TW201307550A - Process to increase the production of a succinyl-CoA derived compound - Google Patents

Abstract

This invention relates to a process to improve the production of a succinyl-CoA derived compound by a eukaryotic cell. This method may be advantageously used in the biological production of e.g. adipic acid.

Description

Method for increasing production of amber sulfhydryl-COA derived compounds Field of invention

This invention relates to a method for improving the production of a succinyl-CoA derived compound by an eukaryotic cell. Furthermore, the present invention relates to an engineered eukaryotic cell which is obtained by the method and which is transformed into a polynucleotide encoding a succinyl-CoA ligase and A method comprising culturing the engineered eukaryotic cells to produce adipic acid or adipic acid ester or adipic acid thioester. The invention also relates to an engineered true comprising an enzyme capable of converting an adipic ester, an adipate or a thiodicarbonate to 5-formylpentanoate. Nuclear cells and a method for producing 5-mercaptovalerate by culturing the cells. The invention also relates to an engineered eukaryotic cell comprising an enzyme capable of converting 5-formylvalerate to 6-amino caproic acid and an engineering by culturing the same A method of producing 6-aminohexanoic acid by a cell and a method of producing caprolactam from the 6-aminocaproic acid.

Background of the invention

Succinyl-coenzyme A is an essential intermediate in many metabolic pathways and is a key precursor in many (industrial-related) valuable compounds [such as fatty acids, carotenoids, Isoprenoids, vitamins, amino acids, fats, wax esters, (poly) sugars, polyhydroxyalkanoates, statins, polyketones Polyketides)]. In particular, amber-based-CoA is also an industrially important precursor for hexamethylene diamine and adipic acid.

A biosynthetic production pathway for such amber-based-CoA-derived compounds may naturally be present in a microorganism, but in order to produce a functional biosynthetic pathway, the microorganism must typically be engineered (eg, by introducing a coding specific enzyme) Gene).

When natural (wild-type) cells are unable to produce the amber-based-CoA-derived compounds of interest, the use of metabolic engineering can provide eukaryotic cells with heterologous genes that support this approach. In these examples, the heterologous gene product is typically targeted to the cytosolic compartment of the cell. Since the biosynthesis of an amber-based-CoA-derived compound will occur completely or partially in the cytosol of the cell, it is important to supply a sufficient amount of the precursor amber-based-CoA in the cytosolic compartment. In eukaryotes, the biosynthesis of amber-based-CoA occurs mainly in mitochondria. Amber thiol-CoA is an intermediate of tricarboxylic acid or Krebs cycle. Two enzymes were produced in the mitochondria and amber thiol-CoA was used. The alpha-ketoglutarate dehydrogenase complex produces amber-based-CoA, and in a subsequent reaction amber-based-CoA is mediated by a reversible amber-based-CoA ligase enzyme. Conversion to succinic acid causes ATP (or GTP) production. Among the industrially related eukaryotic microorganisms, there has been no example in which amber thiol-CoA is formed outside the granule. In heme biosynthesis, for example, the first step involves the reaction of amber-based-CoA with glycine to aminolevulinate (ALA). Due to the location of the amber thiol-CoA, this step occurs in the mitochondria and the ALA is then transported to the cytosol that will occur in subsequent enzyme reactions.

One goal of the invention is to increase the production of an amber-based-CoA derivative compound in a eukaryotic cell.

Detailed description of the invention

In a first aspect, the present invention provides a method for improving the production of an amber-based-CoA-derived compound by a eukaryotic cell capable of producing an amber-based-CoA-derived compound, which comprises encoding a single amber The polynucleotide of the basal-CoA ligase is transformed into the cell.

The terms "a" or "an" as used herein are defined as "at least one" unless otherwise indicated.

When referring to a singular noun (eg, a compound, a cell, etc.), the plural is meant to be included.

When an acid such as adipic acid is mentioned, a conjugate base or a salt is also included.

"Amber thiol-CoA ligase" is defined as a polypeptide which may comprise one, two or more monomers which may each be encoded by a different gene. For example, amber thiol-CoA ligase from E. coli consists of two monomers a and b encoded by the sucC and sucD genes, respectively. Similarly, the amber thiol-CoA ligase from Saccharomyces cerevisiae is also composed of two monomers a and b which are encoded by the lsc1 and lsc 2 genes, respectively. Thus, "a" polynucleotide encoding an amber-based-CoA ligase is also known to comprise a plurality of polynucleotides (such as two, three or more polynucleotides) encoding an amber sulfhydryl group. An example of a -CoA ligase.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer of an amino acid residue. These terms apply to amino acid polymers in which one or more amino acid residues are a corresponding artificially occurring analog of an naturally occurring amino acid and a naturally occurring amino acid polymer. An essential property of such analogs of naturally occurring amino acids is that when incorporated into a protein, that protein is an antibody reaction that is specifically caused by the same protein but consists entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" also encompass modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylate of glutamic acid residues. Gamma-carboxylation, hydroxylation, and ADP-ribosylation.

As used herein, the term "enzyme" is defined as a protein that catalyzes a (bio)chemical reaction in a cell.

As used herein, the term "polynucleotide" includes a deoxyribonucleotide or ribonucleotide polymer in the form of a single or double strand, and unless otherwise limited, includes Known analogs of the essential properties of natural nucleotides (which are hybridized to a single-stranded nucleic acid in a manner similar to naturally occurring nucleotides) (eg, a peptide nucleic acid). A polynucleotide can be a full length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to specific sequences and their complementary sequences.

The term "homologous" or "endogenous" is used when it is used to indicate a relationship between a particular (recombinant) nucleic acid or polypeptide molecule and a particular host organism or host cell. It is meant in essence that the nucleic acid or polypeptide molecule is produced by a host cell or an organism of the same species, in particular the same variant or strain.

The term "heterologous" when used in relation to a nucleic acid (DNA or RNA) or protein means that a part of a biological, cellular, genomic or DNA or RNA sequence that does not occur naturally occurs, or is different from it. A nucleic acid or protein that is naturally found in the location of a cell or genome or DNA or RNA sequence. A heterologous nucleic acid or protein is not endogenous to the cell into which it is introduced, but has been obtained from another cell or produced synthetically or recombinantly.

As used herein, the term "gene" means a nucleic acid sequence comprising a template for a nucleic acid polymerase (in yeast, RNA polymerase II). The gene is transcribed into mRNA and then translated into protein.

In order to increase the likelihood that the introduced protein will be expressed in an activated form in a eukaryotic yeast cell according to the method of the invention, the corresponding coding nucleotide sequence may be directed against the selected yeast host cell. Transform to optimize its codon usage. Several methods for codon optimization are well known in the art. A preferred method for optimizing codon usage of such nucleotide sequences in accordance with the present invention is the codon pair optimization technology as described in WO 2008/000632. Codon pair optimization is a method for producing a polypeptide in a host cell, wherein the nucleotide sequences encoding the polypeptide have been modified for their codon usage (especially the password used) A sub-pair) to obtain improved performance of the nucleotide sequence encoding the polypeptide and/or improved production of the polypeptide.

Typically, a nucleotide sequence encoding a protein is operably linked to a promoter which causes sufficient expression of the corresponding nucleotide sequence in a yeast cell according to the invention to administer the cell to produce a dicarboxylic acid. ability.

As used herein, the term "operably linked" means a link of a polynucleotide element (or coding sequence or nucleic acid sequence) in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

As used herein, the term "promoter" means that one upstream of the transcriptional direction of the transcription initiation site of the gene acts to control the transcription of one or more genes, and by a DNA-dependent RNA polymerase The structurally identified nucleic acid fragments, transcription initiation sites, and any other sequences are known to those skilled in the art. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.

A promoter that can be used to achieve expression of a nucleotide sequence encoding a protein may not be innate for the nucleotide sequence encoding the protein to be expressed, ie, a promoter for which it can be operably linked. The nucleotide sequence (coding sequence) is heterologous. Preferably, the promoter is homologous, i.e. endogenous to the host cell.

Suitable promoters in yeast cells are known to those skilled in the art. Suitable promoters may be, but are not limited to, TDH1, TDH3, GAL7, GAL10, GAL1, CYC1, HIS3, ADH1, PH05, ADC1, ACT1, TRP1, URA3, LEU2, ENO1, TPI1. Other suitable promoters include PDC1, GPD1, PGK1, and TEF1.

Typically a nucleotide sequence encoding a protein comprises a terminator. Any terminator that acts in a cell can be used in the present invention. Preferred terminators are obtained from the native gene of the host cell. Suitable terminator sequences are well known in the art. Preferably, such terminators are combined to prevent mutations in the antisense regulated mRNA decay in the host cells of the invention (see, for example, Shirley et al., 2002, genestics 161:1465-1482).

Applicants have surprisingly discovered that when the cell has been transformed into a polynucleotide encoding an amber-based-CoA ligase, it is produced by a eukaryotic cell capable of producing an amber-based-CoA derivative compound. An amber-based-CoA-derived compound is higher in comparison to a cell that is not transformed into a polynucleotide encoding an amber-based-CoA ligase. In the context of the present invention, "production of a succinyl-CoA derived compound" is defined as the amount of amber thiol-CoA derived compound per fermentation medium. In order to establish a eukaryotic cell, the production of an amber-based-CoA-derived compound is transformed into a coded one after transcoding with a polynucleotide encoding an amber-based-CoA ligase. Whether the production of an amber-based-CoA-derived compound of eukaryotic cells of the amber-based-CoA ligase polynucleotide is improved, and the fermentation conditions (especially for the fermentation length of the two kinds of cells) are preferably similar or even More preferably substantially the same or even advantageously identical. Preferably, the fermentation conditions are selected such that the amount of amber-based-CoA-derived compound is increased to complete the fermentation.

In the context of the specification, "amber-based-CoA ligase" is defined as an enzyme capable of reversibly coupling CoA and succinic acid.

In one embodiment, the amber-based-CoA ligase belongs to the enzyme species of the enzyme E.C.6.2.1.5 or E.C. 6.2.1.4. The EC enzyme species is an enzyme according to the enzyme nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). Classification or categories that can be classified, this nomenclature can be found at http://www.chem.qmul.ac.uk/iubmb/enzyme/. No other suitable enzyme that has been (not yet) classified in a particular classification but can be classified as such is meant to be included.

In a preferred embodiment, the amber-based-CoA ligase is active in the cytosol or in the cell. Biosynthesis of amber-based-CoA-derived compounds can occur wholly or partially in the cytosol. The amino acid sequence of the amber thiol-CoA ligase may comprise a target signal, such as a peroxisomal or mitochondrial target signal. One skilled in the art will be aware of methods to determine the location of a protein, for example, as described in Emanuelsson et al. 2007. Nature Protocols 2: 953-971. As a result, the amber-based-CoA ligase comprises a target signal, and it may be preferred that the eukaryotic cell in the method according to the invention comprises a truncated form of the enzyme, wherein the target signal is removed. . Deletion of the target signal allows the protein to be located in the cytosol, which can result in increased production of the amber-based-CoA-derived compound.

The amber-based-CoA ligase may comprise any suitable ligase and may be derived from any organism. The organism can be a eukaryote, a bacterium or an archeon.

In one embodiment, the amber-based-CoA ligase is derived from a eukaryote, more preferably from Saccharomyces [eg, S. cerevisae]. A suitable eukaryotic amber-based-CoA ligase can be encoded by the S. cerevisiae genes LSC1 and/or LSC2 and/or their homologs.

In another embodiment, the amber-based-CoA ligase is prokaryotic, i.e., derived from a bacterium or archaea, more preferably from E. coli. Polymorphism of amber-based-CoA ligase that replaces a prokaryote with a yeast (such as Saccharomyces) amber-based-CoA ligase, capable of producing an amber-based-CoA derivative compound Eukaryotic cells, in some instances, may not result in improved production of an amber-based-CoA derived compound. A suitable prokaryotic amber-based-CoA ligase can be encoded by the E. coli genes sucC and/or sucD and their homologs.

In a particular embodiment, the amber-based-CoA ligase may comprise an amine group comprising SEQ ID 15 and/or 16 and/or comprising SEQ ID 17 and/or 18 and/or homologs thereof Acid sequence. Such amber-based-CoA ligases can be encoded, for example, by nucleotide sequences comprising SEQ ID 1 and/or 2, respectively, or comprising SEQ ID 3 and 4 and/or homologs thereof. Those skilled in the art will also be able to construct functional analogs of these sequences (which can be used as an alternative) based on common general knowledge.

In a preferred embodiment, the amber-based-CoA ligase comprises an amino acid sequence comprising SEQ ID 15 and/or 16 and/or homologs thereof. This amber thiol-CoA ligase may, for example, be encoded by a polynucleotide sequence comprising SEQ ID 1 and/or 2 and/or homologs thereof, respectively. Those skilled in the art will also be able to construct functional analogs of these sequences (which can be used as an alternative) based on common general knowledge.

The term "homologue" is used herein in particular to have at least 30%, preferably at least 40%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more Preferably at least 75%, more preferably at least 80%, in particular at least 85%, more particularly at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, or at least 99% of sequence identity polynucleotides or polypeptides. The term homolog is also taken to include a nucleic acid sequence (polynucleotide sequence) that differs from another nucleic acid sequence due to degeneracy of the genetic code and encoding the same polypeptide sequence.

Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences as determined by comparison of such sequences. Typically, sequence identity or similarity is compared over the full length of the sequences being compared. In the present technique, "identity" also refers to the degree of relationship between amino acid or nucleic acid sequences as determined by the match between such sequences in such a series.

Preferred methods for determining identity are designed to provide maximum compatibility between the sequences tested. Methods for determining identity and similarity are compiled in publicly available computer programs. The percent identity between the two amino acid sequences or between the two nucleotide sequences can be performed using the Needleman and Wunsch algorithm (Needleman, SB and Wunsch, CD (1970). J. MoI. Biol. 48, 443-453) (here incorporated herein by reference). Both the amino acid sequence and the nucleotide sequence can be aligned by algorithms. The Nederman-Wen algorithm has been implemented in the computer program NEEDLE. For the purposes of this invention, the NEEDLE program from the EMBOSS package software was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in genetics 16, (6) pp 276-277, http://emboss.bioinformatics.nl/), which is incorporated herein by reference. For protein sequences, EBLOSUM62 is used in the substitution matrix. For DNA nucleotide sequences, EDNAFULL is used. The selectivity parameters used are a gap of 10 - a gap-open penalty and a gap extension penalty of 0.5. Those skilled in the art will recognize that all of these different parameters will produce slightly different results when using different algorithms but the total percent identity of the two sequences is not significantly altered.

In the context of the present invention, "an amber-based-CoA-derived product" is understood to include a method of production by a biosynthetic pathway sequence comprising amber-based-CoA or by a process comprising one or more biochemical steps. And any product that is produced. This pathway need not be limited to a biological function. For example, WO 2009/113853, which is hereby incorporated by reference in its entirety, is hereby incorporated by reference in its entirety the entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire portion A polynucleotide which is an enzyme of adipic acid (an amber-based-CoA-derived compound in the context of the present invention). In this biosynthetic pathway, adipic acid is formed by coupling ethylene-CoA (acetyl-CoA) with amber-based-CoA followed by a number of subsequent enzyme reactions. However, the production of adipic acid may not be a biological function of this biosynthetic pathway. In addition to adipic acid, examples of such amber-based-CoA-derived products include fatty acids, carotenoids, isoprenoids, vitamins, amino acids, lipids , wax esters, (poly)saccharides, polyhydroxyalkanoates, statins, polyketides, and haem. In a preferred embodiment, the amber-based-CoA derivative compound is adipic acid.

Suitable eukaryotic cells may in particular be selected from the group consisting of fungi; metazoan; Viridiplantae [especially Arabidopsis and Chlamydomonadales ]; Diplomonads [especially Giardiinae ]; Entamoebidae [especially Entaboeba ]; Euglenozoa [especially Euglena ( Euglena )]; Pelobiontida [especially Mastigamoeba ]; and Alveolata (especially Cryptosporidium ).

Suitable fungi include, in particular selected from among the following group of fungi, and yeasts: Rhizopus (Rhizopus), red Streptomyces (Neurospora), Penicillium (Penicillium), the genus aspergillus (Aspergillus), rumen fungi (Piromyces), hair Bacillus genus (Trichosporon), Candida species (Candida), the genus Hansenula yeast (Hansenula), Kluyveromyces (Kluyveromyces), Saccharomyces, Rhodotorula (Rhodotorula), Schizosaccharomyces , Yarrowia [such as Yarrowia lypolytica ].

In a preferred embodiment, the eukaryotic cell is a yeast, more preferably a brewer's yeast. Compared to bacteria such as E. coli, yeast provides a very suitable choice to produce the amber-based-CoA derivative products mentioned above because yeast-based methods can be run at low pH, yeast Bacteria are not susceptible to phage or other infections. Therefore, the use of yeast does not require a sterile process whereby the cost price of the product of interest is reduced.

Transformation of eukaryotic cells can be made by methods known in the art, for example, by RD Gietz and RH Schiestl, High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method (2007), Nature Protocols 2 , described in 31-34.

In a second aspect, the invention provides a eukaryotic cell engineered into a polynucleotide encoded by an amber-based-CoA ligase, obtainable by a method according to the first aspect of the invention. The engineered eukaryotic cells according to the second aspect of the invention are preferably adapted to produce one or more enzymes whereby an amber-based-CoA derivative compound can be produced in a biosynthetic pathway. One such biosynthetic pathway is the adipic acid biosynthetic pathway. Thus, in another embodiment, the engineered eukaryotic cell of the second aspect of the invention further comprises an adipic acid biosynthetic pathway (preferably a CoA dependent adipic acid biosynthetic pathway).

An engineered eukaryotic cell according to the second aspect of the invention is defined herein as a cell containing or being transformed or genetically modified to a nucleotide sequence that is not naturally occurring in eukaryotic cells, or An additional copy or copy containing the endogenous nucleic acid sequence, or it contains a deletion or disruption of an endogenous nucleic acid sequence. A wild type eukaryotic cell is herein defined as a parental cell of a recombinant eukaryotic cell.

A genetic modification having a nucleotide sequence is used herein to indicate that a gene or a nucleotide sequence is introduced into a (eukaryotic) cell by any available means. A nucleotide sequence or gene can be prepared according to any method of the art, such as extraction from an organism or by chemical means.

The adipic acid biosynthetic pathway preferably comprises an enzyme selected from the group consisting of an enzyme capable of transferring a thiol group (such as, for example, a thiolase), and a catalyzed one 2 a reducing enzyme of a carbon-carbon double bond of a 2-enoate moiety or a 2-enoyl moiety (eg, for example, an allyl thiol reductase) One can catalyze the dehydration of a 3-hydroxyacyl ester or a 3-hydroxyacyl thioester to a 2-enoyl ester or a thioester. An enzyme [such as, for example, a dehydratase], capable of catalyzing the reduction of a carbonyl group to an alcohol group or capable of catalyzing a 3-oxoacyl ester or An enzyme that reduces 3-oxoacyl thioester to the corresponding 3-hydroxydecyl ester or thioester [such as a ketoreductase or a dehydrogenase], and one that can convert one A diester or a thioester of adipate becomes an enzyme of adipic acid [such as an acyl-CoA transferase or a thiol-CoA hydrolase (acyl-CoA hyd) Rolase)].

In a particular embodiment, the chymotrypsin is a beta-ketoadipyl CoA thiolase [e.g., from Acinetobacter]. The chymozyme may comprise a sequence which may comprise a SEQ ID 20 and/or a homolog thereof. This lyase is encoded, for example, by a gene comprising the sequence comprising SEQ ID 9.

In a particular embodiment, the ketyl reductase is an enethio-CoA reductase [eg, from Candida tropicalis]. It may comprise a sequence comprising SEQ ID 23 and/or a homolog thereof.

In a particular embodiment, the dehydratase is an enethio-CoA dehydratase, such as from the genus Acinetobacter. The dehydratase may comprise a sequence comprising SEQ ID 22 and/or a homolog thereof. This dehydratase is encoded, for example, by a gene comprising the sequence comprising SEQ ID 11.

In a particular embodiment, the ketoreductase or dehydrogenase comprises a sequence comprising SEQ ID 21 and/or a homolog thereof. The ketoreductase or a dehydrogenase is encoded, for example, by a gene comprising the sequence comprising SEQ ID 10.

In a particular embodiment, the thiol-CoA transferase comprises a sequence comprising SEQ ID 24 and/or 25 and/or a homolog thereof. This thiol-CoA transferase is encoded, for example, by a gene comprising the sequence comprising SEQ ID 13 and/or 14, respectively.

In a third aspect, the invention provides a method of producing adipic acid or adipate or thiodicarboxylate comprising culturing engineered eukaryotic cells according to the second aspect of the invention.

In a fourth aspect, the engineered eukaryotic cell according to the second aspect of the present invention further comprises a transformable monohexyl ester, an adipate or a thioester of adipate to form 5-mercaptovalerate. Enzyme.

In a fifth aspect, the invention provides a method of producing 5-mercaptovalerate, comprising culturing engineered eukaryotic cells according to the fourth aspect of the invention.

In a sixth aspect, the present invention provides an engineered eukaryotic cell according to the fourth aspect of the present invention further comprising an enzyme capable of converting 5-mercaptovalerate to 6-aminocaproic acid.

In a seventh aspect, the invention provides a method of producing 6-aminocaproic acid comprising culturing engineered eukaryotic cells according to the sixth aspect of the invention.

In an eighth aspect, the present invention provides a process for producing caprolactam comprising the steps of preparing a 6-aminocaproic acid and a cyclized 6-aminocaproic acid according to the method according to the seventh aspect of the present invention, whereby Forming caprolactam.

Example 1. Materials and methods

Oligonucleotides were synthesized by Invitrogen (Carlsbad CA, US). DNA sequencing in SEQLAB (G Ttingen, Germany) or by Baseclear (Leiden, The Netherlands). DNA synthesis was performed in GeneArt (Regensburg, Germany) or DNA 2.0 (Menlo Park, CA, USA). Restricted enzymes are supplied by Invitrogen or New England Biolabs. The strain used for transformation was an E. coli DH10B electromax competent cell (Invitrogen) according to the protocol provided by the manufacturer. All nucleotides are codons that are optimized for transformation in yeast.

2. Gene expression cassettes for the production of adipic acid in the S. cerevisiae integration vector

All DNA fragments used in this example were ordered from DNA 2.0 (Menlo Park, CA, USA) or Geneart as synthetic DNA. The following DNA molecules were used: encoding an Acinetobacter lyase (SEQ ID 9), an Acinetobacter ketoreductase (SEQ ID 10), an Acinetobacter dehydratase (SEQ ID 11), a Candida tropicalis ( Candida tropicalis) DNA of mercaptoreductase (SEQ ID 12) and an Acinetobacter thiol-CoA transferase (SEQ ID 13 and 14). All DNA molecules (10 μg) were restricted using the restriction enzyme SapI. The desired DNA fragment was generated into a promoter-gene-terminator cassette and purified from the agarose gel using a QiaQuick Gel Extraction Kit (Qiagen, Hilden, Germany). Furthermore, the vector backbone pRS414 (SEQ ID 6, Trp1 selection marker), pRS415 (SEQ ID 7, Leu2 selection marker), and pRS416 (SEQ ID 8, Ura3 selection marker) were synthesized from DNA2.0. The three vectors obtained were digested with restriction enzymes XhoI and NotI. The linearized vector is similar to the pathway performance cassette purified from the agarose gel.

3. Producing beer yeast to express plastids and transgenic beer yeast with adipic acid pathway

In vivo homologous recombination in S. cerevisiae, except in more than one fragment inserted in the vector, such as in Kazuko Lida, Tomoko Tada, Hidetoshi Iida, Molecular cloning in yeast by in vivo homologous recombination of the yeast putative al subunit of The voltage-gated calcium channel (2004), FEBS Letters, vol. 576, 291-296 is substantially made. The final expression vector is combined by a linearized DNA fragment. All transformations were made with the strain Saccharomyces cerevisiae CENPK2-1C (genotype MATa; ura3-52; trp1-289; leu2-3, 112; his3Δ1; MAL2-8 ^C ; SUC2). To construct the plastid pADI154, the linearized vector backbone SEQ ID 6 and the expression cassettes for the genes adi21 (SEQ ID 9), adi 22 (SEQ ID 10) and adi23 (SEQ ID 11) were co-transformed. Similarly, to generate plastid pADI155, the linearized vector backbone SEQ ID 7 combination was used with the expression cassettes for genes adi8 (SEQ ID 12), adi24 (SEQ ID 13) and adi25 (SEQ ID 14).

The transformant was placed in Yeast Nitrogen Base (YNB) w/o AA (Difco) + 2% glucose + added compound to overcome the remaining auxotrophies (addition of leucine and histidine to produce pADI154, and addition Tryptophan and histidine were used in combination with pADI155). Culture in liquid medium was carried out using Verduyn medium (C. Verduyn, E. Postma, "Effect of Benzoic Acid on Metabolic Fluxes in Yeasts: A Continuous-Culture Study on the Regulation of Respiration and Alcoholic Fermentation", (1992) Yeast ., vol. 8,501-517). When the transformant was identified according to the screening growth, the plastid in S. cerevisiae was isolated using Zymo Research Yeast Plasmid Isolation Kit (Zymo Research Corporation, USA). The plastid thus obtained was subsequently transformed into E. coli Top10 or XL1-blue cells and subsequently purified to leave E. coli. This step ensures a high quality plastid DNA. Finally, the plastids are sequenced to ensure that only the correct DNA sequence is transformed and used for further research. The sequenced and confirmed plastids pAdDI154 and pADI155 were re-transformed in the S. cerevisiae strain. These strains were restreaked and purified by isolating a single colony. The strains are redrawn on the same type of tray as the one in which the transforms were originally placed. A single colony was cultured in the following medium (Verduyn, 1992) on a 24 microtiter plate: (NH4)2SO4, 5 g; KH2PO4, 3 g; MgS04‧7H2O, 0.5 g; EDTA, 15 mg; ZnS04‧7H2O, 4.5 mg ;CoCl2‧6H2O, 0.3 mg; MnCl2‧4H20, 1 mg; CuSO4‧5H2O, 0.3 mg; CaCl2‧2H20, 4.5 mg; H3BO3, 1 mg; KI, 0.1 mg; and 0.025 ml polyfluorene defoamer (silicone) Antifoam, BDH). The filter-sterilized vitamin is added after heat sterilization (20 ° C) of this medium. The final vitamin concentration per liter is: biotin, 0.05 mg; calcium pantothenate, 1 mg; nicotinic acid, 1 mg; inositol, 25 mg; thiamine HCl (thiamine HCl), 1 mg; pyridoxine HCl, 1 mg; and para-aminobenzoic acid, 0.2 mg. For the first biomass phase, (72 hours), 4% galactose was added, and for the production phase (96 hours) 8% galactose and 1% calcium carbonate were added. The supernatant was separated and analyzed by LC-MS on adipic acid content.

The following LCMS conditions were applied: column, Waters Acquity UPLC HSS T3, 1.8 μm, 30 mm * 2.1 mm; flow rate, 1 mL/min; temperature, 60 °C. Mass spectrometry ESI is in negative mode. Mobile phase A: 0.1% formic acid in water. Injection volume 5 μl, full cycle. Mobile phase B: 0.1% formic acid in acetonitrile.

gradient:

Adipic acid was eluted from the column at 0.98 elution time. Adipic acid production is presented in Table 1 (dual colonies).

Table 1. Adipic acid production

4. Gene-transformed S. cerevisiae with adipic acid pathway including transformation of a polynucleotide encoding amber thiol-CoA ligase

The plastid pADI156 is represented by the linearized vector pRS416 backbone SEQ ID 8 and the related genes sucC (SEQ ID 1), sucD (SEQ ID 2) (sucC and sucD are endogenous E. coli genes) and Lin1129 [SEQ ID 5; Lin1129 A performance cassette corresponding to a gene encoding a acetaldehyde dehydrogenase from Listeria innocua was recombined to be constructed. SEQ ID 1,2,5 and 8 are formed in the co-transfected cerevisiae CENPk2-1c (genotype MATa; ura3-52; trp1-289; leu2-3,112; his3Δ 1; MAL2-8 C; SUC2). The plastid pADI156 was isolated from S. cerevisiae and the sequence was demonstrated. The plastid pADI164 was constructed and re-transformed in S. cerevisiae CENPk2-1c by removing the Lin1129 performance cassette from pADI156 using standard restriction site selection. The resulting transform contains all three plastids pADI154, pADI155 and pADI164. Verduyn medium [containing tryptophan, uracil, and leucine, depending on the auxotrophy present] was used as a medium for transformation. When all three plastids pADI154, pADI155, and pADI164 were transformed, three compounds were not added. This strain was cultured and the supernatant of the culture solution was analyzed for adipic acid production as described in Example 3.

The plastid pADI157 was constructed by the linearized vector pRS416 backbone (SEQ ID 8) and the expression cassette for the genes Lsc1 (SEQ ID 3), Lsc2 (SEQ ID 4) and Lin1129 (SEQ ID 5). Lin1129 corresponds to a Listeria insect gene encoding acetaldehyde dehydrogenase. Lsc1 and Lsc2 are endogenous yeast genes. The S. cerevisiae amber thiol-CoA ligase is located in the mitochondria. In order for the S. cerevisiae amber thiol-CoA ligase to be located in the cytosol, the mitochondrial target sequence (MTS) of the S. cerevisiae amber thiol-CoA ligase subunits was removed. The length of the MTS of the protein encoded by the LSC1 and LSC2 genes is based on Emanuelsson et al. 2007. Nature Protocols 2: 953-971 using a non-plant organism group by TargetP 1.1 server (http://www .cbs.dtu.dk/services/TargetP/) predicted. The MTS of the Lsc1 protein is 24 amino acids at the N-terminus. The MTS of the Lsc 2 protein is 30 amino acids at the N-terminus. Subsequently, the MTS of both proteins (in addition to the N-terminal methionine) was removed, resulting in a protein sequence represented by seq IDs 17 and 18, respectively. Vector pRS416 and SEQ ID 3,4, and 5 are formed in the co-transfected cerevisiae CENPk2-1c (genotype MATa; ura3-52; trp1-289; leu2-3,112; his3Δ 1; MAL2-8 C; SUC2). The plastid pADI157 was isolated from S. cerevisiae and the sequence was demonstrated. The plastid pADI165 was constructed and re-transformed into S. cerevisiae CENPk2-1c by removing the Lin1129 performance cassette from pADI157 using standard restriction site selection. The resulting transforms control all three plastids pADI154, pADI155 and pADIS165. Verduyn medium (containing tryptophan, uracil, and leucine, depending on auxotrophy) was used as a medium for transformation. When all three plastids pADI154, pADI155, and pADI165 were transformed, three compounds were not added. This strain was cultured and the supernatant of the culture solution was analyzed for adipic acid production as described in Example 3. Adipic acid production is presented in Table 2.

Table 2. Adipic acid production

Surprisingly, the strain harboring the adipic acid pathway gene and transformed into yeast amber thiol-CoA Lsc1 and Lsc2 produced no more adipic acid than the strain not transformed with Lsc1 and Lsc2. However, when these yeast cells were transformed into prokaryotic amber-based-CoA ligase genes sucC and sucD, the production of adipic acid was improved.

<110> DSM Smart Property Co., Ltd.

<120> Method for increasing production of amber sulfhydryl-COA derived compounds

<130> 27896-WO-PCT

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Claims (15)

A method for improving production of amber thiol-CoA-derived compounds by eukaryotic cells capable of producing amber thiol-CoA-derived compounds, comprising translating a polynucleotide encoding amber thiol-CoA ligase cell. The method of claim 1, wherein the amber-based-CoA ligase is active in the cytosol of the cell. The method of claim 1 or 2, wherein the amber-based-CoA ligase is a prokaryote. The method of any one of claims 1-3, wherein the amber-coA ligase comprises an amino acid sequence comprising SEQ ID 15 and/or 16 and/or a homolog thereof. The method of any one of claims 1-4, wherein the amber-based-CoA derivative compound is adipic acid. The method of any one of claims 1-5, wherein the eukaryotic cell is a yeast. An engineered eukaryotic cell, which is transformed with a polynucleotide encoding an amber-based-CoA ligase, which can be obtained by the method of any one of claims 1-6. An engineered eukaryotic cell as claimed in claim 7 further comprising an adipic acid biosynthetic pathway. An engineered eukaryotic cell as claimed in claim 8 wherein the adipic acid biosynthetic pathway comprises an enzyme consisting of a group of enzymes capable of transferring a thiol group, one capable of catalyzing An enzyme that reduces the carbon-carbon double bond of a 2,3-enoic acid ester moiety or a 2-ethylenyl moiety, and is capable of catalyzing the dehydration of 3-hydroxydecyl ester or 3-hydroxydecylthioester to a 2-olefin fluorenyl group. An ester or thioester enzyme, capable of catalyzing the reduction of a carbonyl group to an alcohol group or capable of catalyzing the reduction of a 3-oxooxydecyl ester or a 3-oxothiol thioester to the corresponding 3-hydroxydecyl ester or thioester An enzyme, and an enzyme capable of converting an adipate or adipic acid thioester into adipic acid. A method of producing adipic acid or adipate or thiodicarboxylate, comprising culturing an engineered eukaryotic cell according to any one of claims 7-9. An engineered eukaryotic cell according to any one of claims 7-9, further comprising a capable of converting hexamethylenediester, adipate or thiodicarbonate to 5-carbachol Ester enzyme. A method for producing 5-mercaptovalerate by culturing a cell as in claim 11 of the patent application. An engineered eukaryotic cell according to claim 11 further comprising an enzyme capable of converting 5-mercaptovalerate to 6-aminocaproic acid. A method of producing 6-aminocaproic acid comprising culturing engineered cells as in claim 11 of the patent application. A process for producing caprolactam comprising the steps of preparing 6-aminohexanoic acid and cyclizing 6-aminohexanoic acid according to the method of claim 12, thereby forming caprolactam.