPRTTVGWK 120 GLINDPDVNN TFNINKGLQS ARQLFVNLTN IGLP IGSEML DTISPQYLAD LVSFGAIGAR 180 TTESQLHREL ASGLSFPVGF KNGTDGTLNV AVDACQAAAH SHHFMGVTLH GVAAITTTKG 240 NEHCFVILRG GKKGTNYDAK SVAEAKAQLP AGSNGLMIDY SHGNSNKDFR NQPKVNDWC 300 EQIANGENAI TGVMIESNIN EGNQGIPAEG KAGLKYGVS I TDACIGWETT EDVLRKLAAA 360 VRQRREVNKK 370
SEQ ID NO:5
Saccharomyces cerevisiae
atggtgcagt tagccaaagt cccaattcta ggaaatgata ttatccacgt tgggtataac 60 attcatgacc atttggttga aaccataatt aaacattgtc cttcttcgac atacgttatt 120 tgcaatgata cgaacttgag taaagttcca tactaccagc aattagtcct ggaattcaag 180 gcttctttgc cagaaggctc tcgtttactt acttatgttg ttaaaccagg tgagacaagt 240 aaaagtagag aaaccaaagc gcagctagaa gattatcttt tagtggaagg atgtactcgt 300 gatacggtta tggtagcgat cggtggtggt gttattggtg acatgattgg gttcgttgca 360 tctacattta tgagaggtgt tcgtgttgtc caagtaccaa catccttatt ggcaatggtc 420 gattcctcca ttggtggtaa aactgctatt gacactcctc taggtaaaaa ctttattggt 480 gcattttggc aaccaaaatt tgtccttgta gatattaaat ggctagaaac gttagccaag 540 agagagttta tcaatgggat ggcagaagtt atcaagactg cttgtatttg gaacgctgac 600 gaatttacta gattagaatc aaacgcttcg ttgttcttaa atgttgttaa tggggcaaaa 660 aatgtcaagg ttaccaatca attgacaaac gagattgacg agatatcgaa tacagatatt 720 gaagctatgt tggatcatac atataagtta gttcttgaga gtattaaggt caaagcggaa 780 gttgtctctt cggatgaacg tgaatccagt ctaagaaacc ttttgaactt cggacattct 840 attggtcatg cttatgaagc tatactaacc ccacaagcat tacatggtga atgtgtgtcc 900 attggtatgg ttaaagaggc ggaattatcc cgttatttcg gtattctctc ccctacccaa 960 gttgcacgtc tatccaagat tttggttgcc tacgggttgc ctgtttcgcc tgatgagaaa 1020 tggtttaaag agctaacctt acataagaaa acaccattgg atatcttatt gaagaaaatg 1080 agtattgaca agaaaaacga gggttccaaa aagaaggtgg tcattttaga aagtattggt 1140 aagtgctatg gtgactccgc tcaatttgtt agcgatgaag acctgagatt tattctaaca 1200 gatgaaaccc tcgtttaccc cttcaaggac atccctgctg atcaacagaa agttgttatc 1260 ccccctggtt ctaagtccat ctccaatcgt gctttaattc ttgctgccct cggtgaaggt 1320 caatgtaaaa tcaagaactt attacattct gatgatacta aacatatgtt aaccgctgtt 1380 catgaattga aaggtgctac gatatcatgg gaagataatg gtgagacggt agtggtggaa 1440 ggacatggtg gttccacatt gtcagcttgt gctgacccct tatatctagg taatgcaggt 1500 actgcatcta gatttttgac ttccttggct gccttggtca attctacttc aagccaaaag 1560 tatatcgttt taactggtaa cgcaagaatg caacaaagac caattgctcc tttggtcgat 1620 tctttgcgtg ctaatggtac taaaattgag tacttgaata atgaaggttc cctgccaatc 1680 aaagtttata ctgattcggt attcaaaggt ggtagaattg aattagctgc tacagtttct 1740 tctcagtacg tatcctctat cttgatgtgt gccccatacg ctgaagaacc tgtaactttg 1800 gctcttgttg gtggtaagcc aatctctaaa ttgtacgtcg atatgacaat aaaaatgatg 1860 gaaaaattcg gtatcaatgt tgaaacttct actacagaac cttacactta ttatattcca 1920 aagggacatt atattaaccc atcagaatac gtcattgaaa gtgatgcctc aagtgctaca 1980 tacccattgg ccttcgccgc aatgactggt actaccgtaa cggttccaaa cattggtttt 2040 gagtcgttac aaggtgatgc cagatttgca agagatgtct tgaaacctat gggttgtaaa 2100 ataactcaaa cggcaacttc aactactgtt tcgggtcctc ctgtaggtac tttaaagcca 2160 ttaaaacatg ttgatatgga gccaatgact gatgcgttct taactgcatg tgttgttgcc 2220 gctatttcgc acgacagtga tccaaattct gcaaatacaa ccaccattga aggtattgca 2280 aaccagcgtg tcaaagagtg taacagaatt ttggccatgg ctacagagct cgccaaattt 2340 ggcgtcaaaa ctacagaatt accagatggt attcaagtcc atggtttaaa ctcgataaaa 2400 gatttgaagg ttccttccga ctcttctgga cctgtcggtg tatgcacata tgatgatcat 2460 cgtgtggcca tgagtttctc gcttcttgca ggaatggtaa attctcaaaa tgaacgtgac 2520 gaagttgcta atcctgtaag aatacttgaa agacattgta ctggtaaaac ctggcctggc 2580 tggtgggatg tgttacattc cgaactaggt gccaaattag atggtgcaga acctttagag 2640 tgcacatcca aaaagaactc aaagaaaagc gttgtcatta ttggcatgag agcagctggc 2700 aaaactacta taagtaaatg gtgcgcatcc gctctgggtt acaaattagt tgacctagac 2760 gagctgtttg agcaacagca taacaatcaa agtgttaaac aatttgttgt ggagaacggt 2820 tgggagaagt tccgtgagga agaaacaaga attttcaagg aagttattca aaattacggc 2880 gatgatggat atgttttctc aacaggtggc ggtattgttg aaagcgctga gtctagaaaa 2940 gccttaaaag attttgcctc atcaggtgga tacgttttac acttacatag ggatattgag 3000 gagacaattg tctttttaca aagtgatcct tcaagacctg cctatgtgga agaaattcgt 3060 gaagtttgga acagaaggga ggggtggtat aaagaatgct caaatttctc tttctttgct 3120 cctcattgct ccgcagaagc tgagttccaa gctctaagaa gatcgtttag taagtacatt 3180 gcaaccatta caggtgtcag agaaatagaa attccaagcg gaagatctgc ctttgtgtgt 3240 ttaacctttg atgacttaac tgaacaaact gagaatttga ctccaatctg ttatggttgt 3300 gaggctgtag aggtcagagt agaccatttg gctaattact ctgctgattt cgtgagtaaa 3360 cagttatcta tattgcgtaa agccactgac agtattccta tcatttttac tgtgcgaacc 3420 atgaagcaag gtggcaactt tcctgatgaa gagttcaaaa ccttgagaga gctatacgat 3480 attgccttga agaatggtgt tgaattcctt gacttagaac taactttacc tactgatatc 3540 caatatgagg ttattaacaa aaggggcaac accaagatca ttggttccca tcatgacttc 3600 caaggattat actcctggga cgacgctgaa tgggaaaaca gattcaatca agcgttaact 3660 cttgatgtgg atgttgtaaa atttgtgggt acggctgtta atttcgaaga taatttgaga 3720 ctggaacact ttagggatac acacaagaat aagcctttaa ttgcagttaa tatgacttct 3780 aaaggtagca tttctcgtgt tttgaataat gttttaacac ctgtgacatc agatttattg 3840 cctaactccg ctgcccctgg ccaattgaca gtagcacaaa ttaacaagat gtatacatct 3900 atgggaggta tcgagcctaa ggaactgttt gttgttggaa agccaattgg ccactctaga 3960 tcgccaattt tacataacac tggctatgaa attttaggtt tacctcacaa gttcgataaa 4020 tttgaaactg aatccgcaca attggtgaaa gaaaaacttt tggacggaaa caagaacttt 4080 ggcggtgctg cagtcacaat tcctctgaaa ttagatataa tgcagtacat ggatgaattg 4140 actgatgctg ctaaagttat tggtgctgta aacacagtta taccattggg taacaagaag 4200 tttaagggtg ataataccga ctggttaggt atccgtaatg ccttaattaa caatggcgtt 4260 cccgaatatg ttggtcatac cgctggtttg gttatcggtg caggtggcac ttctagagcc 4320 gccctttacg ccttgcacag tttaggttgc aaaaagatct tcataatcaa caggacaact 4380 tcgaaattga agccattaat agagtcactt ccatctgaat tcaacattat tggaatagag 4440 tccactaaat ctatagaaga gattaaggaa cacgttggcg ttgctgtcag ctgtgtacca 4500 gccgacaaac cattagatga cgaactttta agtaagctgg agagattcct tgtgaaaggt 4560 gcccatgctg cttttgtacc aaccttattg gaagccgcat acaaaccaag cgttactccc 4620 gttatgacaa tttcacaaga caaatatcaa tggcacgttg tccctggatc acaaatgtta 4680 gtacaccaag gtgtagctca gtttgaaaag tggacaggat tcaagggccc tttcaaggcc 4740 atttttgatg ccgttacgaa agagtag 4767
SEQ ID NO:6
Saccharomyces cerevisiae
MVQLAKVPIL GNDIIHVGYN IHDHLVETI I KHCPSSTYVI CNDTNLSKVP YYQQLVLEFK 60 ASLPEGSRLL TYWKPGETS KSRETKAQLE DYLLVEGCTR DTVMVAIGGG VIGDMIGFVA 120 STFMRGVRW QVPTSLLAMV DSSIGGKTAI DTPLGKNFIG AFWQPKFVLV DIKWLETLAK 180 REFINGMAEV IKTACIWNAD EFTRLESNAS LFLNVVNGAK NVKVTNQLTN EIDEISNTDI 240 EAMLDHTYKL VLESIKVKAE WSSDERESS LRNLLNFGHS IGHAYEAILT PQALHGECVS 300 IGMVKEAELS RYFGILSPTQ VARLSKILVA YGLPVSPDEK WFKELTLHKK TPLDILLKKM 360 SIDKKNEGSK KKWILESIG KCYGDSAQFV SDEDLRFILT DETLVYPFKD IPADQQKWI 420 PPGSKSISNR ALILAALGEG QCKIKNLLHS DDTKHMLTAV HELKGATISW EDNGETVWE 480 GHGGSTLSAC ADPLYLGNAG TASRFLTSLA ALVNSTSSQK YIVLTGNARM QQRPIAPLVD 540 SLRANGTKIE YLNNEGSLP I KVYTDSVFKG GRIELAATVS SQYVSSILMC APYAEEPVTL 600 ALVGGKPISK LYVDMTIKMM EKFGINVETS TTEPYTYYIP KGHYINPSEY VIESDASSAT 660 YPLAFAAMTG TTVTVPNIGF ESLQGDARFA RDVLKPMGCK ITQTATSTTV SGPPVGTLKP 720 LKHVDMEPMT DAFLTACVVA AISHDSDPNS ANTTTIEGIA NQRVKECNRI LAMATELAKF 780 GVKTTELPDG IQVHGLNSIK DLKVPSDSSG PVGVCTYDDH RVAMSFSLLA GMVNSQNERD 840 EVANPVRILE RHCTGKTWPG WWDVLHSELG AKLDGAEPLE CTSKKNSKKS WIIGMRAAG 900 KTTISKWCAS ALGYKLVDLD ELFEQQHNNQ SVKQFWENG WEKFREEETR IFKEVIQNYG 960 DDGYVFSTGG GIVESAESRK ALKDFASSGG YVLHLHRDIE ETIVFLQSDP SRPAYVEEIR 1020 EVWNRREGWY KECSNFSFFA PHCSAEAEFQ ALRRSFSKYI ATITGVREIE IPSGRSAFVC 1080 LTFDDLTEQT ENLTP ICYGC EAVEVRVDHL ANYSADFVSK QLSILRKATD SIPIIFTVRT 1140 MKQGGNFPDE EFKTLRELYD IALKNGVEFL DLELTLPTD I QYEVINKRGN TKI IGSHHDF 1200 QGLYSWDDAE WENRFNQALT LDVDWKFVG TAVNFEDNLR LEHFRDTHKN KPLIAVNMTS 1260 KGSISRVLNN VLTPVTSDLL PNSAAPGQLT VAQINKMYTS MGGIEPKELF WGKPIGHSR 1320 SPILHNTGYE ILGLPHKFDK FETESAQLVK EKLLDGNKNF GGAAVTIPLK LDIMQYMDEL 1380 TDAAKVIGAV NTVIPLGNKK FKGDNTDWLG IRNALINNGV PEYVGHTAGL VIGAGGTSRA 1440 ALYALHSLGC KKIFI INRTT SKLKPLIESL PSEFNIIGIE STKSIEEIKE HVGVAVSCVP 1500 ADKPLDDELL SKLERFLVKG AHAAFVPTLL EAAYKPSVTP VMTISQDKYQ WHVVPGSQML 1560 VHQGVAQFEK WTGFKGPFKA IFDAVTKE 1588
SEQ ID NO:7
Saccharomyces cerevisiae
atgtcaacgt ttgggaaact gttccgcgtc accacatatg gtgaatcgca ttgtaagtct 60 gtcggttgca ttgtcgacgg tgttcctcca ggaatgtcat taaccgaagc tgacattcag 120 ccacaattga ccagaagaag accgggtcaa tctaagctat cgacccctag agacgaaaag 180 gatagagtgg aaatccagtc cggtaccgag ttcggcaaga ctctaggtac acccatcgcc 240 atgatgatca aaaacgagga ccaaagacct cacgactact ccgacatgga caagttccct 300 agaccttccc atgcggactt cacgtactcg gaaaagtacg gtatcaaggc ctcctctggt 360 ggtggcagag cttctgctag agaaacgatt ggccgtgtcg cttcaggtgc cattgctgag 420 aagttcttag ctcagaactc taatgtcgag atcgtagcct ttgtgacaca aatcggggaa 480 atcaagatga acagagactc tttcgatcct gaatttcagc atctgttgaa caccatcacc 540 agggaaaaag tggactcaat gggtcctatc agatgtccag acgcctccgt tgctggtttg 600 atggtcaagg aaatcgaaaa gtacagaggc aacaaggact ctatcggtgg tgtcgtcact 660 tgtgtcgtga gaaacttgcc taccggtctc ggtgagccat gctttgacaa gttggaagcc 720 atgttggctc atgctatgtt gtccattcca gcatccaagg gtttcgaaat tggctcaggt 780 tttcagggtg tctctgttcc agggtccaag cacaatgacc cattttactt tgaaaaagaa 840 acaaacagat taagaacaaa gaccaacaat tcaggtggtg tacaaggtgg tatctctaat 900 ggtgagaaca tctatttctc tgtcccattc aagtcagtgg ccactatctc tcaagaacaa 960 aaaaccgcca cttacgatgg tgaagaaggt atcttagccg ctaagggtag acatgaccct 1020 gctgtcactc caagagctat tcctattgtg gaagccatga ccgctctggt gttggctgac 1080 gcgcttttga tccaaaaggc aagagatttc tccagatccg tggttcatta a 1131
SEQ ID NO:8
Saccharomyces cerevisiae
MSTFGKLFRV TTYGESHCKS VGCIVDGVPP GMSLTEADIQ PQLTRRRPGQ SKLSTPRDEK 60 DRVEIQSGTE FGKTLGTP IA MMIKNEDQRP HDYSDMDKFP RPSHADFTYS EKYGIKASSG 120 GGRASARETI GRVASGAIAE KFLAQNSNVE IVAFVTQIGE IKMNRDSFDP EFQHLLNTIT 180 REKVDSMGP I RCPDASVAGL MVKE IEKYRG NKDSIGGWT CWRNLPTGL GEPCFDKLEA 240 MLAHAMLSIP ASKGFEIGSG FQGVSVPGSK HNDPFYFEKE TNRLRTKTNN SGGVQGGISN 300 GENIYFSVPF KSVATISQEQ KTATYDGEEG ILAAKGRHDP AVTPRAIPIV EAMTALVLAD 360 ALLIQKARDF SRSWH 376
SEQ ID NO:9
Saccharomyces cerevisiae
atggatttca caaaaccaga aactgtttta aatctacaaa atattagaga tgaattagtt 60 agaatggagg attcgatcat cttcaaattt attgagaggt cgcatttcgc cacatgtcct 120 tcagtttatg aggcaaacca tccaggttta gaaattccga attttaaagg atctttcttg 180 gattgggctc tttcaaatct tgaaattgcg cattctcgca tcagaagatt cgaatcacct 240 gatgaaactc ccttctttcc tgacaagatt cagaaatcat tcttaccgag cattaactac 300 ccacaaattt tggcgcctta tgccccagaa gttaattaca atgataaaat aaaaaaagtt 360 tatattgaaa agattatacc attaatttcg aaaagagatg gtgatgataa gaataacttc 420 ggttctgttg ccactagaga tatagaatgt ttgcaaagct tgagtaggag aatccacttt 480 ggcaagtttg ttgctgaagc caagttccaa tcggatatcc cgctatacac aaagctgatc 540 aaaagtaaag atgtcgaggg gataatgaag aatatcacca attctgccgt tgaagaaaag 600 attctagaaa gattaactaa gaaggctgaa gtctatggtg tggaccctac caacgagtca 660 ggtgaaagaa ggattactcc agaatatttg gtaaaaattt ataaggaaat tgttatacct 720 atcactaagg aagttgaggt ggaatacttg ctaagaaggt tggaagagta a 771
SEQ ID NO: 10
Saccharomyces cerevisiae
MDFTKPETVL NLQNIRDELV RMEDSIIFKF IERSHFATCP SVYEANHPGL EIPNFKGSFL 60
DWALSNLEIA HSRIRRFESP DETPFFPDKI QKSFLPSINY PQILAPYAPE VNYNDKIKKV 120
YIEKIIPLIS KRDGDDKNNF GSVATRD IEC LQSLSRRIHF GKFVAEAKFQ SDIPLYTKLI 180
KSKDVEGIMK NITNSAVEEK ILERLTKKAE VYGVDPTNES GERRITPEYL VKIYKEIVIP 240
ITKEVEVEYL LRRLEE 256
SEQ ID NO: 11
atggatttca caaaaccaga aactgtttta aatctacaaa atattagaga tgaattagtt 60 agaatggagg attcgatcat cttcaaattt attgagaggt cgcatttcgc cacatgtcct 120 tcagtttatg aggcaaacca tccaggttta gaaattccga attttaaagg atctttcttg 180 gattgggctc tttcaaatct tgaaattgcg cattctcgca tcagaagatt cgaatcacct 240 gatgaaactc ccttctttcc tgacaagatt cagaaatcat tcttaccgag cattaactac 300 ccacaaattt tggcgcctta tgccccagaa gttaattaca atgataaaat aaaaaaagtt 360 tatattgaaa agattatacc attaatttcg aaaagagatg gtgatgataa gaataacttc 420 tcttctgttg ccactagaga tatagaatgt ttgcaaagct tgagtaggag aatccacttt 480 ggcaagtttg ttgctgaagc caagttccaa tcggatatcc cgctatacac aaagctgatc 540 aaaagtaaag atgtcgaggg gataatgaag aatatcacca attctgccgt tgaagaaaag 600 attctagaaa gattaactaa gaaggctgaa gtctatggtg tggaccctac caacgagtca 660 ggtgaaagaa ggattactcc agaatatttg gtaaaaattt ataaggaaat tgttatacct 720 atcactaagg aagttgaggt ggaatacttg ctaagaaggt tggaagagta a 771
SEQ ID NO: 12
MDFTKPETVL NLQNIRDELV RMEDSIIFKF IERSHFATCP SVYEANHPGL EIPNFKGSFL 60 DWALSNLEIA HSRIRRFESP DETPFFPDKI QKSFLPSINY PQILAPYAPE VNYNDKIKKV 120 YIEKIIPLIS KRDGDDKNNF SSVATRDIEC LQSLSRRIHF GKFVAEAKFQ SDIPLYTKLI 180 KSKDVEGIMK NITNSAVEEK ILERLTKKAE VYGVDPTNES GERRITPEYL VKIYKEIVIP 240 ITKEVEVEYL LRRLEE 256
SEQ ID NO: 13
Saccharomyces cerevisiae
atggtatcag aggataagat tgagcaatgg aaagccacaa aagtcattgg tataattggt 60 ctgggtgata tgggcctatt atacgctaat aaatttacag atgctggatg gggtgttata 120 tgttgtgata gggaagaata ttatgatgaa ctgaaagaaa aatatgcctc agctaaattc 180 gaactggtga aaaatggtca tttggtatcc aggcaaagcg actatattat ctatagtgtt 240 gaagcatcca atattagtaa gatcgtcgca acgtatggac catcttctaa ggttggaaca 300 attgttgggg gtcaaacgag ttgtaagctg ccggaaatcg aggctttcga aaagtattta 360 cccaaggact gcgacatcat taccgtgcat tcccttcatg ggcctaaagt taatactgaa 420 ggccaaccac tagttattat caatcacaga tcacagtacc cagaatcttt tgagttcgtt 480 aattctgtta tggcatgttt gaaaagtaag caagtttatt tgacatatga agagcatgac 540 aagattaccg ctgatacaca agctgtgaca catgctgctt tcttaagtat gggatctgcg 600 tgggcaaaga taaagattta tccttggact ctgggtgtaa acaaatggta cggtggccta 660 gaaaatgtga aagttaatat atcactaaga atctattcga acaagtggca tgtttacgca 720 ggattagcca taacaaaccc aagtgcacat cagcaaattc ttcaatatgc aaccagtgca 780 acagaactat ttagtttaat gatagataac aaagaacaag aacttactga tagactatta 840 aaagctaagc aatttgtatt tggaaagcat actggtctct tactattgga tgacacgatt 900 ttagagaaat attcgctatc aaaaagcagc attggtaaca gcaacaattg caagccagtg 960 ccgaattcac atttatcatt gttggcgatt gttgattcgt ggtttcaact tggtattgat 1020 ccatatgatc atatgatttg ttcgacgcca ttattcagaa tattcctggg tgtgtccgaa 1080 tatctttttt taaaacctgg cttattagaa cagacaattg atgcagctat ccatgataaa 1140 tcattcataa aagatgattt agaatttgtt atttcggcta gagaatggag ctcggttgtt 1200 tcttttgcca attttgatat atacaaaaag caatttcaga gtgttcaaaa gttctttgag 1260 ccaatgcttc cagaggctaa tctcattggc aacgagatga taaaaaccat tctgagtcat 1320 tctagtgacc gttcggccgc tgaaaaaaga aatacataa 1359
SEQ ID NO: 14
Saccharomyces cerevisiae
MVSEDKIEQW KATKVIGI IG LGDMGLLYAN KFTDAGWGVI CCDREEYYDE LKEKYASAKF 60 ELVKNGHLVS RQSDYIIYSV EASNISKIVA TYGPSSKVGT IVGGQTSCKL PEIEAFEKYL 120 PKDCDI ITVH SLHGPKVNTE GQPLVIINHR SQYPESFEFV NSVMACLKSK QVYLTYEEHD 180 KITADTQAVT HAAFLSMGSA WAKIKIYPWT LGVNKWYGGL ENVKVNI SLR IYSNKWHVYA 240 GLAITNPSAH QQILQYATSA TELFSLMIDN KEQELTDRLL KAKQFVFGKH TGLLLLDDTI 300 LEKYSLSKSS IGNSNNCKPV PNSHLSLLAI VDSWFQLGID PYDHMICSTP LFRIFLGVSE 360 YLFLKPGLLE QTIDAAIHDK SFIKDDLEFV ISAREWSSVV SFANFDIYKK QFQSVQKFFE 420 PMLPEANLIG NEMIKTILSH SSDRSAAEKR NT 452
SEQ ID NO: 15
Saccharomyces cerevisiae
atgactttac ctgaatcaaa agacttttct tacttgtttt cggatgaaac caatgctcgt 60 aaaccatccc cattgaaaac ctgcatccat cttttccaag atcctaacat tatctttttg 120 ggtggtggcc tgccattaaa agattatttc ccatgggata atctatctgt agattcaccc 180 aagcctcctt ttccccaggg tattggagct ccaattgacg agcagaattg cataaaatac 240 accgtcaaca aagattacgc tgataaaagt gccaatcctt ccaacgatat tcctttgtca 300 agagctttgc aatacgggtt cagtgctggt caacctgaac tattaaactt cattagagat 360 cataccaaga ttatccacga tttgaagtat aaggactggg acgttttagc cactgcaggt 420 aacacaaatg cctgggaatc tactttaaga gtcttttgta accgaggtga tgtcatctta 480 gttgaggcac attctttttc ctcttcattg gcttctgcag aggctcaagg tgtcattacc 540 ttccccgtgc caattgacgc tgatggtatc attcctgaaa aattagctaa agtcatggaa 600 aactggacac ctggtgctcc taaaccaaag ttgttataca ctattccaac gggccaaaat 660 ccaactggta cttccattgc agaccataga aaggaggcaa tttacaagat cgctcaaaag 720 tacgacttcc taattgtgga agatgaacct tattatttct tacaaatgaa tccctacatc 780 aaagacttga aggaaagaga gaaggcacaa agttctccaa agcaggacca tgacgaattt 840 ttgaagtcct tggcaaacac tttcctttcc ttggatacag aaggccgtgt tattagaatg 900 gattcctttt caaaagtttt ggccccaggg acaagattgg gttggattac tggttcatcc 960 aaaatcttga agccttactt gagtttgcat gaaatgacga ttcaagcccc agcaggtttt 1020 acacaagttt tggtcaacgc tacgctatcc aggtggggtc aaaagggtta cttggactgg 1080 ttgcttggcc tgcgtcatga atacactttg aaacgtgact gtgccatcga tgccctttac 1140 aagtatctac cacaatctga tgctttcgtg atcaatcctc caattgcagg tatgtttttc 1200 accgtgaaca ttgacgcatc tgtccaccct gagtttaaaa caaaatacaa ctcagaccct 1260 taccagctag aacagagtct ttaccacaaa gtggttgaac gtggtgtttt agtggttccc 1320 ggttcttggt tcaagagtga gggtgagacg gaacctcctc aacccgctga atctaaagaa 1380 gtcagtaatc caaacataat tttcttcaga ggtacctatg cagctgtctc tcctgagaaa 1440 ctgactgaag gtctgaagag attaggtgat actttatacg aagaatttgg tatttccaaa 1500 tag 1503
SEQ ID NO: 16
Saccharomyces cerevisiae
MTLPESKDFS YLFSDETNAR KPSPLKTCIH LFQDPNI IFL GGGLPLKDYF PWDNLSVDSP 60 KPPFPQGIGA PIDEQNCIKY TVNKDYADKS ANPSNDIPLS RALQYGFSAG QPELLNF IRD 120 HTKI IHDLKY KDWDVLATAG NTNAWESTLR VFCNRGDVIL VEAHSFSSSL ASAEAQGVIT 180 FPVP IDADGI IPEKLAKVME NWTPGAPKPK LLYTIPTGQN PTGTSIADHR KEAIYKIAQK 240 YDFLIVEDEP YYFLQMNPYI KDLKEREKAQ SSPKQDHDEF LKSLANTFLS LDTEGRVIRM 300 DSFSKVLAPG TRLGWITGSS KILKPYLSLH EMTIQAPAGF TQVLVNATLS RWGQKGYLDW 360 LLGLRHEYTL KRDCAIDALY KYLPQSDAFV INPP IAGMFF TVNIDASVHP EFKTKYNSDP 420 YQLEQSLYHK WERGVLVVP GSWFKSEGET EPPQPAESKE VSNPNIIFFR GTYAAVSPEK 480 LTEGLKRLGD TLYEEFGI SK 500 SEQ ID NO: 17
Saccharomyces cerevisiae
atgactgctg gttctgcccc ccctgttgat tacacttcct taaagaagaa cttccaaccg 60 tttctctcca gaagagtaga aaatagatct ctgaaaagct tttgggatgc ttctgatatc 120 tcagatgacg tcattgagct agctggtgga atgccaaacg agagattttt tcctatcgaa 180 tctatggatt tgaaaatatc aaaagttcct tttaatgata acccaaaatg gcataattcg 240 tttaccacgg cgcatttgga cttgggatcc cccagtgagc tacccattgc acgttctttc 300 caatatgcag aaaccaaggg tttaccccct ctcttacatt ttgttaaaga ttttgtgtcc 360 agaattaatc gcccagcctt ttccgatgag acggagtcta actgggatgt catcctttct 420 ggcgggtcca acgattcaat gtttaaggtt tttgaaacaa tttgcgacga atcgaccact 480 gtgatgattg aagagtttac tttcaccccg gctatgtcca atgtggaggc tacaggagca 540 aaagtcatcc ccatcaagat gaacctgacc ttcgacagag agtcccaggg tattgatgtc 600 gaatatctaa cgcagttgct cgataattgg tcaactggac catacaaaga cttaaacaag 660 ccaagggtcc tatataccat tgcaacgggc caaaatccta ccgggatgtc tgtcccccag 720 tggaaaagag agaaaattta ccagttggcc caaagacacg atttcctcat tgttgaagat 780 gatccctacg gttatctgta ctttccttcc tataatccgc aagagccatt agaaaaccct 840 taccattcta gcgacctgac tactgaacgg tatttgaatg attttttaat gaaatcattc 900 ttgactttgg atacagatgc ccgtgtcatc cgtttggaga ctttttctaa aatttttgct 960 cctggattaa ggttatcctt catcgttgct aataaattcc ttttgcaaaa aatcttggat 1020 ttggccgaca ttactacaag ggcccccagt ggtacctcac aagctattgt ttattctaca 1080 ataaaggcaa tggctgagtc caacttatcg tcctctcttt ctatgaaaga agcaatgttt 1140 gagggttgga taagatggat aatgcagatt gcttctaaat acaatcatag gaaaaatctt 1200 actttgaaag ccttatacga aacagaatct taccaagctg gtcagtttac cgttatggaa 1260 ccctccgcgg gtatgttcat cattattaaa atcaattggg ggaatttcga tagacctgac 1320 gatttgccgc aacagatgga tattttagat aagttcttgc tgaagaatgg tgttaaagta 1380 gtgcttggtt ataaaatggc tgtttgccca aattattcaa agcagaattc agattttcta 1440 agactcacca tcgcctatgc aagggatgat gatcagttga ttgaagcttc caaaagaatc 1500 ggtagtggca taaaagaatt ttttgacaac tataaaagtt ga 1542
SEQ ID NO: 18
Saccharomyces cerevisiae
MTAGSAPPVD YTSLKKNFQP FLSRRVENRS LKSFWDASD I SDDVIELAGG MPNERFFP IE 60 SMDLKI SKVP FNDNPKWHNS FTTAHLDLGS PSELPIARSF QYAETKGLPP LLHFVKDFVS 120 RINRPAFSDE TESNWDVILS GGSNDSMFKV FETICDESTT VMIEEFTFTP AMSNVEATGA 180 KVIP IKMNLT FDRESQGIDV EYLTQLLDNW STGPYKDLNK PRVLYTIATG QNPTGMSVPQ 240 WKREKIYQLA QRHDFLIVED DPYGYLYFPS YNPQEPLENP YHSSDLTTER YLNDFLMKSF 300 LTLDTDARVI RLETFSKIFA PGLRLSF IVA NKFLLQKILD LADITTRAPS GTSQAIVYST 360 IKAMAESNLS SSLSMKEAMF EGWIRWIMQI ASKYNHRKNL TLKALYETES YQAGQFTVME 420 PSAGMFIIIK INWGNFDRPD DLPQQMDILD KFLLKNGVKV VLGYKMAVCP NYSKQNSDFL 480 RLTIAYARDD DQLIEASKRI GSGIKEFFDN YKS 513
SEQ ID NO: 19
Saccharomyces cerevisiae
atggcacctg ttacaattga aaagttcgta aatcaagaag aacgacacct tgtttccaac 60 cgatcagcaa caattccgtt tggtgaatac atatttaaaa gattgttgtc catcgatacg 120 aaatcagttt tcggtgttcc tggtgacttc aacttatctc tattagaata tctctattca 180 cctagtgttg aatcagctgg cctaagatgg gtcggcacgt gtaatgaact gaacgccgct 240 tatgcggccg acggatattc ccgttactct aataagattg gctgtttaat aaccacgtat 300 ggcgttggtg aattaagcgc cttgaacggt atagccggtt cgttcgctga aaatgtcaaa 360 gttttgcaca ttgttggtgt ggccaagtcc atagattcgc gttcaagtaa ctttagtgat 420 cggaacctac atcatttggt cccacagcta catgattcaa attttaaagg gccaaatcat 480 aaagtatatc atgatatggt aaaagataga gtcgcttgct cggtagccta cttggaggat 540 attgaaactg catgtgacca agtcgataat gttatccgcg atatttacaa gtattctaaa 600 cctggttata tttttgttcc tgcagatttt gcggatatgt ctgttacatg tgataatttg 660 gttaatgttc cacgtatatc tcaacaagat tgtatagtat acccttctga aaaccaattg 720 tctgacataa tcaacaagat tactagttgg atatattcca gtaaaacacc tgcgatcctt 780 ggagacgtac tgactgatag gtatggtgtg agtaactttt tgaacaagct tatctgcaaa 840 actgggattt ggaatttttc cactgttatg ggaaaatctg taattgatga gtcaaaccca 900 acttatatgg gtcaatataa tggtaaagaa ggtttaaaac aagtctatga acattttgaa 960 ctgtgcgact tggtcttgca ttttggagtc gacatcaatg aaattaataa tgggcattat 1020 acttttactt ataaaccaaa tgctaaaatc attcaatttc atccgaatta tattcgcctt 1080 gtggacacta ggcagggcaa tgagcaaatg ttcaaaggaa tcaattttgc ccctatttta 1140 aaagaactat acaagcgcat tgacgtttct aaactttctt tgcaatatga ttcaaatgta 1200 actcaatata cgaacgaaac aatgcggtta gaagatccta ccaatggaca atcaagcatt 1260 attacacaag ttcacttaca aaagacgatg cctaaatttt tgaaccctgg tgatgttgtc 1320 gtttgtgaaa caggctcttt tcaattctct gttcgtgatt tcgcgtttcc ttcgcaatta 1380 aaatatatat cgcaaggatt tttcctttcc attggcatgg cccttcctgc cgccctaggt 1440 gttggaattg ccatgcaaga ccactcaaac gctcacatca atggtggcaa cgtaaaagag 1500 gactataagc caagattaat tttgtttgaa ggtgacggtg cagcacagat gacaatccaa 1560 gaactgagca ccattctgaa gtgcaatatt ccactagaag ttatcatttg gaacaataac 1620 ggctacacta ttgaaagagc catcatgggc cctaccaggt cgtataacga cgttatgtct 1680 tggaaatgga ccaaactatt tgaagcattc ggagacttcg acggaaagta tactaatagc 1740 actctcattc aatgtccctc taaattagca ctgaaattgg aggagcttaa gaattcaaac 1800 aaaagaagcg ggatagaact tttagaagtc aaattaggcg aattggattt ccccgaacag 1860 ctaaagtgca tggttgaagc agcggcactt aaaagaaata aaaaatag 1908
SEQ ID NO:20
Saccharomyces cerevisiae
MAPVTIEKFV NQEERHLVSN RSATIPFGEY IFKRLLSIDT KSVFGVPGDF NLSLLEYLYS 60 PSVESAGLRW VGTCNELNAA YAADGYSRYS NKIGCLITTY GVGELSALNG IAGSFAENVK 120 VLHIVGVAKS IDSRSSNFSD RNLHHLVPQL HDSNFKGPNH KVYHDMVKDR VACSVAYLED 180 IETACDQVDN VIRDIYKYSK PGYIFVPADF ADMSVTCDNL VNVPRISQQD CIVYPSENQL 240 SDI INKITSW IYSSKTPAIL GDVLTDRYGV SNFLNKLICK TGIWNFSTVM GKSVIDESNP 300 TYMGQYNGKE GLKQVYEHFE LCDLVLHFGV DINEINNGHY TFTYKPNAKI IQFHPNYIRL 360 VDTRQGNEQM FKGINFAP IL KELYKRIDVS KLSLQYDSNV TQYTNETMRL EDPTNGQSSI 420 ITQVHLQKTM PKFLNPGDW VCETGSFQFS VRDFAFPSQL KYISQGFFLS IGMALPAALG 480 VGIAMQDHSN AHINGGNVKE DYKPRLILFE GDGAAQMTIQ ELSTILKCNI PLEVIIWNNN 540 GYTIERAIMG PTRSYNDVMS WKWTKLFEAF GDFDGKYTNS TLIQCPSKLA LKLEELKNSN 600 KRSGIELLEV KLGELDFPEQ LKCMVEAAAL KRNKK 635
SEQ ID NO:21
Agaricus bisporus
atggatcacg caacattagc tatgatatta gcaattttgt ttatttcatt tcattttatt 60 aaattattat tctcacaaca aacaacaaaa ttgttacctc caggtcctaa acctttacca 120 attattggta atatcttgga agtaggaaag aagcctcata gatcatttgc aaatttggct 180 aagatccacg gtcctttaat ctctttgagg ttaggatcag ttaccaccat tgttgtatca 240 tctgctgatg tcgccaaaga gatgttcttg aagaaggatc accctttatc aaacaggaca 300 attccaaatt cagtaactgc tggtgaccac cacaaattga ctatgtcttg gttgcctgtt 360 tcacctaaat ggagaaactt cagaaagata actgctgttc atttattgtc tccacaaaga 420 ttagatgcat gccagacctt tagacatgca aaggttcagc aattatatga atacgttcaa 480 gagtgcgctc agaagggtca ggcagttgac attggtaaag ctgccttcac aacttctttg 540 aatttattgt ctaaattatt cttttctgtt gaattggccc atcataaatc acatacctct 600 caagagttta aggaattaat ttggaatatt atggaagaca tcggtaaacc aaactacgcc 660 gattacttcc caattttagg ttgcgtcgat ccatcaggaa ttagaagaag gttggcatgt 720 tcttttgata agttaattgc agtatttcaa ggaataatat gtgagaggtt agctccagat 780 tcatcaacta caactactac aactacagat gatgtcttgg acgtattatt gcaattgttt 840 aagcagaatg aattaactat gggagagatt aaccacttgt tagtcgatat tttcgatgcc 900 ggtactgata ctacatcatc taccttagag tgggttatga ctgagttaat aagaaaccct 960 gaaatgatgg aaaaggccca agaggaaata aaacaggtat tgggtaagga taagcaaatc 1020 caagagtcag acataataaa tttgccatac ttgcaagcaa tcattaagga aactttgagg 1080 ttgcacccac ctacagtttt cttgttgcct agaaaagccg acacagacgt agaattgtac 1140 ggatacattg ttccaaagga tgctcaaatt ttggtaaact tgtgggccat aggtagagat 1200 ccaaatgctt ggcaaaatgc cgatattttc tcacctgaaa gatttatagg ttgtgagatt 1260 gatgtaaagg gaagagattt tggtttattg ccttttggtg ctggtagaag aatttgccct 1320 ggtatgaatt tggccataag gatgttgaca ttgatgttgg ccacattatt gcaattcttc 1380 aactggaaat tagagggaga catttcacct aaagatttgg acatggatga aaagtttggt 1440 atagccttgc agaaaactaa acctttaaaa ttgatcccaa ttccaagata cggatcctaa 1500
SEQ ID NO:22
Agaricus bisporus
MDHATLAMIL AILFISFHFI KLLFSQQTTK LLPPGPKPLP I IGNILEVGK KPHRSFANLA 60 KIHGPLISLR LGSVTTIVVS SADVAKEMFL KKDHPLSNRT IPNSVTAGDH HKLTMSWLPV 120 SPKWRNFRKI TAVHLLSPQR LDACQTFRHA KVQQLYEYVQ ECAQKGQAVD IGKAAFTTSL 180 NLLSKLFFSV ELAHHKSHTS QEFKELIWNI MEDIGKPNYA DYFP ILGCVD PSGIRRRLAC 240 SFDKLIAVFQ GIICERLAPD SSTTTTTTTD DVLDVLLQLF KQNELTMGEI NHLLVDIFDA 300 GTDTTSSTLE WVMTELIRNP EMMEKAQEEI KQVLGKDKQI QESDIINLPY LQAI IKETLR 360 LHPPTVFLLP RKADTDVELY GYIVPKDAQI LVNLWAIGRD PNAWQNADIF SPERFIGCEI 420 DVKGRDFGLL PFGAGRRICP GMNLAIRMLT LMLATLLQFF NWKLEGDISP KDLDMDEKFG 480 IALQKTKPLK LIPIPRYGS 499
SEQ ID NO:23
atggatcatg ctaccttggc tatgattttg gccatcttgt tcattagctt ccacttcatc 60 aagctgttgt tctctcaaca aactaccaag ttgttgccac caggtccaaa accattgcca 120 attattggta acatcttgga ggttggtaag aagccacata gatcttttgc taatttggcc 180 aagattcacg gtccattgat ttctttgaga ttgggttctg ttacgaccat cgttgtttct 240 tcagctgatg ttgctaaaga gatgttcttg aagaaggatc acccattgtc caacagaact 300 attccaaatt ctgttaccgc tggtgatcat cataagttga ctatgtcttg gttgccagtt 360 tctccaaagt ggcgtaattt cagaaagatt accgctgttc atttgttgtc cccacaaaga 420 ttggatgctt gtcaaacttt tagacacgct aaggttcaac agttgtacga atacgttcaa 480 gaatgtgctc aaaaaggtca agccgttgat attggtaaag ctgcttttac tacctccttg 540 aacttgctgt ctaagttgtt cttctctgtt gaattggctc atcacaagtc tcatacctct 600 caagaattca aagagctgat ctggaacatc atggaagata tcggtaagcc aaattacgct 660 gattacttcc caattttggg ttgcgttgat ccatctggta ttagaagaag attggcttgc 720 tctttcgata agttgattgc tgttttccaa ggtatcatct gtgaaagatt agccccagat 780 tcttctacta ctacaactac taccactgat gatgttttgg atgtgttgtt gcagttgttc 840 aagcaaaacg aattgaccat gggtgaaatc aaccacttgt tggttgatat tttcgatgct 900 ggtactgata ccacttcctc tactttggaa tgggttatga ccgaattgat cagaaaccca 960 gaaatgatgg aaaaggccca agaagagatt aagcaagttt tgggtaaaga caagcagatc 1020 caagaatccg atattatcaa cttgccatac ttgcaggcca tcatcaaaga aacattgaga 1080 ttgcatccac caaccgtttt tttgttgcca agaaaagctg ataccgatgt tgagttgtat 1140 ggttacatcg ttccaaagga tgcccaaatc ttggttaatt tgtgggctat tggtagagat 1200 ccaaatgctt ggcaaaacgc cgatattttc tcaccagaaa ggttcattgg ttgcgaaatt 1260 gatgttaagg gtagagactt tggtttgttg ccttttggtg ctggtagaag gatttgtcca 1320 ggtatgaatt tggctatcag aatgttgact ttgatgctgg ctactctgtt gcaatttttc 1380 aactggaaat tggagggtga catctcacca aaagatttgg atatggacga aaagttcggt 1440 atcgccttgc aaaaaactaa gccattgaag ttgatcccca ttcctagata cggttcttga 1500
SEQ ID NO:24
MDHATLAMIL AILFISFHFI KLLFSQQTTK LLPPGPKPLP I IGNILEVGK KPHRSFANLA 60 KIHGPLISLR LGSVTTIVVS SADVAKEMFL KKDHPLSNRT IPNSVTAGDH HKLTMSWLPV 120 SPKWRNFRKI TAVHLLSPQR LDACQTFRHA KVQQLYEYVQ ECAQKGQAVD IGKAAFTTSL 180 NLLSKLFFSV ELAHHKSHTS QEFKELIWNI MEDIGKPNYA DYFP ILGCVD PSGIRRRLAC 240 SFDKLIAVFQ GIICERLAPD SSTTTTTTTD DVLDVLLQLF KQNELTMGEI NHLLVDIFDA 300 GTDTTSSTLE WVMTELIRNP EMMEKAQEEI KQVLGKDKQI QESDIINLPY LQAI IKETLR 360 LHPPTVFLLP RKADTDVELY GYIVPKDAQI LVNLWAIGRD PNAWQNADIF SPERFIGCEI 420 DVKGRDFGLL PFGAGRRICP GMNLAIRMLT LMLATLLQFF NWKLEGDISP KDLDMDEKFG 480 IALQKTKPLK LIPIPRYGS 499 SEQ ID NO:25
Pseudomonas putida
atgactcctg aacaatttag gcaatatggt catcaattaa tcgatttgat cgccgattac 60 agacagaccg ttggagaaag acctgtaatg gctcaagttg aacctggata tttaaaggcc 120 gctttgcctg ctaccgcacc acaacaaggt gaaccattcg ctgccatttt ggatgatgta 180 aacaatttgg tcatgccagg attgtcacat tggcaacacc cagactttta tggttatttc 240 ccttcaaatg gaacattatc atctgttttg ggtgacttct tatctacagg tttaggagta 300 ttgggtttat cttggcaatc ttctccagca ttgtctgaat tggaagaaac caccttagac 360 tggttaagac aattgttggg tttatctgga cagtggtctg gagttataca agacaccgca 420 tctacctcaa ctttggtcgc attgatttca gctagagaga gagcaacaga ttacgcattg 480 gtcaggggtg gtttgcaagc cgaaccaaaa ccattgattg tatatgtctc tgcacacgct 540 cattcatcag ttgacaaagc tgcattattg gctggtttcg gtagagataa cataagatta 600 atcccaacag acgagaggta tgcattaaga cctgaagcat tacaagctgc aattgaacag 660 gacttggcag ctggaaacca gccttgtgct gttgtcgcta ctactggtac aactacaact 720 accgcattag atcctttaag acctgtagga gaaatcgcac aagctaatgg tttatggtta 780 catgtcgatt cagctatggc aggttctgct atgatattac cagagtgtag atggatgtgg 840 gatggtattg aattggcaga ttcagtcgtt gtaaacgcac ataagtggtt gggtgttgca 900 tttgattgtt caatatatta cgtcagagat ccacagcact taattagagt aatgtcaacc 960 aacccatctt acttgcaatc agcagttgac ggtgaagtta agaatttaag ggattggggt 1020 atcccattag gtagaagatt tagagctttg aaattatggt tcatgttgag gtcagagggt 1080 gttgatgctt tgcaagccag attgagaagg gatttagata acgctcaatg gttagcaggt 1140 caagtagaag ccgcagctga gtgggaagtt ttggcacctg ttcaattgca aaccttgtgt 1200 attaggcata gaccagcagg attagagggt gaggctttag atgctcatac taagggttgg 1260 gctgaaagat taaacgcatc aggagcagca tacgtaaccc ctgccacatt agatggtaga 1320 tggatggtca gggtttctat aggtgcattg ccaactgaaa gaggtgatgt acaaagattg 1380 tgggctagat tacaagatgt tattaaggga ggatcctaa 1419
SEQ ID NO:26
Pseudomonas putida
MTPEQFRQYG HQLIDLIADY RQTVGERPVM AQVEPGYLKA ALPATAPQQG EPFAAILDDV 60 NNLVMPGLSH WQHPDFYGYF PSNGTLSSVL GDFLSTGLGV LGLSWQSSPA LSELEETTLD 120 WLRQLLGLSG QWSGVIQDTA STSTLVALIS ARERATDYAL VRGGLQAEPK PLIVYVSAHA 180 HSSVDKAALL AGFGRDNIRL IPTDERYALR PEALQAAIEQ DLAAGNQPCA WATTGTTTT 240 TALDPLRPVG EIAQANGLWL HVDSAMAGSA MILPECRWMW DGIELADSW VNAHKWLGVA 300 FDCSIYYVRD PQHLIRVMST NPSYLQSAVD GEVKNLRDWG IPLGRRFRAL KLWFMLRSEG 360 VDALQARLRR DLDNAQWLAG QVEAAAEWEV LAPVQLQTLC IRHRPAGLEG EALDAHTKGW 420 AERLNASGAA YVTPATLDGR WMVRVSIGAL PTERGDVQRL WARLQDVIKG GS 472
SEQ ID NO:27
atgaccccag aacaattcag acaatacggt caccaattga ttgatttgat cgccgattac 60 agacaaaccg ttggtgaaag accagttatg gctcaagttg aaccaggtta tttgaaagct 120 gctttgccag ctactgctcc acaacaaggt gaaccatttg ctgctatttt ggatgatgtt 180 aacaacttgg ttatgccagg tttgtctcat tggcaacatc cagattttta cggttacttt 240 ccatccaacg gtactttgtc atctgttttg ggtgatttct tgtctactgg tttgggtgtt 300 ttaggtttgt catggcaatc ttctccagct ttgtctgaat tggaagaaac tactttggat 360 tggttgagac agttgttggg tttatctggt caatggtctg gtgttattca agatactgct 420 tctacttcta ccttggttgc tttgatttct gctagagaaa gagctactga ttacgctttg 480 gttagaggtg gtttacaagc tgaacctaaa ccattgatcg tttacgtttc tgctcatgcc 540 cattcttcag ttgataaggc tgctttgttg gctggttttg gtagagataa cattagattg 600 attccaaccg acgaaagata cgctttaaga ccagaagcct tgcaagctgc tattgaacaa 660 gatttggctg ctggtaatca accatgtgct gttgttgcta ctactggtac tactactaca 720 actgctttgg atccattaag acctgtaggt gaaattgctc aagctaatgg tttgtggttg 780 catgttgatt cagctatggc tggttctgct atgattttgc cagaatgtag atggatgtgg 840 gatggtattg aattggctga ttctgttgtt gttaacgccc ataagtggtt gggtgttgct 900 tttgattgct ctatctacta cgttagagat ccacaacact tgatcagagt gatgtctact 960 aatccatcct acttgcaatc agctgttgat ggtgaagtta agaacttgag agattggggt 1020 attccattgg gtagaagatt tagagctttg aagttgtggt ttatgttgag atccgaaggt 1080 gttgatgcat tgcaagctag attgagaaga gatttggata atgctcaatg gttggctgga 1140 caagttgaag ctgctgctga atgggaagtt ttggctccag ttcaattgca aaccttgtgc 1200 attagacata gaccagcagg tttggaaggt gaagccttgg atgctcatac aaaaggttgg 1260 gctgaaagat tgaatgcttc tggtgctgct tatgttactc cagctacttt agatggaaga 1320 tggatggtta gagtttccat tggtgcttta ccaactgaaa gaggtgacgt tcaaagattg 1380 tgggctagat tgcaagatgt tatcaagggt tga 1413
SEQ ID NO:28
Pseudomonas putida
MTPEQFRQYG HQLIDLIADY RQTVGERPVM AQVEPGYLKA ALPATAPQQG EPFAAILDDV 60 NNLVMPGLSH WQHPDFYGYF PSNGTLSSVL GDFLSTGLGV LGLSWQSSPA LSELEETTLD 120 WLRQLLGLSG QWSGVIQDTA STSTLVALIS ARERATDYAL VRGGLQAEPK PLIVYVSAHA 180 HSSVDKAALL AGFGRDNIRL IPTDERYALR PEALQAAIEQ DLAAGNQPCA WATTGTTTT 240 TALDPLRPVG EIAQANGLWL HVDSAMAGSA MILPECRWMW DGIELADSW VNAHKWLGVA 300 FDCSIYYVRD PQHLIRVMST NPSYLQSAVD GEVKNLRDWG IPLGRRFRAL KLWFMLRSEG 360 VDALQARLRR DLDNAQWLAG QVEAAAEWEV LAPVQLQTLC IRHRPAGLEG EALDAHTKGW 420 AERLNASGAA YVTPATLDGR WMVRVSIGAL PTERGDVQRL WARLQDVIKG 470
SEQ ID NO:29
atggaaagat tgatcttcaa tggtagacct ttgttgcaca gagttaccaa agaagaaacc 60 gttatgttgt accacgaatt ggaagttgct gcttctgctg atgaagtttg gtctgttgaa 120 ggttctccag aattgggttt acatttgcca gatttgttgc cagctggtat ttttgccaag 180 ttcgaaatta ctggtgatgg tggtgaaggt tccattttgg atatgacttt tccaccaggt 240 caattcccac atcattacag agaaaagttc gtctttttcg accacaagaa cagatacaag 300 ttggtcgaac aaatcgatgg tgatttcttc gatttgggtg ttacttacta catggacacc 360 attagagttg ttgctactgg tccagattct tgcgttatta agtctactac tgaataccac 420 gtcaagccag aatttgctaa aatcgttaag ccattgatcg ataccgttcc attggctatt 480 atgtctgaag ctattgccaa ggttgtcttg gaaaacaaac acaagtcatc tgaatga 537
SEQ ID NO:30
MERLIFNGRP LLHRVTKEET VMLYHELEVA ASADEVWSVE GSPELGLHLP DLLPAGIFAK 60 FEITGDGGEG SILDMTFPPG QFPHHYREKF VFFDHKNRYK LVEQIDGDFF DLGVTYYMDT 120 IRVVATGPDS CVIKSTTEYH VKPEFAKIVK PLIDTVPLAI MSEAIAKWL ENKHKSSE 178
SEQ ID NO:31
Nandina domestica
gctcttctac aatgagatct ggtattgtct tcttggtctt gttcttcttg ggttgtgaaa 60 tttcccaagg tagacaattg ttagagtcta gattattcag aaagtccacc attagaaagg 120 tcttacacca cgaattatcc gttgccgctt ccgctcaaga agtttgggat gtttactcct 180 ctccagaatt gccaaagcat ttgccagaga ttttaccagg tgctttcaaa aaagtcgtcg 240 ttactggtga cggtggtgtt ggtactgtta ttgagatgat tttcccacca ggtgtcgtcc 300 cacacagata caaagaaaaa ttcgttttaa ttgatgacga aaaatttttg aagaaggttg 360 aaatgatcga aggtggttac ttggatatgg gttgtacttt ttacatggac actattcaaa 420 tcattccaac cggtcctgat tcttgcatta tcaagtcttc tactgaatac tacgtcaagc 480 cagagttcgc tgataaggtt gtcccattaa tttctactgt tccattacaa gctatggctg 540 aagctatcgc taagatcgtt ttggaaaaca aggctaagca taagggtttt atcgaaatcg 600 gctgaagagc 610
SEQ ID NO:32
Nandina domestica
MRSGIVFLVL FFLGCEISQG RQLLESRLFR KSTIRKVLHH ELSVAASAQE VWDVYSSPEL 60 PKHLPE ILPG AFKKVWTGD GGVGTVIEMI FPPGVVPHRY KEKFVLIDDE KFLKKVEMIE 120 GGYLDMGCTF YMDTIQIIPT GPDSCIIKSS TEYYVKPEFA DKWPLISTV PLQAMAEAIA 180 KIVLENKAKH KGFIEIG 197 SEQ ID NO:33
Papaver somniferum
gctcttctac aatgagaaag gttattaagt acgacatgga agtcgccgtc tctgctgatt 60 ccgtctgggc tgtctactct tccccagata tcccaagatt gttgcgtgat gtcttgttgc 120 caggtgtctt cgaaaagttg gacgttattg agggtaacgg tggtgttggt accgttttgg 180 atattgtctt tccacctggt gccgttccaa gatcctacaa ggaaaagttc gtcaatatcg 240 accgtgaaaa gagattgaag gaagttatta tgattgaggg tggttacttg gacatgggtt 300 gtactttcta cttggataga attcacgtcg ttgagaagac taagtcttct tgcgtcattg 360 agtcctccat cgtttacgat gttaaagaag agtgtgccga cgctatgtcc aagttgatta 420 ccaccgaacc attgaaatcc atggccgaag tcatttctaa ctacgttatt caaaaggaat 480 tgttctctgc cagaaacatc ttatctaagc aatccgttgt caagaaggaa attcgttacg 540 atttagaagt tccaatttcc gttgattcta tttggtccgt ttactcctgt ccagacatcc 600 cacgtttatt aggctgaaga gc 622
SEQ ID NO:34
Papaver somniferum
MRKVIKYDME VAVSADSVWA VYSSPDIPRL LRDVLLPGVF EKLDVIEGNG GVGTVLD IVF 60 PPGAVPRSYK EKFVNIDREK RLKEVIMIEG GYLDMGCTFY LDRIHWEKT KSSCVIESSI 120 VYDVKEECAD AMSKLITTEP LKSMAEVISN YVIQKELFSA RNILSKQSW KKE IRYDLEV 180 PISVDSIWSV YSCPDIPRLL G 201
SEQ ID NO:35
Sanguinaria canadensis
gctcttctac aatgagatct ggtattgtct ttttggtttt gttcttctta ggttgtgaaa 60 tttcccaagg tagacaattg ttggaatcta gattgttcag aaagtctact attcaaaagg 120 ttttgcacca cgaattgcca gtcgctgctt ctgctcaaga agtttgggat gtctattctt 180 ccccagaatt gccaaaacac ttgccagaaa ttttgccagg tgccttcgaa aaggttgtcg 240 tcaccggtga tggtggtgtt ggtactgttt tggaaatggt ttttccacca ggtgaagttc 300 caagatccta caaagaaaag ttcgtcttaa ttgacgatga gcaattgttg aagaaggttg 360 aaatgatcga aggtggttac ttggatatgg gttgtacttt ctacatggac actatccaaa 420 ttgtcccaac cggtccagac tcctgtatca tcaagtcttc tactgaatac tatgtcaagc 480 ctgaattcgc tgataaggtt gttccattga tttctactat cccattgcaa gctatggccg 540 aggccatctc taacattgtc ttagctaaca aggccaagaa caagtctatt attatcgaaa 600 ttggctgaag age 613
SEQ ID NO:36
Sanguinaria canadensis
MRSGIVFLVL FFLGCEISQG RQLLESRLFR KSTIQKVLHH ELPVAASAQE VWDVYSSPEL 60 PKHLPE ILPG AFEKVWTGD GGVGTVLEMV FPPGEVPRSY KEKFVLIDDE QLLKKVEMIE 120 GGYLDMGCTF YMDTIQIVPT GPDSCIIKSS TEYYVKPEFA DKWPLISTI PLQAMAEAIS 180 NIVLANKAKN KSIIIEIG 198
SEQ ID NO:37
Stylophorum diphyllum
gctcttctac aatgegtaag gaagteagat acgaaatgga agttccaacc teegetgatt 60 ccatttgggc tgtctactct tctcacgata ttccaagatt gttgaaggaa gttttgttgc 120 caggtgtctt cgaaaagttg gaegteattg aaggtgacgg tggtgttggt actgttttgg 180 atattgeett cccacctggt gctgttccaa gaacttacaa ggaaaaattc gttactatta 240 accacgaaaa gagattgaaa gaggttatta tgategaagg tggttacttg gatatgggtt 300 gtacttttta catggataga atccatgttt tggaaaaagg tccaaactcc tgtgttatcg 360 aatccgctat tatttacgaa gttaaggaag aattcgctga tgtcgtcgtc ccattgatta 420 ccaccgaacc attagettet atggctgaag tcatttctaa ttacgtcttg aagaagcaaa 480 ttcatgtttt cggttacgtt atcaaaccaa agttgggttt atccttattg ttatgtttca 540 tcttgtgttt agtcttgtta ggtgttttgt taattggtgg tgtcccattg ggctgaagag 600 c 601 SEQ ID NO:38
Stylophorum diphyllum
MRKEVRYEME VPTSADSIWA VYSSHDIPRL LKEVLLPGVF EKLDVIEGDG GVGTVLD IAF 60 PPGAVPRTYK EKFVTINHEK RLKEVIMIEG GYLDMGCTFY MDRIHVLEKG PNSCVIESAI 120 IYEVKEEFAD WVPLITTEP LASMAEVISN YVLKKQIHVF GYVIKPKLGL SLLLCFILCL 180 VLLGVLLIGG VPLG 194
SEQ ID NO:39
Thalictrum flavum
gctcttctac aatgagaaag gaattgaccc acgaaatgga agttccagcc tctgctgacg 60 ctatctgggc tgtctacggt tcccctgaca ttccacgttt gttgaaggag gttttgttgc 120 caggtgtttt cgaaaagttg gatgttatcg aaggtgacgg tggtgtcggt actgtcttag 180 acattgcctt cccaccaggt gctgttccac gtgcctacaa ggaaaagttc atgaaagtta 240 accacgaaaa gagattgaaa gaagttgaaa tgattgaagg tggttacttg gacatgggtt 300 gtaccttcta catggataga atccacgttg ttgagaaagg tccaaatgct tgtgttattg 360 aatctgccat catttacgaa gtcaaagatg aatttgctga cgtcgttgtt ccattgatca 420 ctactgaacc tttggcctcc atggctgaag tcatttccaa ctacgtcttg aagaaccaat 480 tcagagtctt cggttatgtc attaagccaa agttaggttt atccttgtta ttgtgtttca 540 tcttgtgttt ggttttgtta ggtggtttgt tgatcggtgg tgttccatta ggctgaagag 600 c 601
SEQ ID NO:40
Thalictrum flavum
MRKELTHEME VPASADAIWA VYGSPDIPRL LKEVLLPGVF EKLDVIEGDG GVGTVLD IAF 60 PPGAVPRAYK EKFMKVNHEK RLKEVEMIEG GYLDMGCTFY MDRIHWEKG PNACVIESAI 120 IYEVKDEFAD WVPLITTEP LASMAEVISN YVLKNQFRVF GYVIKPKLGL SLLLCFILCL 180 VLLGGLLIGG VPLG 194
SEQ ID NO:41
atgagacaat tgttagagtc tagattattc agaaagtcca ccattagaaa ggtcttacac 60 cacgaattat ccgttgccgc ttccgctcaa gaagtttggg atgtttactc ctctccagaa 120 ttgccaaagc atttgccaga gattttacca ggtgctttca aaaaagtcgt cgttactggt 180 gacggtggtg ttggtactgt tattgagatg attttcccac caggtgtcgt cccacacaga 240 tacaaagaaa aattcgtttt aattgatgac gaaaaatttt tgaagaaggt tgaaatgatc 300 gaaggtggtt acttggatat gggttgtact ttttacatgg acactattca aatcattcca 360 accggtcctg attcttgcat tatcaagtct tctactgaat actacgtcaa gccagagttc 420 gctgataagg ttgtcccatt aatttctact gttccattac aagctatggc tgaagctatc 480 gctaagatcg ttttggaaaa caaggctaag cataagggtt ttatcgaaat cggctga 537
SEQ ID NO:42
MRQLLESRLF RKSTIRKVLH HELSVAASAQ EVWDVYSSPE LPKHLPE ILP GAFKKWVTG 60 DGGVGTVIEM IFPPGWPHR YKEKFVLIDD EKFLKKVEMI EGGYLDMGCT FYMDTIQIIP 120 TGPDSCIIKS STEYYVKPEF ADKVVPLIST VPLQAMAEAI AKIVLENKAK HKGFIEIG 178
SEQ ID NO:43
atgagacaat tgttggaatc tagattgttc agaaagtcta ctattcaaaa ggttttgcac 60 cacgaattgc cagtcgctgc ttctgctcaa gaagtttggg atgtctattc ttccccagaa 120 ttgccaaaac acttgccaga aattttgcca ggtgccttcg aaaaggttgt cgtcaccggt 180 gatggtggtg ttggtactgt tttggaaatg gtttttccac caggtgaagt tccaagatcc 240 tacaaagaaa agttcgtctt aattgacgat gagcaattgt tgaagaaggt tgaaatgatc 300 gaaggtggtt acttggatat gggttgtact ttctacatgg acactatcca aattgtccca 360 accggtccag actcctgtat catcaagtct tctactgaat actatgtcaa gcctgaattc 420 gctgataagg ttgttccatt gatttctact atcccattgc aagctatggc cgaggccatc 480 tctaacattg tcttagctaa caaggccaag aacaagtcta ttattatcga aattggctga 540 SEQ ID NO:44
MRQLLESRLF RKSTIQKVLH HELPVAASAQ EVWDVYSSPE LPKHLPE ILP GAFEKWVTG 60 DGGVGTVLEM VFPPGEVPRS YKEKFVLIDD EQLLKKVEMI EGGYLDMGCT FYMDTIQIVP 120 TGPDSCIIKS STEYYVKPEF ADKVVPLIST IPLQAMAEAI SNIVLANKAK NKSIIIEIG 179
SEQ ID NO:45
atggctgatt ccgtctgggc tgtctactct tccccagata tcccaagatt gttgcgtgat 60 gtcttgttgc caggtgtctt cgaaaagttg gacgttattg agggtaacgg tggtgttggt 120 accgttttgg atattgtctt tccacctggt gccgttccaa gatcctacaa ggaaaagttc 180 gtcaatatcg accgtgaaaa gagattgaag gaagttatta tgattgaggg tggttacttg 240 gacatgggtt gtactttcta cttggataga attcacgtcg ttgagaagac taagtcttct 300 tgcgtcattg agtcctccat cgtttacgat gttaaagaag agtgtgccga cgctatgtcc 360 aagttgatta ccaccgaacc attgaaatcc atggccgaag tcatttctaa ctacgttatt 420 caaaaggaat tgttctctgc cagaaacatc ttatctaagc aatccgttgt caagaaggaa 480 attcgttacg atttagaagt tccaatttcc gttgattcta tttggtccgt ttactcctgt 540 ccagacatcc cacgtttatt aggctga 567
SEQ ID NO:46
MADSVWAVYS SPDIPRLLRD VLLPGVFEKL DVIEGNGGVG TVLDIVFPPG AVPRSYKEKF 60 VNIDREKRLK EVIMIEGGYL DMGCTFYLDR IHWEKTKSS CVIESSIVYD VKEECADAMS 120 KLITTEPLKS MAEVI SNYVI QKELFSARNI LSKQSWKKE IRYDLEVPIS VDSIWSVYSC 180 PDIPRLLG 188
SEQ ID NO:47
atggctgatt ccatttgggc tgtctactct tctcacgata ttccaagatt gttgaaggaa 60 gttttgttgc caggtgtctt cgaaaagttg gacgtcattg aaggtgacgg tggtgttggt 120 actgttttgg atattgcctt cccacctggt gctgttccaa gaacttacaa ggaaaaattc 180 gttactatta accacgaaaa gagattgaaa gaggttatta tgatcgaagg tggttacttg 240 gatatgggtt gtacttttta catggataga atccatgttt tggaaaaagg tccaaactcc 300 tgtgttatcg aatccgctat tatttacgaa gttaaggaag aattcgctga tgtcgtcgtc 360 ccattgatta ccaccgaacc attagcttct atggctgaag tcatttctaa ttacgtcttg 420 aagaagcaaa ttcatgtttt cggttacgtt atcaaaccaa agttgggttt atccttattg 480 ttatgtttca tcttgtgttt agtcttgtta ggtgttttgt taattggtgg tgtcccattg 540 ggctga 546
SEQ ID NO:48
MADSIWAVYS SHDIPRLLKE VLLPGVFEKL DVIEGDGGVG TVLD IAFPPG AVPRTYKEKF 60 VTINHEKRLK EVIMIEGGYL DMGCTFYMDR IHVLEKGPNS CVIESAIIYE VKEEFADVW 120 PLITTEPLAS MAEVI SNYVL KKQIHVFGYV IKPKLGLSLL LCFILCLVLL GVLLIGGVPL 180 G 181
SEQ ID NO:49
atggctgacg ctatctgggc tgtctacggt tcccctgaca ttccacgttt gttgaaggag 60 gttttgttgc caggtgtttt cgaaaagttg gatgttatcg aaggtgacgg tggtgtcggt 120 actgtcttag acattgcctt cccaccaggt gctgttccac gtgcctacaa ggaaaagttc 180 atgaaagtta accacgaaaa gagattgaaa gaagttgaaa tgattgaagg tggttacttg 240 gacatgggtt gtaccttcta catggataga atccacgttg ttgagaaagg tccaaatgct 300 tgtgttattg aatctgccat catttacgaa gtcaaagatg aatttgctga cgtcgttgtt 360 ccattgatca ctactgaacc tttggcctcc atggctgaag tcatttccaa ctacgtcttg 420 aagaaccaat tcagagtctt cggttatgtc attaagccaa agttaggttt atccttgtta 480 ttgtgtttca tcttgtgttt ggttttgtta ggtggtttgt tgatcggtgg tgttccatta 540 ggctga 546 SEQ ID NO:50
MADAIWAVYG SPDIPRLLKE VLLPGVFEKL DVIEGDGGVG TVLD IAFPPG AVPRAYKEKF 60 MKVNHEKRLK EVEMIEGGYL DMGCTFYMDR IHWEKGPNA CVIESAIIYE VKDEFADVW 120 PLITTEPLAS MAEVI SNYVL KNQFRVFGYV IKPKLGLSLL LCFILCLVLL GGLLIGGVPL 180 G 181
SEQ ID NO:51
atgcgtaagg aagtcagata cgaaatggaa gttccaacct ccgctgattc catttgggct 60 gtctactctt ctcacgatat tccaagattg ttgaaggaag ttttgttgcc aggtgtcttc 120 gaaaagttgg acgtcattga aggtgacggt ggtgttggta ctgttttgga tattgccttc 180 ccacctggtg ctgttccaag aacttacaag gaaaaattcg ttactattaa ccacgaaaag 240 agattgaaag aggttattat gatcgaaggt ggttacttgg atatgggttg tactttttac 300 atggatagaa tccatgtttt ggaaaaaggt ccaaactcct gtgttatcga atccgctatt 360 atttacgaag ttaaggaaga attcgctgat gtcgtcgtcc cattgattac caccgaacca 420 ttagcttcta tggctgaagt catttctaat tacgtcttga agaagcaaat tcatgttttc 480 ggttacgtta tcaaaccaaa gttgggttga 510
SEQ ID NO:52
MRKEVRYEME VPTSADSIWA VYSSHDIPRL LKEVLLPGVF EKLDVIEGDG GVGTVLD IAF 60 PPGAVPRTYK EKFVTINHEK RLKEVIMIEG GYLDMGCTFY MDRIHVLEKG PNSCVIESAI 120 IYEVKEEFAD WVPLITTEP LASMAEVISN YVLKKQIHVF GYVIKPKLG 169
SEQ ID NO:53
atgagaaagg aattgaccca cgaaatggaa gttccagcct ctgctgacgc tatctgggct 60 gtctacggtt cccctgacat tccacgtttg ttgaaggagg ttttgttgcc aggtgttttc 120 gaaaagttgg atgttatcga aggtgacggt ggtgtcggta ctgtcttaga cattgccttc 180 ccaccaggtg ctgttccacg tgcctacaag gaaaagttca tgaaagttaa ccacgaaaag 240 agattgaaag aagttgaaat gattgaaggt ggttacttgg acatgggttg taccttctac 300 atggatagaa tccacgttgt tgagaaaggt ccaaatgctt gtgttattga atctgccatc 360 atttacgaag tcaaagatga atttgctgac gtcgttgttc cattgatcac tactgaacct 420 ttggcctcca tggctgaagt catttccaac tacgtcttga agaaccaatt cagagtcttc 480 ggttatgtca ttaagccaaa gttaggttga 510
SEQ ID NO:54
MRKELTHEME VPASADAIWA VYGSPDIPRL LKEVLLPGVF EKLDVIEGDG GVGTVLD IAF 60 PPGAVPRAYK EKFMKVNHEK RLKEVEMIEG GYLDMGCTFY MDRIHWEKG PNACVIESAI 120 IYEVKDEFAD WVPLITTEP LASMAEVISN YVLKNQFRVF GYVIKPKLG 169
SEQ ID NO:55
atgcgtaagg aagtcagata cgaaatggaa gttccaacct ccgctgattc catttgggct 60 gtctactctt ctcacgatat tccaagattg ttgaaggaag ttttgttgcc aggtgtcttc 120 gaaaagttgg acgtcattga aggtgacggt ggtgttggta ctgttttgga tattgccttc 180 ccacctggtg ctgttccaag aacttacaag gaaaaattcg ttactattaa ccacgaaaag 240 agattgaaag aggttattat gatcgaaggt ggttacttgg atatgggttg tactttttac 300 atggatagaa tccatgtttt ggaaaaaggt ccaaactcct gtgttatcga atccgctatt 360 atttacgaag ttaaggaaga attcgctgat gtcgtcgtcc cattgattac caccgaacca 420 ttagcttcta tggctgaagt catttctaat tacgtcattc aaaaggaatt gttctctgcc 480 agaaacatct tatctaagca atccgttgtc aagaaggaaa ttcgttacga tttagaagtt 540 ccaatttccg ttgattctat ttggtccgtt tactcctgtc cagacatccc acgtttatta 600 ggctga 606
SEQ ID NO:56
MRKEVRYEME VPTSADSIWA VYSSHDIPRL LKEVLLPGVF EKLDVIEGDG GVGTVLD IAF 60 PPGAVPRTYK EKFVTINHEK RLKEVIMIEG GYLDMGCTFY MDRIHVLEKG PNSCVIESAI 120 IYEVKEEFAD WVPLITTEP LASMAEVISN YVIQKELFSA RNILSKQSW KKE IRYDLEV 180 PISVDSIWSV YSCPDIPRLL G 201 SEQ ID NO:57
atgagaaagg aattgaccca cgaaatggaa gttccagcct ctgctgacgc tatctgggct 60 gtctacggtt cccctgacat tccacgtttg ttgaaggagg ttttgttgcc aggtgttttc 120 gaaaagttgg atgttatcga aggtgacggt ggtgtcggta ctgtcttaga cattgccttc 180 ccaccaggtg ctgttccacg tgcctacaag gaaaagttca tgaaagttaa ccacgaaaag 240 agattgaaag aagttgaaat gattgaaggt ggttacttgg acatgggttg taccttctac 300 atggatagaa tccacgttgt tgagaaaggt ccaaatgctt gtgttattga atctgccatc 360 atttacgaag tcaaagatga atttgctgac gtcgttgttc cattgatcac tactgaacct 420 ttggcctcca tggctgaagt catttccaac tacgtcattc aaaaggaatt gttctctgcc 480 agaaacatct tatctaagca atccgttgtc aagaaggaaa ttcgttacga tttagaagtt 540 ccaatttccg ttgattctat ttggtccgtt tactcctgtc cagacatccc acgtttatta 600 ggctga 606
SEQ ID NO:58
MRKELTHEME VPASADAIWA VYGSPDIPRL LKEVLLPGVF EKLDVIEGDG GVGTVLD IAF 60
PPGAVPRAYK EKFMKVNHEK RLKEVEMIEG GYLDMGCTFY MDRIHWEKG PNACVIESAI 120
IYEVKDEFAD WVPLITTEP LASMAEVISN YVIQKELFSA RNILSKQSW KKE IRYDLEV 180
PISVDSIWSV YSCPDIPRLL G 201
SEQ ID NO:59
atgagatctg gtattgtctt cttggtcttg ttcttcttgg gttgtgaaat ttcccaaggt 60 agacaattgt tagagtctag attattcaga aagtccacca ttagaaaggt cttacaccac 120 gaattatccg ttgccgcttc cgctcaagaa gtttgggatg tttactcctc tccagaattg 180 ccaaagcatt tgccagagat tttaccaggt gctttcaaaa aagtcgtcgt tactggtgac 240 ggtggtgttg gtactgttat tgagatgatt ttcccaccag gtgtcgtccc acacagatac 300 aaagaaaaat tcgttttaat tgatgacgaa aaatttttga agaaggttga aatgatcgaa 360 ggtggttact tggatatggg ttgtactttt tacatggaca ctattcaaat cattccaacc 420 ggtcctgatt cttgcattat caagtcttct actgaatact acgtcaagcc agagttcgct 480 gataaggttg tcccattaat ttctactgtt ccattacaag ctatggctga agctatcgct 540 aagatcgttt tggaaaacaa ggctaagcat aagggtttta tcgaaatcgg catgagtaaa 600 ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat 660 gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc 720 cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact tgtcactact 780 ttcacttatg gtgttcaatg catttcaaga tacccagatc atatgaaacg gcatgacttt 840 ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac 900 gggaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc 960 gagttaaaag gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac 1020 aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagtt 1080 aacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa 1140 caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca 1200 caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt 1260 gtaacagctg ctgggattac ttacattcac gccctccccc cacatccgct ctaa 1314
SEQ ID NO:60
MRSGIVFLVL FFLGCEISQG RQLLESRLFR KSTIRKVLHH ELSVAASAQE VWDVYSSPEL 60
PKHLPE ILPG AFKKVWTGD GGVGTVIEMI FPPGVVPHRY KEKFVLIDDE KFLKKVEMIE 120
GGYLDMGCTF YMDTIQIIPT GPDSCIIKSS TEYYVKPEFA DKWPLISTV PLQAMAEAIA 180
KIVLENKAKH KGFIEIGMSK GEELFTGWP ILVELDGDVN GHKFSVSGEG EGDATYGKLT 240
LKF ICTTGKL PVPWPTLVTT FTYGVQCISR YPDHMKRHDF FKSAMPEGYV QERTIFFKDD 300
GNYKTRAEVK FEGDTLVNRI ELKGIDFKED GNILGHKLEY NYNSHNVYIM ADKQKNGIKV 360
NFKIRHNIED GSVQLADHYQ QNTP IGDGPV LLPDNHYLST QSALSKDPNE KRDHMVLLEF 420
VTAAGITYIH ALPPHPL 437
SEQ ID NO:61
atgagacaat tgttagagtc tagattattc agaaagtcca ccattagaaa ggtcttacac 60 cacgaattat ccgttgccgc ttccgctcaa gaagtttggg atgtttactc ctctccagaa 120 ttgccaaagc atttgccaga gattttacca ggtgctttca aaaaagtcgt cgttactggt 180 gacggtggtg ttggtactgt tattgagatg attttcccac caggtgtcgt cccacacaga 240 tacaaagaaa aattcgtttt aattgatgac gaaaaatttt tgaagaaggt tgaaatgatc 300 gaaggtggtt acttggatat gggttgtact ttttacatgg acactattca aatcattcca 360 accggtcctg attcttgcat tatcaagtct tctactgaat actacgtcaa gccagagttc 420 gctgataagg ttgtcccatt aatttctact gttccattac aagctatggc tgaagctatc 480 gctaagatcg ttttggaaaa caaggctaag cataagggtt ttatcgaaat cggcatgagt 540 aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga tggtgatgtt 600 aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata cggaaaactt 660 acccttaaat ttatttgcac tactggaaaa ctacctgttc catggccaac acttgtcact 720 actttcactt atggtgttca atgcatttca agatacccag atcatatgaa acggcatgac 780 tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt tttcaaagat 840 gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct tgttaataga 900 atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca caaattggaa 960 tacaactata actcacacaa tgtatacatc atggcagaca aacaaaagaa tggaatcaaa 1020 gttaacttca aaattagaca caacattgaa gatggaagcg ttcaactagc agaccattat 1080 caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca ttacctgtcc 1140 acacaatctg ccctttcgaa agatcccaac gaaaagagag accacatggt ccttcttgag 1200 tttgtaacag ctgctgggat tacttacatt cacgccctcc ccccacatcc gctctaa 1257
SEQ ID NO:62
MRQLLESRLF RKSTIRKVLH HELSVAASAQ EVWDVYSSPE LPKHLPE ILP GAFKKWVTG 60 DGGVGTVIEM IFPPGWPHR YKEKFVLIDD EKFLKKVEMI EGGYLDMGCT FYMDTIQIIP 120 TGPDSCIIKS STEYYVKPEF ADKVVPLIST VPLQAMAEAI AKIVLENKAK HKGFIEIGMS 180 KGEELFTGW PILVELDGDV NGHKFSVSGE GEGDATYGKL TLKF ICTTGK LPVPWPTLVT 240 TFTYGVQCIS RYPDHMKRHD FFKSAMPEGY VQERTIFFKD DGNYKTRAEV KFEGDTLVNR 300 IELKGIDFKE DGNILGHKLE YNYNSHNVYI MADKQKNGIK VNFKIRHNIE DGSVQLADHY 360 QQNTPIGDGP VLLPDNHYLS TQSALSKDPN EKRDHMVLLE FVTAAGITYI HALPPHPL 418
SEQ ID NO:63
atgagatctg gtattgtctt tttggttttg ttcttcttag gttgtgaaat ttcccaaggt 60 agacaattgt tggaatctag attgttcaga aagtctacta ttcaaaaggt tttgcaccac 120 gaattgccag tcgctgcttc tgctcaagaa gtttgggatg tctattcttc cccagaattg 180 ccaaaacact tgccagaaat tttgccaggt gccttcgaaa aggttgtcgt caccggtgat 240 ggtggtgttg gtactgtttt ggaaatggtt tttccaccag gtgaagttcc aagatcctac 300 aaagaaaagt tcgtcttaat tgacgatgag caattgttga agaaggttga aatgatcgaa 360 ggtggttact tggatatggg ttgtactttc tacatggaca ctatccaaat tgtcccaacc 420 ggtccagact cctgtatcat caagtcttct actgaatact atgtcaagcc tgaattcgct 480 gataaggttg ttccattgat ttctactatc ccattgcaag ctatggccga ggccatctct 540 aacattgtct tagctaacaa ggccaagaac aagtctatta ttatcgaaat tggcatgagt 600 aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga tggtgatgtt 660 aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata cggaaaactt 720 acccttaaat ttatttgcac tactggaaaa ctacctgttc catggccaac acttgtcact 780 actttcactt atggtgttca atgcatttca agatacccag atcatatgaa acggcatgac 840 tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt tttcaaagat 900 gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct tgttaataga 960 atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca caaattggaa 1020 tacaactata actcacacaa tgtatacatc atggcagaca aacaaaagaa tggaatcaaa 1080 gttaacttca aaattagaca caacattgaa gatggaagcg ttcaactagc agaccattat 1140 caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca ttacctgtcc 1200 acacaatctg ccctttcgaa agatcccaac gaaaagagag accacatggt ccttcttgag 1260 tttgtaacag ctgctgggat tacttacatt cacgccctcc ccccacatcc gctctaa 1317 SEQ ID NO:64
MRSGIVFLVL FFLGCEISQG RQLLESRLFR KSTIQKVLHH ELPVAASAQE VWDVYSSPEL 60 PKHLPE ILPG AFEKVWTGD GGVGTVLEMV FPPGEVPRSY KEKFVLIDDE QLLKKVEMIE 120 GGYLDMGCTF YMDTIQIVPT GPDSCIIKSS TEYYVKPEFA DKWPLISTI PLQAMAEAIS 180 NIVLANKAKN KSIIIEIGMS KGEELFTGW PILVELDGDV NGHKFSVSGE GEGDATYGKL 240 TLKFICTTGK LPVPWPTLVT TFTYGVQCIS RYPDHMKRHD FFKSAMPEGY VQERTIFFKD 300 DGNYKTRAEV KFEGDTLVNR IELKGIDFKE DGNILGHKLE YNYNSHNVYI MADKQKNGIK 360 VNFKIRHNIE DGSVQLADHY QQNTPIGDGP VLLPDNHYLS TQSALSKDPN EKRDHMVLLE 420 FVTAAGITYI HALPPHPL 438
SEQ ID NO:65
atgagacaat tgttggaatc tagattgttc agaaagtcta ctattcaaaa ggttttgcac 60 cacgaattgc cagtcgctgc ttctgctcaa gaagtttggg atgtctattc ttccccagaa 120 ttgccaaaac acttgccaga aattttgcca ggtgccttcg aaaaggttgt cgtcaccggt 180 gatggtggtg ttggtactgt tttggaaatg gtttttccac caggtgaagt tccaagatcc 240 tacaaagaaa agttcgtctt aattgacgat gagcaattgt tgaagaaggt tgaaatgatc 300 gaaggtggtt acttggatat gggttgtact ttctacatgg acactatcca aattgtccca 360 accggtccag actcctgtat catcaagtct tctactgaat actatgtcaa gcctgaattc 420 gctgataagg ttgttccatt gatttctact atcccattgc aagctatggc cgaggccatc 480 tctaacattg tcttagctaa caaggccaag aacaagtcta ttattatcga aattggcatg 540 agtaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat 600 gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa 660 cttaccctta aatttatttg cactactgga aaactacctg ttccatggcc aacacttgtc 720 actactttca cttatggtgt tcaatgcatt tcaagatacc cagatcatat gaaacggcat 780 gactttttca agagtgccat gcccgaaggt tatgtacagg aaagaactat atttttcaaa 840 gatgacggga actacaagac acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat 900 agaatcgagt taaaaggtat tgattttaaa gaagatggaa acattcttgg acacaaattg 960 gaatacaact ataactcaca caatgtatac atcatggcag acaaacaaaa gaatggaatc 1020 aaagttaact tcaaaattag acacaacatt gaagatggaa gcgttcaact agcagaccat 1080 tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa ccattacctg 1140 tccacacaat ctgccctttc gaaagatccc aacgaaaaga gagaccacat ggtccttctt 1200 gagtttgtaa cagctgctgg gattacttac attcacgccc tccccccaca tccgctctaa 1260
SEQ ID NO:66
MRQLLESRLF RKSTIQKVLH HELPVAASAQ EVWDVYSSPE LPKHLPE ILP GAFEKWVTG 60 DGGVGTVLEM VFPPGEVPRS YKEKFVLIDD EQLLKKVEMI EGGYLDMGCT FYMDTIQIVP 120 TGPDSCIIKS STEYYVKPEF ADKVVPLIST IPLQAMAEAI SNIVLANKAK NKSIIIEIGM 180 SKGEELFTGV VP ILVELDGD VNGHKFSVSG EGEGDATYGK LTLKFICTTG KLPVPWPTLV 240 TTFTYGVQCI SRYPDHMKRH DFFKSAMPEG YVQERTIFFK DDGNYKTRAE VKFEGDTLVN 300 RIELKGIDFK EDGNILGHKL EYNYNSHNVY IMADKQKNGI KVNFKIRHNI EDGSVQLADH 360 YQQNTP IGDG PVLLPDNHYL STQSALSKDP NEKRDHMVLL EFVTAAGITY IHALPPHPL 419
SEQ ID NO:67
Lactococcus lactis
atgaaaatcg tagttatcgg tacaaaccac gcaggcattg ctacagcgaa tacattactt 60 gaacaatatc ccgggcatga aattgtcatg attgaccgta atagcaacat gagttatcta 120 ggttgtggca cagcaatttg ggttggaaga caaattgaaa aaccagatga attattttat 180 gccaaagcag aggattttga ggcaaaaggg gtaaaaattt tgactgaaac agaagtttca 240 gaaattgatt ttgctaataa gaaagtttat gcaaaaacta aatctgatga tgaaataatt 300 gaagcttacg acaagcttgt tttagcaaca ggttcacgtc caattattcc taatctacca 360 ggcaaagacc ttaagggaat tcattttctg aaactttttc aagaaggtca agcaattgac 420 gcagaatttg ccaaagaaaa agtcaagcgt atcgcagtca ttggtgcagg atatatcggt 480 acagagattg cggaagcagc taaacgtcgg ggtaaagaag ttcttctctt tgacgctgaa 540 aatacttcac ttgcatcata ttatgatgaa gaatttgcca aaggaatgga tgaaaacctt 600 gctcaacatg gaattgaact tcattttgga gaactggcca aagaatttaa agcgaatgag 660 gaaggttatg tatcacaaat cgtaaccaac aaggcgactt atgatgttga tcttgtcatc 720 aattgtattg gttttactgc caacagtgcc ttggcaagtg ataagttagc taccttcaaa 780 aatggcgcaa tcaaggtgga taagcatcaa caaagtagtg atccagatgt ttacgcggta 840 ggtgatgttg cgacaattta ttctaatgcc ttgcaagatt ttacttatat cgctcttgcc 900 tcaaacgctg ttcggtcagg aattgtcgca ggacacaata ttggtggaaa agaattagaa 960 tctgttggtg ttcaaggttc taatggtatt tcgatttttg gttacaatat gacttctaca 1020 ggactttctg ttaaagctgc taaaaaatta ggtttagaag tttcatttag tgattttgaa 1080 gataaacaaa aagcttggtt tcttcatgaa aacaacgata gtgtgaaaat tcgtatcgta 1140 tatgagacaa aaagtcgcag aattattgga gcacaacttg ctagtaaaag tgagataatt 1200 gcaggaaata taaatatgtt cagtttagcg attcaagaga aaaaaacaat tgatgaacta 1260 gctttgcttg atttattctt tctcccccac ttcaacagtc catataatta tatgacagtt 1320 gcagctttga atgccaaata a 1341
SEQ ID NO:68
Lactococcus lactis
MKIWIGTNH AGIATANTLL EQYPGHEIVM IDRNSNMSYL GCGTAIWVGR QIEKPDELFY 60 AKAEDFEAKG VKILTETEVS EIDFANKKVY AKTKSDDEI I EAYDKLVLAT GSRPIIPNLP 120 GKDLKGIHFL KLFQEGQAID AEFAKEKVKR IAVIGAGYIG TEIAEAAKRR GKEVLLFDAE 180 NTSLASYYDE EFAKGMDENL AQHGIELHFG ELAKEFKANE EGYVSQIVTN KATYDVDLVI 240 NCIGFTANSA LASDKLATFK NGAIKVDKHQ QSSDPDVYAV GDVATIYSNA LQDFTYIALA 300 SNAVRSGIVA GHNIGGKELE SVGVQGSNGI SIFGYNMTST GLSVKAAKKL GLEVSFSDFE 360 DKQKAWFLHE NNDSVKIRIV YETKSRRI IG AQLASKSEI I AGNINMFSLA IQEKKTIDEL 420 ALLDLFFLPH FNSPYNYMTV AALNAK 446
SEQ ID NO:69
Saccharomyces cerevisiae
atgcccgagg ccaagcttaa caataacgtc aacgacgtta ctagctactc ctccgcgtct 60 tcttctactg aaaacgctgc tgatctacac aattataatg ggttcgatga gcatacagaa 120 gctcgaatcc aaaaactggc aaggactctg accgcacaga gtatgcaaaa ctccactcaa 180 tcggcaccca acaaaagtga tgctcagtct atattttcta gcggtgtgga aggtgtaaac 240 ccgatattct ctgatcctga agctccaggc tatgacccaa aattggaccc caactccgaa 300 aatttttcta gtgccgcctg ggttaagaat atggctcacc taagtgcggc agaccctgac 360 ttttataagc cttattcctt aggttgcgct tggaagaact taagtgcttc tggtgcttcc 420 gcagatgtcg cctatcagtc aactgtggtt aatattccat acaaaatcct aaaaagtggg 480 ctgagaaagt ttcaacgttc taaagaaacc aatactttcc aaatcttgaa accaatggat 540 ggttgcctaa acccaggtga attgctagtc gttttaggta gaccaggctc tggctgtact 600 actttattaa aatccatctc ttcaaatact catggttttg atcttggtgc agatactaaa 660 atttcttaca gcggctactc aggtgatgat attaagaaac attttcgtgg tgaagttgtt 720 tacaacgcag aagctgatgt acatctgcct catttaacag tcttcgaaac tttggttaca 780 gtagcgaggt tgaaaacccc acagaaccgt atcaagggtg tcgataggga aagttatgcg 840 aatcatttgg cggaagtagc aatggcaacg tacggtttat cgcatacaag gaatacaaaa 900 gttggtaacg acatcgtcag aggtgtttcc ggtggtgaaa ggaagcgtgt ctccattgct 960 gaagtctcca tctgtggatc caaatttcaa tgctgggata atgctacaag gggtttggat 1020 tccgctaccg ctttggaatt tattcgtgcc ttaaagactc aagctgatat ttccaataca 1080 tctgccacag tggccatcta tcaatgttct caagatgcgt acgacttgtt caataaagtc 1140 tgtgttttgg atgatggtta tcagatctac tatggccccg ccgataaggc caagaagtac 1200 tttgaagata tggggtatgt ttgtccaagc agacaaacca ccgcagattt tttgacctca 1260 gttacaagtc cctctgagag aaccctgaac aaagatatgc taaaaaaagg tattcatata 1320 ccacagaccc cgaaggaaat gaacgattac tgggtaaaat ctccaaatta caaagagcta 1380 atgaaagaag tcgaccaacg attattgaat gacgatgaag caagccgtga agctattaag 1440 gaagcccaca ttgctaagca gtccaagaga gcaagacctt cctctcctta tactgtcagc 1500 tacatgatgc aagttaaata cctattaatc agaaatatgt ggagactgcg aaataatatc 1560 gggtttacat tatttatgat tttgggtaac tgtagtatgg ctttaatctt gggttcaatg 1620 tttttcaaga tcatgaaaaa gggtgatact tctacattct atttccgtgg ttctgctatg 1680 ttttttgcaa ttctattcaa tgcattttct tctctgttag aaatcttttc gttatatgag 1740 gccagaccaa tcactgaaaa acatagaaca tattcgttat accatccaag tgctgacgct 1800 tttgcatcag ttctatcaga aataccctca aagttaatca tcgctgtttg cttcaatata 1860 atcttctatt tcttagtaga ctttagaaga aatggtggtg tattcttttt ctacttatta 1920 ataaacattg tcgcggtttt ctccatgtct cacttgttta gatgtgttgg ttccttaaca 1980 aagacattgt cagaagctat ggttcccgct tctatgttat tgttggctct atccatgtat 2040 accggttttg ctattcctaa gaagaagatc ctacgttggt ctaaatggat ttggtatatc 2100 aatccgttgg cttacttatt cgaatctttg ttaattaacg agtttcatgg tataaaattc 2160 ccctgcgctg aatatgttcc tcgtggtcct gcgtatgcaa acatttctag tacagaatct 2220 gtttgtaccg tggttggagc tgttccaggc caagactatg ttctgggtga tgatttcatt 2280 agaggaactt atcaatacta ccacaaagac aaatggcgtg gtttcggtat tggtatggct 2340 tatgtcgtct tctttttctt tgtctatcta ttcttatgtg aatacaacga gggtgctaaa 2400 caaaaaggtg aaatattagt tttcccacgc agtatagtta aaagaatgaa gaaaagaggt 2460 gtactaactg aaaagaatgc aaatgacccc gaaaacgttg gggaacgtag tgacttatcc 2520 agcgatagga aaatgctaca agaaagctct gaagaggaat ccgatactta cggagaaatt 2580 ggtttatcca agtcagaggc tatatttcac tggagaaacc tttgttacga agttcagatt 2640 aaggccgaaa caagacgtat tttgaacaat gttgatggtt gggttaaacc aggtacttta 2700 acagctttaa tgggtgcttc aggtgctggt aaaaccacac ttctggattg tttggccgaa 2760 agggttacca tgggtgttat aactggtgat atcttggtca atggtattcc ccgtgataaa 2820 tctttcccaa gatccattgg ttattgtcag caacaagatt tgcatttgaa aactgccact 2880 gtgagggagt cattgagatt ttctgcttac ctacgtcaac cagctgaagt ttccattgaa 2940 gaaaagaaca gatatgttga agaagttatt aaaattcttg aaatggaaaa atatgctgat 3000 gctgttgttg gtgttgctgg tgaaggttta aacgttgaac aaagaaaaag attaaccatt 3060 ggtgttgaat taactgccaa accaaaactg ttggtctttt tagatgaacc tacttctggt 3120 ttggattctc aaactgcttg gtctatttgt cagctaatga aaaagttggc aaatcatggt 3180 caagcaattc tatgtactat tcaccaaccc tctgctattt tgatgcaaga attcgatcgt 3240 ttactattta tgcaacgtgg tggtaagact gtctactttg gcgacttggg cgaaggttgt 3300 aaaactatga tcgattattt tgaaagccat ggtgctcata aatgccctgc tgacgccaac 3360 ccagctgaat ggatgctaga agttgttggt gcagctccag gctctcatgc aaatcaagat 3420 tattacgaag tttggaggaa ttctgaagag tacagggccg ttcaatctga attagattgg 3480 atggaaagag aattaccaaa gaaaggttcg ataactgcag ctgaggacaa acacgaattt 3540 tcacaatcaa ttatttatca aacaaaattg gtcagtattc gtctattcca gcaatattgg 3600 agatctccag attatttatg gtcgaagttt attttaacta ttttcaatca attgttcatc 3660 ggtttcactt tcttcaaagc aggaacctcg ctacagggtt tacaaaatca aatgttggct 3720 gtgttcatgt ttacggttat tttcaatcct attctacaac aatacctacc atcttttgtc 3780 cagcaaagag atttgtatga ggccagggaa cgcccctcaa ggactttttc ttggatttca 3840 tttatcttcg ctcaaatatt cgtggaagtt ccatggaata tattggcagg tactattgct 3900 tattttatct actattatcc aattggattt tactccaacg cgtctgcagc tggccagttg 3960 catgaaaggg gtgctttatt ttggttgttc tcttgtgctt tctacgttta tgttggttct 4020 atgggtctgc ttgtcatttc attcaaccaa gttgcagaaa gtgcagctaa cttagcctct 4080 ttgttgttta caatgtcttt gtctttttgt ggtgttatga ctaccccaag tgccatgcct 4140 agattttgga tattcatgta cagggtttca cctttgactt atttcattca ggctctgttg 4200 gctgttggtg ttgctaacgt agacgtcaaa tgcgctgatt acgaattgct agaattcaca 4260 ccaccatccg gtatgacatg tgggcagtac atggaaccat atttacaact agcaaagact 4320 ggttacttaa ctgatgaaaa tgccactgac acctgtagtt tctgtcaaat atctacaacc 4380 aatgattact tagctaatgt caattctttc tacagtgaga gatggagaaa ttatggtatc 4440 ttcatctgtt atattgcatt caattatatc gctggtgtct ttttctactg gttagcaaga 4500 gtgcctaaaa agaacggtaa actctccaag aaataa 4536
SEQ I D NO:70
Saccharomyces cerevisiae
MPEAKLNNNV NDVTSYSSAS SSTENAADLH NYNGFDEHTE ARIQKLARTL TAQSMQNSTQ 60 SAPNKSDAQS IFSSGVEGVN PIFSDPEAPG YDPKLDPNSE NFSSAAWVKN MAHLSAADPD 120 FYKPYSLGCA WKNLSASGAS ADVAYQSTW NIPYKILKSG LRKFQRSKET NTFQILKPMD 180 GCLNPGELLV VLGRPGSGCT TLLKSISSNT HGFDLGADTK ISYSGYSGDD IKKHFRGEW 240 YNAEADVHLP HLTVFETLVT VARLKTPQNR IKGVDRESYA NHLAEVAMAT YGLSHTRNTK 300 VGNDIVRGVS GGERKRVS IA EVSICGSKFQ CWDNATRGLD SATALEFIRA LKTQADISNT 360 SATVAIYQCS QDAYDLFNKV CVLDDGYQIY YGPADKAKKY FEDMGYVCPS RQTTADFLTS 420 VTSPSERTLN KDMLKKGIHI PQTPKEMNDY WVKSPNYKEL MKEVDQRLLN DDEASREAIK 480 EAHIAKQSKR ARPSSPYTVS YMMQVKYLLI RNMWRLRNNI GFTLFMILGN CSMALILGSM 540 FFKIMKKGDT STFYFRGSAM FFAILFNAFS SLLEIFSLYE ARPITEKHRT YSLYHPSADA 600 FASVLSEIPS KLIIAVCFNI IFYFLVDFRR NGGVFFFYLL INIVAVFSMS HLFRCVGSLT 660 KTLSEAMVPA SMLLLALSMY TGFAIPKKKI LRWSKWIWYI NPLAYLFESL LINEFHGIKF 720 PCAEYVPRGP AYANISSTES VCTVVGAVPG QDYVLGDDFI RGTYQYYHKD KWRGFGIGMA 780 YWFFFFVYL FLCEYNEGAK QKGEILVFPR SIVKRMKKRG VLTEKNANDP ENVGERSDLS 840 SDRKMLQESS EEESDTYGEI GLSKSEAIFH WRNLCYEVQI KAETRRILNN VDGWVKPGTL 900 TALMGASGAG KTTLLDCLAE RVTMGVITGD ILVNGIPRDK SFPRSIGYCQ QQDLHLKTAT 960 VRESLRFSAY LRQPAEVSIE EKNRYVEEVI KILEMEKYAD AWGVAGEGL NVEQRKRLTI 1020 GVELTAKPKL LVFLDEPTSG LDSQTAWSIC QLMKKLANHG QAILCTIHQP SAILMQEFDR 1080 LLFMQRGGKT VYFGDLGEGC KTMIDYFESH GAHKCPADAN PAEWMLEWG AAPGSHANQD 1140 YYEVWRNSEE YRAVQSELDW MERELPKKGS ITAAEDKHEF SQSI IYQTKL VSIRLFQQYW 1200 RSPDYLWSKF ILTIFNQLFI GFTFFKAGTS LQGLQNQMLA VFMFTVIFNP ILQQYLPSFV 1260 QQRDLYEARE RPSRTFSWIS FIFAQIFVEV PWNILAGTIA YFIYYYP IGF YSNASAAGQL 1320 HERGALFWLF SCAFYVYVGS MGLLVISFNQ VAESAANLAS LLFTMSLSFC GVMTTPSAMP 1380 RFWIFMYRVS PLTYF IQALL AVGVANVDVK CADYELLEFT PPSGMTCGQY MEPYLQLAKT 1440 GYLTDENATD TCSFCQISTT NDYLANVNSF YSERWRNYGI FICYIAFNYI AGVFFYWLAR 1500 VPKKNGKLSK K 1511
SEQ ID NO:71
Saccharomyces cerevisiae
atgtcttcga ctgacgaaca tattgagaaa gacatttcgt cgagatcgaa ccatgacgat 60 gattatgcta attcggtaca atcctacgct gcctccgaag gccaagttga taatgaggat 120 ttggcagcca cttctcagct atcccgtcac ctttcaaaca ttctttccaa tgaagaaggt 180 attgaaaggt tggagtctat ggcgagagtc atttcacata agacaaagaa ggaaatggac 240 tcttttgaaa ttaatgactt agattttgat ttgcgctcac tattacatta tttgaggtct 300 cgtcaattgg aacagggaat tgaacctggt gattctggta ttgcctttaa aaacctaaca 360 gcagtcggtg ttgatgcctc tgctgcatat gggcctagtg ttgaagagat gtttagaaat 420 attgctagta taccggcaca tctcataagt aaatttacca agaaatctga tgtcccatta 480 aggaatatta ttcaaaattg tacgggtgtc gttgaatctg gtgaaatgtt atttgtcgtc 540 ggtaggccag gtgcaggttg ctccactttc ctaaagtgtc tatctggtga aacttcagaa 600 ttagttgatg tacaaggtga attctcctat gatggtctgg accaaagcga aatgatgtct 660 aagtataaag gttacgttat ttactgtccc gagcttgatt tccatttccc aaaaattact 720 gtgaaggaaa caatcgattt tgccctaaaa tgtaagactc ctcgtgttag aattgacaaa 780 atgacgagaa agcaatacgt tgataacatc agagatatgt ggtgtaccgt ttttggttta 840 agacacacat atgccaccaa agtcggtaac gatttcgtaa gaggtgtttc tggtggtgaa 900 cgtaagcgtg tttccttggt tgaagctcag gcaatgaatg cctccatcta ctcttgggat 960 aacgccacaa gaggtttgga tgcctctact gctttagagt ttgcccaagc cattagaacg 1020 gctacaaata tggtaaacaa ctctgctatt gttgctattt accaagctgg tgaaaatatt 1080 tatgaattat ttgataaaac tactgttcta tataacggta gacagattta cttcggtcct 1140 gctgataaag ctgttggata tttccaaaga atgggttggg ttaaaccaaa cagaatgacc 1200 tctgcggaat ttttaacatc cgtcacggtc gattttgaaa ataggacatt ggatattaaa 1260 cctggctatg aagataaagt tccaaaatct agttcagagt ttgaggaata ctggttgaac 1320 tctgaggatt atcaggaact tttaagaact tatgatgatt atcaaagtag acaccctgtt 1380 aatgaaacga gagatagact ggatgtggcc aagaagcaaa gactgcaaca aggccaaaga 1440 gaaaattctc aatatgttgt caattattgg acacaagttt attattgtat gattcgtggt 1500 tttcaaaggg ttaagggtga ttcaacgtat actaaggtct acttaagttc ttttttgatc 1560 aaagctttga ttatcggttc tatgttccac aaaattgatg acaaaagtca atccaccacg 1620 gcaggtgctt attctcgtgg tggtatgtta ttctatgttt tattgttcgc ttctgttact 1680 tccttggccg aaattggtaa ctctttttct agtagacctg ttattgtcaa acacaaatca 1740 tattccatgt accatttgtc tgcggaatcg ttacaagaga ttatcactga gttccctact 1800 aaatttgtcg ctattgtgat actatgtttg attacttact ggattccatt tatgaaatat 1860 gaagctggtg ctttcttcca gtatatttta tatctactga ctgtgcaaca atgtacttct 1920 ttcattttca agtttgttgc tactatgagt aaatctggtg tggatgccca tgccgtcggt 1980 ggtttatggg tcctgatgct ttgtgtttat gctggttttg tcttgccaat tggtgaaatg 2040 catcattgga ttagatggct tcatttcatt aatcctttaa cttatgcttt tgaaagttta 2100 gtttccactg aatttcacca cagggaaatg ttgtgtagcg ccttagtccc atctggtcct 2160 ggttatgaag gtatttctat tgctaaccaa gtctgtgatg ctgctggtgc ggttaagggt 2220 aacttgtatg ttagcggtga ctcttacatc ttacaccaat atcatttcgc atataagcat 2280 gcttggagaa attggggtgt gaacattgtg tggacttttg gttatattgt gttcaatgtc 2340 atcttatcag aatatttgaa acctgttgag ggaggaggtg acttgctgtt atataagaga 2400 ggtcatatgc cggagttagg taccgaaaat gcagatgcaa gaaccgcttc cagagaggaa 2460 atgatggagg ctctgaatgg tccaaatgtc gatttagaaa aggtcattgc agaaaaggac 2520 gttttcacct ggaaccatct ggactacacc attccatacg acggagctac aagaaaatta 2580 ttatcggatg tctttggtta cgttaagcct ggtaagatga ccgccttgat gggtgaatcc 2640 ggtgctggta aaactacctt gttaaatgtt ttagcacaaa gaatcaatat gggtgtcatc 2700 actggtgata tgttagtcaa tgccaagccc ttgcctgctt ctttcaacag atcatgtggt 2760 tatgttgcgc aagccgataa tcatatggcc gaattatctg ttagggaatc cctgagattt 2820 gcagccgagt taagacagca aagttccgtt ccgttagagg agaaatatga atatgttgaa 2880 aaaattatca cattgctagg tatgcaaaat tacgctgaag ccttagttgg taagactggt 2940 agaggtttga acgttgaaca gagaaagaag ttatctattg gtgttgaact ggttgctaaa 3000 ccatcattat tattgttttt ggatgagcct acctctggtc tggactctca gtctgcttgg 3060 tcaattgttc aattcatgag agccttagct gattctggtc aatccatttt gtgtacgatt 3120 catcaaccct ctgctacctt gtttgaacag tttgacagat tgttgttgtt aaagaaaggt 3180 ggtaagatgg tttactttgg tgacattggt ccaaattctg aaactttgtt gaagtatttt 3240 gaacgtcaat ctggtatgaa gtgtggtgtt tctgaaaatc cagctgaata tattttgaat 3300 tgtattggtg ccggtgccac tgctagtgtt aactctgatt ggcacgactt atggcttgct 3360 tccccagaat gtgccgctgc aagggctgaa gttgaagaat tacatcgtac tttacctggt 3420 agagcagtta atgatgatcc tgagttagct acaagatttg ctgccagtta catgactcaa 3480 atcaaatgtg ttttacgtag aacagctctt caattttgga gatcgcctgt ctatatcagg 3540 gccaaattct ttgaatgtgt cgcatgtgct ttgttcgtcg gtttatcata tgttggtgta 3600 aatcactctg ttggtggtgc cattgaggcc ttttcgtcta ttttcatgct attattgatt 3660 gctctggcta tgatcaatca actgcacgtc ttcgcttatg atagtaggga attatatgag 3720 gttagagaag ccgcttctaa cactttccat tggagtgtct tgttattatg tcatgctgct 3780 gttgaaaact tttggtccac actttgtcag tttatgtgtt tcatttgcta ctactggcca 3840 gctcaattca gtggacgtgc atctcatgca ggtttcttct tcttcttcta tgttttaatt 3900 ttcccattat attttgtcac atatggtcta tggatcctgt acatgtctcc tgatgttccc 3960 tcagcttcta tgattaattc caatttgttt gctgctatgt tactgttctg tggtatttta 4020 caaccaagag agaaaatgcc tgccttctgg agaagattga tgtataatgt atcaccattt 4080 acctacgtgg ttcaagcttt ggttacacca ttagttcaca ataaaaaggt cgtttgtaat 4140 cctcatgaat acaacatcat ggacccacca agcggaaaaa cttgtggtga gtttttatct 4200 acctatatgg acaataatac cggttatttg gtaaatccaa ctgccaccga aaactgtcaa 4260 tattgcccat acactgttca agatcaagtt gtggctaaat acaatgtcaa atgggatcac 4320 agatggagaa actttggttt catgtgggct tatatttgct tcaatattgc cgctatgttg 4380 atttgttact atgttgtaag agttaaggtg tggtctttga agtctgtttt gaatttcaag 4440 aaatggttta atgggccaag aaaggaaaga catgaaaaag ataccaacat tttccaaaca 4500 gttccaggtg acgaaaataa aatcacgaag aaataa 4536
SEQ ID NO:72
Saccharomyces cerevisiae
MSSTDEHIEK DISSRSNHDD DYANSVQSYA ASEGQVDNED LAATSQLSRH LSNILSNEEG 60 IERLESMARV ISHKTKKEMD SFEINDLDFD LRSLLHYLRS RQLEQGIEPG DSGIAFKNLT 120 AVGVDASAAY GP SVEEMFRN IASIPAHLIS KFTKKSDVPL RNIIQNCTGV VESGEMLFW 180 GRPGAGCSTF LKCLSGETSE LVDVQGEFSY DGLDQSEMMS KYKGYVIYCP ELDFHFPKIT 240 VKETIDFALK CKTPRVRIDK MTRKQYVDNI RDMWCTVFGL RHTYATKVGN DFVRGVSGGE 300 RKRVSLVEAQ AMNASIYSWD NATRGLDAST ALEFAQAIRT ATNMVNNSAI VAIYQAGENI 360 YELFDKTTVL YNGRQIYFGP ADKAVGYFQR MGWVKPNRMT SAEFLTSVTV DFENRTLDIK 420 PGYEDKVPKS SSEFEEYWLN SEDYQELLRT YDDYQSRHPV NETRDRLDVA KKQRLQQGQR 480 ENSQYVVNYW TQVYYCMIRG FQRVKGDSTY TKVYLSSFLI KALI IGSMFH KIDDKSQSTT 540 AGAYSRGGML FYVLLFASVT SLAE IGNSFS SRPVIVKHKS YSMYHLSAES LQEIIpTEFPT 600
KFVAIVILCL ITYWIPFMKY EAGAFFQYIL YLLTVQQCTS F IFKFVATMS KSGVDAHAVG 660
GLWVLMLCVY AGFVLP IGEM HHWIRWLHFI NPLTYAFESL VSpTEFHHREM LCSALVPSGP 720
GYEGISIANQ VCDAAGAVKG NLYVSGDSYI LHQYHFAYKH AWRNWGVNIV WTFGYIVFNV 780
ILSEYLKPVE GGGDLLLYKR GHMPELGTEN ADARTASREE MMEALNGPNV DLEKVIAEKD 840
VFTWNHLDYT IPYDGATRKL LSDVFGYVKP GKMTALMGES GAGKTTLLNV LAQRINMGVI 900
TGDMLVNAKP LPASFNRSCG YVAQADNHMA ELSVRESLRF AAELRQQSSV PLEEKYEYVE 960
KIITLLGMQN YAEALVGKTG RGLNVEQRKK LS IGVELVAK P SLLLFLDEP TSGLDSQSAW 1020
SIVQFMRALA DSGQSILCTI HQPSATLFEQ FDRLLLLKKG GKMVYFGDIG PNSETLLKYF 1080
ERQSGMKCGV SENPAEYILN CIGAGATASV NSDWHDLWLA SPECAAARAE VEELHRTLPG 1140
RAVNDDPELA TRFAASYMTQ IKCVLRRTAL QFWRSPVYIR AKFFECVACA LFVGLSYVGV 1200
NHSVGGAIEA FSSIFMLLLI ALAMINQLHV FAYDSRELYE VREAASNTFH WSVLLLCHAA 1260
VENFWSTLCQ FMCFICYYWP AQFSGRASHA GFFFFFYVLI FPLYFVTYGL WILYMSPDVP 1320
SASMINSNLF AAMLLFCGIL QPREKMPAFW RRLMYNVSPF TYWQALVTP LVHNKKVVCN 1380
PHEYNIMDPP SGKTCGEFLS TYMDNNTGYL VNPTATENCQ YCPYTVQDQV VAKYNVKWDH 1440
RWRNFGFMWA YICFNIAAML ICYYWRVKV WSLKSVLNFK KWFNGPRKER HEKDTNIFQT 1500
VPGDENKITK K 1511
SEQ I D NO:73
Saccharomyces cerevisiae
atgtcatcag atatcagaga cgtagaggaa cgaaattcgc ggagctcgag ctcaagctcg 60 agctcgaact ctgccgccca atccattgga cagcatccat accgcggttt cgacagcgaa 120 gccgcggaaa gggtgcatga gttggctaga acactcacat cgcagagttt actatacact 180 gctaactcaa acaatagctc ttccagcaac cataatgcgc acaatgcgga ctcgagatcc 240 gtattttcta cggacatgga aggtgtgaac ccggtgttca ctaacccgga caccccggga 300 tacaatccca aattggaccc taacagtgat caattctcca gtacggcgtg ggtacagaac 360 atggcaaata tttgtacttc ggacccggat ttctataaac catactcact cggttgtgta 420 tggaagaatc taagcgcctc cggagactcc gccgatgtgt cataccagtc aactttcgct 480 aacatcgtac caaagctgct tacgaaaggg ctcagattac tgaagcccag taaagaagag 540 gacacttttc agatcctgaa acccatggat ggttgtctta atcccggtga actgttggtt 600 gttcttggga gaccagggtc aggttgtact acgctgctaa aatccatatc ttctaattcg 660 cacgggttca agatcgcaaa agactccatt gtctcttaca acggtttgtc aagctcggat 720 atcaggaaac attaccgtgg tgaagtcgtt tacaatgcgg aatcagatat tcatttaccg 780 catcttaccg tgtaccagac gcttttcacc gtggcaagga tgaaaacgcc gcaaaatcgt 840 atcaagggtg ttgatagaga agcgtacgcc aatcacgtga cagaagttgc aatggccaca 900 tatggtcttt cgcatacaag ggacaccaag gtcggaaacg atctagtcag aggtgtttct 960 ggtggtgaaa gaaagcgtgt atccattgcg gaagtcgcaa tctgcggcgc cagatttcaa 1020 tgttgggata atgccaccag aggtttggac tctgctactg ctttagaatt catccgtgct 1080 ttaaaaaccc aggctgacat tggaaagacc gcggctactg tggccatcta tcaatgttct 1140 caggatgctt atgatctttt tgataaggtc tgtgtcctgg atgatggtta ccagctttat 1200 tttgggcccg ctaaggatgc aaagaaatat ttccaagaca tgggctacta ttgtcctccc 1260 agacagacca ctgcagattt tttaacttca attacaagtc ctactgaaag aattattagc 1320 aaagaattta tcgaaaaagg tactagagtg cctcaaacgc caaaggatat ggccgaatat 1380 tggctacaat cagaaagcta caaaaattta atcaaggata tagactctac attagagaaa 1440 aacacagatg aagcacgcaa tattatcagg gatgctcacc acgctaaaca ggcaaaaagg 1500 gcaccacctt cctctccata cgttgtcaac tacggaatgc aagtcaaata cttgttgatt 1560 agaaatttct ggagaatgaa gcaaagtgct agtgttactt tgtggcaagt catcggtaac 1620 tctgtcatgg ctttcatctt gggttctatg ttttataaag tgatgaagaa aaacgatact 1680 tccactttct atttccgtgg tgctgcaatg ttttttgcta ttttatttaa tgcattttcg 1740 tgtcttttgg aaatctttag tttgtatgaa acaagaccta taactgaaaa acacagaact 1800 tattccttgt atcatccaag cgctgacgca tttgcgtctg ttttgtcaga aatgccgcca 1860 aaattgatca ctgctgtctg cttcaacatc atcttttatt tcctagttga tttcaggaga 1920 aacggtggtg tctttttctt ttatttttta attaatgtca ttgccacatt cactttatcc 1980 catttattta gatgcgtggg ctccttgacc aaaacattac aggaggccat ggtccccgct 2040 tcaatgttat tattggcaat ttctatgtat acagggtttg ccatccctaa aacgaagatt 2100 ttaggttggt ccatttggat ttggtatatc aacccgctag cctacctgtt tgaatcttta 2160 atgatcaatg aattccatga ccgtagattc ccttgtgccc aatacatccc tgctggccct 2220 gcttatcaga acatcacagg tactcaacgc gtctgttccg cagttggtgc ttatccaggt 2280 aacgactatg tgttgggtga tgacttcttg aaggaaagtt acgattatga gcacaaacat 2340 aagtggcgtg ggttcggtat tggtatggca tatgttgttt tctttttctt tgtttatcta 2400 attctttgtg agtataatga aggtgctaaa caaaagggtg aaatggttgt gttcctaaga 2460 tctaagatca agcaattgaa aaaggaaggt aaattacaag aaaagcatag gcctggagat 2520 attgaaaata atgcaggcag ttctccagat tccgctacaa cagaaaaaaa aatactagat 2580 gatagttctg agggatcgga tagctcttca gataatgccg gattaggact ttccaaatct 2640 gaagcaattt tccactggcg tgatttatgc tatgatgttc ctataaaagg aggtcaaaga 2700 cgtatcttaa ataacgtaga tggttgggta aaaccaggca ctttgactgc cttaatgggg 2760 gcgtcaggtg caggtaaaac aactttactg gattgtttgg ctgaaagagt caccatgggt 2820 gttattactg gtaatatttt tgtcgatggt cgtctccgtg acgaatcatt ccctagatct 2880 attggttatt gtcaacaaca ggatttacat ttgaaaacgg ctacagtaag agaatctttg 2940 agattttcag cttatttacg tcagccttcg tcagtttcta ttgaagaaaa aaataggtac 3000 gtggaagaag ttatcaaaat tttggaaatg caacaatatt cagatgctgt tgttggtgtt 3060 gcaggtgaag gtctaaatgt cgaacaaaga aaaagactta ctattggtgt tgaactagcg 3120 gcaaggccta aacttttggt ttttttggat gaaccaactt caggcctgga ttcacaaact 3180 gcttgggaca cttgccaact tatgaggaaa ctagctaccc acggtcaagc aattttgtgt 3240 actatccatc aaccttctgc tatattaatg cagcagtttg atagattatt atttttacag 3300 aaagggggcc aaactgtata tttcggcgat ttaggtgaag ggtgcaaaac tatgatcgat 3360 tattttgaaa gtaaaggagc tcacaaatgt ccacctgatg caaaccctgc cgaatggatg 3420 ttagaggttg taggtgccgc tcctggttct cacgctacgc aagattataa tgaagtctgg 3480 agaaactcag atgaatacaa agctgttcag gaagaattgg attggatgga aaagaattta 3540 ccaggcaggt caaaagaacc aactgcagaa gaacataaac cttttgctgc atctttatac 3600 taccaattta aaatggttac cattcgtttg ttccaacaat actggagatc accagattat 3660 ttatggtcga aatttatttt gactattttc aaccaagttt ttattgggtt cacttttttc 3720 aaggctgaca gaagtttaca gggactacaa aaccaaatgt tatcaatatt catgtatacg 3780 gttattttca atcctatact acaacagtat ctaccatctt tcgtgcagca aagggatttg 3840 tatgaggcaa gagaacgtcc ttcaagaaca ttttcgtggc ttgcgttttt cctctctcaa 3900 atcattgttg aaatcccatg gaatatttta gcgggtacga ttgcttattg tatttactat 3960 tacgcggttg gattttatgc gaacgcctca gctgctggtc aactccatga gagaggtgct 4020 ttgttttggt tattttctat tgccttctac gtctacattg gttcgatggg tttgttaatg 4080 atttctttca atgaagttgc tgaaacagcg gcacatatgg gaacgctatt gttcacgatg 4140 gcattatctt tctgtggtgt tatggctacc cctaaggtta tgccaagatt ttggatcttt 4200 atgtatagag tgtcacccct aacttatatg atcgatgcat tattagccct tggtgtggct 4260 aatgtggacg ttaagtgttc aaattatgaa atggtaaaat ttactccacc atctggaacc 4320 acctgcggtg actatatggc atcatatatc aagttggccg gaacaggcta cttgagtgac 4380 ccgtctgcaa cagatatatg tagtttctgt gcggtatcca ccaccaatgc ctttttggcc 4440 actttcagtt ctcattatta cagaagatgg agaaattacg gtatttttat ctgctatatt 4500 gcttttgatt atatcgctgc aacattcttg tattggttat ccagggtacc caagaagaac 4560 ggtaagattt ccgaaaaacc caagaagtga 4590
SEQ ID NO:74
Saccharomyces cerevisiae
MSSDIRDVEE RNSRSSSSSS SSNSAAQSIG QHPYRGFDSE AAERVHELAR TLTSQSLLYT 60 ANSNNSSSSN HNAHNADSRS VFSTDMEGVN PVFTNPDTPG YNPKLDPNSD QFSSTAWVQN 120 MANICTSDPD FYKPYSLGCV WKNLSASGDS ADVSYQSTFA NIVPKLLTKG LRLLKPSKEE 180 DTFQILKPMD GCLNPGELLV VLGRPGSGCT TLLKSISSNS HGFKIAKDSI VSYNGLSSSD 240 IRKHYRGEW YNAESDIHLP HLTVYQTLFT VARMKTPQNR IKGVDREAYA NHVTEVAMAT 300 YGLSHTRDTK VGNDLVRGVS GGERKRVSIA EVAICGARFQ CWDNATRGLD SATALEFIRA 360 LKTQAD IGKT AATVAIYQCS QDAYDLFDKV CVLDDGYQLY FGPAKDAKKY FQDMGYYCPP 420 RQTTADFLTS ITSPTERIIS KEFIEKGTRV PQTPKDMAEY WLQSESYKNL IKD IDSTLEK 480 NTDEARNI IR DAHHAKQAKR APPSSPYWN YGMQVKYLLI RNFWRMKQSA SVTLWQVIGN 540 SVMAFILGSM FYKVMKKNDT STFYFRGAAM FFAILFNAFS CLLE IFSLYE TRP ITEKHRT 600 YSLYHPSADA FASVLSEMPP KLITAVCFNI IFYFLVDFRR NGGVFFFYFL INVIATFTLS 660 HLFRCVGSLT KTLQEAMVPA SMLLLAI SMY TGFAIPKTKI LGWSIWIWYI NPLAYLFESL 720 MINEFHDRRF PCAQYIPAGP AYQNITGTQR VCSAVGAYPG NDYVLGDDFL KESYDYEHKH 780 KWRGFGIGMA YVVFFFFVYL ILCEYNEGAK QKGEMWFLR SKIKQLKKEG KLQEKHRPGD 840 IENNAGSSPD SATTEKKILD DSSEGSDSSS DNAGLGLSKS EAIFHWRDLC YDVPIKGGQR 900 RILNNVDGWV KPGTLTALMG ASGAGKTTLL DCLAERVTMG VITGNIFVDG RLRDESFPRS 960 IGYCQQQDLH LKTATVRESL RFSAYLRQPS SVSIEEKNRY VEEVIKILEM QQYSDAVVGV 1020 AGEGLNVEQR KRLTIGVELA ARPKLLVFLD EPTSGLDSQT AWDTCQLMRK LATHGQAILC 1080 TIHQPSAILM QQFDRLLFLQ KGGQTVYFGD LGEGCKTMID YFESKGAHKC PPDANPAEWM 1140 LEVVGAAPGS HATQDYNEVW RNSDEYKAVQ EELDWMEKNL PGRSKEPTAE EHKPFAASLY 1200 YQFKMVTIRL FQQYWRSPDY LWSKFILTIF NQVFIGFTFF KADRSLQGLQ NQMLSIFMYT 1260 VIFNPILQQY LPSFVQQRDL YEARERP SRT FSWLAFFLSQ IIVEIPWNIL AGTIAYCIYY 1320 YAVGFYANAS AAGQLHERGA LFWLFSIAFY VYIGSMGLLM I SFNEVAETA AHMGTLLFTM 1380 ALSFCGVMAT PKVMPRFWIF MYRVSPLTYM IDALLALGVA NVDVKCSNYE MVKFTPP SGT 1440 TCGDYMASYI KLAGTGYLSD PSATDICSFC AVSTTNAFLA TFSSHYYRRW RNYGIFICYI 1500 AFDYIAATFL YWLSRVPKKN GKISEKPKK 1529
SEQ I D NO:75
Saccharomyces cerevisiae
atgagcaata tcaaaagcac gcaagatagc tctcataatg ctgtcgctag aagctcaagc 60 gcttcttttg cagcttcaga agaatcattt acgggcataa cccatgacaa agatgagcag 120 agcgataccc cggcggataa actaacaaaa atgctgacag gacctgcaag agacactgcg 180 agccagatta gtgccactgt gtctgaaatg gcgccagatg tcgtatctaa agtggagtca 240 tttgcagatg cactatcccg tcatacaacg agaagcggtg cctttaatat ggattcagat 300 agtgacgatg ggttcgatgc ccatgccatc tttgaaagtt ttgtaagaga cgctgatgag 360 caaggcatcc atatccgcaa ggctggtgtt accatagagg acgtaagcgc taaaggtgtg 420 gatgcgagtg ccctagaagg tgctaccttt ggtaacattc tttgtttacc gttgaccatc 480 tttaaaggta ttaaggctaa gaggcatcaa aagatgagac agatcataag caatgtcaat 540 gccctggcag aagcgggtga aatgattttg gttcttggaa ggcctggtgc tggttgttcc 600 tcctttttaa aagtaacagc tggtgaaata gatcagtttg ccggtggtgt ttccggtgaa 660 gtagcatatg atggtattcc ccaagaagaa atgatgaaac gatataaagc agatgttatt 720 tacaatggtg agttggatgt tcatttccct tatttaacag ttaagcaaac tttggatttc 780 gctattgcct gcaaaacgcc tgctctcaga gtcaataacg tttccaaaaa ggaatacatt 840 gcatccagaa gagatttata tgcaaccatt ttcggtctaa ggcataccta taataccaaa 900 gttggtaacg atttcgttag aggtgtatct ggtggtgaac gtaagcgtgt ttccattgcc 960 gaggctttgg cagccaaagg ttccatttac tgttgggata atgccactag aggtttggat 1020 gcgtctacgg ccttagaata cgcaaaagcc atccgtatta tgacaaactt attgaaatca 1080 accgcttttg ttacaattta tcaggcaagt gaaaacattt acgaaacatt tgataaagtc 1140 actgtccttt attctggtaa gcaaatttat tttggtttga tccacgaggc aaaaccttat 1200 ttcgcaaaaa tgggttattt gtgtcctcca aggcaagcaa cagctgaatt tttaaccgcg 1260 ttgactgatc caaatggatt ccatctgatc aagccaggtt atgaaaataa agtaccaaga 1320 accgctgagg aattcgaaac atattggtta aattctccag agtttgctca aatgaaaaaa 1380 gatatcgctg cttataaaga gaaggtcaat accgaaaaga ctaaagaagt ttatgacgaa 1440 tcgatggctc aagagaaatc caaatatacg agaaagaagt cttattatac agtgtcatat 1500 tgggaacaag ttaaactgtg tacccaacgt gggttccaaa gaatttacgg taacaagagt 1560 tatacagtca tcaatgtctg ctctgcaata attcaatctt ttattactgg atcattattt 1620 tacaataccc cttcatccac ttccggtgct ttttcaagag gtggtgtgtt gtattttgcg 1680 ctactatatt attctttgat gggactggcg aatatttctt ttgaacatag gccaatctta 1740 caaaagcaca agggctattc tttgtatcat ccttcagctg aggcaattgg ctccactctg 1800 gcatctttcc ccttcagaat gattggtttg acctgtttct ttatcatttt attcttccta 1860 tctgggttgc acagaacagc gggatcattt tttaccatct atttgttctt aaccatgtgt 1920 tcagaggcga tcaatggttt atttgagatg gtttcttcag tatgtgacac tctttctcaa 1980 gctaactcta tctcgggtat tctgatgatg tctatctcaa tgtactctac ctatatgatc 2040 caattgcctt cgatgcatcc atggtttaaa tggatatcgt acgtactacc tatcaggtac 2100 gccttcgagt cgatgttaaa tgccgaattt cacggtaggc atatggattg tgctaacact 2160 ctagtaccca gtggaggaga ctatgataat ttatccgatg actacaaagt atgtgctttt 2220 gttggttcga aaccaggtca gtcttatgtg cttggtgatg actaccttaa aaatcaattt 2280 cagtacgttt ataagcacac gtggagaaac tttggtatct tgtggtgctt tttactgggt 2340 tatgttgttt tgaaagtgat attcacagaa tataagaggc ctgtgaaagg tggtggtgat 2400 gctcttatct tcaagaaagg atcaaaaaga tttatcgcac atgcagatga agaatctcca 2460 gacaatgtca atgatataga tgccaaagag caattctcca gtgaaagtag cggcgcaaat 2520 gatgaagtat ttgatgattt agaagccaaa ggtgttttca tttggaagga cgtatgcttt 2580 actattccat atgaaggcgg taagagaatg cttttggata atgtttcagg ttattgtatt 2640 ccaggtacca tgacggcctt gatgggagag tcaggtgctg gtaaaacaac tttgttaaat 2700 actcttgctc aaagaaatgt cggtatcatt actggtgata tgcttgtcaa tggacgtccc 2760 attgatgcga gtttcgaaag gcgtacaggt tatgtacaac aacaggatat acatatcgca 2820 gagttaactg ttagggaatc gttgcagttt tctgctcgta tgcgtcgccc tcagcatttg 2880 cctgattctg aaaaaatgga ttatgtggaa aaaatcatca gagttttggg aatggaagag 2940 tatgcggaag cccttgttgg tgaggttggt tgtggtttaa acgttgaaca gagaaagaag 3000 ctgtctattg gtgttgaact agtcgccaaa ccagacttat tattattcct cgatgaacct 3060 acatcaggtt tggattctca atcttcatgg gccattattc aattattaag aaagttatca 3120 aaagctggcc aatccattct ttgtacgatc catcaacctt cagctactct gttcgaagag 3180 tttgatagat tactactttt gaggaagggt ggacaaactg tttatttcgg agatattggt 3240 aagaactctg ccaccatttt gaactacttt gaaaggaatg gggcaagaaa atgtgattct 3300 agtgaaaatc ctgctgaata tattttagag gctattggtg ccggtgccac agcatccgtc 3360 aaagaagact ggcacgaaaa atggttgaac tctgtcgagt ttgaacaaac aaaagaaaaa 3420 gtacaggatt taataaatga tttatcgaaa caagaaacta aatccgaagt tggagacaaa 3480 ccttccaaat atgctacttc ttatgcttac cagttcagat atgttttaat cagaacctct 3540 acttcatttt ggagaagtct gaattacatc atgtcaaaga tgatgctaat gctggttggt 3600 ggtctgtata ttggtttcac atttttcaat gttggtaaaa gttatgtcgg cttacaaaat 3660 gcgatgttcg cggcatttat ctctattatc ttgtctgctc ctgcaatgaa ccaaatccaa 3720 ggacgtgcta ttgcctccag agaacttttt gaagttaggg aatcccaatc taacatgttt 3780 cactggtcgc tggtgttgat cactcagtac ttgagcgaac ttccctatca tttatttttt 3840 tcgacaattt tctttgtctc atcgtatttt ccattaagaa tcttcttcga agcgtcaaga 3900 tctgcggtgt actttttgaa ttactgcatt atgttccagt tatactatgt tggtcttggc 3960 ttaatgatcc tatatatgtc accgaacctt ccatccgcta atgttatctt aggtttgtgt 4020 ctgtcattta tgctttcttt ctgtggtgtt acacaacctg tctcattgat gcctggcttc 4080 tggacattca tgtggaaggc ttccccatac acatattttg ttcagaatct ggtcggaatt 4140 atgctgcaca aaaaaccagt cgtatgcaaa aagaaagaac taaactactt caacccacca 4200 aacggctcaa cgtgtggaga gtacatgaaa ccctttttgg aaaaagctac tggttacatc 4260 gaaaatcctg atgctacgtc agattgtgca tactgtattt acgaagttgg agataattat 4320 ttgacacata tcagctctaa gtatagctac ttgtggagaa attttggaat attttggatt 4380 tacattttct tcaatatcat tgctatggtt tgtgtgtatt acctcttcca tgtaagacaa 4440 tcttccttcc taagccccgt atctatactc aataaaatta aaaacataag gaaaaagaag 4500 cagtaa 4506
SEQ I D NO:76
Saccharomyces cerevisiae
MSNIKSTQDS SHNAVARSSS ASFAASEESF TGITHDKDEQ SDTPADKLTK MLTGPARDTA 60 SQI SATVSEM APDWSKVES FADALSRHTT RSGAFNMDSD SDDGFDAHAI FESFVRDADE 120 QGIHIRKAGV TIEDVSAKGV DASALEGATF GNILCLPLTI FKGIKAKRHQ KMRQIISNVN 180 ALAEAGEMIL VLGRPGAGCS SFLKVTAGEI DQFAGGVSGE VAYDGIPQEE MMKRYKADVI 240 YNGELDVHFP YLTVKQTLDF AIACKTPALR VNNVSKKEYI ASRRDLYATI FGLRHTYNTK 300 VGNDFVRGVS GGERKRVS IA EALAAKGSIY CWDNATRGLD ASTALEYAKA IRIMTNLLKS 360 TAFVTIYQAS ENIYETFDKV TVLYSGKQIY FGLIHEAKPY FAKMGYLCPP RQATAEFLTA 420 LTDPNGFHLI KPGYENKVPR TAEEFETYWL NSPEFAQMKK DIAAYKEKVN TEKTKEVYDE 480 SMAQEKSKYT RKKSYYTVSY WEQVKLCTQR GFQRIYGNKS YTVINVCSAI IQSFITGSLF 540 YNTPSSTSGA FSRGGVLYFA LLYYSLMGLA NISFEHRPIL QKHKGYSLYH PSAEAIGSTL 600 ASFPFRMIGL TCFFI ILFFL SGLHRTAGSF FTIYLFLTMC SEAINGLFEM VSSVCDTLSQ 660 ANSISGILMM SISMYSTYMI QLPSMHPWFK WISYVLPIRY AFESMLNAEF HGRHMDCANT 720 LVP SGGDYDN LSDDYKVCAF VGSKPGQSYV LGDDYLKNQF QYVYKHTWRN FGILWCFLLG 780 YWLKVIFTE YKRPVKGGGD ALIFKKGSKR FIAHADEESP DNVNDIDAKE QFSSESSGAN 840 DEVFDDLEAK GVFIWKDVCF TIPYEGGKRM LLDNVSGYCI PGTMTALMGE SGAGKTTLLN 900 TLAQRNVGII TGDMLVNGRP IDASFERRTG YVQQQDIHIA ELTVRESLQF SARMRRPQHL 960 PDSEKMDYVE KIIRVLGMEE YAEALVGEVG CGLNVEQRKK LSIGVELVAK PDLLLFLDEP 1020 TSGLDSQSSW AIIQLLRKLS KAGQSILCTI HQPSATLFEE FDRLLLLRKG GQTVYFGDIG 1080 KNSATILNYF ERNGARKCDS SENPAEYILE AIGAGATASV KEDWHEKWLN SVEFEQTKEK 1140 VQDLINDLSK QETKSEVGDK PSKYATSYAY QFRYVLIRTS TSFWRSLNYI MSKMMLMLVG 1200 GLYIGFTFFN VGKSYVGLQN AMFAAFI SI I LSAPAMNQIQ GRAIASRELF EVRESQSNMF 1260 HWSLVLITQY LSELPYHLFF STIFFVSSYF PLRIFFEASR SAVYFLNYCI MFQLYYVGLG 1320 LMILYMSPNL PSANVILGLC LSFMLSFCGV TQPVSLMPGF WTFMWKASPY TYFVQNLVGI 1380 MLHKKPWCK KKELNYFNPP NGSTCGEYMK PFLEKATGYI ENPDATSDCA YCIYEVGDNY 1440 LTHISSKYSY LWRNFGIFWI YIFFNI IAMV CVYYLFHVRQ SSFLSPVSIL NKIKNIRKKK 1500 Q 1501
SEQ ID NO:77
Saccharomyces cerevisiae
atgtcaattt caaagtactt cactcccgtt gctgacgggt cactcacttt caatggcgcg 60 aacattcaat ttggcgccga tgctcaaggc gagtcaaaaa agagttatga cgctgaggac 120 agcatgccga atccggcaaa tcaactaaat gacataacct tccaagcaga ggctggtgaa 180 atggttttgg ttttgggtta tcccacatcc actctattta agactttgtt tcatggtaaa 240 actagtttgt catactctcc tccaggctcg attaaattta aaaataatga gtttaagagc 300 ttttccgaaa aatgtcccca ccaaatcatt tacaataatg aacaagatgt gcatttccca 360 tttctaacgg tagaacaaac aattgatttt gccttgagtt gtaagttcga tattccaaaa 420 ggtgagcgcg atcaaataag aaatgaactt ctaagagaat tcggcctgtc tcatgtattg 480 aaaactattg taggaaatga ttttttccgt ggtgtttctg gtggtgagcg taaacgtatt 540 tctattattg aaacgtttat tgctaatggt tccgtttatc tatgggataa ttctactaaa 600 ggtttagatt ccgccacagc tctcgatttt ttggaaattc ttagaaaaat ggcaaaagct 660 actcgttctg taaacttagt cagaatttcc caggcaagtg ataaaattgt tgataagttt 720 gacaagattc ttatgctatc cgattcctac cagcttttct atggtacggt cgatgagtgt 780 ttgacttatt ttcgtgacac tttaggtatt gagaaagatc ctaacgattg tattattgaa 840 tatctgacct ctatcttaaa ttttcagttc aaaaataaaa atttggggaa tttatcaaat 900 tcatcatctg ctagcgttct caaaaccgca acaggggaag tcactaagta tacttataat 960 tctgatttcg atttatatga tcaatggaaa cattcttcgt actatagaaa tataaagcag 1020 caaatccagg ggtcttcaat tgatgactcc attaaggaag tggatccctc tgatgtctca 1080 cctattttta atattccgtt gaagaaacaa ttattatttt gcacaaaaag agcttttcaa 1140 cgaagtttgg gtgataaagc ttatatgacg gcacaattta tttctgtcgt tattcaatct 1200 ttggtcattg gttcactgtt ttacgaaatt ccgttgacta ccattggttc atactcaaga 1260 ggttctttaa cctttttctc cattcttttc ttcacttttc tttctcttgc agatatgcct 1320 attgccttcc aaagacaacc cgtcgttaaa aagcaatccc aacttcactt ctatactaac 1380 tgggttgaaa ccctttcaac aacagtgttc gactactgct ttaaactttg tttggtaatt 1440 gtattcagta tcatcctata cttccttgct cacctgcaat acaaggctgc aagatttttc 1500 attttcctcc tgttcctttc cttttacaat ttctgtatgg tgtccttatt cgctttgacg 1560 acactagttg ctccaaccat ttcagttgca aatttatttg caggtattct gctactagct 1620 atagcaatgt atgcttctta cgtcatctac ctgaaaaata tgcatccttg gtttgtatgg 1680 attgcttatc taaatcctgc aatgtacgct atggaggcaa tactgtctaa tgagctttac 1740 aatttgaagc tggattgtag tgagacaatt gttccaagag gtcctactta caacgatgtt 1800 ccattttccc ataaggcctg tgcttggcaa ggtgctactc tgggtaatga ttacgttaga 1860 ggccgtgatt acttgaagca aggtttatcc tacacgtatc atcatgtgtg gagaaacttt 1920 ggtattatca ttggtttcct agtattcttt attgcctgta ctctgtttgc atctcaatat 1980 attaagcctt atttcaataa ggatgaaata gagcgcaaca atagtcgttt gacaagatgg 2040 ctaccatttt tgaataaaaa gagaggtacc aggtcctctg caagaaatga cagtaagtat 2100 gttggtatcc caaagtcgca ctccgtttcg tcttcctcgt ctagcctgtc ggctgttcca 2160 tatcagatat caccctcaaa taaggaaatg gctctaaacg attataacga gcaacctatc 2220 acggaaacag tagagactca aaagcatatc atctcttgga aaaatatcaa ctacacagtt 2280 ggtacaaaga aactgattaa taatgcatcc ggtttcatca gttctggttt gaccgcctta 2340 atgggtgagt ccggtgcagg taaaactact ttgctgaatg tcttatcaca aagagttgag 2400 acaggtgttg ttagcggcga aattttgatc gatggccacc cattgacaga tgaggacgcg 2460 tttaagagaa gtattggttt tgttcaacaa caggatttgc atctagattt gctgtctgtg 2520 aaagaatctc tggaaatatc atgtcttttg agaggtgatg gtgacagggc atacttagac 2580 actgtttcaa atttactgaa attgccatct gacattttag tcgctgattt aaacccgact 2640 caaaggaagc ttttatctat cggtgttgaa cttgttacta agccttcact tttattattc 2700 ctagacgaac caacctctgg gctggacgct gaggctgcac taacaatcgt caaattttta 2760 aaacaacttt ctttacaggg tcaggctatt ttttgtacca ttcatcagcc tagtaaaagt 2820 gtcatcagcc attttgacaa tattttctta ttgaaaaggg gtggtgaatg tgtcttcttt 2880 ggaccaatgg acgatgcctg cggctatttc atgtctcatg acaacacgct cgtttacgat 2940 aaagaacatg ataaccctgc agatttcgtc attgatgcag taggtaacag taactcctcg 3000 gcagggaaag atacagcgga ggaagctctt actttgaaca aagaagccat agattggagt 3060 gcattatggg aatcttctgt agaaaagaaa ctagttaaaa aagaaacagc taggttagaa 3120 gatgatgctc gtgcgtctgg tgttgattac acaacctcat tgtggaagca gccttcctac 3180 ttacagcaat tagctcttat tacaagaaga caatatattt gtacgaaaag agacatgact 3240 tatgtcatgg ccaagtactg tttgaatggt ggcgctggtt tgtttatcgg tttctcattt 3300 tggcacatta agcataatat tatcggtttg caggatagta tcttcttctg tttcatggct 3360 ctttgtgttt catctccatt gatcaatcaa attcaggaca aagccttgaa aacaaaggaa 3420 gtttatgttg ctagggaggc gagatctaac acttatcact ggactgttct tcttttgtca 3480 cagtcaatta ttgaactacc actggcactt acaagttcca cactattttt cgtctgtgcc 3540 tttttctctt gtggatttaa caacgctggc tggagtgccg gtgttttctt tttaaactat 3600 atgcttttcg ctgcttatta ctcgactctt ggtttatggc tgatctatac tgctcctaat 3660 ctacaaactg ctgctgtttt tgttgcgttt atttacagtt ttacagcatc attctgtggt 3720 gttatgcaac catactcttt gtttccaact ttttggaaat tcatgtacag ggtttcgcca 3780 tacacatatt ttgtggaaac ttttgttagt attctattgc ataactggga aatcaagtgt 3840 gacatgtcgg agatggttcc tggtcaacct ctgacggggc aatcttgtgg ccaatttatg 3900 gaagctttca tagaagaata cggtggctat ctacataata agaacacttt cacagtctgc 3960 gcttattgta cgtacactgt tggcgatgac ttcctgaaaa atgagaatat gagttacgac 4020 cacgtttgga ggaactttgg tattgaatgg gcttttgttg gtttcaattt ctttgcgatg 4080 tttgctggtt actacttaac ttacgtggca agaatatggc caaaggtctt taaaattatc 4140 accaaagtaa ttccacacag agggaagaag cctgtacaga actaa 4185
SEQ ID NO:78
Saccharomyces cerevisiae
MSISKYFTPV ADGSLTFNGA NIQFGADAQG ESKKSYDAED SMPNPANQLN DITFQAEAGE 60 MVLVLGYPTS TLFKTLFHGK TSLSYSPPGS IKFKNNEFKS FSEKCPHQII YNNEQDVHFP 120 FLTVEQTIDF ALSCKFDIPK GERDQIRNEL LREFGLSHVL KTIVGNDFFR GVSGGERKRI 180 SIIETFIANG SVYLWDNSTK GLDSATALDF LE ILRKMAKA TRSVNLVRIS QASDKIVDKF 240 DKILMLSDSY QLFYGTVDEC LTYFRDTLGI EKDPNDCI IE YLTS ILNFQF KNKNLGNLSN 300 SSSASVLKTA TGEVTKYTYN SDFDLYDQWK HSSYYRNIKQ QIQGSSIDDS IKEVDPSDVS 360 PIFNIPLKKQ LLFCTKRAFQ RSLGDKAYMT AQFISWIQS LVIGSLFYEI PLTTIGSYSR 420 GSLTFFSILF FTFLSLADMP IAFQRQPWK KQSQLHFYTN WVETLSTTVF DYCFKLCLVI 480 VFS I ILYFLA HLQYKAARFF IFLLFLSFYN FCMVSLFALT TLVAPTISVA NLFAGILLLA 540 IAMYASYVIY LKNMHPWFVW IAYLNPAMYA MEAILSNELY NLKLDCSETI VPRGPTYNDV 600 PFSHKACAWQ GATLGNDYVR GRDYLKQGLS YTYHHVWRNF GI I IGFLVFF IACTLFASQY 660 IKPYFNKDEI ERNNSRLTRW LPFLNKKRGT RSSARNDSKY VGIPKSHSVS SSSSSLSAVP 720 YQISPSNKEM ALNDYNEQP I TETVETQKHI ISWKNINYTV GTKKLINNAS GFISSGLTAL 780 MGESGAGKTT LLNVLSQRVE TGWSGEILI DGHPLTDEDA FKRSIGFVQQ QDLHLDLLSV 840 KESLEISCLL RGDGDRAYLD TVSNLLKLPS DILVADLNPT QRKLLSIGVE LVTKPSLLLF 900 LDEPTSGLDA EAALTIVKFL KQLSLQGQAI FCTIHQPSKS VISHFDNIFL LKRGGECVFF 960 GPMDDACGYF MSHDNTLVYD KEHDNPADFV IDAVGNSNSS AGKDTAEEAL TLNKEAIDWS 1020 ALWESSVEKK LVKKETARLE DDARASGVDY TTSLWKQPSY LQQLALITRR QYICTKRDMT 1080 YVMAKYCLNG GAGLFIGFSF WHIKHNI IGL QDSIFFCFMA LCVSSPLINQ IQDKALKTKE 1140 VYVAREARSN TYHWTVLLLS QSIIELPLAL TSSTLFFVCA FFSCGFNNAG WSAGVFFLNY 1200 MLFAAYYSTL GLWLIYTAPN LQTAAVFVAF IYSFTASFCG VMQPYSLFPT FWKFMYRVSP 1260 YTYFVETFVS ILLHNWEIKC DMSEMVPGQP LTGQSCGQFM EAFIEEYGGY LHNKNTFTVC 1320 AYCTYTVGDD FLKNENMSYD HVWRNFGIEW AFVGFNFFAM FAGYYLTYVA RIWPKVFKII 1380 TKVIPHRGKK PVQN 1394
SEQ ID NO:79 Saccharomyces cerevisiae
atgttgcaag cgccctcaag ttcaaactcg ggtttgaatc aaggaaatgc tgcgccggac 60 ggcccaccta acgaaacaca gccgtacgaa ggcctcgacg cggcagctca agaagaaata 120 aaagaattgg caagaacgct gacgagccaa tcttcccttt tatcgcaaga aaagcgtatt 180 accggcactg gcgacccgaa tacactaact gctgcttctt catcgtcatt gagccggtca 240 attttcgcta gtgatatcaa gggagttaac ccaatccttt tggacgtcaa tgatccagac 300 tacgatgaga cattggatcc tcgatcagaa aatttctcga gtgtcagatg ggtacgaaat 360 atggcacaaa tatgcgagaa tgattcggat ttctataaac cgttttcatt aggttgcgcc 420 tggaaagatt tgagtgcctc cggtgattct gccgatataa catatcaggg aacatttggt 480 aatatgccca tcaaatactt gaaaatgagt tggaggtgca tatcccgacg tcttttccac 540 cggacacatg gcaagagtga agataatgac tccggtttcc aaattttgaa gccgatggac 600 ggatgcatca atccggggga actacttgtt gtgcttggac gacccggtgc aggatgtact 660 acgctgctga aatctatatc tgtaaataca cacggattca agatttctcc ggacacaatc 720 atcacgtaca atggattctc caacaaagag atcaaaaacc attaccgtgg tgaagtggtc 780 tacaatgcag aatcagacat tcacatcccg cacttgacag tattccaaac tttatacaca 840 gtggcaagac tgaagacacc aaggaaccga atcaagggtg tcgataggga cacatttgcc 900 aaacacatga ctgaagtggc aatggcaact tacggactga gccacactgc agatacaaaa 960 gtgggtaatg attttgttcg tggtgtatcc gggggagaaa ggaaaagagt ttccattgct 1020 gaagtgtcta tatgtggctc gaagtttcag tgctgggata atgccacgag gggcttagat 1080 tctgctaccg cgctggagtt catcaaggcc ttgaaaacgc aagccactat cacaaagtct 1140 gcagccactg tggccattta ccaatgttct aaagatgcct atgatttgtt cgataaagtc 1200 tgtgttcttt acgatggtta ccaaatcttc tttggcccaa gtaagcaggc caaaaagtac 1260 tttcaaagaa tgggatatgt gtgtcccgaa aggcagacca cagcagatta tttgacctct 1320 attactagtc cttctgaaag aattaaggac aaagacatgg tcaaacatgg aattatgatc 1380 ccacaaacgg cctatgaaat gaaccagtac tggattcaat cagaggaata caaacaatta 1440 caggtgcagg tgaacaaaca tttagataca gactcttccc agcaaagaga acaaataaaa 1500 aatgcacata ttgccaaaca atctaagaga gcacggccct cctctcctta tacagtaagt 1560 ttctttttgc aagttaagta tattctaatc agggatatat ggaggattaa gaatgaccca 1620 agtattcaac ttttcacggt tttgagtcat gcagcaatgg ctcttatctt gggatcaatg 1680 ttttatgaag tcatgctttc cactaccaca acaacctttt attaccgagg cgcggctatt 1740 ttttttgcta ttctgttcaa tgcgttctca tcacttttag agatcttttc actctatgag 1800 acgaggccca ttaccgaaaa gcacaagact tactcgttat atcgcccaag tgctgatgcc 1860 ttcgcctcta cattttctga tgttccaaca aaattagcca cggcagtaac tttcaacata 1920 ccatattatt ttttaatcaa tctaaaaaga gatgccggtg cttttttctt ttacttttta 1980 ataaacatta taacagtttt tgctatgtcg catttgttca gatgcattgg ctcagtgtcg 2040 aaaactttac ctcaggcaat ggttcccgca tctgttcttc tgttagcttt tgcaatgtac 2100 actgggtttg ccatcccaag agtccaaatg cttggctggt caaaatggat ttcttatatc 2160 aacccattgt cttacctttt tgaatcgcta atgattaacg aatttcatgg tagaaatttc 2220 ccatgtgctc agtatatacc tagtgggcca aattatgtca acgcaacagg tgacgaagtc 2280 acctgctccg ccctcggttc tatcccagga aataattatg taagtggtga tgatttcatt 2340 caaacgaact acggctatcg acataagaac aaatggcggt ctgttggtat tggtttagca 2400 tacattattt ttttcttatt tctgtattta ttcttttgtg aatacaatga aggagcaaag 2460 caaaatggtg agatgttagt atttccccat agtgtggtta aaaagatgaa gaagaagggt 2520 attgtctctg agaagaaaaa gaaaaaccaa ccaacgttgt caacatctga tgctgagaaa 2580 gatgtagaga tgaacaacaa ttcaagtgcg actgattcaa gatttttacg cgattctgat 2640 gcagctataa tgggtaacga taaaactgtc gccaaggagc attattcatc tccttcatct 2700 tctgcgtctc aaagcaatag ttttagtaaa agtgatgaca ttgaattgtc caaatcacaa 2760 gcgatttttc attggaaaaa cttatgctac gatatcccga ttaagaatgg gaagaggaga 2820 attttggata atgttgatgg ttgggtcaag cctggtacac taactgcctt gattggcgca 2880 tctggtgcag gaaaaacaac gttattagat tgtcttgccg agagaaccac gatgggttta 2940 atcactggtg atgtattcgt agatggtaga ccgcgggacc agtctttccc cagatcaatt 3000 ggttattgtc agcagcagga tttgcatttg aaaaccgcaa ctgtgagaga gtcattaagg 3060 ttttccgcgt acctgcgtca agccgatgat gtatccatcg aggagaaaga taaatacgtt 3120 gaagaagtca ttgaggtatt ggagatgaaa ctttatgctg atgccatagt aggcgttcct 3180 ggtgaaggtt taaatgtgga acaaagaaaa aggttaacca taggtgtgga gttagctgcc 3240 aaaccaaaac tgttggtatt tttagatgag cccacgtctg ggcttgattc ccaaactgca 3300 tggtcaactt gtcaattaat gaagaaattg gctagtcgcg gacaagcaat tttatgtacc 3360 attcatcaac cttctgctct cttaatgcaa gagtttgata ggttactatt tttgcaagag 3420 ggcggacaaa ctgtatattt tggagagcta gggaaaggtt gcaaaacaat gattaactat 3480 ttcgaagctc atggtgccca taaatgccca ccagatgcca atccagccga atggatgtta 3540 gaaatagtag gtgctgcacc aggaactcac gctagtcaag attattttgc catttggaga 3600 gattctgaag aatataggga aatgcagaaa gagttggact ggatggaacg agaattgcct 3660 aaacggacgg aaggttcgtc aaatgaggag cagaaggagt tcgctacgtc aactttgtac 3720 cagatcaaat tggtgagtta tcgattattc catcaatatt ggagaacacc attttactta 3780 tggtcaaaat ttttttcaac aattgtgtct gaactcttca taggcttcac ttttttcaag 3840 gcgaatacat cattacaggg cctacagaat caaatgctgg ccatttttat gtttacagtg 3900 gtattcaacc caatattgca acaatattta ccacttttcg ttcagcagag ggaactttat 3960 gaagctagag aaaggccatc aagaaccttt tcgtggaaag catttattgt atcgcagatt 4020 ctcgtggaaa tcccttggaa tttactggcc ggtactatag cttttttcgt ctattattat 4080 cctgttggat tttacagaaa tgcttcttat gcaaatcaac ttcatgaacg aggagcttta 4140 ttttggttat ttgcgtgcgc gttttacgtc tacatcagtt caatgggggt attagtgatt 4200 tcatgcattg aaattgcaga aaatgctgcg aatctcgctt cacttttctt tataatgtca 4260 ttatctttct gcggtgttct ggctacgcca aatattctac caagattctg gatttttatg 4320 tatagagtat cgccactgac atacctcatt gatgctctgc tatcggtagg actggccaat 4380 gctagtgtcg tttgttccag caacgaactt ttaaaaattg ttcctccaag cggtatgaca 4440 tgctcggaat atatggaacc ttatatgcaa tccaccggga cgggttatct gctcgatggg 4500 agttccgaaa ctgaatgtca cttttgtcaa ttcagttcta ccaatgatta tctggcgact 4560 gtgagttctt cgtattcacg tagatggatg aactatggca tttttagtgc atatattgtc 4620 tttgactatt gtgcagcaat atttttatac tggctggtgc gggttccaaa gaaaagcaaa 4680 aaattaaaga aataa 4695
SEQ ID NO:80
Saccharomyces cerevisiae
MLQAPSSSNS GLNQGNAAPD GPPNETQPYE GLDAAAQEE I KELARTLTSQ SSLLSQEKRI 60 TGTGDPNTLT AASSSSLSRS IFASDIKGVN PILLDVNDPD YDETLDPRSE NFSSVRWVRN 120 MAQICENDSD FYKPFSLGCA WKDLSASGDS ADITYQGTFG NMP IKYLKMS WRCISRRLFH 180 RTHGKSEDND SGFQILKPMD GCINPGELLV VLGRPGAGCT TLLKSISVNT HGFKISPDTI 240 ITYNGFSNKE IKNHYRGEW YNAESDIHIP HLTVFQTLYT VARLKTPRNR IKGVDRDTFA 300 KHMTEVAMAT YGLSHTADTK VGNDFVRGVS GGERKRVSIA EVSICGSKFQ CWDNATRGLD 360 SATALEFIKA LKTQATITKS AATVAIYQCS KDAYDLFDKV CVLYDGYQIF FGPSKQAKKY 420 FQRMGYVCPE RQTTADYLTS ITSPSERIKD KDMVKHGIMI PQTAYEMNQY WIQSEEYKQL 480 QVQVNKHLDT DSSQQREQIK NAHIAKQSKR ARPSSPYTVS FFLQVKYILI RDIWRIKNDP 540 SIQLFTVLSH AAMALILGSM FYEVMLSTTT TTFYYRGAAI FFAILFNAFS SLLEIFSLYE 600 TRP ITEKHKT YSLYRPSADA FASTFSDVPT KLATAVTFNI PYYFLINLKR DAGAFFFYFL 660 INI ITVFAMS HLFRCIGSVS KTLPQAMVPA SVLLLAFAMY TGFAIPRVQM LGWSKWISYI 720 NPLSYLFESL MINEFHGRNF PCAQYIPSGP NYVNATGDEV TCSALGSIPG NNYVSGDDFI 780 QTNYGYRHKN KWRSVGIGLA YIIFFLFLYL FFCEYNEGAK QNGEMLVFPH SWKKMKKKG 840 IVSEKKKKNQ PTLSTSDAEK DVEMNNNSSA TDSRFLRDSD AAIMGNDKTV AKEHYSSPSS 900 SASQSNSFSK SDDIELSKSQ AIFHWKNLCY DIPIKNGKRR ILDNVDGWVK PGTLTALIGA 960 SGAGKTTLLD CLAERTTMGL ITGDVFVDGR PRDQSFPRSI GYCQQQDLHL KTATVRESLR 1020 FSAYLRQADD VS IEEKDKYV EEVIEVLEMK LYADAIVGVP GEGLNVEQRK RLTIGVELAA 1080 KPKLLVFLDE PTSGLDSQTA WSTCQLMKKL ASRGQAILCT IHQPSALLMQ EFDRLLFLQE 1140 GGQTVYFGEL GKGCKTMINY FEAHGAHKCP PDANPAEWML E IVGAAPGTH ASQDYFAIWR 1200 DSEEYREMQK ELDWMERELP KRTEGSSNEE QKEFATSTLY QIKLVSYRLF HQYWRTPFYL 1260 WSKFFSTIVS ELFIGFTFFK ANTSLQGLQN QMLAIFMFTV VFNP ILQQYL PLFVQQRELY 1320 EARERPSRTF SWKAFIVSQI LVEIPWNLLA GTIAFFVYYY PVGFYRNASY ANQLHERGAL 1380 FWLFACAFYV YISSMGVLVI SCIE IAENAA NLASLFFIMS LSFCGVLATP NILPRFWIFM 1440 YRVSPLTYLI DALLSVGLAN ASWCSSNEL LKIVPPSGMT CSEYMEPYMQ STGTGYLLDG 1500 SSETECHFCQ FSSTNDYLAT VSSSYSRRWM NYGIFSAYIV FDYCAAIFLY WLVRVPKKSK 1560 KLKK 1564
SEQ ID NO:81
Saccharomyces cerevisiae atgacgatta ccgtggggga tgcagtttcg gagacggagc tggaaaacaa aagtcaaaac 60 gtggtactat ctcccaaggc atctgcttct tcagacataa gcacagatgt tgataaggac 120 acatcgtctt cttgggatga caaatctttg ctgcctacag gtgaatatat tgtggacaga 180 aataagcccc aaacctactt gaatagcgat gatatcgaaa aagtgacaga atctgatatt 240 ttccctcaga aacgtctgtt ttcattcttg cactctaaga aaattccaga agtaccacaa 300 accgatgacg agaggaagat atatcctctg ttccatacaa atattatctc taacatgttt 360 ttttggtggg ttctacccat cctgcgagtt ggttataaga gaacgataca gccgaacgat 420 ctcttcaaaa tggatccgag gatgtctata gagacccttt atgacgactt tgaaaaaaac 480 atgatttact attttgagaa gacgaggaaa aaataccgta aaagacatcc agaagcgaca 540 gaagaagagg ttatggaaaa tgccaaacta cctaaacata cagttctgag agctttatta 600 ttcactttta agaaacagta cttcatgtcg atagtgtttg caattctcgc taattgtaca 660 tccggtttta accccatgat taccaagagg ctaattgagt ttgtcgaaga aaaggctatt 720 tttcatagca tgcatgttaa caaaggtatt ggttacgcta ttggtgcatg tttgatgatg 780 ttcgttaacg ggttgacgtt caaccatttc tttcatacat cccaactgac tggtgtgcaa 840 gctaagtcta ttcttactaa agctgccatg aagaaaatgt ttaatgcatc taattatgcg 900 agacattgtt ttcctaacgg taaagtgact tcttttgtaa caacagatct cgctagaatt 960 gaatttgcct tatcttttca gccgtttttg gctgggttcc ctgcaatttt ggctatttgc 1020 attgttttat tgatcgttaa ccttggaccc attgccttag ttgggattgg tatttttttc 1080 ggtgggtttt tcatatcctt atttgcattt aagttaattc tgggctttag aattgctgcg 1140 aacatcttca ctgatgctag agttaccatg atgagagaag tgctgaataa tataaaaatg 1200 attaaatatt atacgtggga ggatgcgtat gaaaaaaata ttcaagatat taggaccaaa 1260 gagatttcta aagttagaaa aatgcaacta tcaagaaatt tcttgattgc tatggccatg 1320 tctttgccta gtattgcttc attggtcact ttccttgcaa tgtacaaagt taataaagga 1380 ggcaggcaac ctggtaatat ttttgcctct ttatctttat ttcaggtctt gagtttgcaa 1440 atgtttttct tacctattgc tattggtact ggaattgaca tgatcattgg attgggccgt 1500 ttgcaaagct tattggaggc tccagaagat gatccaaatc agatgattga aatgaagcct 1560 tctcctggct ttgatccaaa attggcttta aaaatgacac attgctcatt tgagtgggaa 1620 gattatgaat taaacgacgc tattgaagaa gcaaaaggag aagctaaaga tgaaggtaaa 1680 aagaacaaaa aaaagcgtaa ggatacatgg ggtaagccat ctgcaagtac taataaggcg 1740 aaaagattgg acaatatgtt gaaagacaga gacggcccgg aagatttaga aaaaacttcg 1800 tttaggggtt tcaaggactt gaacttcgat attaaaaagg gcgaatttat tatgattacg 1860 ggacctattg gtactggtaa atcttcatta ttgaatgcga tggcaggatc aatgagaaaa 1920 actgatggta aggttgaagt caacggggac ttattaatgt gtggttatcc atggattcaa 1980 aatgcatctg taagagataa catcatattc ggttcaccat tcaataaaga aaagtatgat 2040 gaagtagttc gtgtttgctc tttgaaagct gatctggata ttttaccggc aggcgatatg 2100 accgaaattg gggaacgtgg tattacttta tctggtggtc aaaaggcacg tatcaattta 2160 gccaggtctg tttataagaa gaaggatatt tatctattcg acgatgtcct aagtgctgtc 2220 gattctcgtg ttggtaaaca catcatggat gaatgtctaa ccggaatgct tgctaataaa 2280 accagaattt tagcaacgca tcaattgtca ctgattgaga gagcttctag agtcatcgtt 2340 ttaggtactg atggccaagt cgatattggt actgttgatg agctaaaagc tcgtaatcaa 2400 actttgataa atcttttaca attctcttct caaaattcgg agaaagagga tgaagaacag 2460 gaagcggttg ttgccggtga attgggacaa ctaaaatatg aatcagaggt aaaggaattg 2520 actgaactga agaaaaaggc tacagaaatg tcacaaactg caaatagtgg taaaattgta 2580 gcggatggtc atactagtag taaagaagaa agagcagtca atagtatcag tctgaaaata 2640 taccgtgaat acattaaagc tgcagtaggt aagtggggtt ttatcgcact accgttgtat 2700 gcaattttag tcgttggaac cacattctgc tcactttttt cttccgtttg gttatcttac 2760 tggactgaga ataaattcaa aaacagacca cccagttttt atatgggtct ttactccttc 2820 tttgtgtttg ctgctttcat attcatgaat ggccagttca ccatactttg cgcaatgggt 2880 attatggcat cgaaatggtt aaatttgagg gctgtgaaaa gaattttaca cactccaatg 2940 tcatacatag ataccacacc tttgggacgt attctgaaca gattcacaaa agatacagat 3000 agcttagata atgagttaac cgaaagttta cggttgatga catctcaatt tgctaatatt 3060 gtaggtgttt gcgtcatgtg tattgtttac ttgccgtggt ttgctatcgc aattccgttt 3120 cttttggtca tctttgttct gattgctgat cattatcaga gttctggtag agaaattaaa 3180 agacttgaag ctgtgcaacg gtcttttgtt tacaataatt taaatgaagt tttgggtggg 3240 atggatacaa tcaaagcata ccgaagtcag gaacgatttt tggcgaaatc agattttttg 3300 atcaacaaga tgaatgaggc gggatacctt gtagttgtcc tgcaaagatg ggtaggtatt 3360 ttccttgata tggttgctat cgcatttgca ctaattatta cgttattgtg tgttacgaga 3420 gcctttccta tttccgcggc ttcagttggt gttttgttga cttatgtatt acaattgcct 3480 ggtctattaa ataccatttt aagggcaatg actcaaacag agaatgacat gaatagtgcc 3540 gaaagattgg taacatatgc aactgaacta ccactagagg catcctatag aaagcccgaa 3600 atgacacctc cagagtcatg gccctcaatg ggcgaaataa tttttgaaaa tgttgatttt 3660 gcctatagac ctggtttacc tatagtttta aaaaatctta acttgaatat caagagtggg 3720 gaaaaaattg gtatctgtgg tcgtacaggt gctggtaagt ccactattat gagtgccctt 3780 tacaggttga atgaattgac cgcaggtaaa attttaattg acaatgttga tataagtcag 3840 ctgggacttt tcgatttaag aagaaaatta gccatcattc cacaagatcc agtattattt 3900 aggggtacga ttcgcaagaa cttagatcca tttaatgagc gtacagatga cgaattatgg 3960 gatgcattgg tgagaggtgg tgctatcgcc aaggatgact tgccggaagt gaaattgcaa 4020 aaacctgatg aaaatggtac tcatggtaaa atgcataagt tccatttaga tcaagcagtg 4080 gaagaagagg gctccaattt ctccttaggt gagagacaac tattagcatt aacaagggca 4140 ttggtccgcc aatcaaaaat attgattttg gatgaggcta catcctcagt ggactacgaa 4200 acggatggca aaatccaaac acgtattgtt gaggaatttg gagattgtac aattttgtgt 4260 attgctcaca gactgaagac cattgtaaat tatgatcgta ttcttgtttt agagaagggt 4320 gaagtcgcag aattcgatac accatggacg ttgtttagtc aagaagatag tattttcaga 4380 agcatgtgtt ctagatctgg tattgtggaa aatgatttcg agaacagaag ttaa 4434
SEQ ID NO:82
Saccharomyces cerevisiae
MTITVGDAVS ETELENKSQN WLSPKASAS SDISTDVDKD TSSSWDDKSL LPTGEYIVDR 60 NKPQTYLNSD DIEKVTESDI FPQKRLFSFL HSKKIPEVPQ TDDERKIYPL FHTNIISNMF 120 FWWVLP ILRV GYKRTIQPND LFKMDPRMSI ETLYDDFEKN MIYYFEKTRK KYRKRHPEAT 180 EEEVMENAKL PKHTVLRALL FTFKKQYFMS IVFAILANCT SGFNPMITKR LIEFVEEKAI 240 FHSMHVNKGI GYAIGACLMM FVNGLTFNHF FHTSQLTGVQ AKSILTKAAM KKMFNASNYA 300 RHCFPNGKVT SFVTTDLARI EFALSFQPFL AGFPAILAIC IVLLIVNLGP IALVGIGIFF 360 GGFFISLFAF KLILGFRIAA NIFTDARVTM MREVLNNIKM IKYYTWEDAY EKNIQDIRTK 420 EISKVRKMQL SRNFLIAMAM SLPSIASLVT FLAMYKVNKG GRQPGNIFAS LSLFQVLSLQ 480 MFFLP IAIGT GIDMI IGLGR LQSLLEAPED DPNQMIEMKP SPGFDPKLAL KMTHCSFEWE 540 DYELNDAIEE AKGEAKDEGK KNKKKRKDTW GKPSASTNKA KRLDNMLKDR DGPEDLEKTS 600 FRGFKDLNFD IKKGEFIMIT GPIGTGKSSL LNAMAGSMRK TDGKVEVNGD LLMCGYPWIQ 660 NASVRDNI IF GSPFNKEKYD EWRVCSLKA DLDILPAGDM TEIGERGITL SGGQKARINL 720 ARSVYKKKDI YLFDDVLSAV DSRVGKHIMD ECLTGMLANK TRILATHQLS LIERASRVIV 780 LGTDGQVDIG TVDELKARNQ TLINLLQFSS QNSEKEDEEQ EAWAGELGQ LKYESEVKEL 840 TELKKKATEM SQTANSGKIV ADGHTSSKEE RAVNSISLKI YREYIKAAVG KWGFIALPLY 900 AILWGTTFC SLFSSVWLSY WTENKFKNRP PSFYMGLYSF FVFAAFIFMN GQFTILCAMG 960 IMASKWLNLR AVKRILHTPM SYIDTTPLGR ILNRFTKDTD SLDNELTESL RLMTSQFANI 1020 VGVCVMCIVY LPWFAIAIPF LLVIFVLIAD HYQSSGREIK RLEAVQRSFV YNNLNEVLGG 1080 MDTIKAYRSQ ERFLAKSDFL INKMNEAGYL WVLQRWVGI FLDMVAIAFA LIITLLCVTR 1140 AFP ISAASVG VLLTYVLQLP GLLNTILRAM TQTENDMNSA ERLVTYATEL PLEASYRKPE 1200 MTPPESWPSM GE I IFENVDF AYRPGLP IVL KNLNLNIKSG EKIGICGRTG AGKSTIMSAL 1260 YRLNELTAGK ILIDNVDISQ LGLFDLRRKL AIIPQDPVLF RGTIRKNLDP FNERTDDELW 1320 DALVRGGAIA KDDLPEVKLQ KPDENGTHGK MHKFHLDQAV EEEGSNFSLG ERQLLALTRA 1380 LVRQSKILIL DEATSSVDYE TDGKIQTRIV EEFGDCTILC IAHRLKTIVN YDRILVLEKG 1440 EVAEFDTPWT LFSQEDSIFR SMCSRSGIVE NDFENRS 1477
SEQ ID NO:83
Saccharomyces cerevisiae
atgtcggatc attctcccat ttctaataag gaaaatcatc tgctgccatc agactcgtca 60 agaagctcat ccagcgatat gcattcgacg ggcaccacgg gaaccacagg cgtcgaacct 120 gtagatttca ccggtgaagg tgccaaatac acaactgcta cggagggcaa tggtggtgca 180 gatttagcga ttcaaagaac gacgactatg aattctgctg cagaatcaga ggttaatatc 240 acgagaagat taactaaaat ccttactggg tctgttaacg agcctgaccg tgtagaggtt 300 gattatacca attgtgcgcc catgggtggt gacagacctt accctccatc gttgccgagc 360 agagacctgt acgaggttac ttttgatggt cctaacgacc cactacatcc atttaactgg 420 cccatgaaga agaaagtgct gctatgtctg gtcttatgtc tggattctat tgccattgct 480 atgtgttctt ccatttttgc ctctgcagtg ccgcaaatct gcgagatata ccacgtcatc 540 gaagttgtcg ccattttggg tatcacgctt tttgttcttg ggtttgcggc ctcaccggtt 600 atctatgctc ctctttctga attgtacggt agaaagggtg ttctggtttt atctgcgttt 660 ggatttgccc ttttccaatt tgctgtcgct actgctgaga acctgcaaac tattttcata 720 tgtagattct ttggtggttt tatcggggca gcacccatgg ccgtcgtccc cgccgcgttt 780 gccgacatgt ttgatactaa tgttagaggt aaagccattg cgctattttc tctaggtgtt 840 tttgtaggcc ccattttatc gcctgtcatg ggttcttata tcgcacaaag aactacctgg 900 agatggttag aatatgttgt cggttgtttc gcttccgcag ttttcgttgc catcgtattg 960 ttctttgaag aaacacatca ccctaccatt ttggttaaca aggccaaaca gatgagaaag 1020 caaagtaata actggggtat tcatgctgct catgaagacg tggagctatc catcaaagac 1080 attgtccaaa aaactgtgac gaggcctatc attatgcttt tcgtggaacc attgctacta 1140 ttcgtgacta tttacaactc ttttgtctac ggtatcttgt atttgttact ggaagcctac 1200 ccacttgtct ttgtggaggg ttatgggttt actgaaaacg gtgagttgcc atacatcgcc 1260 ttgattatcg gtatgatggt gtgtgctgct ttcatttggt atatggacaa cgattatttg 1320 aaaagatgta gagccaaggg gaaattagtg cccgaggcca gattgtacgc aatggtcatt 1380 gcaggtaccg ttttccctat tggtatctta tggttctgtt ggacgggcta ctatcctcac 1440 aagattcatt ggatggtccc cacagtagga ggggccttca tcgggttcgg tttaatgggt 1500 attttcttgc catgtttaaa ctatatcatt gaatcgtatc tattgttggc agcttctgcc 1560 gtcgcagcaa acactttcat gaggtctgca tttggtgcat gcttcccatt gtttgcagga 1620 tatatgttcc gtggcatggg tatcggttgg gctggtttgt tattaggtct atttgccgct 1680 gcgatgattc ccgtgccttt actattctta aaatatggtg aatctatcag aaagaaatcc 1740 aagtatgctt acgccgctta a 1761
SEQ ID NO:84
Saccharomyces cerevisiae
MSDHSP ISNK ENHLLPSDSS RSSSSDMHST GTTGTTGVEP VDFTGEGAKY TTATEGNGGA 60 DLAIQRTTTM NSAAESEVNI TRRLTKILTG SVNEPDRVEV DYTNCAPMGG DRPYPPSLPS 120 RDLYEVTFDG PNDPLHPFNW PMKKKVLLCL VLCLDSIAIA MCSSIFASAV PQICEIYHVI 180 EWAILGITL FVLGFAASPV IYAPLSELYG RKGVLVLSAF GFALFQFAVA TAENLQTIFI 240 CRFFGGFIGA APMAVVPAAF ADMFDTNVRG KAIALFSLGV FVGPILSPVM GSYIAQRTTW 300 RWLEYVVGCF ASAVFVAIVL FFEETHHPTI LVNKAKQMRK QSNNWGIHAA HEDVELS IKD 360 IVQKTVTRP I IMLFVEPLLL FVTIYNSFVY GILYLLLEAY PLVFVEGYGF TENGELPYIA 420 LI IGMMVCAA FIWYMDNDYL KRCRAKGKLV PEARLYAMVI AGTVFP IGIL WFCWTGYYPH 480 KIHWMVPTVG GAFIGFGLMG IFLPCLNYII ESYLLLAASA VAANTFMRSA FGACFPLFAG 540 YMFRGMGIGW AGLLLGLFAA AMIPVPLLFL KYGES IRKKS KYAYAA 586
SEQ ID NO:85
Saccharomyces cerevisiae
atgagtgatc aagaatctgt tgtttcattc aactcacaaa acacttccat ggtggacgtt 60 gagggccaac aacctcaaca gtatgtcccc tcaaaaacca actctcgtgc aaatcaactt 120 aagttaacta agactgagac cgtcaagtct ttacaagatt taggtgtcac ttcagccgcc 180 ccagtgcctg atatcaatgc gccacaaact gctaagaata acattttccc tgaagaatat 240 accatggaaa caccatctgg gctggttcca gtggctacct tacaatctat gggtagaacc 300 gcctctgcct tatctcgtac tagaacaaag caattgaacc gtaccgctac caattcctca 360 tccacaggta aagaagaaat ggaagaggaa gaaactgaag aacgtgaaga ccagagcggt 420 gaaaacgagc tagatccaga gatcgaattc gttacttttg ttactggtga tccagaaaac 480 cctcacaatt ggccctcatg ggttcgttgg agttacactg tcctgttgtc catcttagtt 540 atttgcgttg cctacggttc tgcttgtatc agtggtgggt tgggaaccgt tgaaaagaaa 600 taccatgtag gtatggaagc cgctatttta tcatgttctt taatggttat tgggttctcg 660 ctgggtcctt tgatttggtc tcctgttagt gatctttacg gtagaagagt tgcttacttt 720 gtttctatgg gtctttatgt catcttcaat atcccttgcg ccttagctcc aaatctaggt 780 tgtcttttag cttgtagatt tttatgtggt gtttggtcat catctggttt gtgtttagtt 840 ggtgggtcta ttgccgatat gttcccaagt gaaacaagag gtaaggctat tgctttcttc 900 gcttttgctc cttacgttgg tcccgttgtt ggtccactag ttaacggttt tatttccgtt 960 tctaccggac gtatggacct gattttctgg gtcaatatgg cctttgcagg tgttatgtgg 1020 atcatatctt ctgccatccc agaaacgtac gctccagtta tcttgaagag aaaggctgct 1080 agattaagaa aggaaactgg taatcccaag attatgactg agcaggaagc gcaaggtgtc 1140 agtatgagtg aaatgatgag ggcttgtctg ttgagacctt tgtacttcgc tgtcactgaa 1200 cctgttctag ttgccacttg tttctacgtg tgtttgattt actctctact atatgcgttc 1260 ttctttgcct tccctgtcat tttcggtgaa ctatatggct acaaagataa ccttgtgggt 1320 ttaatgttta ttcctattgt tatcggtgct ctttgggcgt tagccacaac tttctactgt 1380 gaaaacaaat atttacaaat tgtcaaacag cgtaaaccta ctcctgaaga tcgtttgcta 1440 ggtgctaaga tcggtgctcc atttgctgca attgctctat ggatcctggg tgctaccgct 1500 tataaacata ttatttgggt tggtccagct tcagctggtt tagcttttgg tttcggtatg 1560 gtgttgattt attattcatt gaataattac attattgatt gctacgtcca atacgcatcc 1620 agtgctctgg ctacaaaggt tttcttaaga tccgccggtg gtgctgcctt ccccttgttt 1680 accattcaaa tgtaccacaa attgaatttg cactggggtt cttggttgtt ggctttcatc 1740 tccactgcta tgattgcttt accttttgca ttttcttact ggggtaaggg cttgagacat 1800 aagttgtcca agaaggacta ttccatcgac agtgttgaga tgtaa 1845
SEQ ID NO:86
Saccharomyces cerevisiae
MSDQESWSF NSQNTSMVDV EGQQPQQYVP SKTNSRANQL KLTKTETVKS LQDLGVTSAA 60 PVPDINAPQT AKNNIFPEEY TMETPSGLVP VATLQSMGRT ASALSRTRTK QLNRTATNSS 120 STGKEEMEEE ETEEREDQSG ENELDPE IEF VTFVTGDPEN PHNWPSWVRW SYTVLLSILV 180 ICVAYGSACI SGGLGTVEKK YHVGMEAAIL SCSLMVIGFS LGPLIWSPVS DLYGRRVAYF 240 VSMGLYVIFN IPCALAPNLG CLLACRFLCG VWSSSGLCLV GGSIADMFPS ETRGKAIAFF 300 AFAPYVGPW GPLVNGFISV STGRMDLIFW VNMAFAGVMW IISSAIPETY APVILKRKAA 360 RLRKETGNPK IMTEQEAQGV SMSEMMRACL LRPLYFAVTE PVLVATCFYV CLIYSLLYAF 420 FFAFPVIFGE LYGYKDNLVG LMFIPIVIGA LWALATTFYC ENKYLQIVKQ RKPTPEDRLL 480 GAKIGAPFAA IALWILGATA YKHIIWVGPA SAGLAFGFGM VLIYYSLNNY IIDCYVQYAS 540 SALATKVFLR SAGGAAFPLF TIQMYHKLNL HWGSWLLAF I STAMIALPFA FSYWGKGLRH 600 KLSKKDYSID SVEM 614
SEQ ID NO:87
Saccharomyces cerevisiae
atgaacagac aggaatccat aaattcgttt aattcagacg aaacatcttc gttgtctgat 60 gtagaaagtc agcagccgca acaatatatc ccttcagaga gtggatctaa atccaacatg 120 gctcctaatc aactgaagtt gacccggacg gaaaccgtga agtcattgca ggacatgggt 180 gtgagctcca aagcccccgt tcctgatgtt aatgctcctc aatctagcaa gaataagatt 240 tttcctgaag aatatacttt agaaacccct acaggtttag ttcctgtcgc cactctacat 300 tccataggta gaacttctac tgcgatttcc cgtacgagaa ctagacagat cgatggcgct 360 tcttcgcctt cttctaatga agatgcttta gaaagtgata ataacgaaaa gggtaaagaa 420 ggcgactcta gtggtgcgaa tgacgaagct ccagatctag atccggaaat tgaattcgtt 480 acctttgtga ctggcgatcc agaaaacccc cataactggc ctgcatggat ccgttggagt 540 tacactgtcc tactgtcaat cttagttatt tgtgtcgcct acgggtctgc ttgtatcagt 600 ggtgggttgg gaaccgttga aaagaaatac catgtaggta tggaagccgc tattttatcg 660 gtttctttaa tggttattgg gttctcgctg ggtcctttga tttggtctcc tgttagtgat 720 ctttacggta gaagagttgc ttactttgtt tctatgggtc tttatgtcat cttcaatatc 780 ccttgcgcct tagctccaaa tctaggtagt cttttagctt gtagattttt atgtggtgtt 840 tggtcatcat ctggtttgtg tttagttggt gggtctattg ccgatatgtt cccaagtgaa 900 acaagaggta aggctattgc tttcttcgct tttgctcctt acgttggtcc cgttgttggt 960 ccactagtta acggttttat ttccgtttct accggacgta tggacctgat tttctgggtc 1020 aatatggcct ttgcaggtgt tatgtggatc atatcttctg ccatcccaga aacgtacgct 1080 ccagttatct tgaagagaaa ggctgctaga ttaagaaagg aaactggtaa tcccaagatt 1140 atgactgagc aggaagcgca aggtgtcagt atgggtgaaa tgatgagggc ttgtctgttg 1200 agacctttgt acttctctgt cactgaacct gttctagttg ctacttgttt ctacgtgtgt 1260 ttgatttact ctctactata tgcgttcttc tttgccttcc ctgtcatttt cggtgaacta 1320 tatggctaca aagacaacct tgtgggtttg atgtttattc ctattgttat cggtgctctt 1380 tgggcgttag ccacaacttt ctactgtgaa aacaagtatt tacaaattgt caaacagcgt 1440 aaacctactc ctgaagatcg tttgctaggt gctaagatcg gtgctccatt tgctgcaatt 1500 gctttatgga tcctgggtgc taccgcttat aaacatatta tttgggttgg tccagcttca 1560 gctggtttag cttttggttt cggtatggtg ttgatttatt attcattgaa taattacatt 1620 attgattgct acgtccaata cgcatccagt gctctggcta caaaggtttt cttaagatcc 1680 gccggtggtg ctgccttccc cttgtttacc attcaaatgt accacaaatt gaatctgcac 1740 tggggttctt ggttgttggc tttcatctcc actgctatga ttgctttacc ttttgcattt 1800 tcttactggg gcaaaggttt gagacataag ttatccaaga aggattattc catagatagt 1860 attgaataa 1869
SEQ ID NO:88
Saccharomyces cerevisiae
MNRQES INSF NSDETSSLSD VESQQPQQYI PSESGSKSNM APNQLKLTRT ETVKSLQDMG 60 VSSKAPVPDV NAPQSSKNKI FPEEYTLETP TGLVPVATLH SIGRTSTAIS RTRTRQIDGA 120 SSP SSNEDAL ESDNNEKGKE GDSSGANDEA PDLDPEIEFV TFVTGDPENP HNWPAWIRWS 180 YTVLLS ILVI CVAYGSACIS GGLGTVEKKY HVGMEAAILS VSLMVIGFSL GPLIWSPVSD 240 LYGRRVAYFV SMGLYVIFNI PCALAPNLGS LLACRFLCGV WSSSGLCLVG GSIADMFPSE 300 TRGKAIAFFA FAPYVGPVVG PLVNGFISVS TGRMDLIFWV NMAFAGVMWI ISSAIPETYA 360 PVILKRKAAR LRKETGNPKI MTEQEAQGVS MGEMMRACLL RPLYFSVTEP VLVATCFYVC 420 LIYSLLYAFF FAFPVIFGEL YGYKDNLVGL MFIPIVIGAL WALATTFYCE NKYLQIVKQR 480 KPTPEDRLLG AKIGAPFAAI ALWILGATAY KHIIWVGPAS AGLAFGFGMV LIYYSLNNYI 540 IDCYVQYASS ALATKVFLRS AGGAAFPLFT IQMYHKLNLH WGSWLLAFIS TAMIALPFAF 600 SYWGKGLRHK LSKKDYSIDS IE 622
SEQ ID NO:89
Saccharomyces cerevisiae
atgccttctt ctttgacaaa aaccgaatct aactcagatc ctcgaacaaa catacagcag 60 gtaccaaaag ccttggacaa aaatgtaact aatagcggca acttggattc gacttcttct 120 tcgacagggt caataacaga ggacgaaaaa agatcagaac cgaatgcaga ttccaataac 180 atgacagggg gcgagccaat tgacccaaga gatctcgatt gggacgggcc cgatgaccca 240 gacaatccgc ataattggtc ctctttgaag aaatggtata ctactatgac ttcagctttt 300 ctttgtttgg tcgtcacaat gggtagttcc ctatatgttt cttccgtacc agaattggta 360 gaaagatatc acgttagtca gactttggcg cttgctggtt taactttcta tcttctgggt 420 ttatcgactg tcatcggcgc tccattaagt gaagtgtttg gtcgtaagcc tgtctattta 480 ttttccctac ctgtgtcaat gcttttcacc atgggtgttg ggttatccaa tggccacatg 540 agaataattc tacccctaag gtttttatca ggtgtctttg catcccccgc cttatccgtt 600 ggatcaggta ccatcctgga tatctttgac gtggaccagg tctccgtcgc aatgacatac 660 tttgttctat ctccattctt agggccagtt ttatctccta ttatggcagg atttgctact 720 gaagccaaag gctggcgttg gtctgaatgg atccaattga ttgcgggcgg tctaatcttg 780 ccatttattg cgctcatgcc agagacccat aaaggtatta ttttaagaaa gcgtgccaag 840 aagagaaata tagccttgaa gaagttcagc agggaagctc agaaagaatt tttaaaaacc 900 actgtcacta ttacaatctt aaggccttta aaaatgcttg tcgttgaacc tattgtattt 960 gttttcagtg tttatgtcgc tttcatattc gccattttat ttggattttt cgaagcatac 1020 gctgtcattt atcgtggtgt ttaccacatg tctatgggga tatctggctt gccatttatt 1080 ggtattggtg tcgggttatg gataggtgct ttcttttacc tttacatcga cagaaaatat 1140 ctcttcccta aaccaccagc tggcactcaa ccactcaccg aaaaagagag aacttctaaa 1200 aggactaccc cttacagagg tgcaagagac gctgaaacag gtgaattgct acccgttgtt 1260 ccagaaaaat ttttaattgc ctgtaaattt ggttctgtag ctttgcctat cggcctgttt 1320 tggcaagcct ggactgctag atcagatgtc cactggatgg ctcctgtggc tgccggtgtg 1380 ccatttggtt ttggtttaat tttgattttt ttcagcgttt taatgtactt ctctacatgc 1440 tatccgccat taactgtggc atcctgtttg gctgccaata atctattaag atacgttatg 1500 agtagtgtat tcccattgtt taccatccaa atgtacacaa agatgaaaat caaatgggca 1560 agcactttat ttgcattagt ttgcgttgtg atgattccaa ttccatgggt ttttgaaaaa 1620 tgggggagca aattaagaca caagtcacaa tttggttatg ccgccatgga aaaggaggct 1680 gagaccgaag gtggtataga tgacgtaaat gctgtcgatg gtgaattgaa tctaacaagg 1740 atgaccacgt taaggaccat ggaaacagac ccttcgacta gagaaaaacc aggtgaaagg 1800 ctatctctgc gcaggaccca tacgcagccc gttcctgcct cgtttgatcg cgaggacggg 1860 caacatgcgc aaaatcgtaa tgaacccatt tccaatagtt tatactcagc tatcaaggat 1920 aatgaagacg gttattcgta tacggaaatg gccaccgatg cttccgccag aatggtttga 1980 SEQ ID NO:90
Saccharomyces cerevisiae
MPSSLTKTES NSDPRTNIQQ VPKALDKNVT NSGNLDSTSS STGS ITEDEK RSEPNADSNN 60 MTGGEP IDPR DLDWDGPDDP DNPHNWSSLK KWYTTMTSAF LCLVVTMGSS LYVSSVPELV 120 ERYHVSQTLA LAGLTFYLLG LSTVIGAPLS EVFGRKPVYL FSLPVSMLFT MGVGLSNGHM 180 RIILPLRFLS GVFASPALSV GSGTILDIFD VDQVSVAMTY FVLSPFLGPV LSP IMAGFAT 240 EAKGWRWSEW IQLIAGGLIL PFIALMPETH KGI ILRKRAK KRNIALKKFS REAQKEFLKT 300 TVTITILRPL KMLWEPIVF VFSVYVAFIF AILFGFFEAY AVIYRGVYHM SMGISGLPFI 360 GIGVGLWIGA FFYLYIDRKY LFPKPPAGTQ PLTEKERTSK RTTPYRGARD AETGELLPW 420 PEKFLIACKF GSVALP IGLF WQAWTARSDV HWMAPVAAGV PFGFGLILIF FSVLMYFSTC 480 YPPLTVASCL AANNLLRYVM SSVFPLFTIQ MYTKMKIKWA STLFALVCW MIPIPWVFEK 540 WGSKLRHKSQ FGYAAMEKEA ETEGGIDDVN AVDGELNLTR MTTLRTMETD PSTREKPGER 600 LSLRRTHTQP VPASFDREDG QHAQNRNEPI SNSLYSAIKD NEDGYSYTEM ATDASARMV 659
SEQ ID NO:91
Saccharomyces cerevisiae
atgaccaaac aacaaacttc tgtaatgcgt aacgcatcta tagccaagga agaacgtgaa 60 ggatctgata ataataatgt ggacagatcc agctcagatg ccatcagtga caacgacgca 120 gaaagatcta attctcattc tgaaatagat aacgaatcaa attttgatat ggtgccttat 180 tcaaggttca gccataagca aaaaatgctt cttgtcgttc agtgtgcctt cacaggcttt 240 ttttcaaccg tggcagggtc tatctactat ccagtgctta ctattattga gaggaaattt 300 aacatcacag aagagttagc taatgtcact attgtggttt attttatttt ccaaggggtg 360 gctccttcaa ttatgggtgg tttagctgat acatttggaa ggcggcctat tgtattatgg 420 gcaatcttag cctacttctg tgcctgcatc ggactggcat gtgcacataa ttacgcccaa 480 atcctggctt taaggtgttt acaggcagca ggtatttctc ctgttattgc aataaatagc 540 ggaataatgg gagatgtaac gacaaaagta gagaggggtg gatatgttgg attggttgca 600 gggttccaag ttgttggaac tgcattcggt gcattgattg gtgccggatt atcctcgaaa 660 tggggttgga gggcaatttt ttggtttttg gctattggtt ccggaatttg tctagttttt 720 tcaaccttat tgatgccaga gacaaaaaga actttggtgg gaaacggctc cgtaacacca 780 aggtcatttt tgaataggtc tttgatattg catgttggtt ccgtaaaaaa aacattacat 840 ttagacgatc ctgaccctga gaccttagaa ccacgtacta gcgtggattt tttagcacct 900 ttaaaaattt tgcacatacg agaaatagac attttattgt ccattgccgg cctgcaattt 960 tccacgtgga ccactcatca aacagcctta acaattgtat tgagtaaaaa gtacaatcta 1020 tctgttgcaa aaataggcct gtgcttttta cctgcaggaa tctcgacact gacaagtatt 1080 atctctgctg gtcggtactt aaattggagc tacagaacaa gaaaggttaa atacaacaga 1140 tggattaaag aacaagaatt acagctgatg gagaaatata aaggtgataa aaacaaggta 1200 gcagaactta ttcactctaa ttctcattat gcattcaatt tagtggaggc aaggttgcat 1260 ccagcatttg tgacacttct tctcagtagc attggcttta ctgcctttgg atggtgtatt 1320 tcggtgaaaa caccgcttgc tgctgtatta tgcacaagtg catttgcaag tttattttca 1380 aactgtattt tgaccttctc tactactttg atagtagatc tctttccgtc taaggcttct 1440 accgcaacag gttgcttaaa tttatttaga tgtctcctat ccgcaatttt cattgcagca 1500 ctgaccaaga tggtcgagaa gatgagatat ggaggtgttt ttacgttttt gagtgctata 1560 acttcatctt catcgctttt attattctat ctcctgaaga atgggaaaca actatcgttc 1620 gataggatta gggcaaatga taagtcggct ggaaggtcag ttggtaaaaa ttcagaaaaa 1680 gtttcgacgt ga 1692
SEQ ID NO:92
Saccharomyces cerevisiae
MTKQQTSVMR NASIAKEERE GSDNNNVDRS SSDAI SDNDA ERSNSHSEID NESNFDMVPY 60 SRFSHKQKML LVVQCAFTGF FSTVAGS IYY PVLTI IERKF NITEELANVT IWYFIFQGV 120 APSIMGGLAD TFGRRP IVLW AILAYFCACI GLACAHNYAQ ILALRCLQAA GISPVIAINS 180 GIMGDVTTKV ERGGYVGLVA GFQVVGTAFG ALIGAGLSSK WGWRAIFWFL AIGSGICLVF 240 STLLMPETKR TLVGNGSVTP RSFLNRSLIL HVGSVKKTLH LDDPDPETLE PRTSVDFLAP 300 LKILHIREID ILLSIAGLQF STWTTHQTAL TIVLSKKYNL SVAKIGLCFL PAGISTLTSI 360 ISAGRYLNWS YRTRKVKYNR WIKEQELQLM EKYKGDKNKV AELIHSNSHY AFNLVEARLH 420 PAFVTLLLSS IGFTAFGWCI SVKTPLAAVL CTSAFASLFS NCILTFSTTL IVDLFPSKAS 480 TATGCLNLFR CLLSAIFIAA LTKMVEKMRY GGVFTFLSAI TSSSSLLLFY LLKNGKQLSF 540 DRIRANDKSA GRSVGKNSEK VST 563
SEQ ID NO:93
Saccharomyces cerevisiae
atggcaggag caacatcaag tataattcgg gaaaatgatt ttgaggacga gcttgcggag 60 agtatgcagt catataatag agaaactgca gacaaattgg ctttgactag gaccgagagt 120 gtaaagccag aaccggagat aaccgctccg cctcactcac gcttttcccg ttctttcaag 180 acagttttaa tagctcagtg cgctttcact gggtttttct ccacaatagc aggtgccatc 240 tactatccag ttctgagcgt tatagaaaga aaattcgata ttgacgagga attggtgaat 300 gtcactgttg tagtatattt tgtatttcag ggtcttgccc ccacattcat gggcgggttt 360 gccgattcac tgggcaggag accggtggtg cttgtcgcaa tcgtcattta ttttggtgcc 420 tgcatcggtc ttgcttgtgc tcaaacgtat gctcagatca ttgtgctaag atgtctacaa 480 gccgccggta tttcacccgt gattgcgatt aacagcggaa taatgggaga tgttactact 540 agagccgagc gcggcgggta cgttggatat gttgctggat ttcaagttct aggttctgcg 600 ttcggagccc ttatcggtgc cggattatca tctagatggg gatggagagc catcttttgg 660 ttcttagcaa ttggatctgg catttgcttc ctagcctcgt ttttaatttt gccagaaaca 720 aagaggaaca tatccgggaa tggttctgtc accccaaaat catacttaaa tagagctccc 780 attcttgttt tgccaacagt aagaaaatca ttgcatttgg ataatccaga ttacgaaaca 840 ttggaactgc ccacgcaact aaatctactg gcgcccttca agattttgaa agcttatgaa 900 atttgtatac ttatgctagt cgctggatta caatttgcca tgtatactac gcaccttacg 960 gcgttatcca cagctttgag taaacaatat cacttgactg ttgcaaaggt aggattatgc 1020 taccttccct ccggtatctg cacattatgt agtattgtca ttgctggaag atatttgaat 1080 tggaattaca ggcgcagatt aaaatattac caaaattggt tgggcaagaa gagatcaaag 1140 cttttagaag aacacgacaa cgaccttaat ttggtgcaac gtatcataga aaatgatccc 1200 aaatacacct ttaatatctt caaggcgagg ttgcaacccg catttgttac cttgctttta 1260 agcagtagcg gattttgtgc gtacggatgg tgcattactg tcaaagcccc tttagcggcc 1320 gttctttgca tgagtggatt tgcgtcgctg ttttccaatt gcattttgac tttttcaaca 1380 actctaatag ttgatctttt tcccacgaag acatctacgg ctacgggatg tttaaacctt 1440 ttcagatgca ttctatctgc tgtgtttata gctgcattga gtaaaatggt agaaaaaatg 1500 aaattcggag gtgtttttac atttttgggg gctttaacct cctcgtcatc gatcctctta 1560 tttattctct tgagaaaagg taaagaatta gcctttaaga ggaaaaagca agaactggga 1620 gtaaattaa 1629
SEQ ID NO:94
Saccharomyces cerevisiae
MAGATSSIIR ENDFEDELAE SMQSYNRETA DKLALTRTES VKPEPEITAP PHSRFSRSFK 60 TVLIAQCAFT GFFSTIAGAI YYPVLSVIER KFDIDEELVN VTWVYFVFQ GLAPTFMGGF 120 ADSLGRRPW LVAIVIYFGA CIGLACAQTY AQIIVLRCLQ AAGISPVIAI NSGIMGDVTT 180 RAERGGYVGY VAGFQVLGSA FGALIGAGLS SRWGWRAIFW FLAIGSGICF LASFLILPET 240 KRNISGNGSV TPKSYLNRAP ILVLPTVRKS LHLDNPDYET LELPTQLNLL APFKILKAYE 300 ICILMLVAGL QFAMYTTHLT ALSTALSKQY HLTVAKVGLC YLPSGICTLC SIVIAGRYLN 360 WNYRRRLKYY QNWLGKKRSK LLEEHDNDLN LVQRI IENDP KYTFNIFKAR LQPAFVTLLL 420 SSSGFCAYGW CITVKAPLAA VLCMSGFASL FSNCILTFST TLIVDLFPTK TSTATGCLNL 480 FRCILSAVFI AALSKMVEKM KFGGVFTFLG ALTSSSSILL F ILLRKGKEL AFKRKKQELG 540 VN 542
SEQ ID NO:95
Saccharomyces cerevisiae
atgcaagccc aaggttcaca atcgaatgta gggtctttga ggagtaattg ctctgacaat 60 tcactaccga acaatcatgt tatgatgcac tgcgatgaaa gcagcggcag cccgcacagc 120 gagcacaacg attatagtta cgaaaagacc aatctggaaa gtacggcatc aaatagtcgt 180 gaacacagag acaaccagct aagtaggttg aagagtgagg aatacgttgt tccaaagaat 240 caacgtaggg gactattgcc tcaactcgcc attataccgg agttcaagga tgccagagat 300 tatccaccga tgatgaaaaa gatgattgtc ttcttgattg cgttttcctc catgatgggc 360 cccatgggca catctatcat ttttccagcg atcaactcaa tcacaacaga atttaaaaca 420 tcagtgatta tggtaaacgt ttcaattggt gtgtaccttt taagtcttgg tgttttccca 480 ttgtggtggt cttctctatc cgagctagag ggcagaagaa ctacttacat aacttcattt 540 gcattattgt ttgcatttaa tatcgggtct gctctagctc ctgatatcaa ctcatttatt 600 gccttgagaa tgctctgtgg ggctgcttct gccagtgttc aaagtgtagg tgctggaaca 660 gtggctgatt tatatattag cgaagataga ggtaaaaatt tgagttatta ctatttgggt 720 ccactactgg cgccgctact atctccaatt tttggatctt tgttagtgaa tcgctggccc 780 tggagatcca ctcaatggtt tatggttatt ttatccggat gtaatgtcat tcttttgacg 840 gtgttactac ctgaaacatt aagaaaacaa gattctaaag gcgctatcgc tcaaattttg 900 gctgaaagac gtattcaagt agacaataac gaacgtggag agatacaaga agattatcag 960 aggggagaag atgagacaga tcgaattgaa aaccaagttg ccacattatc tactgagaag 1020 cataactacg ttggagaggt aagggatcaa gactcgctag atttagaaag tcactctagc 1080 cccaatactt atgatggtcg agctggagaa acccaattgc aacggattta tacagaggcg 1140 agtagaagtc tgtatgaata tcagctagat gatagcggta tcgatgcaac aacagcacaa 1200 gttacgagaa taagatcaac agatccaaag ttagcgagat cgattcgaga aaatagtctg 1260 agaaaattac aaaccaacct ggaagagcaa gtcaaaaaag tgctatccag taatggaggt 1320 gaaatcgctc ctaaacaggt atcagcggtg aggaaggtct gggacacctt ttttgtttat 1380 tttatcaagc ctttaaaatc attgcacttc ctagaatatc cacccgtggc acttgcaata 1440 acattttccg caatttcctt ttccacagta tactttgtta atatgacagt tgaatataaa 1500 tattcaaggc ctccttacaa ctttaaacca ttatacattg gtctactgta tattccgaat 1560 tctgtaacat actttttcgc ctcaatttac ggtggacgtt gggtggacat gcttttaaaa 1620 agatacaaag agaaatatgg aattcttgct cctgaagctc gtatatcgtg gaatgttgtt 1680 acatctgtaa tatctttccc cattgcgcta ttgatatttg gctggtgcct agataaaaaa 1740 tgccactggg taacgccact aattggaaca gccctctttg gatatgcagc tatgatgaca 1800 attggtgcta ccctttccta tttagtcgat tcattgccgg gaaagggtgc caccggtgtt 1860 gctttgaata atttaataag gcaaatcttg gctgcaaccg cagtctttgt caccacaccc 1920 atgttaaacg gtatgggaac tgggtgggct ttcacaatgc tggcctttat cgtcttgggt 1980 gctagcagtg tacttataat actgaaaaag cacggtgatt actggagaga gaactacgat 2040 ttacaaaaat tgtacgacaa aattgattaa 2070
SEQ ID NO:96
Saccharomyces cerevisiae
MQAQGSQSNV GSLRSNCSDN SLPNNHVMMH CDESSGSPHS EHNDYSYEKT NLESTASNSR 60
EHRDNQLSRL KSEEYWPKN QRRGLLPQLA I IPEFKDARD YPPMMKKMIV FLIAFSSMMG 120
PMGTSI IFPA INSITpTEFKT SVIMVNVSIG VYLLSLGVFP LWWSSLSELE GRRTTYITSF 180
ALLFAFNIGS ALAPDINSFI ALRMLCGAAS ASVQSVGAGT VADLYISEDR GKNLSYYYLG 240
PLLAPLLSP I FGSLLVNRWP WRSTQWFMVI LSGCNVILLT VLLPETLRKQ DSKGAIAQIL 300
AERRIQVDNN ERGEIQEDYQ RGEDETDRIE NQVATLSTEK HNYVGEVRDQ DSLDLESHSS 360
PNTYDGRAGE TQLQRIYTEA SRSLYEYQLD DSGIDATTAQ VTRIRSTDPK LARSIRENSL 420
RKLQTNLEEQ VKKVLSSNGG EIAPKQVSAV RKVWDTFFVY FIKPLKSLHF LEYPPVALAI 480
TFSAISFSTV YFVNMTVEYK YSRPPYNFKP LYIGLLYIPN SVTYFFASIY GGRWVDMLLK 540
RYKEKYGILA PEARISWNW TSVISFPIAL LIFGWCLDKK CHWVTPLIGT ALFGYAAMMT 600
IGATLSYLVD SLpPGKGATGV ALNNLIRQIL AATAVFVTTP MLNGMGTGWA FTMLAF IVLG 660
ASSVLI ILKK HGDYWRENYD LQKLYDKID 689
SEQ ID NO:97
Saccharomyces cerevisiae
atggtataca cttcaacgta cagacacact atcgttgttg accttttaga atatttgggt 60 atagtgtcca acttagaaac tttacagagt gcccgtgaag atgaaacaag aaaacccgag 120 aataccgata aaaaagaatg taaacccgac tatgatatag aatgcggtcc taatagatcg 180 tgctctgaat cctctaccga ttcagactct agtggttcac agatcgaaaa aaatgatcct 240 ttcagggtgg attggaacgg ccccagtgat cctgagaacc cacaaaactg gcccctactg 300 aaaaaatcat tggtagtatt ccaaataatg ttacttactt gcgtcacgta catgggatcc 360 tccatttaca cacctggcca ggaatatatt caagaagagt tccacgttgg tcatgtagtg 420 gcaacattaa atctttcttt atatgttctt ggttatggtc taggtcccat cattttttca 480 ccgctatcag aaactgcacg ctatggccgt ctaaatctgt acatggtgac tttatttttt 540 ttcatgatct ttcaagttgg ttgtgctact gtgcataaca tcggcggttt aatcgtcatg 600 cgtttcatca gtggcatact gtgcagccca tcgttggcca ctggtggcgg tacagtggct 660 gatatcattt caccagaaat ggttcctctc gttttaggta tgtggtcagc cggtgctgtt 720 gctgcgccag tcttggctcc cttactaggc gctgctatgg tcgatgctaa aaattggcga 780 ttcatatttt ggttattaat gtggttaagt gctgccactt ttatcttgtt ggcatttttc 840 ttccctgaaa cacaacacca taatattttg taccgccgtg ctttgaaatt gagaaaagaa 900 actggtgatg acaggtacta tactgaacag gataaactcg atagagaagt tgatgcaaga 960 acttttttga tcaatacttt gtataggcct ctcaaaatga ttatcaaaga gcctgcaatt 1020 ttggcttttg atctctatat cgctgttgct tatggttgtt tctacttatt ctttgaagca 1080 ttccctattg tatttgtagg tatataccac ttcagcttag ttgaagttgg cttggcctat 1140 atggggtttt gcgtagggtg cgtacttgct tatggcttat tcggtatttt aaacatgagg 1200 attattgtac cacgttttag aaacggcaca ttcaccccgg aagctttttt aatcgtggca 1260 atgtgtgtct gctggtgcct gcctctgtct ttgttcttat ttggttggac tgctcgagtg 1320 cattggattt tgccagttat ctcggaagtt ttttttgttt tagctgtctt taacattttc 1380 caagcaactt ttgcatattt ggctacatgc tacccaaagt atgttgcatc cgtttttgca 1440 ggcaatggtt tttgtcgggc ttcgtttgcc tgtgcttttc cgttgtttgg tagagcaatg 1500 tatgacaatt tagctactaa gaactatcct gtggcatggg gttcgtcctt agtggggttc 1560 ctaactttag gtctagctat tatcccgttt atactttata agtatgggcc atcattacgt 1620 acaagatctt cgtacacaga ggagtag 1647
SEQ ID NO:98
Saccharomyces cerevisiae
MVYTSTYRHT IVVDLLEYLG IVSNLETLQS AREDETRKPE NTDKKECKPD YDIECGPNRS 60 CSESSTDSDS SGSQIEKNDP FRVDWNGPSD PENPQNWPLL KKSLWFQIM LLTCVTYMGS 120 SIYTPGQEYI QEEFHVGHW ATLNLSLYVL GYGLGPIIFS PLSETARYGR LNLYMVTLFF 180 FMIFQVGCAT VHNIGGLIVM RFISGILCSP SLATGGGTVA DIISPEMVPL VLGMWSAGAV 240 AAPVLAPLLG AAMVDAKNWR FIFWLLMWLS AATFILLAFF FPETQHHNIL YRRALKLRKE 300 TGDDRYYTEQ DKLDREVDAR TFLINTLYRP LKMI IKEPAI LAFDLYIAVA YGCFYLFFEA 360 FP IVFVGIYH FSLVEVGLAY MGFCVGCVLA YGLFGILNMR I IVPRFRNGT FTPEAFLIVA 420 MCVCWCLPLS LFLFGWTARV HWILPVISEV FFVLAVFNIF QATFAYLATC YPKYVASVFA 480 GNGFCRASFA CAFPLFGRAM YDNLATKNYP VAWGSSLVGF LTLGLAI IPF ILYKYGP SLR 540 TRSSYTEE 548
SEQ ID NO:99
Saccharomyces cerevisiae
atgtcacagc aggagaatgg tgatgtggcc actgaattaa tcgaaaatag actttccttc 60 tcaagaatcc ctagaataag ccttcatgta agggatttgt caatcgttgc atccaaaaca 120 aatacgacgc tagttaatac gttttccatg gacctgccca gtgggtcggt catggcagtt 180 atgggtggtt cggggtcagg gaagactacg ttgctgaatg ttctggcatc caagatcagt 240 ggtgggttaa ctcataatgg ttctatacgg tacgtattgg aagatacagg ttcagaaccc 300 aatgaaacag agcctaagag ggctcacttg gatggccaag accatcccat ccagaaacac 360 gtaataatgg cgtacctacc tcaacaagat gtactctctc ctaggttgac ttgtagagaa 420 actcttaagt ttgcagctga tttgaaacta aactcttctg agcgaaccaa gaaattaatg 480 gtcgagcaat taattgaaga attagggctc aaggattgtg ccgatactct tgtgggggac 540 aactcgcaca ggggtctttc tggtggtgaa aagagaagac taagtattgg tactcaaatg 600 atttcaaacc cttccatcat gttcttagat gagcctacca ctggactgga tgcatattct 660 gccttcttgg ttattaaaac tttgaaaaaa ttagctaaag aagatggcag gacttttatc 720 atgtcaattc atcagccgag atcggatata ttgtttttat tggaccaggt ttgtattttg 780 tcaaagggaa atgtggtata ttgtgacaaa atggataata ctatccctta ttttgagtct 840 attggttatc acgtacccca gctggtaaat ccagcagatt atttcattga tttatcaagc 900 gtagactcta gatctgataa agaagaagct gccacgcaaa gtcggctaaa ttcattgatt 960 gatcattggc atgattatga gagaactcat ttgcaactac aggcagaatc ttatataagc 1020 aacgcaacgg aaattcaaat tcagaatatg actaccagac tgccattttg gaagcaagtt 1080 acggtgctaa caaggcgaaa tttcaaatta aatttttcag attacgttac tttaatatct 1140 acttttgcag aaccgttgat tattggtact gtttgcggtt ggatttacta taaacctgac 1200 aaaagcagta taggtggttt aaggacaaca accgcatgtc tttacgcgtc cacaattttg 1260 caatgttact tgtatttgct ttttgatact tatcgacttt gtgagcagga tattgcgtta 1320 tatgacagag aaagagcaga gggctcggta acacctttgg cattcatcgt agctagaaaa 1380 atttcacttt tcctctctga cgatttcgcg atgaccatga tttttgtcag tataacatat 1440 tttatgtttg gattggaagc agatgcaagg aagttttttt atcagtttgc tgttgtattt 1500 ttatgtcagc tatcatgttc cggtttatct atgttatcgg tggcggtatc gagagatttt 1560 tctaaagctt cattagtagg aaatatgaca tttacggtat tatcgatggg atgtggtttt 1620 ttcgttaatg ctaaagtaat gcccgtgtat gttcgttgga ttaaatatat tgcctttacg 1680 tggtattcat tcggtactct catgtcaagc acctttacga attcgtactg tactacagat 1740 aatctcgatg agtgcttggg taaccagata ttggaagttt acgggtttcc taggaattgg 1800 ataaccgtac ctgccgttgt cttactttgc tggtctgtgg gatattttgt agtaggtgca 1860 attattttat atttgcacaa gattgatata actttacaaa atgaagtgaa atcgaagcaa 1920 aaaaaaatca aaaagaaatc cccaacagga atgaaacccg agattcagct gctagatgac 1980 gtgtatcatc agaaagattt ggaagcggag aaaggaaaaa atatacatat tactataaaa 2040 ttagaagata tagacttacg agtcattttt tctgcccctt tctcaaactg gaaagaaggc 2100 aacttccacc atgaaacaaa agaaattcta caatcagtta atgccatttt caaacctgga 2160 atgattaatg caattatggg gccatcaggg tctggaaagt cttctctgtt aaacctaatc 2220 tccggaagat taaaatcttc ggtctttgcc aaatttgaca cttcaggttc aataatgttc 2280 aatgatattc aagtttcaga gcttatgttt aaaaatgtat gctcgtatgt ctcgcaggat 2340 gatgaccacc ttttggcagc tttaaccgtt aaagaaaccc tgaaatatgc cgctgcatta 2400 agattgcatc atctgactga ggcagaacga atggagagaa ctgacaacct aataaggtct 2460 ttgggtttaa agcattgtga aaacaatatc attggtaatg aatttgttaa aggtataagc 2520 ggtggtgaaa aaagaagagt aactatgggt gtgcaattat taaacgatcc tccgatatta 2580 ttgctagatg aaccaacttc agggttagat agcttcacat ccgctactat actggaaatt 2640 ttggagaagt tatgtaggga acagggcaag acgatcatca ttaccattca tcaaccaagg 2700 tcagaattat tcaagagatt tggtaatgtt ttgctattag ctaaatcggg tagaactgct 2760 ttcaatggat caccagatga aatgattgct tatttcactg aattgggata taactgtcct 2820 tcgtttacga acgtggcaga tttctttctt gatttaattt cagttaatac ccagaacgaa 2880 cagaatgaaa taagctcaag ggcacgagta gaaaagatac ttagtgcatg gaaagctaat 2940 atggataacg aaagcctttc accaacccca atttctgaaa aacaacaata ctctcaggag 3000 tcatttttca cagaatacag cgagtttgta agaaaaccag ctaatttggt tttggcgtac 3060 atagtgaacg ttaaaaggca atttactacg acaaggagaa gttttgactc tttgatggcg 3120 cgtattgcac aaattccagg attaggtgtt attttcgcat tattctttgc cccagtcaag 3180 cataattata caagtattag caatcgtcta ggattggcac aagaatctac agcactatat 3240 tttgtgggca tgctggggaa cttggcatgt tatccaactg aaagagatta cttttacgaa 3300 gaatataatg ataatgttta tggtatagca ccttttttct tagcttatat gacattagag 3360 ttgccgctat ccgcattagc ttcagtgtta tacgcggtat ttacagtact ggcatgtggg 3420 ttaccaagaa ctgcaggcaa cttctttgca accgtctact gttcctttat tgttacctgt 3480 tgtggcgaag ctcttggtat aatgacaaat acatttttcg aaaggccagg cttcgtcgtt 3540 aactgcattt ccattatttt atccattggt actcagatgt cagggttaat gtcactaggc 3600 atgtcgagag tattaaaggg ttttaactat ttgaaccctg tagggtatac atctatgatc 3660 atcattaatt tcgcattccc aggtaatttg aaattaacct gcgaagatgg cgggaaaaat 3720 tcagacggta cttgtgaatt tgcgaatggc catgatgtgt tggtttctta tggtttagtc 3780 agaaatacgc aaaagtacct gggaatcatt gtgtgcgtag ccataattta tcgccttatt 3840 gcatttttta ttttaaaagc aaaattggag tggataaaat ggtga 3885
SEQ ID N0:100
Saccharomyces cerevisiae
MSQQENGDVA TELIENRLSF SRIPRISLHV RDLSIVASKT NTTLVNTFSM DLPSGSVMAV 60 MGGSGSGKTT LLNVLASKIS GGLTHNGSIR YVLEDTGSEP NETEPKRAHL DGQDHPIQKH 120 VIMAYLPQQD VLSPRLTCRE TLKFAADLKL NSSERTKKLM VEQLIEELGL KDCADTLVGD 180 NSHRGLSGGE KRRLSIGTQM ISNPSIMFLD EPTTGLDAYS AFLVIKTLKK LAKEDGRTFI 240 MSIHQPRSDI LFLLDQVCIL SKGNWYCDK MDNTIPYFES IGYHVPQLVN PADYFIDLSS 300 VDSRSDKEEA ATQSRLNSLI DHWHDYERTH LQLQAESYIS NATEIQIQNM TTRLPFWKQV 360 TVLTRRNFKL NFSDYVTLIS TFAEPLI IGT VCGWIYYKPD KSSIGGLRTT TACLYASTIL 420 QCYLYLLFDT YRLCEQDIAL YDRERAEGSV TPLAFIVARK I SLFLSDDFA MTMIFVS ITY 480 FMFGLEADAR KFFYQFAVVF LCQLSCSGLS MLSVAVSRDF SKASLVGNMT FTVLSMGCGF 540 FVNAKVMPVY VRWIKYIAFT WYSFGTLMSS TFTNSYCTTD NLDECLGNQI LEVYGFPRNW 600 ITVPAVVLLC WSVGYFWGA IILYLHKIDI TLQNEVKSKQ KKIKKKSPTG MKPEIQLLDD 660 VYHQKDLEAE KGKNIHITIK LEDIDLRVIF SAPFSNWKEG NFHHETKEIL QSVNAIFKPG 720 MINAIMGPSG SGKSSLLNLI SGRLKSSVFA KFDTSGSIMF NDIQVSELMF KNVCSYVSQD 780 DDHLLAALTV KETLKYAAAL RLHHLTEAER MERTDNLIRS LGLKHCENNI IGNEFVKGIS 840 GGEKRRVTMG VQLLNDPP IL LLDEPTSGLD SFTSATILE I LEKLCREQGK TIIITIHQPR 900 SELFKRFGNV LLLAKSGRTA FNGSPDEMIA YFTELGYNCP SFTNVADFFL DLI SVNTQNE 960 QNEISSRARV EKILSAWKAN MDNESLSPTP ISEKQQYSQE SFFTEYSEFV RKPANLVLAY 1020 IVNVKRQFTT TRRSFDSLMA RIAQIPGLGV IFALFFAPVK HNYTSISNRL GLAQESTALY 1080 FVGMLGNLAC YPTERDYFYE EYNDNVYGIA PFFLAYMTLE LPLSALASVL YAVFTVLACG 1140 LPRTAGNFFA TVYCSFIVTC CGEALGIMTN TFFERPGFVV NCISIILSIG TQMSGLMSLG 1200 MSRVLKGFNY LNPVGYTSMI I INFAFPGNL KLTCEDGGKN SDGTCEFANG HDVLVSYGLV 1260 RNTQKYLGII VCVAIIYRLI AFFILKAKLE WIKW 1294
SEQ ID NO:101
Saccharomyces cerevisiae
atggaatgcg tttcagtaga aggtttggat tcttcttttt tggagggcca aacctttggc 60 gatattttgt gtttaccatg gacaattatc aagggtatcc gtgagcggaa gaatcgcaat 120 aagatgaaga tcattttgaa gaatgtcagt ttgctggcta aatcaggaga gatggtcctt 180 gtcctaggaa gaccaggcgc tggctgtaca tcatttttaa agagcgctgc tggtgagacc 240 agtcagtttg caggtggtgt aacaacagga catatatcgt acgatggtat ccctcagaaa 300 gaaatgatgc aacattacaa gccagatgta atctataatg gtgagcaaga tgttcatttc 360 ccacatttga cagtaaaaca aactctagat tttgctattt cctgtaagat gcccgcaaaa 420 agagtcaata atgtaacgaa agaagagtat attactgcca atagagaatt ctatgctaaa 480 atttttggtt tgacgcatac tttcgatacc aaagttggta acgatttcat cagcggtgta 540 tctggaggtg agcgtaaacg cgtttccatt gctgaagcat tagcagcgaa aggttcaatt 600 tactgctggg ataatgctac aagaggtctt gactcttcta ccgcgctaga atttgcacga 660 gctattcgta ctatgacaaa tctgttaggt acaacggccc ttgttacggt ttaccaagcc 720 agtgaaaaca tttatgaaac ttttgataaa gtcactgttc tatacgctgg aagacaaata 780 ttttgcggca aaactactga agcaaaagat tattttgaaa acatgggtta cttgtgtcca 840 ccgagacaat cgactgctga atatttgacc gcaattactg atcctaatgg tctgcacgaa 900 ataaagcctg gctttgagta tcaagtacct cataccgctg atgaattcga aaaatactgg 960 cttgattccc cagaatatgc ccgcctaaaa ggtgaaattc agaagtacaa acatgaagtg 1020 aatactgaat ggaccaaaaa aacatacaat gagtctatgg cacaagaaaa gtcgaaaggt 1080 acaagaaaaa aatcttatta tacggtctct tattgggagc aaattagact ttgcactatt 1140 agaggttttc taaggattta cggtgataag tcatacaccg ttatcaacac atgcgctgct 1200 atagcacagg cttttatcac tgggtcattg ttctaccaag caccttcttc aactctaggg 1260 gccttttcta gaagtggcgt tctgtttttt tccctcttat attattcttt gatgggttta 1320 gctaatatta gtttcgagca caggccaata ttgcaaaaac acaaggtcta ttcactatat 1380 catccctcag ctgaagcgtt agcaagtacg atttcttctt ttccattcag aatgattggt 1440 ctaacatttt tcataatcat cctgtacttc ttagccggtt tgcatagaag cgccggtgct 1500 ttttttacta tgtatttgtt attgacaatg tgttcagaag ctattacaag tttgtttcag 1560 atggtttcat ctttatgcga tacattgtcc caggccaact ccattgccgg tgttgtgatg 1620 ttatctattg ccatgtattc gacgtacatg atacaattac cttcaatgca tccatggttt 1680 aagtggattt cgtacattct acccattaga tatgcatttg aatcgatgtt aaatgcagaa 1740 tttcatggaa gacatatgga ttgtggtggc actttggttc cttctggacc tgggtttgaa 1800 aacatcttgc cagaaaatca agtgtgtgct tttgttggtt caaggcctgg ccaatcttgg 1860 gtcctaggtg atgattattt gagggcccaa tatcaatatg agtacaaaaa tacttggaga 1920 aacttcggca tcatgtggtg tttcttaatt ggctacatcg tcttgagggc ggttttcact 1980 gagtacaaaa gtcctgtcaa aagtggtggt gatgctctgg tcgtcaagaa gggcacaaag 2040 aatgctatac aaagatcatg gagcagcaaa aatgacgaag agaaccttaa tgcctctata 2100 gcaacacagg atatgaaaga gatagcttca agtaacgacg atagcacaag tgcagacttt 2160 gaaggtttgg aatctaccgg ggtgtttatt tggaaaaatg tttctttcac aattcctcat 2220 tctagcggac aacgtaaact tctggacagc gtaagcggtt actgtgttcc tggtactttg 2280 acagcattaa taggtgagtc cggcgctggt aagaccacat tattaaacac tttggctcaa 2340 aggaacgtgg gaacgattac tggtgatatg ttagttgatg gtctcccaat ggacgctagt 2400 ttcaaaaggc gtaccggtta tgttcaacag caagatcttc atgttgctga acttactgtc 2460 aaagaatcgc tacaatttag tgctcgtatg cgtcggccac agtctattcc tgacgctgaa 2520 aagatggaat atgttgaaaa aattatatct attcttgaaa tgcaggagtt ctcagaagct 2580 cttgtcggcg aaattggtta cggcttgaat gttgaacaga gaaagaaact atcaattggc 2640 gttgagctag ttggcaagcc ggatctgtta ttgtttttgg acgaaccgac ctctggcttg 2700 gattcccaat ccgcgtgggc cgtcgtcaaa atgttaaaaa gattagctct agcaggtcaa 2760 tcaattttat gtactattca tcaaccatca gctactcttt ttgaacagtt tgacagatta 2820 ttgcttttgg gaaaaggcgg tcaaacaatt tattttggtg aaataggtaa aaattcaagt 2880 tctgtcatta agtatttcga aaagaacgga gccaggaaat gtcaacaaaa tgaaaatccg 2940 gcagagtata ttttagaagc cataggagct ggggccaccg cttctgttca acagaactgg 3000 cctgatatat ggcaaaaatc tcacgagtat gcaaacatta acgaaaaaat aaatgacatg 3060 attaaggact tatcttctac aactttgcat aaaacagcta caagggcctc taagtatgca 3120 acatcgtatt cctaccaatt ccatcatgtg ctgaaaagat ccagtttaac attttggaga 3180 aacttgaact acatcatggc caaaatgatg ttattgatga tcagtggctt gttcattggt 3240 tttacctttt tccatgtggg tgtaaatgct attggattac aaaatagctt atttgcctgt 3300 ttcatggcta tcgttatatc agctcctgca acaaaccaaa tacaggagcg tgctaccgtt 3360 gctaaggagc tatatgaagt tcgtgagtcc aaatctaata tgtttcattg gtctttactt 3420 ttgattaccc attatttaaa tgaattgcct taccatttat tgttttcaac aatttttttc 3480 gtttcatcat atttccctct gggtgtcttt accgaagcct ctaggtcaag tgttttttat 3540 ctgaactatg ccatactttt tcaactttac tatattggtc ttgctttaat gattctgtac 3600 atgtctccaa atctacaatc tgccaatgtt attgtaggtt ttatactttc gtttttgctc 3660 tctttctgcg gtgctgtcca acctgcctct ttaatgcctg gtttctggac attcatgtgg 3720 aaactatccc cttacacgta ttttttgcaa aatttagttg gattattgat gcatgacaaa 3780 cccgtaagat gttcaaagaa agagttgtct cttttcaacc ccccggtagg ccaaacatgt 3840 ggtgaattta ccaaaccgtt ttttgaattt gggactgggt atattgcaaa tccagatgca 3900 acagcagatt gcgcctattg tcagtacaaa gtaggtgatg aatacttggc gcgcataaat 3960 gctagcttta gttacttatg gagaaacttc ggtttcattt ag 4002
SEQ ID NO:102
Saccharomyces cerevisiae
MECVSVEGLD SSFLEGQTFG DILCLPWTII KGIRERKNRN KMKI ILKNVS LLAKSGEMVL 60 VLGRPGAGCT SFLKSAAGET SQFAGGVTTG HISYDGIPQK EMMQHYKPDV IYNGEQDVHF 120 PHLTVKQTLD FAISCKMPAK RVNNVTKEEY ITANREFYAK IFGLTHTFDT KVGNDFISGV 180 SGGERKRVSI AEALAAKGSI YCWDNATRGL DSSTALEFAR AIRTMTNLLG TTALVTVYQA 240 SENIYETFDK VTVLYAGRQI FCGKTTEAKD YFENMGYLCP PRQSTAEYLT AITDPNGLHE 300 IKPGFEYQVP HTADEFEKYW LDSPEYARLK GEIQKYKHEV NTEWTKKTYN ESMAQEKSKG 360 TRKKSYYTVS YWEQIRLCTI RGFLRIYGDK SYTVINTCAA IAQAFITGSL FYQAPSSTLG 420 AFSRSGVLFF SLLYYSLMGL ANISFEHRPI LQKHKVYSLY HPSAEALAST ISSFPFRMIG 480 LTFFIIILYF LAGLHRSAGA FFTMYLLLTM CSEAITSLFQ MVSSLCDTLS QANSIAGWM 540 LSIAMYSTYM IQLPSMHPWF KWISYILPIR YAFESMLNAE FHGRHMDCGG TLVPSGPGFE 600 NILPENQVCA FVGSRPGQSW VLGDDYLRAQ YQYEYKNTWR NFGIMWCFLI GYIVLRAVFT 660 EYKSPVKSGG DALWKKGTK NAIQRSWSSK NDEENLNAS I ATQDMKE IAS SNDDSTSADF 720 EGLESTGVFI WKNVSFTIPH SSGQRKLLDS VSGYCVPGTL TALIGESGAG KTTLLNTLAQ 780 RNVGTITGDM LVDGLPMDAS FKRRTGYVQQ QDLHVAELTV KESLQFSARM RRPQSIPDAE 840 KMEYVEKIIS ILEMQEFSEA LVGE IGYGLN VEQRKKLSIG VELVGKPDLL LFLDEPTSGL 900 DSQSAWAWK MLKRLALAGQ SILCTIHQPS ATLFEQFDRL LLLGKGGQTI YFGEIGKNSS 960 SVIKYFEKNG ARKCQQNENP AEYILEAIGA GATASVQQNW PDIWQKSHEY ANINEKINDM 1020 IKDLSSTTLH KTATRASKYA TSYSYQFHHV LKRSSLTFWR NLNYIMAKMM LLMISGLFIG 1080 FTFFHVGVNA IGLQNSLFAC FMAIVISAPA TNQIQERATV AKELYEVRES KSNMFHWSLL 1140 LITHYLNELP YHLLFSTIFF VSSYFPLGVF TEASRSSVFY LNYAILFQLY YIGLALMILY 1200 MSPNLQSANV IVGFILSFLL SFCGAVQPAS LMPGFWTFMW KLSPYTYFLQ NLVGLLMHDK 1260 PVRCSKKELS LFNPPVGQTC GEFTKPFFEF GTGYIANPDA TADCAYCQYK VGDEYLARIN 1320 ASFSYLWRNF GFI 1333
SEQ ID NO:103
Saccharomyces cerevisiae atgggaagcg aaccgtttca gaaaaagaat ttgggtctgc aaattaattc gcaagaaagt 60 ggaacaaccc gctcaacatt tcattcgcta gaagatctag gagatgatgt aattaatgaa 120 agctgggatc aggtgaacca aaagagagcc aatatagatc atgatgtctt tcatgagcac 180 cctgactctt ccccatcatt gtcagcacag aaagcaaaaa caaaagaaga ggaagttgct 240 gtaaagtcat cgaactccca gtcaagagac ccttctcctg atactcaagc acatattcca 300 tatacttatt tttccaagga tcaaagacta atcatttttg gaattatcat ttttatagga 360 tttttgggcc caatgtctgg aaacatatat ataccggctt taccattgct gcaaagggaa 420 tatgatgtaa gtgcaacaac aataaacgct acagtttctg tatttatggc tgttttttcc 480 gttggtccat tgttttgggg cgcactggcg gattttggtg gaaggaaatt cttatatatg 540 gtgtcgttat cactaatgtt aattgttaat atacttttgg ccgctgtacc agtcaatatt 600 gctgcccttt ttgttttaag aattttccaa gctttcgctt ccagctctgt gatttctctg 660 ggagctggca ctgtaacaga cgttgttcct ccaaaacaca ggggaaaggc catagcgtat 720 ttcatgatgg gtccaaacat gggtcctatt atagcaccca ttgttgctgg gcttatttta 780 atgaaaggaa attactggag atggcttttt ggtttcactt ctatcatgac aggaatagca 840 ttgatcttgg ttactgcttt acttccagaa acgctacgtt gtatagttgg taatggagac 900 cctaaatggg gtgacaaaaa agatgaacgt gaaaataacg aatctccatt cttcgaaggt 960 aataaaatat cacatcggcg tctgttccca gacattggta tccgtaaacc agtcaataat 1020 gatgctttct tccaagaaaa ttttccaaag ccgcctaaag caggtttgac actatattgg 1080 aaaatgatta aatgtcctcc aataataatc acgtccgtca gtactgcact cctgttctcc 1140 agttactatg cgttcagcgt cacgttttcg tattaccttg aacatgacta ccgttttact 1200 atgttagaaa ttggtgctgc ttatgtctgc ccaggtgtag ctatgttact aggatctcaa 1260 tctggtgggc acctctcaga ttatcttcgt tcacgctgga tcaaaagtca tcctaaaaag 1320 aaattcccgg cagagtttcg tttattactg aacctaattg gaattttact aacaatatgt 1380 ggcacaatag gatacggatg ggcaatcttc tttcattatc attttgtggt tcttttagtt 1440 ttttccgctc tcactgcctt tggtatgacc tggtgcagca acacatcaat gacatattta 1500 actgagttat tccccaaaag agctgctgga actgttgctg ttagtagttt ctttcgaaat 1560 gtgggcgctg ccattagttc cgctatcatt ttacagctct gtaacgcaat gggaattgga 1620 tggtgtttta cagggctcgg tctctgcagt tcaatttcat tgattggtat attatatctt 1680 ctcatttttc aaagaaaata tactgccaaa gaattttaa 1719
SEQ ID NO:104
Saccharomyces cerevisiae
MGSEPFQKKN LGLQINSQES GTTRSTFHSL EDLGDDVINE SWDQVNQKRA NIDHDVFHEH 60 PDSSPSLSAQ KAKTKEEEVA VKSSNSQSRD PSPDTQAHIP YTYFSKDQRL IIFGIIIFIG 120 FLGPMSGNIY IPALPLLQRE YDVSATTINA TVSVFMAVFS VGPLFWGALA DFGGRKFLYM 180 VSLSLMLIVN ILLAAVPVNI AALFVLRIFQ AFASSSVISL GAGTVTDWP PKHRGKAIAY 240 FMMGPNMGP I IAPIVAGLIL MKGNYWRWLF GFTSIMTGIA LILVTALLPE TLRCIVGNGD 300 PKWGDKKDER ENNESPFFEG NKISHRRLFP DIGIRKPVNN DAFFQENFPK PPKAGLTLYW 360 KMIKCPP I I I TSVSTALLFS SYYAFSVTFS YYLEHDYRFT MLEIGAAYVC PGVAMLLGSQ 420 SGGHLSDYLR SRWIKSHPKK KFPAEFRLLL NLIGILLTIC GTIGYGWAIF FHYHFWLLV 480 FSALTAFGMT WCSNTSMTYL TELFPKRAAG TVAVSSFFRN VGAAISSAII LQLCNAMGIG 540 WCFTGLGLCS SISLIGILYL LIFQRKYTAK EF 572
SEQ ID NO:105
Saccharomyces cerevisiae
atggttgcag aatttcaaat agcgagtgca cagtcatcgg cattgacatc tactgaagaa 60 gagcattgtt caataaactc agacaaagcc gcaaaactgg atctggaatt gaccagtgaa 120 cgaaaaaatg acggcaaaca atcgcatgaa gtgaccttca acgaggacat cgcggaccca 180 gaagatatag cacggcatat gagcaccgcc cgccggtatt atatttcctc gttaattaca 240 ttcacatcga tggtaattac gatgatctcg tccagctgga cgcttccttc aacgcacata 300 atcgaacact ttcatatttc gcacgaagtc agcactctgg gaattacgct ttatgtgttt 360 gggctcggta taggaccttt atttctgtct ccactaagtg aactatatgg acgaaggatc 420 acatttttat acgcccttac gctcagtatc atatggcagt gtttgaccat ttggtccaag 480 accattacgg gtgtcatgtt tggcagattt ctatccgggt tttttggttc agcctttcta 540 agtgtggctg gcggcgccat tgccgatata ttcgacaaag accaaattgg tattcctatg 600 gcaatataca ctacatcggc ctttttgggg ccctctttag gaccaatcat tggtggagcc 660 ttataccatc aaagttataa atggacattt atcacacttc tcatcacttc tggatgttgt 720 ctcgttatga tcatctttac catacctgaa acctacaaac caatgctgtt aatacgtaaa 780 gctaagagat tgaggaaaga gaaaaatgat caacggtatt atgctgtcct ggaagtcacc 840 cgcgaacaga cttccttact ttccgcaatc tttctctcaa ccaaaagacc ctttggcctg 900 ttacttcgag accgaatgat gggtgtgctt tgcttctata ctggattgga acttgccata 960 atatatttat atttcgtagc gtttccttat gtgttcaaga aactttacaa ctttgggccc 1020 atggagattg catgctccta tatcggtatt atggtcggca tgatactctc ggctcctact 1080 tgcttgttat tccaaaaaac gtttgagtgg agagtcaaaa gaaataatgg cgtcaagact 1140 cccgaaatga gattcgagcc tctgttctac ggcgcctttt tgaccccagt tgggctcttc 1200 atttttgcat tcacctgcta caagcatgtc cattggattg caccaattat aggcagtgca 1260 attttcggtt caggtgttta tttcgtcttt accggtgttt ttgcgtatac agtggatgcc 1320 tatagaagat atgcagcttc tggaatggct tgtaacacat ttgtgagatg cataatggcc 1380 ggtgtcttcc ctcttttcgg actccagatg tacaagtcaa tgggagtgaa ttgggccggc 1440 tttctcttgg caatggtgac cgtagcgatg attcctgtcc catttctgtt taccaaatac 1500 ggtgcccgac tgagagccaa gtcgccgtat gcatgggatg attga 1545
SEQ ID NO:106
Saccharomyces cerevisiae
MVAEFQIASA QSSALTSTEE EHCS INSDKA AKLDLELTSE RKNDGKQSHE VTFNEDIADP 60 EDIARHMSTA RRYYISSLIT FTSMVITMIS SSWTLPSTHI IEHFHISHEV STLGITLYVF 120 GLGIGPLFLS PLSELYGRRI TFLYALTLSI IWQCLTIWSK TITGVMFGRF LSGFFGSAFL 180 SVAGGAIADI FDKDQIGIPM AIYTTSAFLG PSLGPIIGGA LYHQSYKWTF ITLLITSGCC 240 LVMIIFTIPE TYKPMLLIRK AKRLRKEKND QRYYAVLEVT REQTSLLSAI FLSTKRPFGL 300 LLRDRMMGVL CFYTGLELAI IYLYFVAFPY VFKKLYNFGP MEIACSYIGI MVGMILSAPT 360 CLLFQKTFEW RVKRNNGVKT PEMRFEPLFY GAFLTPVGLF IFAFTCYKHV HWIAPIIGSA 420 IFGSGVYFVF TGVFAYTVDA YRRYAASGMA CNTFVRCIMA GVFPLFGLQM YKSMGVNWAG 480 FLLAMVTVAM IPVPFLFTKY GARLRAKSPY AWDD 514
SEQ ID NO:107
Saccharomyces cerevisiae
atgataaaaa atggtacatg cccctattgg gaacgtgatg atctttcgga atgtgctagg 60 cgagaatata tcgagtttaa atttcctcta tttatattgt tgactggtat gatatacgcg 120 ttttgcaaag tctttcgagc cttttatcta agggggaaaa atcatacaaa tgaagcgcca 180 gaatttgaag aacaaggtaa tggaaaccac gaatatgcaa ggttctcagt tttaagacta 240 aaatctgcat gggaaagccg tagcttttgt aatgttaata atagatctac tttcgacaaa 300 ttcaaaaaat ttatagaggg tgccttcatt gttttgcagc ttactatcca cctgtatatt 360 ttatctagta tgccaatgga taacaaaaag ttctttcacc aaggttttct ggttcaaatg 420 tttctctgga ttttactgct tgttgttatt acacttcgtt taatttcagc aagccagtca 480 tttcggtggg ttttagcatg taagcgtgat ttatgggctg tttcgtttta ctcatatgca 540 tccctgttta ctctttcgat tttacctctt cgttccgtct tcattggaaa gataaaagat 600 aaaattatgg tcaaatatat catttccgag actttcattg atttagcgct tttattatta 660 ttatcaacgt caagtataga aggaactagg tactcatttt tggttgaaaa tgagaacaaa 720 aagttgccac cggctcctac cgtctttggc cttcttactt tttctaggat tgatcggctt 780 atttggaaag catacaaaca ctgccttgga aacgctgata tttgggattt ggatattaat 840 aataagtcta tagcaatttt ggcgaacttt gaaatgtctt ctaaaaaagg aaggctcctt 900 ccaaacatta tttgttattt taaggctgtt ttcatctccc aattatttct ggcttttgta 960 tctagttttt tgaactttgt accctcattg ttgatgccaa gaatactatc atacgttaat 1020 gacccaaagt caaaatcatg gaacttggtg tctttatacg tctcatctat gctcgtcagt 1080 aaaatcattg ccacgacttg tagaggtcaa ggattgtttt taggtgaaaa gggtactatg 1140 caactaagaa cagttttgat atccaatatc tattccaaaa ccttaagaag aacaattcta 1200 aaagactcaa caacgtcact tcaaaaaaat gcgtcaacat cctttgaaga aaatcctgac 1260 tcttctgaag cggaacctag aaaaaagtct agtaggaaag acaactctgt gaacaatgtt 1320 atgtcaattg acgctttcaa ggtttctgaa gctatgaaca ctttttatct ggcatgtgaa 1380 gcagttttca tgacagttac ggccctaatg atactatatt ccctactggg atggtccgcc 1440 tttgctggta cctttgctct tcttgccatg attcctttga atttttggtg tgcaactttt 1500 tacgggaact atcaggccga tcaattaatc ctaactgaca agcgtacctc tgggattagt 1560 gaggctctga actcaatacg tgtaataaag ctactggcat gggaaaatct gttttatcaa 1620 aagattataa acgtaagaga tggggaaata agactcctta aaaagaaggc aacaattttc 1680 tttttgaacc atctcatttg gttctttggg cccactttgg tctctgcaat aacattctca 1740 gtgtttatta aattccaaaa tcagacactt actcctacaa tagcatttac agctctttct 1800 ttatttgcaa tattgaggac acccatggac caaattgctt cgactgtcag ccttttgata 1860 cagtctttta tttctcttga aagaatccag gattatctta atgaatcaga aacaagaaaa 1920 tatgagattt tggaacaaag caatactaaa tttggctttg aagatgcaag catggaatgg 1980 gaagctgctg aaacaagttt taaacttaag aacatttcta ttgattttaa gctgaatagt 2040 ctcaacgcaa ttataggtcc gactgggtca ggaaagtctt cgctattact tggactattg 2100 ggagaattga accttctttc tggaaaaata tacgtaccta cagttgaatc ccgcgacgac 2160 ttagagattg gtaaagacgg aatgacgaat tcaatggcat attgttctca aactccgtgg 2220 ttgattagtg gaacaattaa agataacgtt gtttttggag aaatcttcaa caaacaaaaa 2280 tttgatgatg taatgaaatc ctgttgtctt gacaaagata tcaaagcaat gacagctggc 2340 ataagaacag acgtgggtga tggaggattt tccttatctg gcggacagca gcaaaggatt 2400 gctttagcca gagcaattta ctcctcttcc aggtatttga tccttgatga ttgcttgagt 2460 gcagtagatc ctgaaactgc actttatata tatgaagagt gtttatgcgg ccccatgatg 2520 aaaggaagga cctgcatcat tacgagtcat aatatttctt tagttacgaa acgggctgac 2580 tggcttgtga ttttagatcg tggcgaggtg aaatcacagg gtaaaccatc ggacctcatt 2640 aaatctaatg agtttttgag ggaaagcata aacaatgatt caaaaaatac aactcacaat 2700 caaattgact tgaaaagatc aacaacatca aagaaaacta agaatggaga tcccgaggga 2760 gggaactcgc aagacgaagt gtgtgctgaa gttgaaaatt ttgaggaaac aaaaatggaa 2820 ggatcagtta aattttcagc ttacaaatgg ttagcagact atttcggggg actgggagtt 2880 gtttttgttt tcacctcgtc ctccatcctt attcatggaa tcacactgtc tcagggcttt 2940 tggctcagat actggctgga cactggatct tcagggagta agtctacctg gctttataga 3000 atagttgagg gtcactctaa catctatttc ttactgactt atattatcat aggtcttgtt 3060 tcttcatttt taacttctgg taaagtttgg atagcaataa tttcaggtac caacgtcacc 3120 aagaaaatat ttgcgaagct tctatccagt atcttatatg ccaagttacg ttttcataat 3180 gtcacgccga ctggaagaat aatgaacaga tttagcaagg atatggatat tattgatcaa 3240 caattgatcc ctaatttcga aggactctct tatagcgtcg ttgtttgtct gtggattata 3300 cttttaattg gatatgttac tcctcaattt ctgttatttg ctattccttt atgcgctctt 3360 tattataccg tatgtacgtt atatcttcgt gcatctagag agttgaagag aatagataac 3420 atcaatatct ctccaataca ccagttattc gctgaagcca tcaaaggagt aactacaatt 3480 agagcattag cagatgagcg taggtttatc actcaatctt tggttgcaat tgacagaagt 3540 aatgctccat ttttttatct caatatggcc accgagtgga tcacatatag agtggatata 3600 attgggacac ttgttctttt tagttcttct gtaatgatca taatgaaggc ctcatag 3657
SEQ ID NO:108
Saccharomyces cerevisiae
MIKNGTCPYW ERDDLSECAR REYIEFKFPL FILLTGMIYA FCKVFRAFYL RGKNHTNEAP 60 EFEEQGNGNH EYARFSVLRL KSAWESRSFC NVNNRSTFDK FKKFIEGAFI VLQLTIHLYI 120 LSSMPMDNKK FFHQGFLVQM FLWILLLWI TLRLISASQS FRWVLACKRD LWAVSFYSYA 180 SLFTLS ILPL RSVFIGKIKD KIMVKYIISE TF IDLALLLL LSTSSIEGTR YSFLVENENK 240 KLPPAPTVFG LLTFSRIDRL IWKAYKHCLG NADIWDLDIN NKSIAILANF EMSSKKGRLL 300 PNI ICYFKAV FISQLFLAFV SSFLNFVPSL LMPRILSYVN DPKSKSWNLV SLYVSSMLVS 360 KIIATTCRGQ GLFLGEKGTM QLRTVLISNI YSKTLRRTIL KDSTTSLQKN ASTSFEENPD 420 SSEAEPRKKS SRKDNSVNNV MSIDAFKVSE AMNTFYLACE AVFMTVTALM ILYSLLGWSA 480 FAGTFALLAM IPLNFWCATF YGNYQADQLI LTDKRTSGI S EALNSIRVIK LLAWENLFYQ 540 KIINVRDGEI RLLKKKATIF FLNHLIWFFG PTLVSAITFS VFIKFQNQTL TPTIAFTALS 600 LFAILRTPMD QIASTVSLLI QSFISLERIQ DYLNESETRK YEILEQSNTK FGFEDASMEW 660 EAAETSFKLK NISIDFKLNS LNAI IGPTGS GKSSLLLGLL GELNLLSGKI YVPTVESRDD 720 LEIGKDGMTN SMAYCSQTPW LISGTIKDNV VFGEIFNKQK FDDVMKSCCL DKD IKAMTAG 780 IRTDVGDGGF SLSGGQQQRI ALARAIYSSS RYLILDDCLS AVDPETALYI YEECLCGPMM 840 KGRTCI ITSH NISLVTKRAD WLVILDRGEV KSQGKPSDLI KSNEFLRESI NNDSKNTTHN 900 QIDLKRSTTS KKTKNGDPEG GNSQDEVCAE VENFEETKME GSVKFSAYKW LADYFGGLGV 960 VFVFTSSSIL IHGITLSQGF WLRYWLDTGS SGSKSTWLYR IVEGHSNIYF LLTYIIIGLV 1020 SSFLTSGKVW IAIISGTNVT KKIFAKLLSS ILYAKLRFHN VTPTGRIMNR FSKDMDI IDQ 1080 QLIPNFEGLS YSVWCLWII LLIGYVTPQF LLFAIPLCAL YYTVCTLYLR ASRELKRIDN 1140 INISPIHQLF AEAIKGVTTI RALADERRFI TQSLVAIDRS NAPFFYLNMA TEWITYRVDI 1200 IGTLVLFSSS VMIIMKAS 1218
SEQ ID NO:109
Saccharomyces cerevisiae
atgaactttt taagttttaa gactacaaaa cactatcaca ttttcaggta cgtgaacata 60 cggaatgact acaggctgtt aatgataatg ataataggta ccgtggcaac aggcctagtg 120 ccggcaatta cttctatcct gacgggcaga gtgttcgatc tactatcagt tttcgtggct 180 aatgggtcac atcaaggttt gtattcccaa ctagtacaga ggtcaatggc agtaatggca 240 cttggtgcgg cttctgtgcc agtaatgtgg ctttctctaa caagttggat gcacatcggc 300 gagagacaag gctttagaat acggtcacag atattggagg catatttgga ggaaaagcca 360 atggaatggt acgacaataa tgaaaaattg ttaggagatt ttactcaaat caacagatgt 420 gtggaagagc taagatcaag ctccgcagag gcatcagcca taactttcca gaatttagtt 480 gcaatatgtg cgcttctggg gacgtcattc tactattctt ggtcattaac tttaattatt 540 ctttgcagct ctccaataat cacatttttt gcagtggtgt tttccagaat gattcatgta 600 tattcagaga aggagaattc tgaaacgagt aaagcagccc aattacttac atggtcgatg 660 aatgccgctc aattagtgag attatattgt acacaacgtc tagaaaggaa aaaattcaag 720 gaaatcatac taaattgtaa cactttcttc atcaagagtt gcttttttgt tgctgcaaac 780 gctgggatct tgagattttt gacgttgact atgtttgtac agggattctg gtttggttcc 840 gcaatgatca aaaagggcaa gctgaacatt aacgatgtaa tcacttgctt ccattcatgc 900 attatgctgg gctcgacttt aaataataca ttacaccaaa tagttgttct tcaaaaaggc 960 ggagtggcta tggaaaaaat catgactcta ttaaaagatg gatccaagcg aaatccttta 1020 aataaaactg tagcccacca atttccacta gattatgcca ccagtgatct aacatttgct 1080 aatgtttcgt tttcttatcc aagcagacct tcggaagcag ttttaaagaa cgttagttta 1140 aatttctctg caggacaatt tactttcata gtaggaaaat caggctcagg taaatctaca 1200 ttatccaact tattattaag gttctacgat ggctataatg gatcgatatc tatcaatggc 1260 cacaatatcc aaacaatcga ccaaaaattg ctaattgaaa atatcaccgt cgtagaacag 1320 cgttgtaccc tatttaatga tactcttaga aaaaatattc ttttaggttc aacagattcg 1380 gtaagaaatg ccgattgcag caccaatgaa aatagacatt taatcaaaga tgcctgtcaa 1440 atggctcttc tggatagatt tattctagac cttcccgatg gactggaaac attaattggg 1500 actgggggtg tcacactaag tggcgggcaa caacaaagag ttgctatagc acgtgcattc 1560 atcagagata ctccaatatt attcttagac gaagctgtat cggctctaga tattgttcat 1620 cgcaacctgt tgatgaaggc aattaggcat tggaggaaag gaaagactac aatcatattg 1680 acgcatgagt tgagccaaat tgaatctgat gactatttat atttaatgaa ggaaggtgaa 1740 gttgttgaaa gcggcaccca gtctgaactt ctagccgatc cgaccactac atttagcaca 1800 tggtatcacc tacagaatga ctactctgat gcgaaaacta ttgtagatac agagactgaa 1860 gaaaaatcta tacacactgt ggaaagtttt aactctcaat tggaaacacc aaaacttgga 1920 tcatgcttaa gtaatctggg atatgatgag acagatcagt tgtcctttta cgaagcaatc 1980 tatcaaaaaa gatcgaacgt tagaacaaga agggttaaag ttgaagagga aaatattggg 2040 tatgcactaa aacaacaaaa gaacaccgaa agttcaacag ggccacaact tctgagcatt 2100 attcagatta tcaaaagaat gattaaaagc ataagataca aaaaaattct aatcttggga 2160 ctgctatgtt ctcttatcgc aggcgccaca aatcccgtct tttcatacac attcagtttc 2220 ttactagaag gaattgtccc atccacggat ggaaaaactg gctcttcaca ttatttggcg 2280 aaatggtcgc ttcttgttct tggtgtggct gcggcagatg gtattttcaa ttttgctaaa 2340 ggattcctat tagattgctg cagtgaatac tgggttatgg atcttagaaa tgaagttatg 2400 gaaaaactga cgagaaagaa tatggactgg ttttctggtg aaaacaacaa ggcttctgaa 2460 atttctgctc tagtcttgaa tgatttgcga gatttgaggt ctttggtctc tgaatttttg 2520 agtgcaatga ctagtttcgt taccgtatca acgattggac taatttgggc gttagtatcg 2580 ggctggaagt taagtttggt ttgtatttcg atgtttccac tcataattat attttcagca 2640 atatatggag gcattttaca aaaatgcgaa acagattata agacatctgt tgctcagtta 2700 gaaaactgcc tgtaccagat tgtcactaac attaaaacca ttaagtgctt acaagctgaa 2760 tttcattttc aattgaccta ccatgacttg aagataaaaa tgcaacaaat tgcctccaaa 2820 cgcgccattg ccacaggatt tggtatatct atgacaaaca tgattgtcat gtgtatccaa 2880 gctattattt actactatgg cctaaagctg gttatgattc acgagtacac ctcaaaggaa 2940 atgtttacga ctttcacttt gttattattc actattatgt catgcactag cctagtaagt 3000 cagatacccg atataagtag aggccaacgt gctgccagtt ggatctatag gattcttgat 3060 gaaaagcata ataccctaga ggttgaaaac aataatgcta gaacagtggg aatagctggt 3120 cacacctacc atggcaaaga aaaaaaacca atcgtttcaa ttcaaaattt gacatttgcc 3180 tatccatctg cacctaccgc ctttgtttac aaaaacatga attttgacat gttttgcgga 3240 cagacgttag gtatcattgg tgaatcaggc acaggaaagt ctacacttgt gcttttatta 3300 acaaaacttt ataattgtga agtaggcaaa attaaaatag acggtacgga cgtaaatgac 3360 tggaatttga caagtttaag aaaagaaatt tcagtggttg agcaaaaacc tttattattc 3420 aatggaacca tcagagataa cctaacttat ggtttacaag atgaaatact tgaaattgaa 3480 atgtatgatg cattaaaata cgtaggaatc catgactttg taatttcatc acctcagggc 3540 ttggatacac gtattgatac aactttactg tcaggtggac aagcgcaaag gctttgcata 3600 gccagagcac ttctgagaaa atcaaaaatt ctgattttag atgagtgtac ttcagccttg 3660 gattctgtca gctcctctat catcaatgag atcgtcaaaa aaggtccacc tgctctacta 3720 acaatggtta taacgcatag tgaacaaatg atgaggtctt gtaactcgat tgcagttctt 3780 aaagatggta aagtggttga gcgaggtaac ttcgacactt tatataataa tcgcggggaa 3840 ttattccaaa ttgtttccaa ccaaagcagt taa 3873
SEQ ID NO:1 10
Saccharomyces cerevisiae
MNFLSFKTTK HYHIFRYVNI RNDYRLLMIM I IGTVATGLV PAITSILTGR VFDLLSVFVA 60 NGSHQGLYSQ LVQRSMAVMA LGAASVPVMW LSLTSWMHIG ERQGFRIRSQ ILEAYLEEKP 120 MEWYDNNEKL LGDFTQINRC VEELRSSSAE ASAITFQNLV AICALLGTSF YYSWSLTLII 180 LCSSPIITFF AVVFSRMIHV YSEKENSETS KAAQLLTWSM NAAQLVRLYC TQRLERKKFK 240 EIILNCNTFF IKSCFFVAAN AGILRFLTLT MFVQGFWFGS AMIKKGKLNI NDVITCFHSC 300 IMLGSTLNNT LHQIVVLQKG GVAMEKIMTL LKDGSKRNPL NKTVAHQFPL DYATSDLTFA 360 NVSFSYPSRP SEAVLKNVSL NFSAGQFTFI VGKSGSGKST LSNLLLRFYD GYNGSISING 420 HNIQTIDQKL LIENITWEQ RCTLFNDTLR KNILLGSTDS VRNADCSTNE NRHLIKDACQ 480 MALLDRFILD LPDGLETLIG TGGVTLSGGQ QQRVAIARAF IRDTP ILFLD EAVSALD IVH 540 RNLLMKAIRH WRKGKTTI IL THELSQIESD DYLYLMKEGE VVESGTQSEL LADPTTTFST 600 WYHLQNDYSD AKTIVDTETE EKSIHTVESF NSQLETPKLG SCLSNLGYDE TDQLSFYEAI 660 YQKRSNVRTR RVKVEEENIG YALKQQKNTE SSTGPQLLSI IQIIKRMIKS IRYKKILILG 720 LLCSLIAGAT NPVFSYTFSF LLEGIVP STD GKTGSSHYLA KWSLLVLGVA AADGIFNFAK 780 GFLLDCCSEY WVMDLRNEVM EKLTRKNMDW FSGENNKASE I SALVLNDLR DLRSLVSEFL 840 SAMTSFVTVS TIGLIWALVS GWKLSLVCIS MFPLIIIFSA IYGGILQKCE TDYKTSVAQL 900 ENCLYQIVTN IKTIKCLQAE FHFQLTYHDL KIKMQQIASK RAIATGFGIS MTNMIVMCIQ 960 AI IYYYGLKL VMIHEYTSKE MFTTFTLLLF TIMSCTSLVS QIPDISRGQR AASWIYRILD 1020 EKHNTLEVEN NNARTVGIAG HTYHGKEKKP IVSIQNLTFA YPSAPTAFVY KNMNFDMFCG 1080 QTLGIIGESG TGKSTLVLLL TKLYNCEVGK IKIDGTDVND WNLTSLRKEI SWEQKPLLF 1140 NGTIRDNLTY GLQDEILEIE MYDALKYVGI HDFVISSPQG LDTRIDTTLL SGGQAQRLCI 1200 ARALLRKSKI LILDECTSAL DSVSSSIINE IVKKGPPALL TMVITHSEQM MRSCNSIAVL 1260 KDGKWERGN FDTLYNNRGE LFQIVSNQSS 1290
SEQ ID NO:1 1 1
Saccharomyces cerevisiae
atggctggta atcttgtttc atgggcctgc aagctctgta gatctcctga agggtttgga 60 cctatatcct tttacggtga ctttactcaa tgcttcatcg acggtgtgat cctaaatcta 120 tcagcaattt tcatgataac cttcggtatc agagatttag ttaacctttg caagaaaaaa 180 cactctggca tcaaatatag gcggaattgg attattgtct ctaggatggc actagttctg 240 ttggagatag cgtttgtttc acttgcgtct ttaaatattt ctaaagaaga agcggaaaac 300 tttaccattg taagtcaata tgcttctaca atgttatctt tatttgttgc tttagcctta 360 cactggatag aatacgatag atcagttgta gccaatacgg tacttttatt ctattggctt 420 tttgaaacat tcggtaattt tgctaaacta ataaatattc taattagaca cacctacgaa 480 ggcatttggt attccggaca aacgggtttc atactaacgt tattccaagt aataacatgt 540 gccagtatcc tgttacttga agctcttcca aagaagccgc taatgccaca tcaacacata 600 catcaaactt taacaagaag aaaaccaaat ccatacgata gcgcaaacat attttccagg 660 attaccttct cttggatgtc aggtttgatg aaaactggct atgaaaaata cttagtggaa 720 gcagatttat ataaattacc gaggaacttt agtagtgaag aactctctca aaaattggag 780 aaaaactggg aaaatgagtt gaagcaaaaa tcaaatcctt cattatcatg ggctatatgc 840 agaacttttg gatctaaaat gcttttagcc gcattcttta aagcaattca tgatgttcta 900 gcatttactc aaccacaact actaaggatt ttaatcaagt tcgtcacaga ctataacagt 960 gagagacagg atgaccattc ttctcttcaa gggtttgaaa ataaccaccc acaaaaatta 1020 cccattgtaa gagggttttt gattgcgttt gctatgtttc tggtgggctt tactcagaca 1080 tctgtcctgc atcaatattt cctgaatgtc ttcaacacag gcatgtatat taagagcgcc 1140 ctaacggctt taatatatca aaaatcctta gtgctatcta atgaggcttc tggactttcc 1200 tctaccggtg acattgtcaa tctcatgagt gtggatgttc aaaaattaca agatttaaca 1260 caatggctaa atttaatatg gtcagggcct tttcaaatca ttatttgctt atattctctg 1320 tataagttgt tgggaaattc catgtgggtt ggcgtgatta tactagttat tatgatgcca 1380 ttgaactcat ttttgatgag gatacaaaag aagttgcaaa aatcccagat gaagtacaaa 1440 gatgaaagga cccgtgttat aagtgaaata ctaaacaata ttaaatcttt gaagttatat 1500 gcatgggaga agccttatag ggaaaagcta gaagaagtaa gaaataacaa agagttaaaa 1560 aatcttacaa aactaggatg ttatatggct gtgacaagtt ttcagttcaa tatagtacca 1620 ttccttgttt catgttgtac ctttgctgta tttgtttata ctgaggatag agcattgact 1680 actgacttag ttttccctgc tttgactctg ttcaacctgc tctcattccc actaatgatt 1740 attcctatgg ttttaaattc ttttatcgaa gcttctgttt ctattggtag attatttaca 1800 ttctttacca atgaagagct acaaccagat tcggttcagc gtttaccaaa agtaaaaaat 1860 attggcgatg tagccattaa cattggagat gatgctacct ttttatggca acggaaaccg 1920 gaatacaaag tagccttaaa gaatattaat ttccaagcta aaaaaggaaa tttgacctgt 1980 attgttggta aagttggcag tggtaaaaca gctctattgt catgcatgtt aggtgatcta 2040 ttcagggtta aaggtttcgc caccgttcat ggttctgttg cttatgtttc acaagttcca 2100 tggataatga atggtactgt aaaggaaaac attttatttg ggcatagata cgacgcggaa 2160 ttttacgaaa aaacgatcaa ggcctgtgcg ttaactattg atcttgcaat tttgatggat 2220 ggagataaga cattagttgg cgagaaaggg atctccttat ctggaggaca aaaagctcgt 2280 ttgtctttag caagagcagt ttatgcgaga gctgacactt atttacttga tgatcctttg 2340 gcagctgttg atgaacacgt tgccaggcac ttgatcgaac atgtgttggg tccaaatggt 2400 ttattacata caaaaacgaa ggtattagcc actaataagg tgagcgcgtt atccatcgca 2460 gattctattg cattattaga taatggagaa atcacacagc agggtacata tgatgagatt 2520 acgaaggacg ctgattcgcc attatggaaa ttgctcaaca actatggtaa aaaaaataac 2580 ggtaagtcga atgaattcgg tgactcctct gaaagctcag ttcgagaaag tagtatacct 2640 gtagaaggag agctggaaca actgcagaaa ttaaatgatt tggattttgg caactctgac 2700 gccataagtt taaggagggc cagtgatgca actttgggaa gcatcgattt tggtgacgat 2760 gaaaatattg ctaaaagaga gcatcgtgaa cagggaaaag taaagtggaa catttaccta 2820 gagtacgcta aagcttgcaa cccgaaaagc gtttgtgtat tcatattgtt tattgttata 2880 tcgatgttcc tctctgttat gggtaacgtt tggttgaaac attggtctga agttaatagc 2940 cgctatggat ctaatccaaa tgccgcgcgt tacttggcca tttattttgc acttggtatt 3000 ggttcagcac tggcaacatt aatccagaca atcgttctct gggttttttg taccattcat 3060 gcctccaaat atttacacaa cttgatgaca aactctgtgt tgagagcccc aatgacgttt 3120 tttgaaacaa caccaatcgg tagaattcta aacagattct caaatgacat atacaaagtg 3180 gatgctttat taggaagaac attttctcag tttttcgtca atgcagtgaa agtcacattc 3240 actattacgg ttatctgtgc gacgacatgg caatttatct tcattatcat tccactaagt 3300 gtgttttaca tctactacca gcagtattac ctgagaacat caagggagtt gcgtcgttta 3360 gactctatta ctaggtctcc aatctactct catttccaag agactttggg tggccttgca 3420 acggttagag gttattctca acagaaaagg ttttcccaca ttaatcaatg ccgcattgat 3480 aataacatga gtgcgttcta tccctctatc aatgctaacc gttggctagc atataggttg 3540 gaacttattg gttcaattat cattctaggt gctgcaactt tatccgtttt tagactaaaa 3600 caaggcacat taacggcagg tatggtgggt ttatcattaa gctatgcttt acaaatcact 3660 caaacgttaa attggattgt tagaatgact gtggaagttg aaacgaatat tgtttcagtg 3720 gaaagaataa aggaatatgc tgatttgaag agcgaggcac ctttaatagt tgaaggccac 3780 agaccaccca aagaatggcc gagccagggt gatataaagt ttaataatta ttccactcgt 3840 tataggccgg agcttgatct tgttctgaag cacattaata tacacattaa accaaatgaa 3900 aaagttggta tcgtgggtag aacgggtgcg ggaaaatcct cattaacgct agcattattc 3960 aggatgattg aggctagcga gggaaacatc gtaatcgaca acattgccat caacgagatt 4020 gggttatatg atttgagaca taaattgtca atcatacctc aggattctca agtttttgag 4080 ggcactgttc gtgagaacat tgatcccatt aaccaataca ctgatgaagc tatttggagg 4140 gcattggaac tttctcattt gaaagaacac gtgctatcaa tgagcaatga cggattagat 4200 gcccaactaa ccgaaggtgg tggcaactta agtgttggac aaagacaatt attatgtctt 4260 gcaagagcaa tgttggttcc atcaaagatt ttggtgcttg atgaagccac ggccgcagtc 4320 gacgtggaga cagataaagt cgtccaagag acgattcgta ctgctttcaa ggacagaact 4380 atcttgacca tcgcgcatag actgaacacg ataatggaca gtgatagaat catagtgttg 4440 gacaatggta aagtagccga gtttgactct ccgggccagt tattaagtga taacaaatca 4500 ttgttctatt cactgtgcat ggaggctggt ttggtcaatg aaaattaa 4548
SEQ ID NO:1 12
Saccharomyces cerevisiae
MAGNLVSWAC KLCRSPEGFG PISFYGDFTQ CF IDGVILNL SAIFMITFGI RDLVNLCKKK 60 HSGIKYRRNW IIVSRMALVL LEIAFVSLAS LNISKEEAEN FTIVSQYAST MLSLFVALAL 120 HWIEYDRSW ANTVLLFYWL FETFGNFAKL INILIRHTYE GIWYSGQTGF ILTLFQVITC 180 ASILLLEALP KKPLMPHQHI HQTLTRRKPN PYDSANIFSR ITFSWMSGLM KTGYEKYLVE 240 ADLYKLPRNF SSEELSQKLE KNWENELKQK SNPSLSWAIC RTFGSKMLLA AFFKAIHDVL 300 AFTQPQLLRI LIKFVTDYNS ERQDDHSSLQ GFENNHPQKL P IVRGFLIAF AMFLVGFTQT 360 SVLHQYFLNV FNTGMYIKSA LTALIYQKSL VLSNEASGLS STGD IVNLMS VDVQKLQDLT 420 QWLNLIWSGP FQIIICLYSL YKLLGNSMWV GVIILVIMMP LNSFLMRIQK KLQKSQMKYK 480 DERTRVISEI LNNIKSLKLY AWEKPYREKL EEVRNNKELK NLTKLGCYMA VTSFQFNIVP 540 FLVSCCTFAV FVYTEDRALT TDLVFPALTL FNLLSFPLMI IPMVLNSFIE ASVSIGRLFT 600 FFTNEELQPD SVQRLPKVKN IGDVAINIGD DATFLWQRKP EYKVALKNIN FQAKKGNLTC 660 IVGKVGSGKT ALLSCMLGDL FRVKGFATVH GSVAYVSQVP WIMNGTVKEN ILFGHRYDAE 720 FYEKTIKACA LTIDLAILMD GDKTLVGEKG ISLSGGQKAR LSLARAVYAR ADTYLLDDPL 780 AAVDEHVARH LIEHVLGPNG LLHTKTKVLA TNKVSALSIA DSIALLDNGE ITQQGTYDEI 840 TKDADSPLWK LLNNYGKKNN GKSNEFGDSS ESSVRESSIP VEGELEQLQK LNDLDFGNSD 900 AISLRRASDA TLGSIDFGDD ENIAKREHRE QGKVKWNIYL EYAKACNPKS VCVFILFIVI 960 SMFLSVMGNV WLKHWSEVNS RYGSNPNAAR YLAIYFALGI GSALATLIQT IVLWVFCTIH 1020 ASKYLHNLMT NSVLRAPMTF FETTPIGRIL NRFSNDIYKV DALLGRTFSQ FFVNAVKVTF 1080 TITVICATTW QFIFIIIPLS VFYIYYQQYY LRTSRELRRL DSITRSPIYS HFQETLGGLA 1140 TVRGYSQQKR FSHINQCRID NNMSAFYPSI NANRWLAYRL ELIGSIIILG AATLSVFRLK 1200 QGTLTAGMVG LSLSYALQIT QTLNWIVRMT VEVETNIVSV ERIKEYADLK SEAPLIVEGH 1260 RPPKEWPSQG DIKFNNYSTR YRPELDLVLK HINIHIKPNE KVGIVGRTGA GKSSLTLALF 1320 RMIEASEGNI VIDNIAINEI GLYDLRHKLS IIPQDSQVFE GTVRENIDP I NQYTDEAIWR 1380 ALELSHLKEH VLSMSNDGLD AQLTEGGGNL SVGQRQLLCL ARAMLVP SKI LVLDEATAAV 1440 DVETDKWQE TIRTAFKDRT ILTIAHRLNT IMDSDRIIVL DNGKVAEFDS PGQLLSDNKS 1500 LFYSLCMEAG LVNEN 1515
SEQ ID NO:1 13
Saccharomyces cerevisiae
atgcatcacg tactcaattc aacgagacct gaccatcggt tttggtttta cgatgacgta 60 actcagtacg gcagaacaaa gtaccttaat tattatactc ccttagtgct tttaatattc 120 acggtgttgt tcataactta taacatatgg aaacattatt actactatga cgttctacac 180 ttgaagcaaa aaaatccaat tgatgaactt ttatactcat ctaccgacga agacgaacag 240 agtccgttga taaataataa caccatcaca actaattacg tggacaataa ttgtacaaaa 300 gacgcactaa aaaatagaca cttttctctg gaaaagctga aatcagttaa ggtaaatgga 360 gaacctcatg gtacacctga aatagttaga agaggcttca ttgaaaaatc aagaattatc 420 cttgaatttt ttttggtact ttctcaagtt ataatacatt cttttatact attacactat 480 gtcaataaaa acccagagtt cacccaacaa ggtacgatta ctggtttggt agaatggtgt 540 gcattgttta tcattgtctc tttacgttta gcaaatgtca accaaaattt caaattcata 600 aacaaatatc ccggaaatct atggtcggta tcgttcataa actatttggc cttgtttatc 660 tctatgattt taccctttcg ttccattttc attcaccata taaacagtcc tatttcgaga 720 aagtattaca tttcacaaat atcgattaac cttgcccttt tcttgctact cttttttgcg 780 aggataagaa ataattttgc catcatctat aaaactgaca gctggattac tccatctcca 840 gaaccagtca cttctattgc tggattcata tgctgggctt ggttagacag ttttgtttgg 900 aaagcacaca aagttagtat caaagttaag gatatttggg gattgatgat gcaagattat 960 tctttctttg tagtgaaaaa attcagatat tttgttgacc ataaggttaa aaggaagcgt 1020 attttttcac taaacctttt ctttttcttt tcgaattatt tggtactaca atgcttttgg 1080 gcatttttag gcagcgtcct ttcttttatt ccaacagttt tactaaaaag gatcctggaa 1140 tatgttgaag atcagtcatc tgctccctca aatttagctt ggttttacgt cactgtcatg 1200 tttgtaggta ggatattagt ggctatttgt caagcacagg cactattctt tggaaggaga 1260 gtttgtatca gaatgaaaag tattataatc tctgagattt atactaaagc tttgagaaga 1320 aagatttcca ccaacaagac taaaccttct aatgaggatc cacaggaaat taatgatcaa 1380 aaaagtatca acggcgatga ggagtccact tcttctgcca accttggagc gattattaat 1440 ctcatggcta ttgacgcttt caaggtttct gaaatttgtg gttaccttca ttcatttttg 1500 gaggcttttg ttatgactgt tgtcgcactg gcgctgttat accgtctgct gggttttgct 1560 gcgattgtgg gtgtcttaat cattgtcgca atgcttccac tgaattataa gcttgccaag 1620 tacatcggcg atttgcaaaa gaaaaacttg gctgtcacag ataatcgtat tcaaaagttg 1680 aatgaagctt ttcaagctat cagaatcatc aaatactttt catgggagga gaattttgaa 1740 aaagatatca atactattag agaaaatgaa ttatctctac tgctaatgag atccattgtt 1800 tggtctatca gttcctttct ttggtttgtt accccaacta ttgtaacagc cgcctcattt 1860 gcttactaca tttacgttca aggcgaggta ttaaccactc cggttgcctt tactgcactt 1920 tctttattca cgttattaag agatcctttg gatcgtttgt ctgatatgtt aagttttgtc 1980 gttcaatcaa aggtctcgtt agatagagtt caggatttct taaatgaaaa tgatacaaag 2040 aaatacgatc aactgaccat agatccaaat ggaaacagat ttgcctttga gaattccacc 2100 atctcttggg ataaggacaa tcaagatttc aaattgaaag acttgaacat tgaattcaaa 2160 actggtaaac taaatgtcgt tattggcccc actggttctg gtaagacatc cctactaatg 2220 gcattattag gtgaaatgta tctactcaat ggtaaggttg tcgtcccagc tctagaacca 2280 aggcaggaac taatagtaga tgcgaatgga accactaact ctatcgctta ttgttctcag 2340 gccgcttggc ttctaaatga tactgtaaaa aacaacattt tattcaatag tccattcaat 2400 gaggcaagat ataaagctgt tgtcgaagca tgcggattga aacgtgattt tgagatcttg 2460 aaagccggtg acctaacaga aatcggtgag aagggtatta ctttatccgg tggtcaaaag 2520 cagagagtct cccttgctag agccttgtat tccaatgcta ggcatgtcct tttggatgat 2580 tgtttaagtg cagttgattc acatactgca tcttggattt acgataattg tattactgga 2640 ccattaatgg aagataggac atgtattttg gtttcacaca acatcgcctt gaccttgaga 2700 aatgctgaat tggtagtctt actagaagat ggtagggtta aagaccaagg tgatccaata 2760 gacatgttac agaagggttt gtttggtgaa gacgagttag tgaaaagtag tatcttatcg 2820 cgtgcaaact cttctgctaa cttagccgca aaaagtagca caagcttaag taacctaccc 2880 gctgtcaaag aacaacaagt tagcgtgaac aacaactcct cccactttga agccaagaaa 2940 cttcaaaaat cattgaggac agaagctgaa cgcactgaag atggtaaatt aataaaagag 3000 gaaacaaaag aagaaggtgt ggttggtctg gatgtctata aatggtattt gaaaattttt 3060 ggtggttgga agattgtttc atttttagct tccttgttct tgatagccca acttttgtat 3120 atcggacaat catggtgggt tcgtgcctgg gcgtcacaca atgtcattgc taaaattatt 3180 cccagagcac aacgcgctat tgcatttatc tccaagaagg ctagtcatct aattgattgg 3240 aggggctcgt cccaaatcag tatggcgtca gctgaaaacc aaccttccag tggacactct 3300 acaatgtatt atctggtttt gtacctaatc attggttttg ctcaggcttt attgggagct 3360 ggtaaaacta ttttaaattt cgttgctggt atcaacgcgt caagaaagat atttaatatg 3420 attctaaaca aggttttaca ctccaagata agattttttg atgctactcc aacaggtaga 3480 attatgaaca ggttttccaa ggatatcgaa gctattgatc aggaactgac accttatata 3540 caaggcgcct tctattcgct tattgaatgt ttatccaccg tcatactgat tacatttatt 3600 actccacagt tcttatctgt tgctatagtt gtttcaatat tgtattattt tgttgggtat 3660 ttctacatgg caggttctcg tgaattgaaa agatttgaat ctatttctag atcaccgatc 3720 tatcaacatt tttccgagac tcttgtcggt gttaccacca ttcgtgcgtt tggtgatgaa 3780 gggaggttta tgcaagagaa tttacacaaa attgatgaaa ataataaacc attcttctac 3840 ttgtgggttg ctaatcgttg gttggccttt aggatagata tgattggatc cttggtcatt 3900 tttggagctg gtttgttcat tctattcaat attaataatc tggattcggg tatggctggt 3960 atatctttga cgtatgctat ttccttcact gaaggggcat tgtggttggt aagattgtac 4020 tctgaagtgg agatgaatat gaattctgtt gaaagagtca aagaatatat ggaaattgag 4080 caagagccat acaacgaaca caaagaaata ccgccaccac aatggccaca agacggtaaa 4140 atcgaggtta atgatttatc attacggtat gctccaaatc tacctagagt aattaaaaat 4200 gtttcatttt ctgtcgacgc tcagtctaag atcggtattg ttggtagaac cggtgccggt 4260 aaatcaacta ttattaccgc cttgttcaga tttttggaac ctgagacagg ccatatcaaa 4320 attgataata tcgatatttc cggtgttgat ttacaaagac tgcgtcgttc tataactatt 4380 attccacaag atccaacact attctcaggg acgattaaaa caaacctaga cccatatgat 4440 gaatttagtg acagacagat ctttgaagct ttgaaacgtg taaatctaat tagtgaagaa 4500 caattacaac aaggcgccac aagagaaaca agcaatgagg cttcgtcgac taactccgaa 4560 aatgttaata aattcttgga tttaagtagt gaaattagcg aaggtggatc caatttgtct 4620 caaggacagc gccaattgat gtgccttgct agatctttac tgaggagtcc aaagatcata 4680 ttgctggacg aagctacagc gtccatcgac tacagttccg atgcgaagat ccaagaaacc 4740 ataagaaaag agttccaagg tagtacaatt ttgacaattg cacataggtt gagatctgtt 4800 atcgattatg ataaaatttt ggtgatggat gcaggtgagg taaaagagta tgatcaccca 4860 tattctttgt tacttaacaa acaaagtgcc ttttatagca tgtgtgagca tagtggagaa 4920 ttggatatcc taattgaatt ggctaaaaaa gcctttgtgg aaaaattgaa ctctaaaaag 4980 gactag 4986
SEQ ID NO:1 14
Saccharomyces cerevisiae
MHHVLNSTRP DHRFWFYDDV TQYGRTKYLN YYTPLVLLIF TVLF ITYNIW KHYYYYDVLH 60 LKQKNP IDEL LYSSTDEDEQ SPLINNNTIT TNYVDNNCTK DALKNRHFSL EKLKSVKVNG 120 EPHGTPEIVR RGFIEKSRII LEFFLVLSQV IIHSFILLHY VNKNPEFTQQ GTITGLVEWC 180 ALF I IVSLRL ANVNQNFKFI NKYPGNLWSV SF INYLALF I SMILPFRSIF IHHINSPISR 240 KYYISQISIN LALFLLLFFA RIRNNFAI IY KTDSWITPSP EPVTSIAGFI CWAWLDSFVW 300 KAHKVS IKVK DIWGLMMQDY SFFVVKKFRY FVDHKVKRKR IFSLNLFFFF SNYLVLQCFW 360 AFLGSVLSFI PTVLLKRILE YVEDQSSAPS NLAWFYVTVM FVGRILVAIC QAQALFFGRR 420 VCIRMKSIII SE IYTKALRR KISTNKTKPS NEDPQEINDQ KSINGDEEST SSANLGAI IN 480 LMAIDAFKVS EICGYLHSFL EAFVMTVVAL ALLYRLLGFA AIVGVLIIVA MLPLNYKLAK 540 YIGDLQKKNL AVTDNRIQKL NEAFQAIRI I KYFSWEENFE KDINTIRENE LSLLLMRSIV 600 WSISSFLWFV TPTIVTAASF AYYIYVQGEV LTTPVAFTAL SLFTLLRDPL DRLSDMLSFV 660 VQSKVSLDRV QDFLNENDTK KYDQLTIDPN GNRFAFENST I SWDKDNQDF KLKDLNIEFK 720 TGKLNVVIGP TGSGKTSLLM ALLGEMYLLN GKVWPALEP RQELIVDANG TTNSIAYCSQ 780 AAWLLNDTVK NNILFNSPFN EARYKAVVEA CGLKRDFEIL KAGDLTE IGE KGITLSGGQK 840 QRVSLARALY SNARHVLLDD CLSAVDSHTA SWIYDNCITG PLMEDRTCIL VSHNIALTLR 900 NAELWLLED GRVKDQGDPI DMLQKGLFGE DELVKSSILS RANSSANLAA KSSTSLSNLP 960 AVKEQQVSVN NNSSHFEAKK LQKSLRTEAE RTEDGKLIKE ETKEEGVVGL DVYKWYLKIF 1020 GGWKIVSFLA SLFLIAQLLY IGQSWWVRAW ASHNVIAKII PRAQRAIAFI SKKASHLIDW 1080 RGSSQISMAS AENQPSSGHS TMYYLVLYLI IGFAQALLGA GKTILNFVAG INASRKIFNM 1140 ILNKVLHSKI RFFDATPTGR IMNRFSKDIE AIDQELTPYI QGAFYSLIEC LSTVILITFI 1200 TPQFLSVAIV VS ILYYFVGY FYMAGSRELK RFESISRSPI YQHFSETLVG VTTIRAFGDE 1260 GRFMQENLHK IDENNKPFFY LWVANRWLAF RIDMIGSLVI FGAGLFILFN INNLDSGMAG 1320 ISLTYAISFT EGALWLVRLY SEVEMNMNSV ERVKEYMEIE QEPYNEHKEI PPPQWPQDGK 1380 IEVNDLSLRY APNLPRVIKN VSFSVDAQSK IGIVGRTGAG KSTI ITALFR FLEPETGHIK 1440 IDNIDISGVD LQRLRRSITI IPQDPTLFSG TIKTNLDPYD EFSDRQIFEA LKRVNLI SEE 1500 QLQQGATRET SNEASSTNSE NVNKFLDLSS EISEGGSNLS QGQRQLMCLA RSLLRSPKII 1560 LLDEATASID YSSDAKIQET IRKEFQGSTI LTIAHRLRSV IDYDKILVMD AGEVKEYDHP 1620 YSLLLNKQSA FYSMCEHSGE LDILIELAKK AFVEKLNSKK D 1661
SEQ ID NO:1 15
Saccharomyces cerevisiae
atgtcttcac tagaagtggt agatgggtgc ccctatggat accgaccata tccagatagt 60 ggcacaaatg cattaaatcc atgttttata tcagtaatat ccgcctggca agccgtcttt 120 ttcctattga ttggtagcta tcaattgtgg aaactttata agaacaataa agtaccaccc 180 agatttaaga actttcctac attaccaagt aaaatcaaca gtcgacatct aacgcatttg 240 accaatgttt gctttcagtc cacgcttata atttgtgaac tggccttggt atcccaatct 300 agcgataggg tttatccatt tatactaaag aaggctctgt acttgaatct ccttttcaat 360 ttgggtattt ctctccctac tcaatactta gcttatttta aaagtacatt ttcaatgggc 420 aaccagcttt tctattacat gtttcaaatt cttctacagc tcttcttgat attgcagagg 480 tactatcatg gttctagtaa cgaaaggctt actgttatta gcggacaaac tgctatgatt 540 ttagaagtgc tccttctttt caattctgtg gcaattttta tttatgatct atgcattttt 600 gagccaatta acgaattatc tgaatactac aagaaaaatg ggtggtatcc ccccgttcat 660 gtactatcct atattacatt tatctggatg aacaaactga ttgtggaaac ttaccgtaac 720 aagaaaatca aagatcctaa ccagttacca ttgccgccag tagatctgaa tattaagtcg 780 ataagtaagg aatttaaggc taactgggaa ttggaaaaat ggttgaatag aaattctctt 840 tggagggcca tttggaagtc atttggtagg actatttctg tggctatgct gtatgaaacg 900 acatctgatt tactttctgt agtacagccc cagtttctac ggatattcat agatggtttg 960 aacccggaaa catcttctaa atatcctcct ttaaatggtg tatttattgc tctaaccctt 1020 ttcgtaatca gcgtggtttc tgtgttcctc accaatcaat tttatattgg aatttttgag 1080 gctggtttgg ggataagagg ctctttagct tctttagtgt atcagaagtc cttaagattg 1140 acgctagcag agcgtaacga aaaatctact ggtgacatct taaatttgat gtctgtggat 1200 gtgttaagga tccagcggtt tttcgaaaat gcccaaacca ttattggcgc tcctattcag 1260 attattgttg tattaacttc cctgtactgg ttgctaggaa aggctgttat tggagggttg 1320 gttactatgg ctattatgat gcctatcaat gccttcttat ctagaaaggt aaaaaagcta 1380 tcaaaaactc aaatgaagta taaggacatg agaatcaaga ctattacaga gcttttgaat 1440 gctataaaat ctattaaatt atacgcctgg gaggaaccta tgatggcaag attgaatcat 1500 gttcgtaatg atatggagtt gaaaaatttt cggaaaattg gtatagtgag caatctgata 1560 tattttgcgt ggaattgtgt acctttaatg gtgacatgtt ccacatttgg cttattttct 1620 ttatttagtg attctccgtt atctcctgcc attgtcttcc cttcattatc tttatttaat 1680 attttgaaca gtgccatcta ttccgttcca tccatgataa ataccattat agagacaagc 1740 gtttctatgg aaagattaaa gtcattccta cttagtgacg aaattgatga ttcgttcatc 1800 gaacgtattg atccttcagc ggatgaaaga gcgttacctg ctatagagat gaataatatt 1860 acatttttat ggaaatcaaa agaagtatta acatctagcc aatctggaga taatttgagg 1920 acagatgaag agtctattat cggatcttct caaattgcgt tgaagaatat cgatcatttt 1980 gaagcaaaaa ggggtgattt agtttgtgtt gttggtcggg taggagctgg taaatcaaca 2040 tttttgaagg caattcttgg tcaacttcct tgcatgagtg gttctaggga ctcgatacca 2100 cctaaactga tcattagatc atcgtctgta gcctactgtt cacaagaatc ctggataatg 2160 aacgcatctg taagagaaaa cattctattt ggtcacaagt tcgaccaaga ttattatgac 2220 ctcactatta aagcatgtca attgctaccc gatttgaaaa tactaccaga tggtgatgaa 2280 actttggtag gtgaaaaggg catttcccta tcaggcggtc agaaggcccg tctttcatta 2340 gccagagcgg tgtactcgag agcagatatt tatttgttgg atgacatttt atctgctgtt 2400 gatgcagaag ttagtaaaaa tattattgaa tatgttttga tcggaaagac ggctttatta 2460 aaaaataaaa caattatttt aactaccaat actgtatcaa ttttaaaaca ttcgcagatg 2520 atatatgcgc tagaaaacgg tgaaattgtt gaacaaggga attatgagga tgtaatgaac 2580 cgtaagaaca atacttcaaa actgaaaaaa ttactagagg aatttgattc tccgattgat 2640 aatggaaatg aaagcgatgt ccaaactgaa caccgatccg aaagtgaagt ggatgaacct 2700 ctgcagctta aagtaactga atcagaaact gaggatgagg ttgttactga gagtgaatta 2760 gaactaatca aagccaattc tagaagagct tctctagcta cgctaagacc tagacccttt 2820 gtgggagcac aattggattc cgtgaagaaa acggcgcaaa aggccgagaa gacagaggtg 2880 ggaagagtca aaacaaagat ttatcttgcg tatattaagg cttgtggagt tttaggtgtt 2940 gttttatttt tcttgtttat gatattaaca agggttttcg acttagcaga gaatttttgg 3000 ttaaagtact ggtcagaatc taatgaaaaa aatggttcaa atgaaagggt ttggatgttt 3060 gttggtgtgt attccttaat cggagtagca tcggccgcat tcaataattt acggagtatt 3120 atgatgctac tgtattgttc tattaggggt tctaagaaac tgcatgaaag catggccaaa 3180 tctgtaatta gaagtcctat gactttcttt gagactacac cagttggaag gatcataaac 3240 aggttctcat ctgatatgga tgcagtggac agtaatctac agtacatttt ctcctttttt 3300 ttcaaatcaa tactaaccta tttggttact gttatattag tcgggtacaa tatgccatgg 3360 tttttagtgt tcaatatgtt tttggtggtt atctatattt actatcaaac attttacatt 3420 gtgctatcta gggagctaaa aagattgatc agtatatctt actctccgat tatgtcctta 3480 atgagtgaga gcttgaacgg ttattctatt attgatgcat acgatcattt tgagagattc 3540 atctatctaa attatgaaaa aatccaatac aacgttgatt ttgtcttcaa ctttagatca 3600 acgaatagat ggttatccgt gagattgcaa actattggtg ctacaattgt tttggctact 3660 gcaatcttag cactagcaac aatgaatact aaaaggcaac taagttcggg tatggttggt 3720 ctactaatga gctattcatt agaggttaca ggttcattga cttggattgt aaggacaact 3780 gtgacgattg aaaccaacat tgtatcagtg gagagaattg ttgagtactg cgaattacca 3840 cctgaagcac agtccattaa ccctgaaaag aggccagatg aaaattggcc atcaaagggt 3900 ggtattgaat tcaaaaacta ttccacaaaa tacagagaaa atttggatcc agtgctgaat 3960 aatattaacg tgaagattga gccatgtgaa aaggttggga ttgttggcag aacaggtgca 4020 gggaagtcta cactgagcct ggcattattt agaatactag aacctaccga aggtaaaatt 4080 attattgacg gcattgatat atccgacata ggtctgttcg atttaagaag ccatttggca 4140 attattcctc aggatgcaca agcttttgaa ggtacagtaa agaccaattt ggaccctttc 4200 aatcgttatt cagaagatga acttaaaagg gctgttgagc aggcacattt aaagcctcat 4260 ctggaaaaaa tgctgcacag taaaccaaga ggtgatgatt ctaatgaaga ggatggcaat 4320 gttaatgata ttctggatgt caagattaat gagaacggta gtaacttgtc agtggggcaa 4380 agacaactac tatgtttggc aagagcgctg ctaaaccgtt ccaaaatatt ggtccttgat 4440 gaagcaacgg cttctgtgga tatggaaacc gataaaatta tccaagacac tataagaaga 4500 gaatttaagg accgtaccat cttaacaatt gcacatcgta tcgacactgt attggacagt 4560 gataagataa ttgttcttga ccagggtagt gtgagggaat tcgattcacc ctcgaaattg 4620 ttatccgata aaacgtctat tttttacagt ctttgtgaga aaggtgggta tttgaaataa 4680
SEQ ID NO:1 16
Saccharomyces cerevisiae
MSSLEVVDGC PYGYRPYPDS GTNALNPCFI SVISAWQAVF FLLIGSYQLW KLYKNNKVPP 60 RFKNFPTLPS KINSRHLTHL TNVCFQSTLI ICELALVSQS SDRVYPF ILK KALYLNLLFN 120 LGISLPTQYL AYFKSTFSMG NQLFYYMFQI LLQLFLILQR YYHGSSNERL TVISGQTAMI 180 LEVLLLFNSV AIFIYDLCIF EP INELSEYY KKNGWYPPVH VLSYITFIWM NKLIVETYRN 240 KKIKDPNQLP LPPVDLNIKS ISKEFKANWE LEKWLNRNSL WRAIWKSFGR TISVAMLYET 300 TSDLLSWQP QFLRIFIDGL NPETSSKYPP LNGVF IALTL FVISWSVFL TNQFYIGIFE 360 AGLGIRGSLA SLVYQKSLRL TLAERNEKST GD ILNLMSVD VLRIQRFFEN AQTIIGAPIQ 420 IIVVLTSLYW LLGKAVIGGL VTMAIMMP IN AFLSRKVKKL SKTQMKYKDM RIKTITELLN 480 AIKSIKLYAW EEPMMARLNH VRNDMELKNF RKIGIVSNLI YFAWNCVPLM VTCSTFGLFS 540 LFSDSPLSPA IVFPSLSLFN ILNSAIYSVP SMINTIIETS VSMERLKSFL LSDEIDDSFI 600 ERIDPSADER ALPAIEMNNI TFLWKSKEVL TSSQSGDNLR TDEESIIGSS QIALKNIDHF 660 EAKRGDLVCV VGRVGAGKST FLKAILGQLP CMSGSRDSIP PKLIIRSSSV AYCSQESWIM 720 NASVRENILF GHKFDQDYYD LTIKACQLLP DLKILPDGDE TLVGEKGISL SGGQKARLSL 780 ARAVYSRADI YLLDD ILSAV DAEVSKNI IE YVLIGKTALL KNKTIILTTN TVSILKHSQM 840 IYALENGEIV EQGNYEDVMN RKNNTSKLKK LLEEFDSP ID NGNESDVQTE HRSESEVDEP 900 LQLKVTESET EDEWTESEL ELIKANSRRA SLATLRPRPF VGAQLDSVKK TAQKAEKTEV 960 GRVKTKIYLA YIKACGVLGV VLFFLFMILT RVFDLAENFW LKYWSESNEK NGSNERVWMF 1020 VGVYSLIGVA SAAFNNLRSI MMLLYCS IRG SKKLHESMAK SVIRSPMTFF ETTPVGRI IN 1080 RFSSDMDAVD SNLQYIFSFF FKSILTYLVT VILVGYNMPW FLVFNMFLW IYIYYQTFYI 1140 VLSRELKRLI SISYSPIMSL MSESLNGYSI IDAYDHFERF IYLNYEKIQY NVDFVFNFRS 1200 TNRWLSVRLQ TIGATIVLAT AILALATMNT KRQLSSGMVG LLMSYSLEVT GSLTWIVRTT 1260 VTIETNIVSV ERIVEYCELP PEAQSINPEK RPDENWPSKG GIEFKNYSTK YRENLDPVLN 1320 NINVKIEPCE KVGIVGRTGA GKSTLSLALF RILEPTEGKI IIDGIDISDI GLFDLRSHLA 1380 IIPQDAQAFE GTVKTNLDPF NRYSEDELKR AVEQAHLKPH LEKMLHSKPR GDDSNEEDGN 1440 VND ILDVKIN ENGSNLSVGQ RQLLCLARAL LNRSKILVLD EATASVDMET DKI IQDTIRR 1500 EFKDRTILTI AHRIDTVLDS DKIIVLDQGS VREFDSPSKL LSDKTSIFYS LCEKGGYLK 1559
SEQ ID NO:1 17
Saccharomyces cerevisiae
atgggaacgg atccccttat tatccgaaat aatggttcat tttgggaagt tgatgatttt 60 actcgtttag gaagaactca gctattgagc tactatttac cattggctat catagcctca 120 attggcattt tcgcactttg tcgcagtgga ttatctcgtt atgtaagatc tgccgagtgc 180 gatttagtga acgaatatct atttggcgca caagaagaga gaaaagaaga taatagtata 240 gaaagacttc tacggaactc aaatacccaa gccaattacg tcaacgtcaa aaagcaagga 300 aggattttga aacttagaca ttttgatata acaactatag atgtcaagca aatcgatgct 360 aaaaatcatg gtggactaac gtttagtaga ccgtctacta gtgaccactt aagaaaatca 420 tctgaaattg tattaatgtc tttacaaata attggccttt cctttttaag agtaacaaaa 480 atcaatattg aattaacgaa cagagatgtt acaactttac tattattttg gttaatacta 540 ctttccctaa gtatcttaag agtttacaag cgttcaacga atctttgggc catctgtttt 600 actgcccata caactatttg gatttcaacc tggattccaa ttcgttcggt ctatattggt 660 aatatcgatg atgtaccctc acagatattt tacatctttg aattcgtaat tacttcaacc 720 ttacagccaa taaagctcac ttcaccgatt aaagacaact catccatcat ctacgttaga 780 gacgaccata cgtctccttc gagggaacac atatcctcaa ttttaagttg cattacttgg 840 agctggatta ccaattttat atgggaggcc caaaagaaca ctattaagtt aaaggatatt 900 tggggcttat caatggaaga ctatagcatt ttcattctaa aagggtttac caggagaaac 960 aagcacatta ataatttgac gctagcactg tttgaatctt tcaaaacata tttactcata 1020 ggaatgttat gggttctggt gaacagtatt gtaaaccttc ttcccacaat tttaatgaaa 1080 agatttttag aaattgtgga taacccaaac cgttcctcat catgcatgaa tttggcgtgg 1140 ctttatatta ttggtatgtt catttgtaga ttgacattag caatttgtaa ttcccaaggt 1200 caatttgttt ctgataagat ttgtttaaga ataagagcca tactcatagg agaaatttat 1260 gcaaaaggct tacgtaggag gctgtttaca tctccaaaaa ccagctctga ttcagatagt 1320 atctccgcaa accttggtac cataattaat ctcatttcta ttgactcatt taaggtatcg 1380 gaactagcaa actaccttta tgtgacagtt caggcagtaa ttatgataat agttgttgta 1440 ggactacttt tcaacttttt aggtgtttca gcttttgcag gaatttcaat tatcttagtg 1500 atgttcccat tgaatttctt gttagcgaat ttgttaggta agtttcaaaa gcaaacactg 1560 aaatgtactg accaaagaat ctcaaaattg aacgagtgct tacagaacat aagaattgtc 1620 aaatattttg cttgggaaag gaatattata aatgaaatca aatcaataag gcaaaaggaa 1680 ttaagatcct tattaaaaaa atctttggtg tggtccgtaa cttcttttct ttggttcgtg 1740 acaccgacct tggtgacagg tgtcactttc gccatctgta catttgttca acatgaagat 1800 ttgaatgccc cgcttgcttt cactactttg tcactcttca ctttgttaaa gacacccctg 1860 gatcaattat caaatatgct aagtttcata aatcaatcaa aagtctctct aaaaagaata 1920 agcgattttt taaggatgga cgatacagaa aaatataatc aactaaccat atctccagac 1980 aaaaataaaa ttgaatttaa aaatgcgact ttaacctgga atgaaaatga cagcgatatg 2040 aatgcattca aattatgtgg tttgaatatt aaatttcaaa ttggtaagtt aaatttgatt 2100 ttgggttcta caggatctgg taaaagtgca ttgctgctgg gtttactggg tgaactaaat 2160 ctaattagtg gctctatcat tgttccgagc ttagaaccaa agcatgattt aattcccgac 2220 tgcgaaggtt taaccaattc cttcgcatat tgttcacaaa gtgcgtggct attaaatgac 2280 acggtaaaaa acaatattat ctttgataac ttctataacg aggataggta caacaaagta 2340 attgatgcat gtgggctgaa aagagacctg gagattttac cagcaggtga cctaacagaa 2400 attggtgaaa agggtataac tttatcagga gggcagaaac agagaatttc cttggcgaga 2460 gctgtttatt cgagtgctaa gcatgtctta ctagatgatt gtttgagcgc tgtcgattca 2520 catactgctg tatggatcta tgaaaattgc atcacaggtc cactaatgaa aaatagaacc 2580 tgcattttag ttacgcacaa tgtttcatta acacttagaa atgcccattt cgcgattgtg 2640 ttggaaaatg gcaaagtgaa gaatcaagga actattacag aattacaaag caaagggctt 2700 tttaaggaaa aatatgttca actttcttct cgagatagca ttaatgaaaa gaacgctaat 2760 agattaaaag ctcccagaaa aaatgactct cagaaaatcg aacctgtcac cgagaacata 2820 aattttgatg caaattttgt caatgatggc cagctaatag aagaggaaga aaaatcaaac 2880 ggtgccataa gccccgatgt ttataaatgg tacctgaaat tttttggagg cttcaaagct 2940 ttaacagccc tgttcgctct ttatatcaca gctcaaattt tgttcatcag tcagtcttgg 3000 tggatacgac attgggtcaa cgataccaat gtacgaataa atgctccagg ttttgcgatg 3060 gacacgctgc cattaaaagg gatgaccgac tcttcgaaaa ataaacataa tgcattttat 3120 tacttaaccg tatattttct tattggtatc attcaggcaa tgctaggtgg ttttaaaaca 3180 atgatgacgt ttttatccgg tatgcgagcc tccagaaaga tctttaataa tctgctagat 3240 ctagttctac atgcccaaat acgatttttc gacgtgacgc cggttggtag aatcatgaat 3300 cgcttttcaa aggacatcga aggtgttgat caagaattga ttccatactt agaagtaact 3360 atattttgcc taattcaatg cgcatcaatt atatttctca ttaccgtaat aactcctcgc 3420 tttttgacag tcgccgttat cgtttttgtt ttatatttct ttgtggggaa atggtactta 3480 acggcaagta gagaattgaa aaggttagat tcaataacca aatcacccat ttttcaacat 3540 ttctcagaga ccttggtagg cgtttgcaca attcgtgcat ttggcgacga gaggagattc 3600 attttagaaa atatgaacaa aattgaccaa aataacagag cattctttta tttatcagtt 3660 actgtcaaat ggttttcttt tagagtcgac atgattggcg cattcattgt tttagcatca 3720 ggttctttta ttctgctcaa tattgcaaat attgactcgg gtcttgccgg catttctttg 3780 acatatgcca ttttgtttac agatggtgct ttatggttag ttagactgta ctcaacattt 3840 gaaatgaaca tgaactctgt tgaaagacta aaagaatatt ctagcattga acaagagaac 3900 tatcttggcc atgatgaagg ccgcattcta cttctaaacg aaccatcgtg gccaaaagat 3960 ggagaaattg aaattgaaaa cttatcttta cgttacgcgc caaatttgcc tcctgtcata 4020 agaaatgtta gtttcaaagt ggatcctcaa agtaagattg ggattgtcgg aagaactggc 4080 gcaggcaaat ctaccataat aacggcatta ttcagattac tagaaccaat aaccggatgt 4140 atcaaaatag atgggcagga tataagtaaa attgatctcg ttacattacg tcgttccatt 4200 actatcatcc ctcaggaccc tattctattt gcaggtacaa tcaaaagtaa tgttgatcca 4260 tatgatgaat atgatgaaaa aaaaatattc aaagcacttt cacaagtaaa tctaatttct 4320 tcacatgaat ttgaagaagt gcttaactcg gaggaacgct ttaacagcac tcataataaa 4380 tttttaaatc ttcacacaga aatagctgag ggcggcttaa atctgtccca aggtgaaagg 4440 caattgcttt ttattgcacg atcattgtta cgcgagccaa agataatact tttggacgag 4500 gctacttcct ctattgatta cgattctgac catttaattc agggtattat aagaagtgag 4560 tttaataaaa gcacaattct tactattgca catcgtttga gatctgttat cgattacgac 4620 aggataattg tgatggatgc cggtgaggta aaagaatatg atcgccctag tgaactgttg 4680 aaagatgaac gcggtatatt ttatagtatg tgtcgtgaca gtgggggcct agagcttttg 4740 aagcaaatag ccaagcaatc aagtaagatg atgaaataa 4779
SEQ ID NO:1 18
Saccharomyces cerevisiae
MGTDPLIIRN NGSFWEVDDF TRLGRTQLLS YYLPLAI IAS IGIFALCRSG LSRYVRSAEC 60 DLVNEYLFGA QEERKEDNSI ERLLRNSNTQ ANYVNVKKQG RILKLRHFDI TTIDVKQIDA 120 KNHGGLTFSR PSTSDHLRKS SEIVLMSLQI IGLSFLRVTK INIELTNRDV TTLLLFWLIL 180 LSLSILRVYK RSTNLWAICF TAHTTIWIST WIPIRSVYIG NIDDVPSQIF YIFEFVITST 240 LQP IKLTSP I KDNSSIIYVR DDHTSPSREH ISSILSCITW SWITNFIWEA QKNTIKLKDI 300 WGLSMEDYSI FILKGFTRRN KHINNLTLAL FESFKTYLLI GMLWVLVNSI VNLLPTILMK 360 RFLEIVDNPN RSSSCMNLAW LYIIGMFICR LTLAICNSQG QFVSDKICLR IRAILIGEIY 420 AKGLRRRLFT SPKTSSDSDS ISANLGTIIN LISIDSFKVS ELANYLYVTV QAVIMIIVW 480 GLLFNFLGVS AFAGISIILV MFPLNFLLAN LLGKFQKQTL KCTDQRI SKL NECLQNIRIV 540 KYFAWERNI I NE IKS IRQKE LRSLLKKSLV WSVTSFLWFV TPTLVTGVTF AICTFVQHED 600 LNAPLAFTTL SLFTLLKTPL DQLSNMLSFI NQSKVSLKRI SDFLRMDDTE KYNQLTI SPD 660 KNKIEFKNAT LTWNENDSDM NAFKLCGLNI KFQIGKLNLI LGSTGSGKSA LLLGLLGELN 720 LISGSI IVPS LEPKHDLIPD CEGLTNSFAY CSQSAWLLND TVKNNI IFDN FYNEDRYNKV 780 IDACGLKRDL EILPAGDLTE IGEKGITLSG GQKQRISLAR AVYSSAKHVL LDDCLSAVDS 840 HTAVWIYENC ITGPLMKNRT CILVTHNVSL TLRNAHFAIV LENGKVKNQG TITELQSKGL 900 FKEKYVQLSS RDSINEKNAN RLKAPRKNDS QKIEPVTENI NFDANFVNDG QLIEEEEKSN 960 GAI SPDVYKW YLKFFGGFKA LTALFALYIT AQILFISQSW WIRHWVNDTN VRINAPGFAM 1020 DTLPLKGMTD SSKNKHNAFY YLTVYFLIGI IQAMLGGFKT MMTFLSGMRA SRKIFNNLLD 1080 LVLHAQIRFF DVTPVGRIMN RFSKDIEGVD QELIPYLEVT IFCLIQCASI IFLITVITPR 1140 FLTVAVIVFV LYFFVGKWYL TASRELKRLD SITKSPIFQH FSETLVGVCT IRAFGDERRF 1200 ILENMNKIDQ NNRAFFYLSV TVKWFSFRVD MIGAFIVLAS GSFILLNIAN IDSGLAGISL 1260 TYAILFTDGA LWLVRLYSTF EMNMNSVERL KEYSSIEQEN YLGHDEGRIL LLNEPSWPKD 1320 GEIEIENLSL RYAPNLPPVI RNVSFKVDPQ SKIGIVGRTG AGKSTIITAL FRLLEPITGC 1380 IKIDGQDISK IDLVTLRRSI TIIPQDPILF AGTIKSNVDP YDEYDEKKIF KALSQVNLIS 1440 SHEFEEVLNS EERFNSTHNK FLNLHTE IAE GGLNLSQGER QLLF IARSLL REPKI ILLDE 1500 ATSSIDYDSD HLIQGIIRSE FNKSTILTIA HRLRSVIDYD RIIVMDAGEV KEYDRPSELL 1560 KDERGIFYSM CRDSGGLELL KQIAKQSSKM MK 1592
SEQ ID NO:1 19
Saccharomyces cerevisiae
atgcctacct tgtatactga tatcgaaatc ccacaattga aaatctcttt aaagcaaccg 60 ctagggttgt ttatcaacaa tgagttttgt ccatcatcag atggaaagac catcgaaact 120 gtgaacccag ctactggcga accgataaca tccttccaag cagctaacga aaaggatgta 180 gacaaagctg tgaaagctgc cagggctgct tttgataacg tttggtcgaa gacatcttct 240 gagcaacgtg gtatttatct ttcaaactta ttaaaactta ttgaggagga gcaagacaca 300 cttgccgcat tagagacttt agacgctgga aagccttacc attcaaatgc caaaggtgat 360 ttggcacaaa ttttacagct taccagatat tttgctgggt ccgctgataa gtttgacaaa 420 ggtgcaacca taccattgac ttttaacaag tttgcatata ctctaaaagt tccttttggc 480 gttgttgctc aaatcgttcc atggaattat cctctagcta tggcttgttg gaaattgcaa 540 ggtgccttag cagccggtaa cacggttatc atcaaacctg ctgagaatac ctctctatct 600 ctactttatt ttgctacttt aattaaaaaa gcaggttttc cacctggtgt tgtcaatatc 660 gttcctggtt atggatcact tgtaggccaa gccctagcat ctcacatgga tatcgacaaa 720 atatctttta cgggaagcac caaggtcggt ggatttgtgt tggaagcttc cggccaatcg 780 aaccttaaag acgttacact agaatgcggt ggtaagtctc ctgctctcgt atttgaagat 840 gcagaccttg ataaggctat cgattggata gcagctggca ttttctacaa ttcaggacag 900 aattgtaccg caaactcaag agtttatgtt caaagttcga tctacgacaa gtttgttgaa 960 aagtttaaag aaactgcaaa gaaggagtgg gatgttgcag gaaaatttga tccgtttgat 1020 gagaaatgca tcgttggtcc agttatatca agtacacagt atgaccgcat caaaagttac 1080 atagaacgtg gtaaaaggga ggaaaagttg gacatgttcc agacctctga atttcctatt 1140 ggtggagcta aaggctactt cattccccca accatcttca ctgatgtccc gcaaacatcg 1200 aaactgttac aggatgagat atttggcccg gttgtggttg ttagcaagtt cacaaattat 1260 gatgacgctc tgaagctggc taatgatact tgctacgggc tcgcctctgc ggtcttcaca 1320 aaagatgtca agaaagcgca catgtttgct cgcgatatta aagcaggaac tgtttggatc 1380 aactcatcta acgatgaaga tgttaccgtt ccttttggcg ggtttaaaat gagtggtatt 1440 ggtagagaac tggggcaaag tggtgttgat acctatcttc aaacaaaagc agttcacata 1500 aatctctctt tggacaacta a 1521
SEQ ID NO:120
Saccharomyces cerevisiae
MPTLYTDIEI PQLKISLKQP LGLF INNEFC PSSDGKTIET VNPATGEP IT SFQAANEKDV 60
DKAVKAARAA FDNVWSKTSS EQRGIYLSNL LKLIEEEQDT LAALETLDAG KPYHSNAKGD 120
LAQILQLTRY FAGSADKFDK GATIPLTFNK FAYTLKVPFG VVAQIVPWNY PLAMACWKLQ 180
GALAAGNTVI IKPAENTSLS LLYFATLIKK AGFPPGWNI VPGYGSLVGQ ALASHMD IDK 240
ISFTGSTKVG GFVLEASGQS NLKDVTLECG GKSPALVFED ADLDKAIDWI AAGIFYNSGQ 300
NCTANSRVYV QSSIYDKFVE KFKETAKKEW DVAGKFDPFD EKCIVGPVIS STQYDRIKSY 360
IERGKREEKL DMFQTSEFPI GGAKGYF IPP TIFTDVPQTS KLLQDEIFGP VWVSKFTNY 420
DDALKLANDT CYGLASAVFT KDVKKAHMFA RD IKAGTVWI NSSNDEDVTV PFGGFKMSGI 480
GRELGQSGVD TYLQTKAVHI NLSLDN 506
SEQ ID NO:121
Saccharomyces cerevisiae
atgcctacct tgtatactga tatcgaaatc ccacaattga aaatctcttt aaagcaaccg 60 ctagggttgt ttatcaacaa tgagttttgt ccatcatcag atggaaagac catcgaaact 120 gtgaacccag ctactggcga accgataaca tccttccaag cagctaacga aaaggatgta 180 gacaaagctg tgaaagctgc cagggctgct tttgataacg tttggtcgaa gacatcttct 240 gagcaacgtg gtatttatct ttcaaactta ttaaaactta ttgaggagga gcaagacaca 300 cttgccgcat tagagacttt agacgctggt aagcctttcc attccaatgc taaacaagac 360 ttagcccaga ttatagaact tacaagatac tatgcggggg cggtcgacaa gttcaatatg 420 ggtgaaacca ttccattgac ttttaacaag tttgcatata ctctaaaagt tccttttggc 480 gttgttgctc aaatcgttcc atggaattat cctctagcta tggcttgtag aaaaatgcaa 540 ggtgccttag cggccggtaa cacggttatc atcaaacctg ctgaaaatac ctctctatct 600 ctactttatt ttgctacttt aattaaaaaa gcaggttttc cacctggtgt tgtcaatgtc 660 attcctggtt atggttccgt tgtggggaaa gctttaggaa cccacatgga tatcgacaaa 720 atatctttta cgggaagtac taaggttggc ggctcagtat tggaagcttc cggccaatcg 780 aaccttaagg atatcacact agaatgcggt ggtaagtctc ctgctcttgt atttgaagat 840 gcagaccttg ataaggctat agaatgggta gcaaatggta ttttttttaa ttcgggacag 900 atctgcactg caaactcaag agtttatgtt caaagttcga tctacgacaa gtttgttgaa 960 aagtttaaag aaactgcaaa gaaggagtgg gatgttgcag gaaaatttga tccgtttgat 1020 gagaaatgca tcgttggtcc agttatatca agtacacagt atgaccgcat caaaagttac 1080 atagaacgtg gtaaaaagga ggaaaagttg gacatgttcc agacctctga atttcctatt 1140 ggtggagcta aaggctactt cattccccca accatcttca ctgatgtacc agaaacatct 1200 aagttgctgc gtgatgaaat atttggcccg gttgtggttg ttagcaagtt cacaaattat 1260 gatgacgctc tgaagctggc taatgatact tgctacgggc tcgcctctgc ggtcttcacc 1320 aaagatgtca agaaagcgca catgtttgct cgcgatatta aagcaggaac tgtttggatc 1380 aatcaaacca atcaagaaga agctaaagtt ccttttggcg gatttaagat gagtggtatt 1440 ggtagagaat caggcgacac cggcgttgat aactatttac aaataaaatc agtccatgtg 1500 gatctttcat tggataaata a 1521
SEQ ID NO:122
Saccharomyces cerevisiae
MPTLYTDIEI PQLKISLKQP LGLF INNEFC PSSDGKTIET VNPATGEP IT SFQAANEKDV 60 DKAVKAARAA FDNVWSKTSS EQRGIYLSNL LKLIEEEQDT LAALETLDAG KPFHSNAKQD 120 LAQIIELTRY YAGAVDKFNM GETIPLTFNK FAYTLKVPFG VVAQIVPWNY PLAMACRKMQ 180 GALAAGNTVI IKPAENTSLS LLYFATLIKK AGFPPGWNV IPGYGSVVGK ALGTHMD IDK 240 ISFTGSTKVG GSVLEASGQS NLKD ITLECG GKSPALVFED ADLDKAIEWV ANGIFFNSGQ 300 ICTANSRVYV QSSIYDKFVE KFKETAKKEW DVAGKFDPFD EKCIVGPVIS STQYDRIKSY 360 IERGKKEEKL DMFQTSEFPI GGAKGYF IPP TIFTDVPETS KLLRDEIFGP VWVSKFTNY 420 DDALKLANDT CYGLASAVFT KDVKKAHMFA RD IKAGTVWI NQTNQEEAKV PFGGFKMSGI 480 GRESGDTGVD NYLQIKSVHV DLSLDK 506
SEQ ID NO:123
Saccharomyces cerevisiae
atgttcagta gatctacgct ctgcttaaag acgtctgcat cctccattgg gagacttcaa 60 ttgagatatt tctcacacct tcctatgaca gtgcctatca agctgcccaa tgggttggaa 120 tatgagcaac caacggggtt gttcatcaac aacaagtttg ttccttctaa acagaacaag 180 accttcgaag tcattaaccc ttccacggaa gaagaaatat gtcatattta tgaaggtaga 240 gaggacgatg tggaagaggc cgtgcaggcc gccgaccgtg ccttctctaa tgggtcttgg 300 aacggtatcg accctattga caggggtaag gctttgtaca ggttagccga attaattgaa 360 caggacaagg atgtcattgc ttccatcgag actttggata acggtaaagc tatctcttcc 420 tcgagaggag atgttgattt agtcatcaac tatttgaaat cttctgctgg ctttgctgat 480 aaaattgatg gtagaatgat tgatactggt agaacccatt tttcttacac taagagacag 540 cctttgggtg tttgtgggca gattattcct tggaatttcc cactgttgat gtgggcctgg 600 aagattgccc ctgctttggt caccggtaac accgtcgtgt tgaagactgc cgaatccacc 660 ccattgtccg ctttgtatgt gtctaaatac atcccacagg cgggtattcc acctggtgtg 720 atcaacattg tatccgggtt tggtaagatt gtgggtgagg ccattacaaa ccatccaaaa 780 atcaaaaagg ttgccttcac agggtccacg gctacgggta gacacattta ccagtccgca 840 gccgcaggct tgaaaaaagt gactttggag ctgggtggta aatcaccaaa cattgtcttc 900 gcggacgccg agttgaaaaa agccgtgcaa aacattatcc ttggtatcta ctacaattct 960 ggtgaggtct gttgtgcggg ttcaagggtg tatgttgaag aatctattta cgacaaattc 1020 attgaagagt tcaaagccgc ttctgaatcc atcaaggtgg gcgacccatt cgatgaatct 1080 actttccaag gtgcacaaac ctctcaaatg caactaaaca aaatcttgaa atacgttgac 1140 attggtaaga atgaaggtgc tactttgatt accggtggtg aaagattagg tagcaagggt 1200 tacttcatta agccaactgt ctttggtgac gttaaggaag acatgagaat tgtcaaagag 1260 gaaatctttg gccctgttgt cactgtaacc aaattcaaat ctgccgacga agtcattaac 1320 atggcgaacg attctgaata cgggttggct gctggtattc acacctctaa tattaatacc 1380 gccttaaaag tggctgatag agttaatgcg ggtacggtct ggataaacac ttataacgat 1440 ttccaccacg cagttccttt cggtgggttc aatgcatctg gtttgggcag ggaaatgtct 1500 gttgatgctt tacaaaacta cttgcaagtt aaagcggtcc gtgccaaatt ggacgagtaa 1560
SEQ ID NO:124
Saccharomyces cerevisiae
MFSRSTLCLK TSASSIGRLQ LRYFSHLPMT VP IKLPNGLE YEQPTGLFIN NKFVPSKQNK 60 TFEVINPSTE EEICHIYEGR EDDVEEAVQA ADRAFSNGSW NGIDP IDRGK ALYRLAELIE 120 QDKDVIASIE TLDNGKAI SS SRGDVDLVIN YLKSSAGFAD KIDGRMIDTG RTHFSYTKRQ 180 PLGVCGQI IP WNFPLLMWAW KIAPALVTGN TVVLKTAEST PLSALYVSKY IPQAGIPPGV 240 INIVSGFGKI VGEAITNHPK IKKVAFTGST ATGRHIYQSA AAGLKKVTLE LGGKSPNIVF 300 ADAELKKAVQ NIILGIYYNS GEVCCAGSRV YVEES IYDKF IEEFKAASES IKVGDPFDES 360 TFQGAQTSQM QLNKILKYVD IGKNEGATLI TGGERLGSKG YFIKPTVFGD VKEDMRIVKE 420 EIFGPVVTVT KFKSADEVIN MANDSEYGLA AGIHTSNINT ALKVADRVNA GTVWINTYND 480 FHHAVPFGGF NASGLGREMS VDALQNYLQV KAVRAKLDE 519
SEQ ID NO:125
Saccharomyces cerevisiae
atgctttctc gcacaagagc tgcagctccg aattccagaa tattcactag aagcttgtta 60 cgtctttatt ctcaagcacc attacgcgtt ccaattactc ttccaaatgg tttcacctac 120 gaacagccaa cagggttatt catcaatggt gaatttgttg cctcgaagca aaagaaaacg 180 tttgacgtga tcaatccatc taacgaagaa aagataacaa ctgtatacaa ggctatggaa 240 gatgatgttg atgaagccgt tgcagcggct aaaaaagctt ttgaaacgaa gtggtctatt 300 gtagagccgg aggttcgcgc taaagcttta ttcaatctcg ctgacttggt tgagaaacac 360 caagaaacac tggctgccat tgagtcaatg gataatggta agtcattgtt ttgtgcgcgc 420 ggtgacgtcg ctttagtatc taaatacttg cgttcttgcg gtggttgggc agataaaatc 480 tacggtaacg ttattgacac aggtaaaaac cattttacct actcaattaa ggaaccatta 540 ggcgtttgcg gccaaataat cccttggaac ttccctttat tgatgtggtc atggaaaatt 600 gggcctgctc tggctacagg taacaccgtc gtattgaaac ccgctgaaac aacaccttta 660 tctgcccttt tcgcttccca gttgtgtcag gaagcaggca tacccgctgg tgtagtcaat 720 atccttccgg gttccggtag agttgttgga gaaagattga gtgcacaccc agacgtgaag 780 aagattgctt ttacaggctc tactgccacc ggccgccata ttatgaaggt cgctgccgat 840 actgtcaaga aagtcacttt ggagctggga ggtaaatcac caaatattgt gtttgctgac 900 gctgatctag ataaagccgt caagaacatt gccttcggta ttttttacaa ctctggtgaa 960 gtttgctgcg ctggttccag aatatacatt caagatacag tatacgagga ggtgttgcaa 1020 aaactaaagg attacaccga gtcactaaag gtcggtgacc catttgatga ggaagttttc 1080 caaggtgctc aaacatctga caaacagctg cataaaattt tagactatgt cgatgtagca 1140 aaatcagagg gggctcgtct tgtgactgga ggggccagac atggcagtaa aggttatttt 1200 gtcaagccaa cagtgtttgc tgatgtcaaa gaagatatga gaattgttaa ggaggaagtg 1260 tttggtccca ttgtaactgt atccaagttt tctactgttg atgaagtgat tgctatggca 1320 aatgattctc aatatgggtt agccgcaggt attcacacta acgatattaa caaggctgtt 1380 gatgtgtcca aaagagtgaa agctggtact gtttggataa atacctataa caacttccac 1440 caaaatgttc ctttcggtgg cttcggccag tcaggtattg gccgtgaaat gggtgaggct 1500 gctttaagta actacactca aacaaaatct gtcagaattg ccattgacaa gccaattcgt 1560 tga 1563
SEQ ID NO:126
Saccharomyces cerevisiae
MLSRTRAAAP NSRIFTRSLL RLYSQAPLRV PITLPNGFTY EQPTGLFING EFVASKQKKT 60 FDVINP SNEE KITTVYKAME DDVDEAVAAA KKAFETKWS I VEPEVRAKAL FNLADLVEKH 120 QETLAAIESM DNGKSLFCAR GDVALVSKYL RSCGGWADKI YGNVIDTGKN HFTYSIKEPL 180 GVCGQI IPWN FPLLMWSWKI GPALATGNTV VLKPAETTPL SALFASQLCQ EAGIPAGWN 240 ILPGSGRWG ERLSAHPDVK KIAFTGSTAT GRHIMKVAAD TVKKVTLELG GKSPNIVFAD 300 ADLDKAVKNI AFGIFYNSGE VCCAGSRIYI QDTVYEEVLQ KLKDYTESLK VGDPFDEEVF 360 QGAQTSDKQL HKILDYVDVA KSEGARLVTG GARHGSKGYF VKPTVFADVK EDMRIVKEEV 420 FGP IVTVSKF STVDEVIAMA NDSQYGLAAG IHTND INKAV DVSKRVKAGT VWINTYNNFH 480 QNVPFGGFGQ SGIGREMGEA ALSNYTQTKS VRIAIDKPIR 520
SEQ ID NO:127
Saccharomyces cerevisiae
atgactaagc tacactttga cactgctgaa ccagtcaaga tcacacttcc aaatggtttg 60 acatacgagc aaccaaccgg tctattcatt aacaacaagt ttatgaaagc tcaagacggt 120 aagacctatc ccgtcgaaga tccttccact gaaaacaccg tttgtgaggt ctcttctgcc 180 accactgaag atgttgaata tgctatcgaa tgtgccgacc gtgctttcca cgacactgaa 240 tgggctaccc aagacccaag agaaagaggc cgtctactaa gtaagttggc tgacgaattg 300 gaaagccaaa ttgacttggt ttcttccatt gaagctttgg acaatggtaa aactttggcc 360 ttagcccgtg gggatgttac cattgcaatc aactgtctaa gagatgctgc tgcctatgcc 420 gacaaagtca acggtagaac aatcaacacc ggtgacggct acatgaactt caccacctta 480 gagccaatcg gtgtctgtgg tcaaattatt ccatggaact ttccaataat gatgttggct 540 tggaagatcg ccccagcatt ggccatgggt aacgtctgta tcttgaaacc cgctgctgtc 600 acacctttaa atgccctata ctttgcttct ttatgtaaga aggttggtat tccagctggt 660 gtcgtcaaca tcgttccagg tcctggtaga actgttggtg ctgctttgac caacgaccca 720 agaatcagaa agctggcttt taccggttct acagaagtcg gtaagagtgt tgctgtcgac 780 tcttctgaat ctaacttgaa gaaaatcact ttggaactag gtggtaagtc cgcccatttg 840 gtctttgacg atgctaacat taagaagact ttaccaaatc tagtaaacgg tattttcaag 900 aacgctggtc aaatttgttc ctctggttct agaatttacg ttcaagaagg tatttacgac 960 gaactattgg ctgctttcaa ggcttacttg gaaaccgaaa tcaaagttgg taatccattt 1020 gacaaggcta acttccaagg tgctatcact aaccgtcaac aattcgacac aattatgaac 1080 tacatcgata tcggtaagaa agaaggcgcc aagatcttaa ctggtggcga aaaagttggt 1140 gacaagggtt acttcatcag accaaccgtt ttctacgatg ttaatgaaga catgagaatt 1200 gttaaggaag aaatttttgg accagttgtc actgtcgcaa agttcaagac tttagaagaa 1260 ggtgtcgaaa tggctaacag ctctgaattc ggtctaggtt ctggtatcga aacagaatct 1320 ttgagcacag gtttgaaggt ggccaagatg ttgaaggccg gtaccgtctg gatcaacaca 1380 tacaacgatt ttgactccag agttccattc ggtggtgtta agcaatctgg ttacggtaga 1440 gaaatgggtg aagaagtcta ccatgcatac actgaagtaa aagctgtcag aattaagttg 1500 taa 1503
SEQ ID NO:128
Saccharomyces cerevisiae
MTKLHFDTAE PVKITLPNGL TYEQPTGLFI NNKFMKAQDG KTYPVEDPST ENTVCEVSSA 60
TTEDVEYAIE CADRAFHDTE WATQDPRERG RLLSKLADEL ESQIDLVSSI EALDNGKTLA 120
LARGDVTIAI NCLRDAAAYA DKVNGRTINT GDGYMNFTTL EPIGVCGQII PWNFP IMMLA 180
WKIAPALAMG NVCILKPAAV TPLNALYFAS LCKKVGIPAG VVNIVPGPGR TVGAALTNDP 240
RIRKLAFTGS TEVGKSVAVD SSESNLKKIT LELGGKSAHL VFDDANIKKT LPNLVNGIFK 300
NAGQICSSGS RIYVQEGIYD ELLAAFKAYL ETEIKVGNPF DKANFQGAIT NRQQFDTIMN 360
YID IGKKEGA KILTGGEKVG DKGYFIRPTV FYDVNEDMRI VKEEIFGPW TVAKFKTLEE 420
GVEMANSSEF GLGSGIETES LSTGLKVAKM LKAGTVWINT YNDFDSRVPF GGVKQSGYGR 480
EMGEEVYHAY TEVKAVRIKL 500
SEQ ID NO:129
Saccharomyces cerevisiae
atgtctatcc cagaaactca aaaaggtgtt atcttctacg aatcccacgg taagttggaa 60 tacaaagata ttccagttcc aaagccaaag gccaacgaat tgttgatcaa cgttaaatac 120 tctggtgtct gtcacactga cttgcacgct tggcacggtg actggccatt gccagttaag 180 ctaccattag tcggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca acaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ccgacttggc ccaagtcgcc cccatcttgt gtgctggtat caccgtctac 480 aaggctttga agtctgctaa cttgatggcc ggtcactggg ttgctatctc cggtgctgct 540 ggtggtctag gttctttggc tgttcaatac gccaaggcta tgggttacag agtcttgggt 600 attgacggtg gtgaaggtaa ggaagaatta ttcagatcca tcggtggtga agtcttcatt 660 gacttcacta aggaaaagga cattgtcggt gctgttctaa aggccactga cggtggtgct 720 cacggtgtca tcaacgtttc cgtttccgaa gccgctattg aagcttctac cagatacgtt 780 agagctaacg gtaccaccgt tttggtcggt atgccagctg gtgccaagtg ttgttctgat 840 gtcttcaacc aagtcgtcaa gtccatctct attgttggtt cttacgtcgg taacagagct 900 gacaccagag aagctttgga cttcttcgcc agaggtttgg tcaagtctcc aatcaaggtt 960 gtcggcttgt ctaccttgcc agaaatttac gaaaagatgg aaaagggtca aatcgttggt 1020 agatacgttg ttgacacttc taaataa 1047
SEQ ID NO:130
Saccharomyces cerevisiae
MSIPETQKGV IFYESHGKLE YKDIPVPKPK ANELLINVKY SGVCHTDLHA WHGDWPLPVK 60
LPLVGGHEGA GVWGMGENV KGWKIGDYAG IKWLNGSCMA CEYCELGNES NCPHADLSGY 120
THDGSFQQYA TADAVQAAHI PQGTDLAQVA PILCAGITVY KALKSANLMA GHWVAISGAA 180
GGLGSLAVQY AKAMGYRVLG IDGGEGKEEL FRSIGGEVFI DFTKEKD IVG AVLKATDGGA 240
HGVINVSVSE AAIEASTRYV RANGTTVLVG MPAGAKCCSD VFNQWKSIS IVGSYVGNRA 300
DTREALDFFA RGLVKSPIKV VGLSTLPEIY EKMEKGQIVG RYWDTSK 348
SEQ ID NO:131
Saccharomyces cerevisiae
atgtctattc cagaaactca aaaagccatt atcttctacg aatccaacgg caagttggag 60 cataaggata tcccagttcc aaagccaaag cccaacgaat tgttaatcaa cgtcaagtac 120 tctggtgtct gccacaccga tttgcacgct tggcatggtg actggccatt gccaactaag 180 ttaccattag ttggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca agaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ctgacttggc tgaagtcgcg ccaatcttgt gtgctggtat caccgtatac 480 aaggctttga agtctgccaa cttgagagca ggccactggg cggccatttc tggtgctgct 540 ggtggtctag gttctttggc tgttcaatat gctaaggcga tgggttacag agtcttaggt 600 attgatggtg gtccaggaaa ggaagaattg tttacctcgc tcggtggtga agtattcatc 660 gacttcacca aagagaagga cattgttagc gcagtcgtta aggctaccaa cggcggtgcc 720 cacggtatca tcaatgtttc cgtttccgaa gccgctatcg aagcttctac cagatactgt 780 agggcgaacg gtactgttgt cttggttggt ttgccagccg gtgcaaagtg ctcctctgat 840 gtcttcaacc acgttgtcaa gtctatctcc attgtcggct cttacgtggg gaacagagct 900 gataccagag aagccttaga tttctttgcc agaggtctag tcaagtctcc aataaaggta 960 gttggcttat ccagtttacc agaaatttac gaaaagatgg agaagggcca aattgctggt 1020 agatacgttg ttgacacttc taaataa 1047
SEQ ID NO:132
Saccharomyces cerevisiae
MSIPETQKAI IFYESNGKLE HKDIPVPKPK PNELLINVKY SGVCHTDLHA WHGDWPLPTK 60
LPLVGGHEGA GVWGMGENV KGWKIGDYAG IKWLNGSCMA CEYCELGNES NCPHADLSGY 120
THDGSFQEYA TADAVQAAHI PQGTDLAEVA PILCAGITVY KALKSANLRA GHWAAISGAA 180
GGLGSLAVQY AKAMGYRVLG IDGGpPGKEEL FTSLGGEVFI DFTKEKDIVS AVVKATNGGA 240
HGI INVSVSE AAIEASTRYC RANGTWLVG LPAGAKCSSD VFNHWKSIS IVGSYVGNRA 300
DTREALDFFA RGLVKSPIKV VGLSSLPEIY EKMEKGQIAG RYWDTSK 348
SEQ ID NO:133
Saccharomyces cerevisiae
atgttgagaa cgtcaacatt gttcaccagg cgtgtccaac caagcctatt ttctagaaac 60 attcttagat tgcaatccac agctgcaatc cctaagactc aaaaaggtgt catcttttat 120 gagaataagg ggaagctgca ttacaaagat atccctgtcc ccgagcctaa gccaaatgaa 180 attttaatca acgttaaata ttctggtgta tgtcacaccg atttacatgc ttggcacggc 240 gattggccat tacctgttaa actaccatta gtaggtggtc atgaaggtgc tggtgtagtt 300 gtcaaactag gttccaatgt caagggctgg aaagtcggtg atttagcagg tatcaaatgg 360 ctgaacggtt cttgtatgac atgcgaattc tgtgaatcag gtcatgaatc aaattgtcca 420 gatgctgatt tatctggtta cactcatgat ggttctttcc aacaatttgc gaccgctgat 480 gctattcaag ccgccaaaat tcaacagggt accgacttgg ccgaagtagc cccaatatta 540 tgtgctggtg ttactgtata taaagcacta aaagaggcag acttgaaagc tggtgactgg 600 gttgccatct ctggtgctgc aggtggcttg ggttccttgg ccgttcaata tgcaactgcg 660 atgggttaca gagttctagg tattgatgca ggtgaggaaa aggaaaaact tttcaagaaa 720 ttggggggtg aagtattcat cgactttact aaaacaaaga atatggtttc tgacattcaa 780 gaagctacca aaggtggccc tcatggtgtc attaacgttt ccgtttctga agccgctatt 840 tctctatcta cggaatatgt tagaccatgt ggtaccgtcg ttttggttgg tttgcccgct 900 aacgcctacg ttaaatcaga ggtattctct catgtggtga agtccatcaa tatcaagggt 960 tcttatgttg gtaacagagc tgatacgaga gaagccttag acttctttag cagaggtttg 1020 atcaaatcac caatcaaaat tgttggatta tctgaattac caaaggttta tgacttgatg 1080 gaaaagggca agattttggg tagatacgtc gtcgatacta gtaaataa 1128
SEQ ID NO:134
Saccharomyces cerevisiae
MLRTSTLFTR RVQPSLFSRN ILRLQSTAAI PKTQKGVIFY ENKGKLHYKD IPVPEPKPNE 60
ILINVKYSGV CHTDLHAWHG DWPLPVKLPL VGGHEGAGVV VKLGSNVKGW KVGDLAGIKW 120
LNGSCMTCEF CESGHESNCP DADLSGYTHD GSFQQFATAD AIQAAKIQQG TDLAEVAP IL 180
CAGVTVYKAL KEADLKAGDW VAISGAAGGL GSLAVQYATA MGYRVLGIDA GEEKEKLFKK 240
LGGEVF IDFT KTKNMVSD IQ EATKGGPHGV INVSVSEAAI SLSTEYVRPC GTVVLVGLPA 300 NAYVKSEVFS HVVKSINIKG SYVGNRADTR EALDFFSRGL IKSPIKIVGL SELPKVYDLM 360
EKGKILGRYV VDTSK 375
SEQ ID NO:135
Saccharomyces cerevisiae
atgtcttccg ttactgggtt ttacattcca ccaatctctt tctttggtga aggtgcttta 60 gaagaaaccg ctgattacat caaaaacaag gattacaaaa aggctttgat cgttactgat 120 cctggtattg cagctattgg tctctccggt agagtccaaa agatgttgga agaacgtgac 180 ttaaacgttg ctatctatga caaaactcaa ccaaacccaa atattgccaa tgtcacagct 240 ggtttgaagg ttttgaagga acaaaactct gaaattgttg tttccattgg tggtggttct 300 gctcacgaca atgctaaggc cattgcttta ttggctacta acggtgggga aatcggagac 360 tatgaaggtg tcaatcaatc taagaaggct gctttaccac tatttgccat caacactact 420 gctggtactg cttccgaaat gaccagattc actattatct ctaatgaaga aaagaaaatc 480 aagatggcta tcattgacaa caacgtcact ccagctgttg ctgtcaacga tccatctacc 540 atgtttggtt tgccacctgc tttgactgct gctactggtc tagatgcttt gactcactgt 600 atcgaagctt atgtttccac cgcctctaac ccaatcaccg atgcctgtgc tttgaagggt 660 attgatttga tcaatgaaag cttagtcgct gcatacaaag acggtaaaga caagaaggcc 720 agaactgaca tgtgttacgc tgaatacttg gcaggtatgg ctttcaacaa tgcttctcta 780 ggttatgttc atgcccttgc tcatcaactt ggtggtttct accacttgcc tcatggtgtt 840 tgtaacgctg tcttgttgcc tcatgttcaa gaggccaaca tgcaatgtcc aaaggccaag 900 aagagattag gtgaaattgc tttgcatttc ggtgcttctc aagaagatcc agaagaaacc 960 atcaaggctt tgcacgtttt aaacagaacc atgaacattc caagaaactt gaaagaatta 1020 ggtgttaaaa ccgaagattt tgaaattttg gctgaacacg ccatgcatga tgcctgccat 1080 ttgactaacc cagttcaatt caccaaagaa caagtggttg ccattatcaa gaaagcctat 1140 gaatattaa 1149
SEQ ID NO:136
Saccharomyces cerevisiae
MSSVTGFYIP P I SFFGEGAL EETADYIKNK DYKKALIVTD tPGIAAIGLSG RVQKMLEERD 60
LNVAIYDKTQ PNPNIANVTA GLKVLKEQNS EIWSIGGGS AHDNAKAIAL LATNGGE IGD 120
YEGVNQSKKA ALPLFAINTT AGTASEMTRF TIISNEEKKI KMAI IDNNVT PAVAVNDPST 180
MFGLPPALTA ATGLDALTHC IEAYVSTASN PITDACALKG IDLINESLVA AYKDGKDKKA 240
RTDMCYAEYL AGMAFNNASL GYVHALAHQL GGFYHLPHGV CNAVLLPHVQ EANMQCPKAK 300
KRLGEIALHF GASQEDPEET IKALHVLNRT MNIPRNLKEL GVKTEDFEIL AEHAMHDACH 360
LTNPVQFTKE QVVAI IKKAY EY 382
SEQ ID NO:137
Saccharomyces cerevisiae
atgccttcgc aagtcattcc tgaaaaacaa aaggctattg tcttttatga gacagatgga 60 aaattggaat ataaagacgt cacagttccg gaacctaagc ctaacgaaat tttagtccac 120 gttaaatatt ctggtgtttg tcatagtgac ttgcacgcgt ggcacggtga ttggccattt 180 caattgaaat ttccattaat cggtggtcac gaaggtgctg gtgttgttgt taagttggga 240 tctaacgtta agggctggaa agtcggtgat tttgcaggta taaaatggtt gaatgggact 300 tgcatgtcct gtgaatattg tgaagtaggt aatgaatctc aatgtcctta tttggatggt 360 actggcttca cacatgatgg tacttttcaa gaatacgcaa ctgccgatgc cgttcaagct 420 gcccatattc caccaaacgt caatcttgct gaagttgccc caatcttgtg tgcaggtatc 480 actgtttata aggcgttgaa aagagccaat gtgataccag gccaatgggt cactatatcc 540 ggtgcatgcg gtggcttggg ttctctggca atccaatacg cccttgctat gggttacagg 600 gtcattggta tcgatggtgg taatgccaag cgaaagttat ttgaacaatt aggcggagaa 660 atattcatcg atttcacgga agaaaaagac attgttggtg ctataataaa ggccactaat 720 ggcggttctc atggagttat taatgtgtct gtttctgaag cagctatcga ggcttctacg 780 aggtattgta ggcccaatgg tactgtcgtc ctggttggta tgccagctca tgcttactgc 840 aattccgatg ttttcaatca agttgtaaaa tcaatctcca tcgttggatc ttgtgttgga 900 aatagagctg atacaaggga ggctttagat ttcttcgcca gaggtttgat caaatctccg 960 atccacttag ctggcctatc ggatgttcct gaaatttttg caaagatgga gaagggtgaa 1020 attgttggta gatatgttgt tgagacttct aaatga 1056 SEQ ID NO:138
Saccharomyces cerevisiae
MPSQVIPEKQ KAIVFYETDG KLEYKDVTVP EPKPNEILVH VKYSGVCHSD LHAWHGDWPF 60 QLKFPLIGGH EGAGVWKLG SNVKGWKVGD FAGIKWLNGT CMSCEYCEVG NESQCPYLDG 120 TGFTHDGTFQ EYATADAVQA AHIPPNVNLA EVAPILCAGI TVYKALKRAN VIPGQWVTIS 180 GACGGLGSLA IQYALAMGYR VIGIDGGNAK RKLFEQLGGE IFIDFTEEKD IVGAI IKATN 240 GGSHGVINVS VSEAAIEAST RYCRPNGTW LVGMPAHAYC NSDVFNQWK SISIVGSCVG 300 NRADTREALD FFARGLIKSP IHLAGLSDVP EIFAKMEKGE IVGRYWETS K 351
SEQ ID NO:139
Saccharomyces cerevisiae
atgtcttatc ctgagaaatt tgaaggtatc gctattcaat cacacgaaga ttggaaaaac 60 ccaaagaaga caaagtatga cccaaaacca ttttacgatc atgacattga cattaagatc 120 gaagcatgtg gtgtctgcgg tagtgatatt cattgtgcag ctggtcattg gggcaatatg 180 aagatgccgc tagtcgttgg tcatgaaatc gttggtaaag ttgtcaagct agggcccaag 240 tcaaacagtg ggttgaaagt cggtcaacgt gttggtgtag gtgctcaagt cttttcatgc 300 ttggaatgtg accgttgtaa gaatgataat gaaccatact gcaccaagtt tgttaccaca 360 tacagtcagc cttatgaaga cggctatgtg tcgcagggtg gctatgcaaa ctacgtcaga 420 gttcatgaac attttgtggt gcctatccca gagaatattc catcacattt ggctgctcca 480 ctattatgtg gtggtttgac tgtgtactct ccattggttc gtaacggttg cggtccaggt 540 aaaaaagttg gtatagttgg tcttggtggt atcggcagta tgggtacatt gatttccaaa 600 gccatggggg cagagacgta tgttatttct cgttcttcga gaaaaagaga agatgcaatg 660 aagatgggcg ccgatcacta cattgctaca ttagaagaag gtgattgggg tgaaaagtac 720 tttgacacct tcgacctgat tgtagtctgt gcttcctccc ttaccgacat tgacttcaac 780 attatgccaa aggctatgaa ggttggtggt agaattgtct caatctctat accagaacaa 840 cacgaaatgt tatcgctaaa gccatatggc ttaaaggctg tctccatttc ttacagtgct 900 ttaggttcca tcaaagaatt gaaccaactc ttgaaattag tctctgaaaa agatatcaaa 960 atttgggtgg aaacattacc tgttggtgaa gccggcgtcc atgaagcctt cgaaaggatg 1020 gaaaagggtg acgttagata tagatttacc ttagtcggct acgacaaaga attttcagac 1080 tag 1083
SEQ ID NO:140
Saccharomyces cerevisiae
MSYPEKFEGI AIQSHEDWKN PKKTKYDPKP FYDHDIDIKI EACGVCGSDI HCAAGHWGNM 60 KMPLWGHEI VGKWKLGPK SNSGLKVGQR VGVGAQVFSC LECDRCKNDN EPYCTKFVTT 120 YSQPYEDGYV SQGGYANYVR VHEHFWPIP ENIPSHLAAP LLCGGLTVYS PLVRNGCGPG 180 KKVGIVGLGG IGSMGTLI SK AMGAETYVIS RSSRKREDAM KMGADHYIAT LEEGDWGEKY 240 FDTFDLIWC ASSLTDIDFN IMPKAMKVGG RIVSISIPEQ HEMLSLKPYG LKAVSISYSA 300 LGSIKELNQL LKLVSEKD IK IWVETLPVGE AGVHEAFERM EKGDVRYRFT LVGYDKEFSD 360
SEQ ID NO:141
Saccharomyces cerevisiae
atgctttacc cagaaaaatt tcagggcatc ggtatttcca acgcaaagga ttggaagcat 60 cctaaattag tgagttttga cccaaaaccc tttggcgatc atgacgttga tgttgaaatt 120 gaagcctgtg gtatctgcgg atctgatttt catatagccg ttggtaattg gggtccagtc 180 ccagaaaatc aaatccttgg acatgaaata attggccgcg tggtgaaggt tggatccaag 240 tgccacactg gggtaaaaat cggtgaccgt gttggtgttg gtgcccaagc cttggcgtgt 300 tttgagtgtg aacgttgcaa aagtgacaac gagcaatact gtaccaatga ccacgttttg 360 actatgtgga ctccttacaa ggacggctac atttcacaag gaggctttgc ctcccacgtg 420 aggcttcatg aacactttgc tattcaaata ccagaaaata ttccaagtcc gctagccgct 480 ccattattgt gtggtggtat tacagttttc tctccactac taagaaatgg ctgtggtcca 540 ggtaagaggg taggtattgt tggcatcggt ggtattgggc atatggggat tctgttggct 600 aaagctatgg gagccgaggt ttatgcgttt tcgcgaggcc actccaagcg ggaggattct 660 atgaaactcg gtgctgatca ctatattgct atgttggagg ataaaggctg gacagaacaa 720 tactctaacg ctttggacct tcttgtcgtt tgctcatcat ctttgtcgaa agttaatttt 780 gacagtatcg ttaagattat gaagattgga ggctccatcg tttcaattgc tgctcctgaa 840 gttaatgaaa agcttgtttt aaaaccgttg ggcctaatgg gagtatcaat ctcaagcagt 900 gctatcggat ctaggaagga aatcgaacaa ctattgaaat tagtttccga aaagaatgtc 960 aaaatatggg tggaaaaact tccgatcagc gaagaaggcg tcagccatgc ctttacaagg 1020 atggaaagcg gagacgtcaa atacagattt actttggtcg attatgataa gaaattccat 1080 aaatag 1086
SEQ ID NO:142
Saccharomyces cerevisiae
MLYPEKFQGI GISNAKDWKH PKLVSFDPKP FGDHDVDVE I EACGICGSDF HIAVGNWGPV 60
PENQILGHEI IGRWKVGSK CHTGVKIGDR VGVGAQALAC FECERCKSDN EQYCTNDHVL 120
TMWTPYKDGY ISQGGFASHV RLHEHFAIQI PENIPSPLAA PLLCGGITVF SPLLRNGCGP 180
GKRVGIVGIG GIGHMGILLA KAMGAEVYAF SRGHSKREDS MKLGADHYIA MLEDKGWTEQ 240
YSNALDLLW CSSSLSKVNF DSIVKIMKIG GSIVSIAAPE VNEKLVLKPL GLMGVSISSS 300
AIGSRKEIEQ LLKLVSEKNV KIWVEKLPIS EEGVSHAFTR MESGDVKYRF TLVDYDKKFH 360
K 361
SEQ ID NO:143
Saccharomyces cerevisiae
atgtccgccg ctactgttgg taaacctatt aagtgcattg ctgctgttgc gtatgatgcg 60 aagaaaccat taagtgttga agaaatcacg gtagacgccc caaaagcgca cgaagtacgt 120 atcaaaattg aatatactgc tgtatgccac actgatgcgt acactttatc aggctctgat 180 ccagaaggac ttttcccttg cgttctgggc cacgaaggag ccggtatcgt agaatctgta 240 ggcgatgatg tcataacagt taagcctggt gatcatgtta ttgctttgta cactgctgag 300 tgtggcaaat gtaagttctg tacttccggt aaaaccaact tatgtggtgc tgttagagct 360 actcaaggga aaggtgtaat gcctgatggg accacaagat ttcataatgc gaaaggtgaa 420 gatatatacc atttcatggg ttgctctact ttttccgaat atactgtggt ggcagatgtc 480 tctgtggttg ccatcgatcc aaaagctccc ttggatgctg cctgtttact gggttgtggt 540 gttactactg gttttggggc ggctcttaag acagctaatg tgcaaaaagg cgataccgtt 600 gcagtatttg gctgcgggac tgtaggactc tccgttatcc aaggtgcaaa gttaaggggc 660 gcttccaaga tcattgccat tgacattaac aataagaaaa aacaatattg ttctcaattt 720 ggtgccacgg attttgttaa tcccaaggaa gatttggcca aagatcaaac tatcgttgaa 780 aagttaattg aaatgactga tgggggtctg gattttactt ttgactgtac tggtaatacc 840 aaaattatga gagatgcttt ggaagcctgt cataaaggtt ggggtcaatc tattatcatt 900 ggtgtggctg ccgctggtga agaaatttct acaaggccgt tccagctggt cactggtaga 960 gtgtggaaag gctctgcttt tggtggcatc aaaggtagat ctgaaatggg cggtttaatt 1020 aaagactatc aaaaaggtgc cttaaaagtc gaagaattta tcactcacag gagaccattc 1080 aaagaaatca atcaagcctt tgaagatttg cataacggtg attgcttaag aaccgtcttg 1140 aagtctgatg aaataaaata g 1161
SEQ ID NO:144
Saccharomyces cerevisiae
MSAATVGKP I KCIAAVAYDA KKPLSVEEIT VDAPKAHEVR IKIEYTAVCH TDAYTLSGSD 60
PEGLFPCVLG HEGAGIVESV GDDVITVKPG DHVIALYTAE CGKCKFCTSG KTNLCGAVRA 120
TQGKGVMPDG TTRFHNAKGE DIYHFMGCST FSEYTWADV SWAIDPKAP LDAACLLGCG 180
VTTGFGAALK TANVQKGDTV AVFGCGTVGL SVIQGAKLRG ASKIIAIDIN NKKKQYCSQF 240
GATDFVNPKE DLAKDQTIVE KLIEMTDGGL DFTFDCTGNT KIMRDALEAC HKGWGQSIII 300
GVAAAGEEIS TRPFQLVTGR VWKGSAFGGI KGRSEMGGLI KDYQKGALKV EEF ITHRRPF 360
KEINQAFEDL HNGDCLRTVL KSDEIK 386
SEQ ID NO:145
Saccharomyces cerevisiae
atgactactg ataccactgt tttcgtttct ggcgcaaccg gtttcattgc tctacacatt 60 atgaacgatc tgttgaaagc tggctataca gtcatcggct caggtagatc tcaagaaaaa 120 aatgatggct tgctcaaaaa atttaataac aatcccaaac tatcgatgga aattgtggaa 180 gatattgctg ctccaaacgc ctttgatgaa gttttcaaaa aacatggtaa ggaaattaag 240 attgtgctac acactgcctc cccattccat tttgaaacta ccaattttga aaaggattta 300 ctaacccctg cagtgaacgg tacaaaatct atcttggaag cgattaaaaa atatgctgca 360 gacactgttg aaaaagttat tgttacttcg tctactgctg ctctggtgac acctacagac 420 atgaacaaag gagatttggt gatcacggag gagagttgga ataaggatac atgggacagt 480 tgtcaagcca acgccgttgc cgcatattgt ggctcgaaaa agtttgctga aaaaactgct 540 tgggaatttc ttaaagaaaa caagtctagt gtcaaattca cactatccac tatcaatccg 600 ggattcgttt ttggtcctca aatgtttgca gattcgctaa aacatggcat aaatacctcc 660 tcagggatcg tatctgagtt aattcattcc aaggtaggtg gagaatttta taattactgt 720 ggcccattta ttgacgtgcg tgacgtttct aaagcccacc tagttgcaat tgaaaaacca 780 gaatgtaccg gccaaagatt agtattgagt gaaggtttat tctgctgtca agaaatcgtt 840 gacatcttga acgaggaatt ccctcaatta aagggcaaga tagctacagg tgaacctgcg 900 accggtccaa gctttttaga aaaaaactct tgcaagtttg acaattctaa gacaaaaaaa 960 ctactgggat tccagtttta caatttaaag gattgcatag ttgacaccgc ggcgcaaatg 1020 ttagaagttc aaaatgaagc ctaa 1044
SEQ ID NO:146
Saccharomyces cerevisiae
MTTDTTVFVS GATGF IALHI MNDLLKAGYT VIGSGRSQEK NDGLLKKFNN NPKLSME IVE 60 DIAAPNAFDE VFKKHGKE IK IVLHTASPFH FETTNFEKDL LTPAVNGTKS ILEAIKKYAA 120 DTVEKVIVTS STAALVTPTD MNKGDLVITE ESWNKDTWDS CQANAVAAYC GSKKFAEKTA 180 WEFLKENKSS VKFTLSTINP GFVFGPQMFA DSLKHGINTS SGIVSELIHS KVGGEFYNYC 240 GPF IDVRDVS KAHLVAIEKP ECTGQRLVLS EGLFCCQEIV D ILNEEFPQL KGKIATGEPA 300 TGP SFLEKNS CKFDNSKTKK LLGFQFYNLK DCIVDTAAQM LEVQNEA 347
SEQ ID NO:147
Saccharomyces cerevisiae
atgactactg aaaaaaccgt tgtttttgtt tctggtgcta ctggtttcat tgctctacac 60 gtagtggacg atttattaaa aactggttac aaggtcatcg gttcgggtag gtcccaagaa 120 aagaatgatg gattgctgaa aaaatttaag agcaatccca acctttcaat ggagattgtc 180 gaagacattg ctgctccaaa cgcttttgac aaagtttttc aaaagcacgg caaagagatc 240 aaggttgtct tgcacatagc ttctccggtt cacttcaaca ccactgattt cgaaaaggat 300 ctgctaattc ctgctgtgaa tggtaccaag tccattctag aagcaatcaa aaattatgcc 360 gcagacacag tcgaaaaagt cgttattact tcttctgttg ctgcccttgc atctcccgga 420 gatatgaagg acactagttt cgttgtcaat gaggaaagtt ggaacaaaga tacttgggaa 480 agttgtcaag ctaacgcggt ttccgcatac tgtggttcca agaaatttgc tgaaaaaact 540 gcttgggatt ttctcgagga aaaccaatca agcatcaaat ttacgctatc aaccatcaac 600 ccaggatttg tttttggccc tcagctattt gccgactctc ttagaaatgg aataaatagc 660 tcttcagcca ttattgccaa tttggttagt tataaattag gcgacaattt ttataattac 720 agtggtcctt ttattgacgt tcgcgatgtt tcaaaagctc atttacttgc atttgagaaa 780 cccgaatgcg ctggccaaag actattctta tgtgaagata tgttttgctc tcaagaagcg 840 ctggatatct tgaatgagga atttccacag ttaaaaggca agatagcaac tggcgaacct 900 ggtagcggct caaccttttt gacaaaaaac tgctgcaagt gcgacaaccg caaaaccaaa 960 aatttattag gattccaatt taataagttc agagattgca ttgtcgatac tgcctcgcaa 1020 ttactagaag ttcaaagtaa aagctaa 1047
SEQ ID NO:148
Saccharomyces cerevisiae
MTTEKTWFV SGATGFIALH WDDLLKTGY KVIGSGRSQE KNDGLLKKFK SNPNLSMEIV 60 EDIAAPNAFD KVFQKHGKEI KWLHIASPV HFNTTDFEKD LLIPAVNGTK SILEAIKNYA 120 ADTVERWIT SSVAALASPG DMKDTSFWN EESWNKDTWE SCQANAVSAY CGSKKFAEKT 180 AWDFLEENQS SIKFTLSTIN PGFVFGPQLF ADSLRNGINS SSAIIANLVS YKLGDNFYNY 240 SGPFIDVRDV SKAHLLAFEK PECAGQRLFL CEDMFCSQEA LDILNEEFPQ LKGKIATGEP 300 GSGSTFLTKN CCKCDNRKTK NLLGFQFNKF RDCIVDTASQ LLEVQSKS 348 SEQ ID NO:149
Saccharomyces cerevisiae
atgtctaata cagttctagt ttctggcgct tcaggtttta ttgccttgca tatcctgtca 60 caattgttaa aacaagatta taaggttatt ggaactgtga gatcccatga aaaagaagca 120 aaattgctaa gacaatttca acataaccct aatttaactt tagaaattgt tccggacatt 180 tctcatccaa atgctttcga taaggttctg cagaaacgtg gacgtgagat taggtatgtt 240 ctacacacgg cctctccttt tcattatgat actaccgaat atgaaaaaga cttattgatt 300 cccgcgttag aaggtacaaa aaacatccta aattctatca agaaatatgc agcagacact 360 gtagagcgtg ttgttgtgac ttcttcttgt actgctatta taacccttgc aaagatggac 420 gatcccagtg tggtttttac agaagagagt tggaacgaag caacctggga aagctgtcaa 480 attgatggga taaatgctta ctttgcatcc aagaagtttg ctgaaaaggc tgcctgggag 540 ttcacaaaag agaatgaaga tcacatcaaa ttcaaactaa caacagtcaa cccttctctt 600 ctttttggtc ctcaactttt cgatgaagat gtgcatggcc atttgaatac ttcttgcgaa 660 atgatcaatg gcctaattca taccccagta aatgccagtg ttcctgattt tcattccatt 720 tttattgatg taagggatgt ggccctagct catctgtatg ctttccagaa ggaaaatacc 780 gcgggtaaaa gattagtggt aactaacggt aaatttggaa accaagatat cctggatatt 840 ttgaacgaag attttccaca attaagaggt ctcattcctt tgggtaagcc tggcacaggt 900 gatcaagtca ttgaccgcgg ttcaactaca gataatagtg caacgaggaa aatacttggc 960 tttgagttca gaagtttaca cgaaagtgtc catgatactg ctgcccaaat tttgaagaag 1020 cagaacagat tatga 1035
SEQ ID NO:150
Saccharomyces cerevisiae
MSNTVLVSGA SGFIALHILS QLLKQDYKVI GTVRSHEKEA KLLRQFQHNP NLTLEIVPDI 60 SHPNAFDKVL QKRGREIRYV LHTASPFHYD TTEYEKDLLI PALEGTKNIL NSIKKYAADT 120 VERVWTSSC TAI ITLAKMD DPSVVFTEES WNEATWESCQ IDGINAYFAS KKFAEKAAWE 180 FTKENEDHIK FKLTTVNP SL LFGPQLFDED VHGHLNTSCE MINGLIHTPV NASVPDFHSI 240 FIDVRDVALA HLYAFQKENT AGKRLWTNG KFGNQDILDI LNEDFPQLRG LIPLGKPGTG 300 DQVIDRGSTT DNSATRKILG FEFRSLHESV HDTAAQILKK QNRL 344
SEQ ID NO:151
Saccharomyces cerevisiae
atgtcagttt tcgtttcagg tgctaacggg ttcattgccc aacacattgt cgatctcctg 60 ttgaaggaag actataaggt catcggttct gccagaagtc aagaaaaggc cgagaattta 120 acggaggcct ttggtaacaa cccaaaattc tccatggaag ttgtcccaga catatctaag 180 ctggacgcat ttgaccatgt tttccaaaag cacggcaagg atatcaagat agttctacat 240 acggcctctc cattctgctt tgatatcact gacagtgaac gcgatttatt aattcctgct 300 gtgaacggtg ttaagggaat tctccactca attaaaaaat acgccgctga ttctgtagaa 360 cgtgtagttc tcacctcttc ttatgcagct gtgttcgata tggcaaaaga aaacgataag 420 tctttaacat ttaacgaaga atcctggaac ccagctacct gggagagttg ccaaagtgac 480 ccagttaacg cctactgtgg ttctaagaag tttgctgaaa aagcagcttg ggaatttcta 540 gaggagaata gagactctgt aaaattcgaa ttaactgccg ttaacccagt ttacgttttt 600 ggtccgcaaa tgtttgacaa agatgtgaaa aaacacttga acacatcttg cgaactcgtc 660 aacagcttga tgcatttatc accagaggac aagataccgg aactatttgg tggatacatt 720 gatgttcgtg atgttgcaaa ggctcattta gttgccttcc aaaagaggga aacaattggt 780 caaagactaa tcgtatcgga ggccagattt actatgcagg atgttctcga tatccttaac 840 gaagacttcc ctgttctaaa aggcaatatt ccagtgggga aaccaggttc tggtgctacc 900 cataacaccc ttggtgctac tcttgataat aaaaagagta agaaattgtt aggtttcaag 960 ttcaggaact tgaaagagac cattgacgac actgcctccc aaattttaaa atttgagggc 1020 agaatataa 1029
SEQ ID NO:152
Saccharomyces cerevisiae
MSVFVSGANG FIAQHIVDLL LKEDYKVIGS ARSQEKAENL TEAFGNNPKF SMEWPDISK 60 LDAFDHVFQK HGKDIKIVLH TASPFCFDIT DSERDLLIPA VNGVKGILHS IKKYAADSVE 120 RWLTSSYAA VFDMAKENDK SLTFNEESWN PATWESCQSD PVNAYCGSKK FAEKAAWEFL 180 EENRDSVKFE LTAVNPVYVF GPQMFDKDVK KHLNTSCELV NSLMHLSPED KIPELFGGYI 240 DVRDVAKAHL VAFQKRETIG QRLIVSEARF TMQDVLDILN EDFPVLKGNI PVGKPGSGAT 300 HNTLGATLDN KKSKKLLGFK FRNLKETIDD TASQILKFEG RI 342
SEQ ID NO:153
Saccharomyces cerevisiae
atgcctgcta cgttaaagaa ttcttctgct acattaaaac taaatactgg tgcctccatt 60 ccagtgttgg gtttcggcac ttggcgttcc gttgacaata acggttacca ttctgtaatt 120 gcagctttga aagctggata cagacacatt gatgctgcgg ctatctattt gaatgaagaa 180 gaagttggca gggctattaa agattccgga gtccctcgtg aggaaatttt tattactact 240 aagctttggg gtacggaaca acgtgatccg gaagctgctc taaacaagtc tttgaaaaga 300 ctaggcttgg attatgttga cctatatctg atgcattggc cagtgccttt gaaaaccgac 360 agagttactg atggtaacgt tctgtgcatt ccaacattag aagatggcac tgttgacatc 420 gatactaagg aatggaattt tatcaagacg tgggagttga tgcaagagtt gccaaagacg 480 ggcaaaacta aagccgttgg tgtctctaat ttttctatta acaacattaa agaattatta 540 gaatctccaa ataacaaggt ggtaccagct actaatcaaa ttgaaattca tccattgcta 600 ccacaagacg aattgattgc cttttgtaag gaaaagggta ttgttgttga agcctactca 660 ccatttggga gtgctaatgc tcctttacta aaagagcaag caattattga tatggctaaa 720 aagcacggcg ttgagccagc acagcttatt atcagttgga gtattcaaag aggctacgtt 780 gttctggcca aatcggttaa tcctgaaaga attgtatcca attttaagat tttcactctg 840 cctgaggatg atttcaagac tattagtaac ctatccaaag tgcatggtac aaagagagtc 900 gttgatatga agtggggatc cttcccaatt ttccaatga 939
SEQ ID NO:154
Saccharomyces cerevisiae
MPATLKNSSA TLKLNTGASI PVLGFGTWRS VDNNGYHSVI AALKAGYRHI DAAAIYLNEE 60 EVGRAIKDSG VPREEIFITT KLWGTEQRDP EAALNKSLKR LGLDYVDLYL MHWPVPLKTD 120 RVTDGNVLCI PTLEDGTVDI DTKEWNF IKT WELMQELPKT GKTKAVGVSN FSINNIKELL 180 ESPNNKWPA TNQIEIHPLL PQDELIAFCK EKGIVVEAYS PFGSANAPLL KEQAI IDMAK 240 KHGVEPAQLI ISWSIQRGYV VLAKSVNPER IVSNFKIFTL PEDDFKTISN LSKVHGTKRV 300 VDMKWGSFP I FQ 312
SEQ ID NO:155
Saccharomyces cerevisiae
atgcctgcta ctttacatga ttctacgaaa atcctttctc taaatactgg agcccaaatc 60 cctcaaatag gtttaggtac gtggcagtcg aaagagaacg atgcttataa ggctgtttta 120 accgctttga aagatggcta ccgacacatt gatactgctg ctatttaccg taatgaagac 180 caagtcggtc aagccatcaa ggattcaggt gttcctcggg aagaaatctt tgttactaca 240 aagttatggt gtacacaaca ccacgaacct gaagtagcgc tggatcaatc actaaagagg 300 ttaggattgg actacgtaga cttatatttg atgcattggc ctgccagatt agatccagcc 360 tacatcaaaa atgaagacat cttgagtgtg ccaacaaaga aggatggttc tcgtgcagtg 420 gatatcacca attggaattt catcaaaacc tgggaattaa tgcaggaact accaaagact 480 ggtaaaacta aggccgttgg agtctccaac ttttctataa ataacctgaa agatctatta 540 gcatctcaag gtaataagct tacgccagct gctaaccaag tcgaaataca tccattacta 600 cctcaagacg aattgattaa tttttgtaaa agtaaaggca ttgtggttga agcttattct 660 ccgttaggta gtaccgatgc tccactattg aaggaaccgg ttatccttga aattgcgaag 720 aaaaataacg ttcaacccgg acacgttgtt attagctggc acgtccaaag aggttatgtt 780 gtcttgccaa aatctgtgaa tcccgatcga atcaaaacga acaggaaaat atttactttg 840 tctactgagg actttgaagc tatcaataac atatcgaagg aaaagggcga aaaaagggtt 900 gtacatccaa attggtctcc tttcgaagta ttcaagtaa 939
SEQ ID NO:156
Saccharomyces cerevisiae
MPATLHDSTK ILSLNTGAQI PQIGLGTWQS KENDAYKAVL TALKDGYRHI DTAAIYRNED 60 QVGQAIKDSG VPREE IFVTT KLWCTQHHEP EVALDQSLKR LGLDYVDLYL MHWPARLDPA 120 YIKNED ILSV PTKKDGSRAV DITNWNFIKT WELMQELPKT GKTKAVGVSN FSINNLKDLL 180 ASQGNKLTPA ANQVEIHPLL PQDELINFCK SKGIVVEAYS PLGSTDAPLL KEPVILE IAK 240 KNNVQPGHW ISWHVQRGYV VLPKSVNPDR IKTNRKIFTL STEDFEAINN ISKEKGEKRV 300 VHPNWSPFEV FK 312
SEQ ID NO:157
Saccharomyces cerevisiae
atgactgact tgtttaaacc tctacctgaa ccacctaccg aattgggacg tctcagggtt 60 ctttctaaaa ctgccggcat aagggtttca ccgctaattc tgggaggagc ttcaatcggc 120 gacgcatggt caggctttat gggctctatg aataaggaac aggcctttga acttcttgat 180 gctttttatg aagctggagg taattgtatt gatactgcaa acagttacca aaatgaagag 240 tcagagattt ggataggtga atggatggca tcaagaaaac tgcgtgacca gattgtaatt 300 gccaccaagt ttaccggaga ttataagaag tatgaagtag gtggtggtaa aagtgccaac 360 tactgtggta atcacaagcg tagtttacat gtgagtgtga gggattctct ccgcaaattg 420 caaactgatt ggattgatat actttacatt cactggtggg attatatgag ttcaatcgaa 480 gaagttatgg atagtttgca tattttagtt cagcagggca aggtcctata tttaggagta 540 tctgatacac ctgcttgggt tgtttctgcg gcaaattact acgctacatc tcatggtaaa 600 actcctttta gcgtctatca aggtaaatgg aatgtattga acagggactt tgagcgtgat 660 attattccaa tggctaggca ttttggtatg gctctagccc catgggatgt catgggaggt 720 ggaagatttc agagtaaaaa agcaatggaa gaacggaaga agaatggaga gggtctgcgt 780 acttttgtgg gtggccccga acaaacagaa ttggaggtta aaatcagcga agcattgact 840 aaaattgctg aggaacatgg aacagagtct gttactgcta tcgctattgc ctatgttcgc 900 tctaaagcga aaaatgtttt cccattgatt ggaggaagga aaattgaaca tctcaagcag 960 aacattgagg ctttgagtat taaattaaca ccggaacaaa tagaatacct ggaaagtatt 1020 gttccttttg atgttggctt tcccaaaagt ttaataggag atgacccagc ggtaaccaag 1080 aagctttcac ccctcacatc gatgtctgcc aggatagctt ttgacaatta g 1131
SEQ ID NO:158
Saccharomyces cerevisiae
MTDLFKPLPE PPTELGRLRV LSKTAGIRVS PLILGGASIG DAWSGFMGSM NKEQAFELLD 60 AFYEAGGNCI DTANSYQNEE SEIWIGEWMA SRKLRDQIVI ATKFTGDYKK YEVGGGKSAN 120 YCGNHKRSLH VSVRDSLRKL QTDWIDILYI HWWDYMSSIE EVMDSLHILV QQGKVLYLGV 180 SDTPAWWSA ANYYATSHGK TPFSVYQGKW NVLNRDFERD I IPMARHFGM ALAPWDVMGG 240 GRFQSKKAME ERKKNGEGLR TFVGGPEQTE LEVKISEALT KIAEEHGTES VTAIAIAYVR 300 SKAKNVFPLI GGRKIEHLKQ NIEALSIKLT PEQIEYLESI VPFDVGFPKS LIGDDPAVTK 360 KLSPLTSMSA RIAFDN 376
SEQ ID NO:159
Saccharomyces cerevisiae
atgattgggt ccgcgtccga ctcatctagc aagttaggac gcctccgatt tctttctgaa 60 actgccgcta ttaaagtatc cccgttaatc ctaggagaag tctcatacga tggagcacgt 120 tcggattttc tcaaatcaat gaacaagaat cgagcttttg aattgcttga tactttttac 180 gaggcaggtg gaaatttcat tgatgccgca aacaactgcc aaaacgagca atcagaagaa 240 tggattggtg aatggataca gtccagaagg ttacgtgatc aaattgtcat tgcaaccaag 300 tttataaaaa gcgataaaaa gtataaagca ggtgaaagta acactgccaa ctactgtggt 360 aatcacaagc gtagtttaca tgtgagtgtg agggattctc tccgcaaatt gcaaactgat 420 tggattgata tactttacgt tcactggtgg gattatatga gttcaatcga agaatttatg 480 gatagtttgc atattctggt ccagcagggc aaggtcctct atttgggtgt atctgataca 540 cctgcttggg ttgtttctgc ggcaaactac tacgctacat cttatggtaa aactcccttt 600 agtatctacc aaggtaaatg gaacgtgttg aacagagatt ttgagcgtga tattattcca 660 atggctaggc atttcggtat ggccctcgcc ccatgggatg tcatgggagg tggaagattt 720 cagagtaaaa aagcaatgga ggaacggagg aagaatggag agggtattcg ttctttcgtt 780 ggcgcctccg aacaaacaga tgcagaaatc aagattagtg aagcattggc caagattgct 840 gaggaacatg gcactgagtc tgttactgct attgctattg cctatgttcg ctctaaggcg 900 aaaaattttt ttccgtcggt tgaaggagga aaaattgagg atctcaaaga gaacattaag 960 gctctcagta tcgatctaac gccagacaat ataaaatact tagaaagtat agttcctttt 1020 gacatcggat ttcctaataa ttttatcgtg ttaaattcct tgactcaaaa atatggtacg 1080 aataatgttt ag 1092
SEQ ID NO:160
Saccharomyces cerevisiae
MIGSASDSSS KLGRLRFLSE TAAIKVSPLI LGEVSYDGAR SDFLKSMNKN RAFELLDTFY 60
EAGGNF IDAA NNCQNEQSEE WIGEWIQSRR LRDQIVIATK F IKSDKKYKA GESNTANYCG 120
NHKRSLHVSV RDSLRKLQTD WIDILYVHWW DYMSS IEEFM DSLHILVQQG KVLYLGVSDT 180
PAWWSAANY YATSYGKTPF SIYQGKWNVL NRDFERDIIP MARHFGMALA PWDVMGGGRF 240
QSKKAMEERR KNGEGIRSFV GASEQTDAEI KISEALAKIA EEHGTESVTA IAIAYVRSKA 300
KNFFPSVEGG KIEDLKENIK ALSIDLTPDN IKYLESIVPF DIGFPNNFIV LNSLTQKYGT 360
NNV 363
SEQ ID NO:161
Saccharomyces cerevisiae
atgtctatcc cagaaactca aaaaggtgtt atcttctacg aatcccacgg taagttggaa 60 tacaaagata ttccagttcc aaagccaaag gccaacgaat tgttgatcaa cgttaaatac 120 tctggtgtct gtcacactga cttgcacgct tggcacggtg actggccatt gccagttaag 180 ctaccattag tcggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca acaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ccgacttggc ccaagtcgcc cccatcttgt gtgctggtat caccgtctac 480 aaggctttga agtctgctaa cttgatggcc ggtcactggg ttgctatctc cggtgctgct 540 ggtggtctag gttctttggc tgttcaatac gccaaggcta tgggttacag agtcttgggt 600 attgacggtg gtgaaggtaa ggaagaatta ttcagatcca tcggtggtga agtcttcatt 660 gacttcacta aggaaaagga cattgtcggt gctgttctaa aggccactga cggtggtgct 720 cacggtgtca tcaacgtttc cgtttccgaa gccgctattg aagcttctac cagatacgtt 780 agagctaacg gtaccaccgt tttggtcggt atgccagctg gtgccaagtg ttgttctgat 840 gtcttcaacc aagtcgtcaa gtccatctct attgttggtt cttacgtcgg taacagagct 900 gacaccagag aagctttgga cttcttcgcc agaggtttgg tcaagtctcc aatcaaggtt 960 gtcggcttgt ctaccttgcc agaaatttac gaaaagatgg aaaagggtca aatcgttggt 1020 agatacgttg ttgacacttc taaataa 1047
SEQ ID NO:162
Saccharomyces cerevisiae
MSIPETQKGV IFYESHGKLE YKDIPVPKPK ANELLINVKY SGVCHTDLHA WHGDWPLPVK 60
LPLVGGHEGA GVWGMGENV KGWKIGDYAG IKWLNGSCMA CEYCELGNES NCPHADLSGY 120
THDGSFQQYA TADAVQAAHI PQGTDLAQVA PILCAGITVY KALKSANLMA GHWVAISGAA 180
GGLGSLAVQY AKAMGYRVLG IDGGEGKEEL FRSIGGEVFI DFTKEKD IVG AVLKATDGGA 240
HGVINVSVSE AAIEASTRYV RANGTTVLVG MPAGAKCCSD VFNQWKSIS IVGSYVGNRA 300
DTREALDFFA RGLVKSPIKV VGLSTLPEIY EKMEKGQIVG RYWDTSK 348
SEQ ID NO:163
Saccharomyces cerevisiae
atggccagca agactttgag ggttcttttt ctgggtccca aaggtacgta ttcccatcaa 60 gctgcattac aacaatttca atcaacatct gatgttgagt acctcccagc agcctctatc 120 ccccaatgtt ttaaccaatt ggagaacgac actagtatag attattcagt ggtaccgttg 180 gaaaattcca ccaatggaca agtagttttt tcctatgatc tcttgcgtga taggatgatc 240 aaaaaagccc tatccttacc tgctccagca gatactaata gaattacacc agatatagaa 300 gttatagcgg agcaatatgt acccattacc cattgtctaa tcagcccaat ccaactacca 360 aatggtattg catcccttgg aaattttgaa gaagtcataa tacactcaca tccgcaagta 420 tggggccagg ttgaatgtta cttaaggtcc atggcagaaa aatttccgca ggtcaccttt 480 ataagattgg attgttcttc cacatctgaa tcagtgaacc aatgcattcg gtcatcaacg 540 gccgattgcg acaacattct gcatttagcc attgctagtg aaacagctgc ccaattgcat 600 aaggcgtaca tcattgaaca ttcgataaat gataagctag gaaatacaac aagattttta 660 gtattgaaga gaagggagaa cgcaggcgac aatgaagtag aagacactgg attactacgg 720 gttaacctac tcacctttac tactcgtcaa gatgaccctg gttctttggt agatgttttg 780 aacatactaa aaatccattc actcaacatg tgttctataa actctagacc attccatttg 840 gacgaacatg atagaaactg gcgatattta tttttcattg aatattacac cgagaagaat 900 accccaaaga ataaagaaaa attctatgaa gatatcagcg acaaaagtaa acagtggtgc 960 ctgtggggta cattccccag aaatgagaga tattatcaca aataa 1005
SEQ ID NO:164
Saccharomyces cerevisiae
MASKTLRVLF LGPKGTYSHQ AALQQFQSTS DVEYLPAAS I PQCFNQLEND TSIDYSVVPL 60 ENSTNGQWF SYDLLRDRMI KKALSLPAPA DTNRITPDIE VIAEQYVPIT HCLISPIQLP 120 NGIASLGNFE EVIIHSHPQV WGQVECYLRS MAEKFPQVTF IRLDCSSTSE SVNQCIRSST 180 ADCDNILHLA IASETAAQLH KAYI IEHSIN DKLGNTTRFL VLKRRENAGD NEVEDTGLLR 240 VNLLTFTTRQ DDPGSLVDVL NILKIHSLNM CSINSRPFHL DEHDRNWRYL FFIEYYTEKN 300 TPKNKEKFYE DISDKSKQWC LWGTFPRNER YYHK 334
SEQ ID NO:165
Saccharomyces cerevisiae
atgaccgctt ccatcaaaat tcaaccggat attgactctc taaagcaatt acagcagcaa 60 aatgacgata gttccataaa tatgtatccc gtgtatgcgt atttgccatc attggatctg 120 actcctcacg tggcatatct aaaattggca caattgaaca accctgatag aaaggaatca 180 tttctgttgg aaagtgctaa gacaaataat gaattagatc gttattcatt cataggtatc 240 tcgccacgca agaccatcaa aaccggtcct actgaaggca ttgaaacaga tcctttggaa 300 attttggaaa aggagatgtc tacctttaaa gtagccgaaa atgttcctgg tttaccgaaa 360 ttaagtggtg gtgctattgg ttatatttct tatgactgtg ttcgttattt cgagccaaaa 420 acaagaaggc ctttgaaaga tgtcctaaga cttccagagg catatttaat gctttgtgat 480 accattattg cctttgataa tgtttttcag agatttcaaa tcattcataa cattaatacc 540 aatgaaactt cgttggagga aggttaccaa gctgcagcac aaataatcac tgatatcgta 600 tcaaagctaa ccgacgattc ctcgccaata ccatatccag aacaacctcc tattaaattg 660 aatcaaactt ttgaatcgaa tgtgggcaag gaaggttacg aaaatcacgt ctccactttg 720 aagaagcata ttaagaaagg tgatattatt caaggtgtgc catcgcaaag agtggcaagg 780 ccaacttcgt tacatccttt caatatttac agacatttac gtacagtgaa cccatctcct 840 tacctgtttt atattgattg tttggatttc caaatcattg gtgcatctcc agaattgttg 900 tgcaaatcgg attccaaaaa tagagtcatt acccatccaa ttgctggtac tgtcaaacgt 960 ggggctacta ctgaagagga tgatgcttta gcggaccaat tacgtggctc gttaaaagac 1020 cgtgcagaac atgttatgct ggtagattta gcaagaaacg atattaacag aatttgtgac 1080 ccattaacaa caagtgtcga taaactgtta actattcaaa aattttctca tgtccaacat 1140 ctggtttctc aagtcagcgg tgttctccgc ccagaaaaga caagatttga tgcattcaga 1200 tcgattttcc ctgcaggtac tgtcagtggt gctccaaagg ttagagccat ggaattgatt 1260 gccgaactag aaggagaaag gcgtggggtt tatgcaggcg ccgtaggtca ttggtcatac 1320 gacggtaaaa caatggacaa ttgtatcgct ttaaggacta tggtctataa agatggcatt 1380 gcttacttgc aagctggcgg tggtattgtt tacgattcag atgagtacga tgaatatgtc 1440 gaaaccatga ataaaatgat ggccaatcac agtactattg tgcaagcaga agaattgtgg 1500 gccgatatcg taggatcagc ttaa 1524
SEQ ID NO:166
Saccharomyces cerevisiae
MTASIKIQPD IDSLKQLQQQ NDDSSINMYP VYAYLPSLDL TPHVAYLKLA QLNNPDRKES 60 FLLESAKTNN ELDRYSFIGI SPRKTIKTGP TEGIETDPLE ILEKEMSTFK VAENVPGLPK 120 LSGGAIGYIS YDCVRYFEPK TRRPLKDVLR LPEAYLMLCD TIIAFDNVFQ RFQIIHNINT 180 NETSLEEGYQ AAAQIITDIV SKLTDDSSPI PYPEQPPIKL NQTFESNVGK EGYENHVSTL 240 KKHIKKGDII QGVPSQRVAR PTSLHPFNIY RHLRTVNPSP YLFYIDCLDF QIIGASPELL 300 CKSDSKNRVI THP IAGTVKR GATTEEDDAL ADQLRGSLKD RAEHVMLVDL ARNDINRICD 360 PLTTSVDKLL TIQKFSHVQH LVSQVSGVLR PEKTRFDAFR SIFPAGTVSG APKVRAMELI 420 AELEGERRGV YAGAVGHWSY DGKTMDNCIA LRTMVYKDGI AYLQAGGGIV YDSDEYDEYV 480 ETMNKMMANH STIVQAEELW ADIVGSA 507 SEQ ID NO:167
Saccharomyces cerevisiae
atgtctgtgc acgctgcaac aaacccaatc aataagcatg tggttctaat tgacaactac 60 gattccttta cctggaacgt ttacgagtac ttgtgccagg agggcgccaa agtgagcgtc 120 taccgtaacg atgcaattac agttccagaa attgccgcct tgaatcccga cacattgctt 180 atctcgcctg gaccaggcca cccaaagaca gattctggca tttcaagaga ctgtatccgg 240 tactttactg ggaaaattcc tgtatttgga atctgtatgg gccagcaatg catgtttgac 300 gtatttggtg gtgaagttgc ctacgctggt gagattgtcc acggtaaaac gtccccaatc 360 tctcacgaca actgtggaat tttcaagaac gtgccgcaag gtattgctgt gacaagatac 420 cattcattgg ccgggacaga atcgtcccta ccatcctgct tgaaggttac tgcgagtacc 480 gaaaatggaa ttatcatggg tgtaagacac aagaagtaca ctgtagaagg tgtgcaattt 540 catccggaat ccatcttgac cgaggaaggt catctgatga tcaggaacat tttaaacgtc 600 agtggaggca cttgggagga aaacaaatca tctccttcaa attctatttt ggaccgtatc 660 tatgctcggc gtaaaataga cgtcaatgag cagtctaaaa tcccaggttt cacctttcaa 720 gacttacaat ctaactatga tttaggtctt gccccaccgt tacaggattt ctacacggtg 780 ttgtcatcat cccataaaag agccgttgtt cttgctgaag tcaagcgtgc ctctccatcg 840 aagggaccca tttgtttaaa agctgttgct gctgaacagg ctctcaaata cgcagaggct 900 ggtgcatccg caatttccgt attgaccgaa cctcattggt ttcacggttc gttacaggat 960 ttagtaaatg tgaggaaaat cctagatttg aaatttcctc ccaaggagag gccttgtgtt 1020 ttgagaaaag aatttatttt cagcaagtat caaatactag aagcaagatt agctggagct 1080 gacactgtcc ttcttatagt caagatgcta tctcaaccct tattgaagga actgtacagc 1140 tacagtaaag atttgaacat ggaacctctc gttgaggtga actccaaaga ggaattacaa 1200 agggctctag aaattggtgc taaagttgta ggtgtcaata atagggacct gcattcattc 1260 aacgtagacc taaataccac cagtaacttg gtagaatcta ttccaaagga tgttcttcta 1320 attgctctat cgggaattac caccagggac gatgctgaaa aatacaaaaa agaaggtgtc 1380 catggatttt tagtgggtga agccctaatg aaatcaaccg atgtgaagaa gttcattcat 1440 gaattatgcg aataa 1455
SEQ ID NO:168
Saccharomyces cerevisiae
MSVHAATNP I NKHWLIDNY DSFTWNVYEY LCQEGAKVSV YRNDAITVPE IAALNPDTLL 60 ISPGPGHPKT DSGISRDCIR YFTGKIPVFG ICMGQQCMFD VFGGEVAYAG EIVHGKTSPI 120 SHDNCGIFKN VPQGIAVTRY HSLAGTESSL PSCLKVTAST ENGI IMGVRH KKYTVEGVQF 180 HPESILTEEG HLMIRNILNV SGGTWEENKS SPSNSILDRI YARRKIDVNE QSKIPGFTFQ 240 DLQSNYDLGL APPLQDFYTV LSSSHKRAW LAEVKRASP S KGP ICLKAVA AEQALKYAEA 300 GASAISVLTE PHWFHGSLQD LVNVRKILDL KFPPKERPCV LRKEFIFSKY QILEARLAGA 360 DTVLLIVKML SQPLLKELYS YSKDLNMEPL VEVNSKEELQ RALE IGAKW GVNNRDLHSF 420 NVDLNTTSNL VESIPKDVLL IALSGITTRD DAEKYKKEGV HGFLVGEALM KSTDVKKFIH 480 ELCE 484
SEQ ID NO:169
gatctatgcg actgggtgag catatgttcc gctgatgtga tgtgcaagat aaacaagcaa 60 ggcagaaact aacttcttct tcatgtaata aacacacccc gcgtttattt acctatctct 120 aaacttcaac accttatatc ataactaata tttcttgaga taagcacact gcacccatac 180 cttccttaaa aacgtagctt ccagtttttg gtggttccgg cttccttccc gattccgccc 240 gctaaacgca tatttttgtt gcctggtggc atttgcaaaa tgcataacct atgcatttaa 300 aagattatgt atgctcttct gacttttcgt gtgatgaggc tcgtggaaaa aatgaataat 360 ttatgaattt gagaacaatt ttgtgttgtt acggtatttt actatggaat aatcaatcaa 420 ttgaggattt tatgcaaata tcgtttgaat atttttccga ccctttgagt acttttcttc 480 ataattgcat aatattgtcc gctgcccctt tttctgttag acggtgtctt gatctacttg 540 ctatcgttca acaccacctt attttctaac tatttttttt ttagctcatt tgaatcagct 600 tatggtgatg gcacattttt gcataaacct agctgtcctc gttgaacata ggaaaaaaaa 660 atatataaac aaggctcttt cactctcctt gcaatcagat ttgggtttgt tccctttatt 720 ttcatatttc ttgtcatatt cctttctcaa ttattatttt ctactcataa cctcacgcaa 780 aataacacag tcaaatctat caaaa 805 SEQ ID NO:170
gatctgggcc gtatacttac atatagtaga tgtcaagcgt aggcgcttcc cctgccggct 60 gtgagggcgc cataaccaag gtatctatag accgccaatc agcaaactac ctccgtacat 120 tcatgttgca cccacacatt tatacaccca gaccgcgaca aattacccat aaggttgttt 180 gtgacggcgt cgtacaagag aacgtgggaa ctttttaggc tcaccaaaaa agaaagaaaa 240 aatacgagtt gctgacagaa gcctcaagaa aaaaaaaatt cttcttcgac tatgctggag 300 gcagagatga tcgagccggt agttaactat atatagctaa attggttcca tcaccttctt 360 ttctggtgtc gctccttcta gtgctatttc tggcttttcc tatttttttt tttccatttt 420 tctttctctc tttctaatat ataaattctc ttgcattttc tatttttctc tctatctatt 480 ctacttgttt attcccttca aggttttttt ttaaggagta cttgttttta gaatatacgg 540 tcaacgaact ataattaact aaaca 565
SEQ ID NO:171
tcgagtttat cattatcaat actgccattt caaagaatac gtaaataatt aatagtagtg 60 attttcctaa ctttatttag tcaaaaaatt agccttttaa ttctgctgta acccgtacat 120 gcccaaaata gggggcgggt tacacagaat atataacatc gtaggtgtct gggtgaacag 180 tttattcctg gcatccacta aatataatgg agcccgcttt ttaagctggc atccagaaaa 240 aaaaagaatc ccagcaccaa aatattgttt tcttcaccaa ccatcagttc ataggtccat 300 tctcttagcg caactacaga gaacaggggc acaaacaggc aaaaaacggg cacaacctca 360 atggagtgat gcaacctgcc tggagtaaat gatgacacaa ggcaattgac ccacgcatgt 420 atctatctca ttttcttaca ccttctatta ccttctgctc tctctgattt ggaaaaagct 480 gaaaaaaaag gttgaaacca gttccctgaa attattcccc tacttgacta ataagtatat 540 aaagacggta ggtattgatt gtaattctgt aaatctattt cttaaacttc ttaaattcta 600 cttttatagt tagtcttttt tttagtttta aaacaccaag aacttagttt cgaataaaca 660 cacataaaca aacaaa 676
SEQ ID NO:172
gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60 gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120 ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180 aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc 240 tcgacttcct gtcatcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctag 300 cgacggctca caggttttgt aacaagcaat cgaaggttct ggaatggcgg gaaagggttt 360 agtaccacat gctatgatgc ccactgtgat ctccagagca aagttcgttc gatcgtactg 420 ttactctctc tctttcaaac agaattgtcc gaatcgtgtg acaacaacag cctgttctca 480 cacactcttt tcttctaacc aagggggtgg tttagtttag tagaacctcg tgaaacttac 540 atttacatat atataaactt gcataaattg gtcaatgcaa gaaatacata tttggtcttt 600 tctaattcgt agtttttcaa gttcttagat gctttctttt tctctttttt acagatcatc 660 aaggaagtaa ttatctactt tttacaacaa atataaaaca agatct 706
SEQ ID NO:173
ggacttttaa ttttcgagga ccgcgaatcc ttacatcaca cccaatcccc cacaagtgat 60 cccccacaca ccatagcttc aaaatgtttc tactcctttt ttactcttcc agattttctc 120 ggactccgcg catcgccgta ccacttcaaa acacccaagc acagcatact aaatttcccc 180 tctttcttcc tctagggtgt cgttaattac ccgtactaaa ggtttggaaa agaaaaaaga 240 gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa aaatttttat cacgtttctt 300 tttcttgaaa attttttttt ttgatttttt tctctttcga tgacctccca ttgatattta 360 agttaataaa cggtcttcaa tttctcaagt ttcagtttca tttttcttgt tctattacaa 420 ctttttttac ttcttgctca ttagaaagaa agcatagcaa tctaatctaa gttttaataa 480 aca 483
SEQ ID NO:174
aacaaatcgc tcttaaatat atacctaaag aacattaaag ctatattata agcaaagata 60 cgtaaatttt gcttatatta ttatacacat atcatatttc tatattttta agatttggtt 120 atataatgta cgtaatgcaa aggaaataaa ttttatacat tattgaacag cgtccaagta 180 actacattat gtgcactaat agtttagcgt cgtgaagact ttattgtgtc gcgaaaagta 240 aaaattttaa aaattagagc accttgaact tgcgaaaaag gttctcatca actgtttaaa 300 aggaggatat caggtcctat ttctgacaaa caatatacaa atttagtttc aaagatgaat 360 cagtgcgcga aggacataac tcatgaagcc tccagtatac c 401
SEQ ID NO:175
atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 60 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 120 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 180 acgctcgaag 190
SEQ ID NO:176
gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 60 gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 120 tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 180 caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 240 gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 300 ttggagcatg 310
SEQ ID NO:177
ggagtgcttt taactaagaa ttattagtct tttctgctta ttttttcatc atagtttaga 60 acactttata ttaacgaata gtttatgaat ctatttaggt ttaaaaattg atacagtttt 120 ataagttact ttttcaaaga ctcgtgctgt ctattgcata atgcactgga aggggaaaaa 180 aaaggtgcac acgcgtggct ttttcttgaa tttgcagttt gaaaaataac tacatggatg 240 ataagaaaac atggagtaca gtcactttga gaaccttcaa tcagctggta acgtcttcgt 300 taattgg 307
SEQ ID NO:178
gtagatacgt tgttgacact tctaaataag cgaatttctt atgatttatg atttttatta 60 ttaaataagt tataaaaaaa ataagtgtat acaaatttta aagtgactct taggttttaa 120 aacgaaaatt cttattcttg agtaactctt tcctgtaggt caggttgctt tctcaggtat 180 agcatgaggt cgctc 195
SEQ ID NO:179
gagttataat aatcctacgt tagtgtgagc gggatttaaa ctgtgaggac cttaatacat 60 tcagacactt ctgcggtatc accctactta ttcccttcga gattatatct aggaacccat 120 caggttggtg gaagattacc cgttctaaga cttttcagct tcctctattg atgttacacc 180 tggacacccc ttttctggca tccagttttt aatcttcagt ggcatgtgag attctccgaa 240 attaattaaa gcaatcacac aattctctcg gataccacct cggttgaaac tgacaggtgg 300 tttgttacgc atgctaatgc aaaggagcct atataccttt ggctcggctg ctgtaacagg 360 gaatataaag ggcagcataa tttaggagtt tagtgaactt gcaacattta ctattttccc 420 ttcttacgta aatatttttc tttttaattc taaatcaatc tttttcaatt ttttgtttgt 480 attcttttct tgcttaaatc tataactaca aaaaacacat acataaacta 530
SEQ ID NO:180
cacccatgaa ccacacggtt agtccaaaag gggcagttca gattccagat gcgggaatta 60 gcttgctgcc accctcacct cactaacgct gcggtgtgcg gatacttcat gctatttata 120 gacgcgcgtg tcggaatcag cacgcgcaag aaccaaatgg gaaaatcgga atgggtccag 180 aactgctttg agtgctggct attggcgtct gatttccgtt ttgggaatcc tttgccgcgc 240 gcccctctca aaactccgca caagtcccag aaagcgggaa agaaataaaa cgccaccaaa 300 aaaaaaaaaa taaaagccaa tcctcgaagc gtgggtggta ggccctggat tatcccgtac 360 aagtatttct caggagtaaa aaaaccgttt gttttggaat ttcccatttc gcggccacct 420 acgccgctat ctttgcaaca actatctgcg ataactcagc aaattttgca tattcgtgtt 480 gcagtattgc gataatggga gtcttacttc caacataacg gcagaaagaa atgtgagaaa 540 attttgcatc ctttgcctcc gttcaagtat ataaagtcgg catgcttgat aatctttctt 600 tccatcctac attgttctaa ttattcttat tctcctttat tctttcctaa cataccaaga 660 aattaatctt ctgtcattcg cttaaacact atatcaataa agatct 706
SEQ ID NO:181
ataaagcaat cttgatgagg ataatgattt ttttttgaat atacataaat actaccgttt 60 ttctgctaga ttttgtgaag acgtaaataa gtacatatta ctttttaagc caagacaaga 120 ttaagcatta actttaccct tttctcttct aagtttcaat actagttatc actgtttaaa 180 agttatggcg agaacgtcgg cggttaaaat atattaccct gaacgtggtg aattgaagtt 240 ctaggatgg 249
SEQ ID NO:182
ggatcctaac tcgagattga attgaattga aatcgataga tcaatttttt tcttttctct 60 ttccccatcc tttacgctaa aataatagtt tattttattt tttgaatatt ttttatttat 120 atacgtatat atagactatt atttatcttt taatgattat taagattttt attaaaaaaa 180 aattcgctcc tcttttaatg cctttatgca gttttttttt cccattcgat atttctatgt 240
SEQ ID NO:183
Saccharomyces cerevisiae
atgcgaggct tgacacctaa gaacggtgta catattgaga cgggtccgga tacagaatcg 60 tccgcggact ccagcaactt ttctacaggt ttcagcggca agattcgtaa gccaaggtcg 120 aaagtaagta aagcgtgcga taactgtaga aaaagaaaga taaaatgtaa tgggaagttt 180 ccctgcgcaa gctgtgagat atattcatgt gagtgcacgt tcagcactag acaaggtggc 240 gctcgaataa aaaaccttca caagacgagt ttggaaggta caaccgtaca agtcaaagag 300 gaaacagatt ccagttcgac ttctttttct aatcctcagc gatgtacaga cgggccatgc 360 gcagtggaac aaccaacgaa attttttgag aatttcaagc taggtggtcg tagtagtggt 420 gataatagcg gaagtgatgg gaagaatgac gacgatgtga atagaaacgg tttttatgaa 480 gacgatagcg aatcacaggc aactttgaca tctctacaaa ccactctgaa aaatttgaag 540 gagatggctc atttaggtac acatgtaacc tcagccatcg agagcattga acttcagata 600 agtgacttgc ttaagcgatg ggaacccaaa gtgaggacca aagaattagc aacaacgaaa 660 ttttacccta ataaatcaat agaaactcaa ttgatgaaaa acaaatactg tgatgtggta 720 catttgacaa gatatgccgc ttggtcaaac aataagaagg accaagatac ttctagtcaa 780 cctttgatcg acgaaatctt tggtttgtac tctcctttcc aatttttgtc acttcaaggt 840 attggtaaat gtttccaaaa ttatagatcc aaatccaaat gcgagatttt tcccaggacc 900 gccaaggaaa ccatatacat catgttaagg tttttcgacg tttgttttca tcacatcaac 960 caaggctgtg tctccatagc taatcccctg gaaaactacc tacagaaaat gaatctttta 1020 ccttcaaccc catcttcgat atcatctgca gggagcccga atacagcaca cacaaaatcg 1080 catgttgcgt tagtgataaa tcacttaccg cagccctttg tgagaaatat aaccggaata 1140 agtaattctg agctactaag cgaaatgaat aatgatatca gcatgtttgg cattttattg 1200 aagatgttgg atatgcataa aaattcctac cagaacttcc taatggaaat cacatctaac 1260 ccctccgtgg ctaaaaacac gcagtcaatt gatgtcttgc aggaattcat ccattattgt 1320 caagctggag aggcgctaat cgccttatgt tacagctatt ataattctac attatacaac 1380 tacgtggact tcacatgcga cataacgcat ttagagcaac ttctatactt tttggatctg 1440 ctattttggc tatcggaaat atacggtttt gaaaaagtct tgaacgtagc agttcatttt 1500 gtttcaagag ttggtttatc aaggtgggaa ttttacgtgg gtcttgatga aaactttgct 1560 gaaagaagaa gaaatctctg gtggaaagct ttctatttcg agaaaacttt agcctctaaa 1620 cttggctatc cttcgaacat tgatgactcc aaaatcaatt gtttattacc taagaatttt 1680 cgagatgttg ggttcttgga taatagagat tttatcgaga acgtccactt agttcgcagg 1740 agcgaagctt ttgacaacat gtgcatatcc gacttaaaat actatggcga attagctgtc 1800 ttacaaatag tcagccattt ctcatcatct gttttattca atgaaaaatt cacgtcaatt 1860 aggaatacat caaaaccatc tgttgttaga gagaaactac tcttcgaggt actcgagatt 1920 tttaacgaaa cagaaatgaa atatgatgct atcaaagagc aaactggaaa attgttcgat 1980 attgcattct ctaaagatag tactgaactc aaagtttcaa gagaggataa gattatggct 2040 tcaaaatttg ttctatttta cgagcaccat ttctgcagaa tggttaacga atctgataat 2100 attgtcgcca gattatgcgt gcacagaagg ccctcaatac taattgaaaa cttgaaaata 2160 taccttcaca agatttacaa atcatggacc gatatgaaca aaatcttatt agactttgac 2220 aacgattatt ccgtataccg ttcgttcgcc cattattcta tcagttgcat tattttggta 2280 tcacaagcat tctcagtggc cgaattcatt aaagtaaatg acgttgtgaa tatgattaga 2340 gtcttcaaaa gatttttgga tataaaaatt ttttccgaaa atgagaccaa tgagcatgtc 2400 ttcaacagcc aaagctttaa agactataca agagcttttt ctttcctgac aatcgtcact 2460 cgtatcatgc ttttggcata cggagagtcc tctagcacca acctcgatgt tatctccaaa 2520 tatattgatg agaatgcgcc agatttaaaa ggtataatag aacttgtctt ggatacaaac 2580 tcttgcgctt atcgattttt attagaacct gttcagaagt caggattcca tttaacggtc 2640 tcacaaatgt tgaaaaatag aaagtttcag gaaccactaa tgtcaaatga agataataag 2700 cagatgaagc ataattcggg aaaaaatttg aatccggatc tcccaagtct taagacgggc 2760 acttcatgct tactaaatgg cattgaatcg cctcaattgc cattcaatgg tcgctcagca 2820 ccttccccag tgagaaacaa ctcactaccg gagtttgcac aattgccttc atttaggtca 2880 ttatccgtgt ctgatatgat caatcccgat tacgcgcaac caacaaatgg gcaaaataat 2940 acgcaagtcc aatctaataa accaatcaat gctcagcagc aaatacccac ttcagtacaa 3000 gtaccattta tgaacacaaa tgaaatcaat aacaacaaca acaacaacaa caacaataaa 3060 aacaatatta acaatattaa caacaacaac agtaacaatt tttctgcaac tagttttaat 3120 ttggggacac tagatgaatt tgttaataac ggtgatcttg aggacctcta cagtatcctg 3180 tggagcgacg tttatccaga tagttaa 3207
SEQ ID NO:184
Saccharomyces cerevisiae
MRGLTPKNGV HIETGPDTES SADSSNFSTG FSGKIRKPRS KVSKACDNCR KRKIKCNGKF 60 PCASCEIYSC ECTFSTRQGG ARIKNLHKTS LEGTTVQVKE ETDSSSTSFS NPQRCTDGPC 120 AVEQPTKFFE NFKLGGRSSG DNSGSDGKND DDVNRNGFYE DDSESQATLT SLQTTLKNLK 180 EMAHLGTHVT SAIESIELQI SDLLKRWEPK VRTKELATTK FYPNKSIETQ LMKNKYCDW 240 HLTRYAAWSN NKKDQDTSSQ PLIDEIFGLY SPFQFLSLQG IGKCFQNYRS KSKCEIFPRT 300 AKETIYIMLR FFDVCFHHIN QGCVSIANPL ENYLQKMNLL PSTPSSISSA GSPNTAHTKS 360 HVALVINHLP QPFVRNITGI SNSELLSEMN NDISMFGILL KMLDMHKNSY QNFLMEITSN 420 PSVAKNTQSI DVLQEFIHYC QAGEALIALC YSYYNSTLYN YVDFTCD ITH LEQLLYFLDL 480 LFWLSE IYGF EKVLNVAVHF VSRVGLSRWE FYVGLDENFA ERRRNLWWKA FYFEKTLASK 540 LGYPSNIDDS KINCLLPKNF RDVGFLDNRD FIENVHLVRR SEAFDNMCIS DLKYYGELAV 600 LQIVSHFSSS VLFNEKFTSI RNTSKPSWR EKLLFEVLE I FNETEMKYDA IKEQTGKLFD 660 IAFSKDSTEL KVSREDKIMA SKFVLFYEHH FCRMVNESDN IVARLCVHRR PSILIENLKI 720 YLHKIYKSWT DMNKILLDFD NDYSVYRSFA HYSISCIILV SQAFSVAEFI KVNDWNMIR 780 VFKRFLDIKI FSENETNEHV FNSQSFKDYT RAFSFLTIVT RIMLLAYGES SSTNLDVISK 840 YIDENAPDLK GIIELVLDTN SCAYRFLLEP VQKSGFHLTV SQMLKNRKFQ EPLMSNEDNK 900 QMKHNSGKNL NPDLP SLKTG TSCLLNGIES PQLPFNGRSA PSPVRNNSLP EFAQLPSFRS 960 LSVSDMINPD YAQPTNGQNN TQVQSNKPIN AQQQIPTSVQ VPFMNTNEIN NNNNNNNNNK 1020 NNINNINNNN SNNFSATSFN LGTLDEFVNN GDLEDLYSIL WSDVYPDS 1068
SEQ ID NO:185
Saccharomyces cerevisiae
atgaaagtga agaaatcaac tagatcaaaa gtttcgacag catgtgtcaa ttgcagaaaa 60 aggaaaatca aatgcacagg taaatatcca tgtaccaact gcatttctta cgattgtacg 120 tgtgtattcc taaaaaaaca tttaccgcag aaggaggata gttcccagtc tttgcctact 180 acagctgttg ctccaccctc ttcccacgcc aatgtagagg cttcagcaga tgtacagcat 240 ctggacactg cgattaagct agataatcaa tattacttca aactgatgaa cgacctgata 300 cagactccag tctctccgag tgcgacgcat gctcctgata cttccaataa tcctactaat 360 gataataata ttctctttaa agatgattcc aaatatcaaa atcaactggt tacgtatcaa 420 aatattctga caaatttgta cgctctgccg ccttgtgatg acactcagct cttgattgat 480 aaaacgaagt cgcagttgaa taacctgatt aacagttgga atcccgaaat aaactacccc 540 aagctttcca gtttctctcc tcgcccacaa agatcgatag aaacgtatct tttaaccaac 600 aagtatagaa ataaaataca catgacgagg ttctcctttt ggacagacca aatggttaaa 660 tcacaaagtc cagattcatt tctagccacc actccactag tagatgaagt atttggtctt 720 ttctctccaa tacaggcttt ttcactaaga ggtataggat atttaattaa aaaaaatatc 780 gaaaacacgg gttcatcgat gttaatagat acaaaggaaa ctatttatct aatattaaga 840 ttgtttgatt tgtgttatga acatttgatc caaggttgca tctctatttc taatccatta 900 gagaactatc ttcaaaaaat aaagcaaact cctactacga cggcatctgc tagtttgcct 960 acttccccag cacctttatc taacgattta gtcatttctg ttattcatca actacctcag 1020 ccatttatac aatcgattac cgggtttacg actactcaat tgatagaaaa tttacatgat 1080 tcattttcga tgtttcgaat agttactcaa atgtatgctc aacataggaa gcgctttgcg 1140 gaatttttaa accaagcttt ctccttgccc catcaagaaa agagtgtttt attctcgtca 1200 ttctgctcat cagaatatct tctatctact ctttgttacg catactacaa tgttacccta 1260 tatcacatgt tggacataaa cactttagat tacctagaga ttttagtgtc attgctagaa 1320 atccaaaatg aaattgatga gcgttttgga tttgaaaaaa tgctagaagt tgcggttaca 1380 tgctccacta agatgggatt gtctcgttgg gagtattatg ttggaataga cgaaaatact 1440 gccgaacgga gaagaaaaat atggtggaaa atatacagtc tggaaaagcg ttttttaact 1500 gatcttggtg atttatcctt aataaatgaa catcaaatga attgtctctt gccgaaggat 1560 ttcagggaca tgggattcat taaccataaa gaatttttaa cgaaaattgg tacgtcctct 1620 ttatcaccgt catcgcccaa gctaaaaaac ttgtcattgt ccaggcttat tgaatatggt 1680 gagttagcga tagcccaaat tgttggagat tttttttcag agactcttta taatgagaaa 1740 ttcacgtctt tagaagtatc cgttaaaccc acaattatca gacaaaagtt attggagaaa 1800 gtttttgagg acattgaatc ttttaggtta aaattggcca aaataaagct tcacacctca 1860 agagtttttc aagtagctca ctgcaaatat ccagaatatc caaaaaacga tctaattgaa 1920 gcagctaaat ttgtaagtta ccataaaaat acatggttct ccatcttggg tgctgttaac 1980 aatcttattg ctaggctatc tgaagatcca gaggtgataa ctgagcaaag catgaaatat 2040 gcgaatgaaa tgtttcaaga atggagggaa attaatcaat tcttaataca ggttgatact 2100 gattttattg tttgggcatg tttggacttt tatgaactga tatttttcgt gatggcttca 2160 aaattttatg tggaagaccc gcacatcact ttagaggatg ttatcaacac tttgaaagtt 2220 tttaagagaa taactaacat tatttctttt tttaataata atttggacga gaaggattat 2280 gattgtcaaa ctttcaggga gttttcgaga agttcgagtt tggttgccat atccataaga 2340 atcatatttt taaaatactg ctatgccgaa caaattgata gagccgaatt catcgaacgt 2400 ttgaaagaag ttgaaccggg tctaagtgac cttttgcgtg agttttttga tacccgctct 2460 tttatttaca ggtacatgtt gaaatccgtt gaaaaatcag gctttcattt aataattaga 2520 aaaatgttag aaagcgacta taaatttttg tatagagaca aattggccac tggtaatatt 2580 ccagaccaag gaaattcaag ccaaatttct cagttgtatg acagtactgc tccttcatac 2640 aacaatgctt ctgcctcagc agcaaactca ccgttgaagt tatcgtcttt gttgaactct 2700 ggagaggaat cgtacactca agacgcatca gaaaatgttc catgtaatct gcggcatcaa 2760 gatcgatcgt tacaacagac aaaaagacaa cattctgcgc ctagccaaat aagcgctaat 2820 gagaataata tatacaactt gggtacttta gaggagtttg tcagcagtgg tgacctgact 2880 gatttatatc atactctgtg gaatgacaat acttcatatc ccttcttatg a 2931
SEQ ID NO:186
Saccharomyces cerevisiae
MKVKKSTRSK VSTACVNCRK RKIKCTGKYP CTNCISYDCT CVFLKKHLPQ KEDSSQSLPT 60
TAVAPP SSHA NVEASADVQH LDTAIKLDNQ YYFKLMNDLI QTPVSPSATH APDTSNNPTN 120
DNNILFKDDS KYQNQLVTYQ NILTNLYALP PCDDTQLLID KTKSQLNNLI NSWNPEINYP 180
KLSSFSPRPQ RS IETYLLTN KYRNKIHMTR FSFWTDQMVK SQSPDSFLAT TPLVDEVFGL 240
FSP IQAFSLR GIGYLIKKNI ENTGSSMLID TKETIYLILR LFDLCYEHLI QGCISISNPL 300
ENYLQKIKQT PTTTASASLP TSPAPLSNDL VISVIHQLPQ PFIQSITGFT TTQLIENLHD 360
SFSMFRIVTQ MYAQHRKRFA EFLNQAFSLP HQEKSVLFSS FCSSEYLLST LCYAYYNVTL 420
YHMLDINTLD YLEILVSLLE IQNEIDERFG FEKMLEVAVT CSTKMGLSRW EYYVGIDENT 480
AERRRKIWWK IYSLEKRFLT DLGDLSLINE HQMNCLLPKD FRDMGFINHK EFLTKIGTSS 540
LSP SSPKLKN LSLSRLIEYG ELAIAQIVGD FFSETLYNEK FTSLEVSVKP TIIRQKLLEK 600
VFEDIESFRL KLAKIKLHTS RVFQVAHCKY PEYPKNDLIE AAKFVSYHKN TWFSILGAVN 660
NLIARLSEDP EVITEQSMKY ANEMFQEWRE INQFLIQVDT DFIVWACLDF YELIFFVMAS 720
KFYVEDPHIT LEDVINTLKV FKRITNI ISF FNNNLDEKDY DCQTFREFSR SSSLVAISIR 780
I IFLKYCYAE QIDRAEFIER LKEVEPGLSD LLREFFDTRS F IYRYMLKSV EKSGFHLIIR 840
KMLESDYKFL YRDKLATGNI PDQGNSSQIS QLYDSTAPSY NNASASAANS PLKLSSLLNS 900
GEESYTQDAS ENVPCNLRHQ DRSLQQTKRQ HSAPSQISAN ENNIYNLGTL EEFVSSGDLT 960
DLYHTLWNDN TSYPFL 976
SEQ ID NO:187
atgagaatgg aagtcgtctt ggtcgttttc ttgatgttca ttggtactat caactgcgaa 60 agattgatct tcaatggtag acctttgttg cacagagtta ccaaagaaga aaccgttatg 120 ttgtaccacg aattggaagt tgctgcttct gctgatgaag tttggtctgt tgaaggttct 180 ccagaattgg gtttacattt gccagatttg ttgccagctg gtatttttgc caagttcgaa 240 attactggtg atggtggtga aggttccatt ttggatatga cttttccacc aggtcaattc 300 ccacatcatt acagagaaaa gttcgtcttt ttcgaccaca agaacagata caagttggtc 360 gaacaaatcg atggtgattt cttcgatttg ggtgttactt actacatgga caccattaga 420 gttgttgcta ctggtccaga ttcttgcgtt attaagtcta ctactgaata ccacgtcaag 480 ccagaatttg ctaaaatcgt taagccattg atcgataccg ttccattggc tattatgtct 540 gaagctattg ccaaggttgt cttggaaaac aaacacaagt catctgaatg a 591
SEQ ID NO:188
Coptis japonica
MRMEWLWF LMFIGTINCE RLIFNGRPLL HRVTKEETVM LYHELEVAAS ADEVWSVEGS 60
PELGLHLPDL LPAGIFAKFE ITGDGGEGSI LDMTFPPGQF PHHYREKFVF FDHKNRYKLV 120
EQIDGDFFDL GVTYYMDTIR WATGPDSCV IKSTTEYHVK PEFAKIVKPL IDTVPLAIMS 180
EAIAKVVLEN KHKSSE 196
SEQ ID NO:189
atggaaagat tgatcttcaa tggtagacct ttgttgcaca gagttaccaa agaagaaacc 60 gttatgttgt accacgaatt ggaagttgct gcttctgctg atgaagtttg gtctgttgaa 120 ggttctccag aattgggttt acatttgcca gatttgttgc cagctggtat ttttgccaag 180 ttcgaaatta ctggtgatgg tggtgaaggt tccattttgg atatgacttt tccaccaggt 240 caattcccac atcattacag agaaaagttc gtctttttcg accacaagaa cagatacaag 300 ttggtcgaac aaatcgatgg tgatttcttc gatttgggtg ttacttacta catggacacc 360 attagagttg ttgctactgg tccagattct tgcgttatta agtctactac tgaataccac 420 gtcaagccag aatttgctaa aatcgttaag ccattgatcg ataccgttcc attggctatt 480 atgtctgaag ctattgccaa ggttgtcttg gaaaacaaac acaagtcatc tgaatga 537
SEQ ID NO:190
MERLIFNGRP LLHRVTKEET VMLYHELEVA ASADEVWSVE GSPELGLHLP DLLPAGIFAK 60
FEITGDGGEG SILDMTFPPG QFPHHYREKF VFFDHKNRYK LVEQIDGDFF DLGVTYYMDT 120
IRVVATGPDS CVIKSTTEYH VKPEFAKIVK PLIDTVPLAI MSEAIAKWL ENKHKSSE 178
SEQ ID NO:191
atgagaatgg aagtcgtctt ggtcgttttc ttgatgttca ttggtactat caactgcgaa 60 agattgatct tcaatggtag acctttgttg cacagagtta ccaaagaaga aaccgttatg 120 ttgtaccacg aattggaagt tgctgcttct gctgatgaag tttggtctgt tgaaggttct 180 ccagaattgg gtttacattt gccagatttg ttgccagctg gtatttttgc caagttcgaa 240 attactggtg atggtggtga aggttccatt ttggatatga cttttccacc aggtcaattc 300 ccacatcatt acagagaaaa gttcgtcttt ttcgaccaca agaacagata caagttggtc 360 gaacaaatcg atggtgattt cttcgatttg ggtgttactt actacatgga caccattaga 420 gttgttgcta ctggtccaga ttcttgcgtt attaagtcta ctactgaata ccacgtcaag 480 ccagaatttg ctaaaatcgt taagccattg atcgataccg ttccattggc tattatgtct 540 gaagctattg ccaaggttgt cttggaaaac aaacacaagt catctgaaca tgatgaattg 600 tga 603
SEQ ID NO:192
MRMEWLWF LMFIGTINCE RLIFNGRPLL HRVTKEETVM LYHELEVAAS ADEVWSVEGS 60
PELGLHLPDL LPAGIFAKFE ITGDGGEGSI LDMTFPPGQF PHHYREKFVF FDHKNRYKLV 120
EQIDGDFFDL GVTYYMDTIR WATGPDSCV IKSTTEYHVK PEFAKIVKPL IDTVPLAIMS 180
EAIAKVVLEN KHKSSEHDEL 200
SEQ ID NO:193
atgagaatgg aagtcgtctt ggtcgttttc ttgatgttca ttggtactat caactgcgaa 60 agattgatct tcaatggtag acctttgttg cacagagtta ccaaagaaga aaccgttatg 120 ttgtaccacg aattggaagt tgctgcttct gctgatgaag tttggtctgt tgaaggttct 180 ccagaattgg gtttacattt gccagatttg ttgccagctg gtatttttgc caagttcgaa 240 attactggtg atggtggtga aggttccatt ttggatatga cttttccacc aggtcaattc 300 ccacatcatt acagagaaaa gttcgtcttt ttcgaccaca agaacagata caagttggtc 360 gaacaaatcg atggtgattt cttcgatttg ggtgttactt actacatgga caccattaga 420 gttgttgcta ctggtccaga ttcttgcgtt attaagtcta ctactgaata ccacgtcaag 480 ccagaatttg ctaaaatcgt taagccattg atcgataccg ttccattggc tgatatgtct 540 gaagctattg ccaaggttgt cttggaaaac aaacacaagt catctgaatg a 591
SEQ ID NO:194
MRMEWLWF LMFIGTINCE RLIFNGRPLL HRVTKEETVM LYHELEVAAS ADEVWSVEGS 60 PELGLHLPDL LPAGIFAKFE ITGDGGEGSI LDMTFPPGQF PHHYREKFVF FDHKNRYKLV 120 EQIDGDFFDL GVTYYMDTIR WATGPDSCV IKSTTEYHVK PEFAKIVKPL IDTVPLADMS 180 EAIAKVVLEN KHKSSE 196
SEQ ID NO:195
atgagaatgg aagtcgtctt ggtcgttttc ttgatgttca ttggtactat caactgcgaa 60 agattgatct tcaatggtag acctttgttg cacagagtta ccaaagaaga aaccgttatg 120 ttgtaccacg aattggaagt tgctgcttct gctgatgaag tttggtctgt tgaaggttct 180 ccagaattgg gtttacattt gccagatttg ttgccagctg gtatttttgc caagttcgaa 240 attactggtg atggtggtga aggttccatt ttggatatga cttttccacc aggtcaattc 300 ccacatcatt acagagaaaa gttcgtcttt ttcgaccaca agaacagata caagttggtc 360 gaacaaatcg atggtgattt cttcgatttg ggtgttactt actacatgga caccattaga 420 gttgttgcta ctggtccaga ttcttgcgtt attaagtcta ctactgaata ccacgtcaag 480 ccagaatttg ctaaaatcgt taagccattg atcgataccg ttccattggc tgatatgtct 540 gaagctattg ccaaggttgt cttggaaaac aaacacaagt catctgaaca tgatgaattg 600 tga 603
SEQ ID NO:196
MRMEWLWF LMFIGTINCE RLIFNGRPLL HRVTKEETVM LYHELEVAAS ADEVWSVEGS 60 PELGLHLPDL LPAGIFAKFE ITGDGGEGSI LDMTFPPGQF PHHYREKFVF FDHKNRYKLV 120 EQIDGDFFDL GVTYYMDTIR WATGPDSCV IKSTTEYHVK PEFAKIVKPL IDTVPLADMS 180 EAIAKVVLEN KHKSSEHDEL 200
SEQ ID NO:197
atggaaagat tgatcttcaa tggtagacct ttgttgcaca gagttaccaa agaagaaacc 60 gttatgttgt accacgaatt ggaagttgct gcttctgctg atgaagtttg gtctgttgaa 120 ggttctccag aattgggttt acatttgcca gatttgttgc cagctggtat ttttgccaag 180 ttcgaaatta ctggtgatgg tggtgaaggt tccattttgg atatgacttt tccaccaggt 240 caattcccac atcattacag agaaaagttc gtctttttcg accacaagaa cagatacaag 300 ttggtcgaac aaatcgatgg tgatttcttc gatttgggtg ttacttacta catggacacc 360 attagagttg ttgctactgg tccagattct tgcgttatta agtctactac tgaataccac 420 gtcaagccag aatttgctaa aatcgttaag ccattgatcg ataccgttcc attggctgat 480 atgtctgaag ctattgccaa ggttgtcttg gaaaacaaac acaagtcatc tgaatga 537
SEQ ID NO:198
MERLIFNGRP LLHRVTKEET VMLYHELEVA ASADEVWSVE GSPELGLHLP DLLPAGIFAK 60 FEITGDGGEG SILDMTFPPG QFPHHYREKF VFFDHKNRYK LVEQIDGDFF DLGVTYYMDT 120 IRVVATGPDS CVIKSTTEYH VKPEFAKIVK PLIDTVPLAD MSEAIAKWL ENKHKSSE 178
SEQ ID NO:199
Saccharomyces cerevisiae
atgggctcta tgaataagga acaggctttt gaacttcttg atgcttttta tgaagcagga 60 ggtaattgca ttgatactgc aaacagttac caaaatgaag agtcagagat ttggataggt 120 gaatggatga aatcaagaaa gttgcgtgac caaattgtaa ttgccaccaa gtttaccgga 180 gattataaga agtatgaagt aggtggcggt aaaagtgcca actattgtgg taatcacaag 240 catagtttac atgtgagtgt gagggattct ctccgcaaat tgcaaactga ttggattgat 300 atactttacg ttcactggtg ggattatatg agttcaatcg aagaagttat ggatagtttg 360 catattttag ttcagcaggg caaagtcctc tatttgggtg tgtctgatac acctgcttgg 420 gttgtttctg cggcaaacta ctacgccaca tctcatggga aaactccttt tagtatctat 480 caaggtaaat ggaatgtgtt gaacagggac tttgagcgcg atatcattcc aatggccaga 540 cattttggta tggctctagc cccatgggat gttatgggag gtggaagatt tcagagtaaa 600 aaagcaatgg aggaacggaa gaagaatgga gagggtctgc gtactgtttc gggtacttct 660 aaacagacgg ataaagaggt taagatcagt gaagcattgg ccaaggttgc tgaggaacat 720 ggcactgagt ctgttactgc tattgctatt gcctatgttc gctctaaggc gaaaaatgtt 780 ttcccattgg ttggtggaag gaaaattgaa cacctcaaac agaacattga ggctttaagt 840 atcaaactga caccagaaca gatagaatac ttagaaagta ttattccttt tgatgttggt 900 tttcctacta attttatcgg tgatgatccg gctgttacca agaaggcttc acttctcacg 960 gcaatgtctg cgcagatttc cttcgattaa 990
SEQ ID N0:200
Saccharomyces cerevisiae
MGSMNKEQAF ELLDAFYEAG GNCIDTANSY QNEESEIWIG EWMKSRKLRD QIVIATKFTG 60 DYKKYEVGGG KSANYCGNHK HSLHVSVRDS LRKLQTDWID ILYVHWWDYM SSIEEVMDSL 120 HILVQQGKVL YLGVSDTPAW WSAANYYAT SHGKTPFSIY QGKWNVLNRD FERDIIPMAR 180 HFGMALAPWD VMGGGRFQSK KAMEERKKNG EGLRTVSGTS KQTDKEVKIS EALAKVAEEH 240 GTESVTAIAI AYVRSKAKNV FPLVGGRKIE HLKQNIEALS IKLTPEQIEY LESIIPFDVG 300 FPTNFIGDDP AVTKKASLLT AMSAQISFD 329
SEQ ID NO:201
Papaver somniferum
METVSKIDQQ NQAKIWKQIY GFAESLVLKC AVQLE IAETL HNNVKPMSLS ELASKLPVAQ 60 PVNEDRLFRI MRYLVHMELF KIDATTQKYS LAPPAKYLLR GWEKSMVDSI LCINDKDFLA 120 PWHHLGDGLT GNCDAFEKAL GKSIWVYMSV NPEKNQLFNA AMACDTRLVT SALANECKSI 180 FSDGISTLVD VGGGTGTAVK AISKAFPDIK CTIYDLPHVI ADSPEIPNIT KISGDMFKSI 240 PSADAIFMKC ILHDWNDDEC IQILKRCKEA LPKGGKVIIV DWIDMDSTH PYAKIRLTLD 300 LDMMLNTGGK ERTKEEWKTL FDAAGFASHK VTQISAVQSV IEAYPY 346
SEQ ID NO:202
Eschscholzia californica
MEVVTVALIA VIISSILYLL FGSSGHKNLP PGPKPWPIVG NLLQLGEKPH AQFAELAQTY 60 GDIFTLKMGT ETVWASTSS AASE ILKTHD RILSARYVFQ SFRVKGHVEN SIVWSDCTET 120 WKNLRKVCRT ELFTQKMIES QAHVREKKCE EMVEYLMKKQ GEEVKIVEVI FGTLVNIFGN 180 LIFSQNIFEL GXPNSGSSEF KEYLWRMLEL GNSTNPADYF PMLGKFDLFG QRKEVAECLK 240 GIYAIWGAML QERKLAKKVD GYQSKNDFVD VCLDSGLNDY QINALLMELF GAGTETSAST 300 IEWAMTELTK NPKITAKLRS ELQTWGERS VKESDFPNLP YLEATVKETL RLHPPTPLLL 360 PRRALETCTI LNYTIPKDCQ IMVNAWGIGR DPKTWIDPLT FSPERFLNSS VDFRGNDFSL 420 IPFGAGRRIC PGLPIANQFI ALLVATFVQN LDWCLPNGMS VDHLIVEEKF GLTLQKEPPL 480 FIVPKSRV 488
SEQ ID NO:203
Papaver somniferum
MQLKAKEELL RNMELGLIPD QEIRQLIRVE LEKRLQWGYK ETHEEQLSQL LDLVHSLKGM 60 KMATEMENLD LKLYEAPMEF LKIQHGSNMK QSAGYYTDES TTLDEAE IAM LDLYMERAQI 120 KDGQSVLDLG CGLGAVALFG ANKFKKCQFT GVTSSVEQKD YIEGKCKELK LTNVKVLLAD 180 ITTYETEERF DRIFAVELIE HMKNYQLLLK KISEWMKDDG LLFVEHVCHK TLAYHYEPVD 240 AEDWYTNYIF PAGTLTLSSA SMLLYFQDDV SVVNQWTLSG KHYSRSHEEW LKNMDKNIVE 300 FKEIMRSITK TEKEAIKLLN FWRIFCMCGA ELFGYKNGEE WMLTHLLFKK 350 SEQ ID NO:204
Papaver somniferum
MGSLDAKPAA ATQEVSIKDQ AQLWNIIYGF ADSLVLRCAV EIGIADIIKN NDGAITLAQL 60 AAKLPITNVS SDYLYRMVRY LVHLNI IEQE TCNGGVEKVY SLKPVGTLLL RDAERSMVPM 120 ILGMTQKDFM VSWHFMKEGL GNGSTTAFEK GMGMD IWKYL EGNPDQSQLF NEGMAGETRL 180 LTKTLIEDCR DTFQGLDSLV DIGGGNGTTI KAIYEAFPHI KCTLYDLPHV VANSHDLPNI 240 EKVPGDMFKS VP SAQAILLK LILHDWTDEE CVNILKKCKE AIPKETGKVI IVDVALEEES 300 NHELTKTRLI LD IDMLVNTG GRERTADDWE NLLKRAGFRS HKIRPIRAIQ SVIEAFP 357
SEQ ID NO:205
Papaver bracteatum
MELQYFSYFQ PTSSVVALLL ALVSILFSW VLRKTFSNNY SSPASSTETA VLCHQRQQSC 60 ALP ISGLLHV FMNKNGLIHV TLGNMADKYG PIFSFPTGSH RTLVVSSWEM VKECFTGNND 120 TFFSNRPIPL AFKI IFYAGG VDSYGLALVP YGKYWRELRK ICVHNLLSNQ QLLKFRHLI I 180 SQVDTSFNKL YELCKNSEDN QGMVRMDDWL AQLSFSVIGR IVCGFQSDPK TGAPSRVEQF 240 KEAINEASYF MSTSPVSDNV PMLGWIDQLT GLTRNMTHCG KKLDLWESI INDHRQKRRF 300 SRTKGGDEKD DEQDDFIDIC LSIMEQPQLP GNNNPPKIPI KSIVLDMIGG GTDTTKLTTI 360 WTLSLLLNNP HVLDKAKQEV DAHFLTKRRS TNDAAWDFD DIRNLVYIQA I IKESMRLYP 420 ASPWERLSG EDCWGGFHV PAGTRLWVNV WKMQRDPNVW ADPMVFRPER FLSHGQKKMV 480 DVRGKNYELL PFGAGRRICP GISFSLDLMQ LVLTRLILEF EMKSPSGKVD MTATPGLMSY 540 KWPLDILLT HRRIKSCVQL ASSERDMESS GVPVITLRSG KVMPVLGMGT FEKAGKGSER 600 ERLAILKAIE VGYRYFDTAA AYETEEVLGE AIAEALQLGL IKSRDELFIS SMLWCTDAHP 660 DRVLLALQNS LRNLKLEYVD LYMLPFPASL KPGKITMDIP EEDICPMDYR SVWSAMEECQ 720 NLGLTKSIGV SNFSCKKLEE LMATANIPPA VNQVEMSPAF QQKKLREYCN ANNILVSAVS 780 ILGSNGTPWG SNAVLGSEVL KKIAMAKGKS VAQVSMRWVY EQGASLVVKS FSEERLRENL 840 NIFDWQLTKE DNEKIGEIPQ CRILSAYFLV SPKGPFKSQE ELWDDKA 887
SEQ ID NO:206
Papaver bracteatum
MAP INIEEND FWMIACTVII VFALMKFMVS FYQSANTTEW PEGPKTLPII GNLHQLGGGV 60 PLQVALANLA KVYGGAFTIW IGSWVPMIVI SD IDNAREVL VNKSADYSAR DVPDILKIIT 120 ANGKNIADCD SGPFWHHLKK GLQSCINPSN VMSLSRLQEK DMQNLIKSMQ ERASQQNGIL 180 KPLDHAKEAS IRLLSRVIFG QDFSNEDLVI GVKDALDEMV RISGLASLAD AFKIAKYLPS 240 QKKNIRDMYA TRDRVYNLIQ PHIVSNLPAN SFLHFLTSQD YSDEIIYSMV LEIFGLGVDS 300 TAATAVWALS FLVGEQEIQE KLYREINNLT GGQRPVKWD LKELPYLQAV MKETLRMKP I 360 APLAVPHVAA KDTTFKGRRI VKGTKVMVNL YAIHHDPNVF PAPYKFMPER FLKGVNSDGR 420 YGD INTMESS LIPFGAGMRI CGGVELAKQM VGFALASMVN EFKWDCVSEG NLPDLSEAIS 480 FILYMKNPLE AKVTPRTKPF DSR 503
SEQ ID NO:207
Papaver bracteatum
MPETCPNTVT KMRCAWTGG NKGIGFE ICK QLSSSGIMVV LTCRDVTRGL EAVEKLKNSN 60 HENWFHQLD VTDPITTMSS LADF IKARFG KLDILVNNAG VAGFSVDADR FKAMISDIGE 120 DSEEWKIYE KPEAQELMSE TYELAEECLK INYYGVKSVT EVLLPLLQLS DSPRIVNVSS 180 STGSLKYVSN ETALE ILGDG DALTEERIDM WNMLLKDFK ENLIETNGWP SFGAAYTTSK 240 ACLNAYTRVL AKKIPKFQVN CVCPGLVKTE MNYGIGNYTA DEGAKHVVRI ALFPDDGPSG 300 FFYDCSELSA F 311 SEQ ID NO:208
Papaver somniferum
MATMYSAAVE VISKETIKPT TPTP SQLKNF NLSLLDQCFP LYYYVP I ILF YPATAANSTG 60 SSNHHDDLDL LKSSLSKTLV HFYPMAGRMI DNILVDCHDQ GINFYKVKIR GKMCEFMSQP 120 DVPLSQLLPS EVVSASVPKE ALVIVQVNMF DCGGTAICSS VSHKIADAAT MSTFIRSWAS 180 TTKTSRSGGS TAAVTDQKLI PSFDSASLFP PSERLTSPSG MSEIPFSSTP EDTEDDKTVS 240 KRFVFDFAKI TSVREKLQVL MHDNYKSRRQ TRVEVVTSLI WKSVMKSTPA GFLPWHHAV 300 NLRKKMDPPL QDVSFGNLSV TVSAFLPATT TTTTNAVNKT INSTSSESQV VLHELHDFIA 360 QMRSEIDKVK GDKGSLEKVI QNFASGHDAS IKKINDVEVI NFWISSWCRM GLYEIDFGWG 420 KP IWVTVDPN IKPNKNCFFM NDTKCGEGIE VWASFLEDDM AKFELHLSEI LELI 474
SEQ ID NO:209
Papaver somniferum
MAPLGVSGLV GKLSTELEVD CDAEKYYNMY KHGEDVKKAV PHLCVDVKII SGDPTSSGCI 60 KEWNVNIDGK TIRSVEETTH DDETKTLRHR VFEGDVMKDF KKFDTIMWN PKPDGNGCW 120 TRS IEYEKTN ENSPTPFDYL QFGHQAIEDM NKYLRDSESN 160

Claims

WHAT IS CLAIMED IS:
1 . A recombinant S. cerevisiae host cell capable of producing one or more
benzylisoquinoline alkaloids, comprising a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine and having reduced expression of:
(a) one or more endogenous transporter genes or one or more endogenous transcription factor genes that regulates expression of the one or more endogenous transporter genes; and/or
(b) one or more endogenous genes encoding one or more polypeptides
capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor or one or more transcription factor genes that regulate expression of the one or more endogenous gene; and/or
(c) one or more endogenous genes encoding one or more NCS-compatible substrate pathway polypeptide or one or more transcription factor genes that regulate expression of the one or more endogenous genes.
2. The recombinant host cell of claim 1 , wherein the one or more endogenous transporter genes encode a polypeptide having at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:70 or 74, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 76, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72.
3. The recombinant host cell of claim 1 , wherein the one or more endogenous transcription factor genes encode a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:184 or 186.
4. The recombinant host cell of claim 1 , comprising reduced expression of the one or more transporter genes encoding the one or more polypeptides having at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:70 or 74, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 76, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72; and the one or more endogenous transporter genes encoding one or more polypeptides having at least 90% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 86 or 88, or at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 102, 1 12, or at least 70% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 78, 80, 98, 1 14, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 92, 94, 106, or 1 16, or at least 60% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 100, 104, or 1 10, or at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 18, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:82, 84, 90, 96, or 108.
5. The recombinant host cell of claim 1 , comprising reduced expression of the transporter gene encoding a polypeptide having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:70 and further comprising:
(a) reduced expression of the transporter gene encoding the polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72;
(b) reduced expression of the transporter gene encoding the polypeptide having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:74; (c) reduced expression of the transporter gene encoding the polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:76;
(d) reduced expression of the transporter gene encoding the polypeptide having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:74 and reduced expression of the transporter gene encoding the polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:76; or
(e) reduced expression of the transporter gene encoding the polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72, reduced expression of the transporter gene encoding the polypeptide having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:74, and reduced expression of the transporter gene encoding the polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:76.
6. The recombinant host cell of any one of claims 1 -5, comprising reduced expression of the endogenous gene encoding the one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:154, 152, or 150.
7. The recombinant host cell of claim 6, comprising reduced expression of:
(a) the one or more transporter genes encoding the one or more polypeptides having at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:70 or 74, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 76, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72; and
(b) the one or more polypeptides capable of reducing or oxidizing a
benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:146.
8. A recombinant S. cerevisiae host cell capable of producing one or more
benzylisoquinoline alkaloids, comprising a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine, wherein:
(a) the gene has a copy number of 2 or more and the host cell further
comprises reduced expression of one or more endogenous genes encoding one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor or one or more transcription factor genes that regulate expression of the one or more endogenous gene; wherein the one or more polypeptides comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:154, 152, or 150; and/or (b) the host cell has reduced expression of one or more endogenous genes encoding one or more NCS-compatible substrate pathway polypeptide or one or more transcription factor genes that regulate expression of the one or more endogenous genes.
9. The recombinant host cell of any of claims 1 -8, wherein the gene encoding the
polypeptide capable of synthesizing (S)-norcoclaurine has a copy number of 2 or more.
10. The recombinant host cell of any of claims 1 -8, wherein the polypeptide capable of synthesizing (S)-norcoclaurine comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, or 198.
1 1 . The recombinant host cell of any of claims 1 -10, wherein the benzylisoquinoline alkaloid precursor is 4-hydroxyphenylacetaldehyde (4-HPAA) or 3,4- dihydroxyphenylacetaldehyde (3,4-DHPAA).
12. The recombinant host cell of any of claims 1 -1 1 , wherein the one or more endogenous genes encode the one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:154,
152, or 150.
13. The recombinant host cell of any of claims 1 -12, wherein the NCS-compatible substrate pathway is a tryptophan biosynthesis pathway or a phenylalanine biosynthesis pathway.
14. The recombinant host cell of claim 13, wherein the endogenous gene encodes an NCS- compatible pathway polypeptide having at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:166 or 168, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 164.
15. The recombinant host cell of any of claims 1 -14, wherein the host cell further comprises:
(a) a gene encoding a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:2 or 4;
(b) a gene encoding a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:6;
(c) a gene encoding a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:8;
(d) a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:10 or 12;
(e) a gene encoding a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:14;
(f) a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:16 or 18; and
(g) a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:20;
wherein at least one of the genes is overexpressed.
16. The recombinant host cell of any of claims 1 -15, comprising:
(a) a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, the polypeptide lacking an N-terminal signal peptide; and
(b) reduced expression of one or more endogenous genes encoding one or more polypeptides capable of synthesizing tyrosol or 4-HPAC from 4- HPAA, each comprising a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:154, 152, or
150.
17. The recombinant host cell of any one of claims 1 -16, further comprising:
(c) reduced expression of at least one endogenous gene encoding a
polypeptide capable of synthesizing phenylpyruvate from prephenate comprising a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:164; and/or at least one endogenous gene encoding a polypeptide capable of synthesizing anthranilate from chorismate comprising a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:166 or 168.
18. The recombinant host cell of any of claims 1 -17, comprising:
(a) a polypeptide having the amino acid sequence set forth in SEQ ID NO:4;
(b) a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:24; and (c) a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:28.
19. The recombinant host cell of any one of claims 1 -18, wherein the one or more
benzylisoquinoline alkaloids is (S)-norcoclaurine or (S)-norlaudanosoline.
20. The recombinant host cell of any of claims 1 -19, wherein the host cell is further capable of producing a natural or a non-natural benzylisoquinoline alkaloid derivative of (S)- norcoclaurine.
21 . The recombinant host cell of any one of claims 1 -20, wherein the derivative is (S)- reticuline, (R)-reticuline, salutaridinol, thebaine neopinone, and codeinone.
22. A method of producing one or more benzylisoquinoline alkaloids in a cell culture,
comprising culturing the host cell of any one of claims 1 -21 in the cell culture, under conditions in which the genes are expressed; wherein the one or more
benzylisoquinoline alkaloids is produced by the host cell.
23. The method of claim 22, wherein the genes are constitutively expressed.
24. The method of claim 22, wherein expression of the genes is induced.
25. The method of any one of claims 22-24, wherein the host cell further produces a natural or a non-natural benzylisoquinoline alkaloid derivative of (S)-norcoclaurine,
26. The method of any one of claims 22-25, wherein the derivative is (S)-reticuline, (R)- reticuline, berberine, papaverine, morphine, sanguinarine, noscapine, codeine, thebaine, northebaine, oripavine, nororipavine, neopinone, codeinone, oxycodone, or
buprenorphine.
27. The method of any one of claims 22-26, wherein the host cell is cultured in a fermentor with a feed profile and at a temperature for a period of time, wherein the feed profile, the temperature, and the period of time facilitate reduced formation of an acetaldehyde.
28. The method of claim 27, wherein formation of the acetaldehyde is reduced compared to a corresponding host cell cultured under conditions that do not facilitate reduced formation of an acetaldehyde.
29. The method of claim 27 or 28, wherein the cell culture comprises a non-fermentable carbon source, and wherein the cell culture has a total level of acetaldehyde that is lower than the total level of acetaldehyde in a corresponding cell culture including a glucose carbon source and/or a sucrose carbon source.
30. The method of claim 29, wherein the non-fermentable carbon source is an acetate
carbon source or a glycerol carbon source.
31 . The method of any one of claims 22-30, further comprising isolating one or more
benzylisoquinoline alkaloids produced by the recombinant host cell.
32. The method of any one of claims 22-31 , further comprising enzymatically or chemically converting one or more benzylisoguinoline alkaloids produced by the recombinant host cell to provide a benzylisoquinoline alkaloid derivative.
33. The method of claim 32, wherein the benzylisoquinoline alkaloid derivative is berberine, papaverine, morphine, sanguinarine, noscapine, neomorphine, hydrocodone, codeine, oxycodone, oxymorphone, dihydromorphine, or buprenorphine.
34. The method of any one of claims 22-33, wherein the host cell is cultured in a fermentor with a feed profile and at a temperature for a period of time, wherein the feed profile, the temperature, and the period of time facilitate production of the one or more
benzylisoquinoline alkaloids.
35. The method of any one of claims 22-34, wherein the one or more benzylisoquinoline alkaloids is (S)-norcoclaurine.
36. A cell culture, comprising the host cell of any one of claims 1 -21 and further comprising:
(a) one or more benzylisoquinoline alkaloids produced by the recombinant host cell;
(b) dextrose, sucrose, fructose, glycerol, xylose, ethanol, glucose, and/or acetate; and
(c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), nucleobases, and/or amino acids; and optionally,
(d) malt extract, corn steep liquor, casein hydrosylate, yeast extract, urea, amino acids, ammonia and/or ammonium salts; wherein the one or more benzylisoquinoline alkaloids are present at a concentration of at least 100 mg/liter of the cell culture.
37. The cell culture of claim 36, comprising less than 10 g/L of ethanol.
38. A cell lysate from the host cell of any one of claims 1 -21 grown in the cell culture, comprising:
(a) one or more benzylisoquinoline alkaloids produced by the recombinant host cell;
(b) dextrose, sucrose, fructose, glycerol, xylose, ethanol, glucose, and/or acetate; and
(c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), nucleobases, and/or amino acids; and optionally,
(d) malt extract, corn steep liquor, casein hydrosylate, yeast extract, urea, amino acids, ammonia and/or ammonium salts;
wherein the one or more benzylisoquinoline alkaloids are present at a concentration of at least 1 mg/liter of the cell culture.
PCT/EP2019/066561 2018-06-22 2019-06-21 Production of benzylisoquinoline alkaloids in recombinant hosts Ceased WO2019243624A1 (en)

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WO2022234005A1 (en) 2021-05-07 2022-11-10 River Stone Biotech Aps Glycosylated opioids
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