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WO2008008256A2 - Procédés permettant d'améliorer la production de composés isoprénoïdes par des cellules hôtes - Google Patents

Procédés permettant d'améliorer la production de composés isoprénoïdes par des cellules hôtes Download PDF

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WO2008008256A2
WO2008008256A2 PCT/US2007/015498 US2007015498W WO2008008256A2 WO 2008008256 A2 WO2008008256 A2 WO 2008008256A2 US 2007015498 W US2007015498 W US 2007015498W WO 2008008256 A2 WO2008008256 A2 WO 2008008256A2
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host cell
synthetic
igr
nucleotide sequence
pathway
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WO2008008256A3 (fr
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Jay D. Keasling
Douglas J. Pitera
Chris J. Paddon
Brian Pfleger
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University of California Berkeley
University of California San Diego UCSD
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University of California San Diego UCSD
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    • 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
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Isoprenoids are a highly diverse class of natural products from which numerous commercial flavors, fragrances, chemicals, and medicines are derived. Isoprenoids constitute an extremely large and diverse group of natural products that have a common biosynthetic origin, i.e., a single metabolic precursor, isopentenyl diphosphate (IPP). At least 20,000 isoprenoids have been described. The number of C-atoms present in the isoprenoids is typically divisible by five (C5, ClO, C15, C20, C25, C30 and C40), although irregular isoprenoids and polyterpenes have been reported.
  • IPP isopentenyl diphosphate
  • Isoprenoid compounds are also referred to as "terpenes” or “terpenoids.”
  • Important members of the isoprenoids include the carotenoids, monoterpenoids, sesquiterpenoids, diterpenoids, and hemiterpenes.
  • Carotenoids include, e.g., lycopene, ⁇ -carotene, and the like, many of which function as antioxidants.
  • Monoterpenoids include, e.g., menthol and camphor, which are flavor and fragrance agents.
  • Sesquiterpenoids include, e.g., artemisinin, a compound having anti-malarial activity.
  • Diterpenoids include, e.g., taxol, a cancer chemotherapeutic agent.
  • the present invention provides methods of producing an isoprenoid or an isoprenoid precursor in a host cell that comprises a biosynthetic pathway that converts a substrate to isopentenyl pyrophosphate, where the biosynthetic pathway is modified to include a synthetic intergenic region (IGR) between at least two coding regions encoding enzymes in the biosynthetic pathway.
  • the present invention further provides recombinant nucleic acid constructs comprising a synthetic IGR, and genetically modified host cells comprising a synthetic IGR.
  • Figures 1 A-E depict Tunable InterGenic Regions (TIGR) assembly and reporter operon.
  • TIGR InterGenic Regions
  • Figures 2A-C depict expression from TIGR RG library.
  • Figures 3A-I depict TIGR effects on expression.
  • Figures 4 A-F depict mevalonate pathway optimization using the TIGR method.
  • Figures 5A-C, 6A-C, 7A-C, and 8A-C depict TIGRs from various constructs.
  • FIG. 9 is a schematic representation of the mevalonate (MEV) pathway for the production of
  • FIG 10 is a schematic representation of the DXP pathway for the production of IPP and dimethylallyl pyrophosphate (DMAPP).
  • Figure 11 is a schematic representation of isoprenoid metabolic pathways that result in the production of the isoprenoid biosynthetic pathway intermediates polyprenyl diphosphates geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP), from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).
  • GPP polyprenyl diphosphate
  • FPP farnesyl diphosphate
  • GGPP geranylgeranyl diphosphate
  • IPP isopentenyl diphosphate
  • DMAPP dimethylallyl diphosphate
  • isoprenoid isoprenoid compound
  • terpene isoprenoid compound
  • terpenoid compound refers to any compound that is capable of being derived from IPP.
  • the number of C-atoms present in the isoprenoids is typically evenly divisible by five (e.g., C5, ClO, Cl 5, C20, C25, C30 and C40).
  • Isoprenoid compounds include, but are not limited to, monoterpenes, diterpenes, triterpenes, sesquiterpenes, and polyterpenes.
  • prenyl diphosphate is used interchangeably with “prenyl pyrophosphate,” and includes monoprenyl diphosphates having a single prenyl group (e.g., IPP and DMAPP), as well as polyprenyl diphosphates that include 2 or more prenyl groups.
  • monoprenyl diphosphates include isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP).
  • terpene synthase refers to any enzyme that enzymatically modifies IPP, DMAPP, or a polyprenyl pyrophosphate, such that a terpenoid precursor compound is produced.
  • the term “terpene synthase” includes enzymes that catalyze the conversion of a prenyl diphosphate into an isoprenoid or isoprenoid precursor.
  • pyrophosphate is used interchangeably herein with “diphosphate.”
  • prenyl diphosphate and “prenyl pyrophosphate” are interchangeable;
  • famesyl diphosphate” and farnesyl pyrophosphate are interchangeable; etc.
  • mevalonate pathway or "MEV pathway” is used herein to refer to the biosynthetic pathway that converts acetyl-CoA to IPP.
  • the mevalonate pathway comprises enzymes that catalyze the following steps: (a) condensing two molecules of acetyl-CoAto acetoacetyl-CoA(e.g., by action of acetoacetyl-CoA thiolase); (b) condensing acetoacetyl-CoAwith acetyl-CoAto form hydroxymethylglutaryl-CoenzymeA (HMG-CoA) (e.g., by action of HMG-CoA synthase (HMGS)); (c) converting HMG-CoA to mevalonate (e.g., by action of HMG-Co A reductase (HMGR)); (d) phosphorylating mevalonate to mevalonate 5-phosphate (e.g., by
  • DXP pathway The term "1 -deoxy-D-xylulose 5-diphosphate pathway” or "DXP pathway” is used herein to refer to the pathway that converts glyceraldehyde-3 -phosphate and pyruvate to IPP and DMAPP through a DXP pathway intermediate, where DXP pathway comprises enzymes that catalyze the reactions depicted schematically in Figure 10.
  • prenyl transferase is used interchangeably with the terms “isoprenyl diphosphate synthase” and “polyprenyl synthase” (e.g., “GPP synthase,” “FPP synthase,” “GGPP synthase,” etc.) to refer to an enzyme that catalyzes the consecutive 1 '-4 condensation of isopentenyl diphosphate with allylic primer substrates, resulting in the formation of prenyl diphosphates of various chain lengths.
  • GPP synthase e.g., "GPP synthase,” “FPP synthase,” “GGPP synthase,” etc.
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides.
  • this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • peptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • nucleic acid refers to a nucleic acid, cell, or organism that is found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring.
  • isolated is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs.
  • An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.
  • exogenous nucleic acid refers to a nucleic acid that is not normally or naturally found in and/or produced by a given bacterium, organism, or cell in nature.
  • endogenous nucleic acid refers to a nucleic acid that is normally found in and/or produced by a given bacterium, organism, or cell in nature.
  • An “endogenous nucleic acid” is also referred to as a “native nucleic acid” or a nucleic acid that is “native” to a given bacterium, organism, or cell.
  • nucleic acids encoding HMGS, mevalonate kinase, and phosphomevalonate kinase in represent exogenous nucleic acids to E. coli.
  • mevalonate pathway nucleic acids were cloned from Sacchromyces cerevisiae.
  • SI cerevisiae the gene sequences encoding HMGS, MK, and PMK on the chromosome would be "endogenous" nucleic acids.
  • heterologous nucleic acid refers to a nucleic acid wherein at least one of the following is true: (a) the nucleic acid is foreign ("exogenous") to (i.e., not naturally found in) a given host microorganism or host cell; (b) the nucleic acid comprises a nucleotide sequence that is naturally found in (e.g., is "endogenous to") a given host microorganism or host cell (e.g., the nucleic acid comprises a nucleotide sequence that is endogenous to the host microorganism or host cell) but is either produced in an unnatural (e.g., greater than expected or greater than naturally found) amount in the cell, or differs in sequence from the endogenous nucleotide sequence such that the same encoded protein (having the same or substantially the same amino acid sequence) as found endogenously is produced in an unnatural (e.g., greater than expected or greater than naturally found) amount in
  • heterologous polypeptide refers to a polypeptide that is not naturally associated with a given polypeptide.
  • an isoprenoid precursor-modifying enzyme that comprises a "heterologous transmembrane domain” refers to an isoprenoid precursor-modifying enzyme that comprises a transmembrane domain that is not normally associated with (e.g., not normally contiguous with; not normally found in the same polypeptide chain with) the isoprenoid precursor- modifying enzyme in nature.
  • Recombinant means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems.
  • DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system.
  • sequences can be provided in the form of an open reading frame uninterrupted by internal non- translated sequences, or introns, which are typically present in eukaryotic genes.
  • Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5 ' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see “DNA regulatory sequences", below).
  • the term "recombinant" polynucleotide or “recombinant” nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention.
  • This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions.
  • This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • the term “recombinant” polypeptide refers to a polypeptide which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequence through human intervention.
  • a polypeptide that comprises a heterologous amino acid sequence is recombinant.
  • construct or “vector” is meant a recombinant nucleic acid, generally recombinant DNA, which has been generated for the purpose of the expression and/or propagation of a specific nucleotide sequence(s), or is to be used in the construction of other recombinant nucleotide sequences.
  • the terms “operon” and “single transcription unit” are used interchangeably to refer to two or more contiguous coding regions (nucleotide sequences that encode a gene product such as an RNA or a protein) that are coordinately regulated by one or more controlling elements (e.g., a promoter).
  • the term “gene product” refers to RNA encoded by DNA (or vice versa) or protein that is encoded by an RNA or DNA, where a gene will typically comprise one or more nucleotide sequences that encode a protein, and may also include introns and other non-coding nucleotide sequences.
  • DNA regulatory sequences refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate expression of a coding sequence and/or production of an encoded polypeptide in a host cell.
  • transformation is used interchangeably herein with “genetic modification” and refers to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (i.e., DNA exogenous to the cell). Genetic change (“modification”) can be accomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. Where the cell is a eukaryotic cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.
  • chromosomes In prokaryotic cells, permanent changes can be introduced into the chromosome or via extrachromosomal elements such as plasmids and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell.
  • Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like.
  • the choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e. in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.
  • operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • heterologous promoter and “heterologous control regions” refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature.
  • a “transcriptional control region heterologous to a coding region” is a transcriptional control region that is not normally associated with the coding region in nature.
  • a "host cell,” as used herein, denotes an in vivo or in vitro eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells can be, or have been, used as recipients for a nucleic acid (e.g., an expression vector that comprises a nucleotide sequence encoding one or more biosynthetic pathway gene products such as mevalonate pathway gene products), and include the progeny of the original cell which has been genetically modified by the nucleic acid.
  • a nucleic acid e.g., an expression vector that comprises a nucleotide sequence encoding one or more biosynthetic pathway gene products such as mevalonate pathway gene products
  • a “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector.
  • a subject genetically modified prokaryotic host cell is a prokaryotic host cell that, by virtue of introduction into a suitable prokaryotic host cell a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to (not normally found in nature in) the prokaryotic host cell, or a recombinant nucleic acid that is not normally found in the prokaryotic host cell; and a subject genetically modified eukaryotic host cell is a eukaryotic host cell that, by virtue of introduction into a suitable eukaryotic host cell a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.
  • a heterologous nucleic acid e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a
  • a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide- containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains consists of cysteine and methionine.
  • Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine
  • Synthetic nucleic acids can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene.
  • “Chemically synthesized,” as related to a sequence of DNA means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines.
  • the nucleotide sequence of the nucleic acids can be modified for optimal expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST. See, e.g., Altschul et al. (1990), /. MoI. Biol. 215:403-10.
  • FASTA is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • GCG Genetics Computing Group
  • Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA.
  • alignment programs that permit gaps in the sequence.
  • the Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. SeeMeth. MoI. Biol. 70: 173-187 (1997).
  • the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. MoI. Biol. 48: 443-453 (1970).
  • a nucleic acid is "hybridizable" to another nucleic acid, such as a cDNA, genomic DNA, or
  • RNA when a single stranded form of the nucleic acid can anneal to the other nucleic acid under the appropriate conditions of temperature and solution ionic strength.
  • Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001).
  • the conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms.
  • Hybridization conditions and post-hybridization washes are useful to obtain the desired determine stringency conditions of the hybridization.
  • One set of illustrative post-hybridization washes is a series of washes starting with 6 x SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer), 0.5% SDS at room temperature for 15 minutes, then repeated with 2 x SSC, 0.5% SDS at 45°C for 30 minutes, and then repeated twice with 0.2 x SSC, 0.5% SDS at 50 0 C for 30 minutes.
  • stringent conditions are obtained by using higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 minute washes in 0.2 x SSC, 0.5% SDS, which is increased to 60 0 C.
  • Another set of highly stringent conditions uses two final washes in 0.1 x SSC, 0.1% SDS at 65°C.
  • Another example of stringent hybridization conditions is hybridization at 50 0 C or higher and 0.1 xSSC (15 mM sodium chloride/1.5 inM sodium citrate).
  • stringent hybridization conditions is overnight incubation at 42°C in a solution: 50% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 * Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 * SSC at about 65°C.
  • Stringent hybridization conditions and post-hybridization wash conditions are hybridization conditions and post-hybridization wash conditions that are at least as stringent as the above representative conditions.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences.
  • Tm melting temperature
  • the relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • the length for a hybridizable nucleic acid is at least about 10 nucleotides.
  • Illustrative minimum lengths for a hybridizable nucleic acid are: at least about 15 nucleotides; at least about 20 nucleotides; and at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
  • the present invention provides methods of producing an isoprenoid or an isoprenoid precursor in a host cell that comprises a biosynthetic pathway that converts a substrate to isopentenyl pyrophosphate (EPP), where the biosynthetic pathway is modified to include a synthetic intergenic region (IGR) between at least two coding regions encoding enzymes in the biosynthetic pathway.
  • EPP isopentenyl pyrophosphate
  • IGR synthetic intergenic region
  • One method of making an isoprenoid or an isoprenoid precursor is to culture a host cell, where the host cell is capable of making the isoprenoid or isoprenoid precursor. Because the biosynthetic pathway for making an isoprenoid or an precursor involves multiple enzymes, the flux through the pathway may not be optimum or properly balanced. One method of correcting such imbalance is to modulate the number or stability of either the RNA transcript or the resulting enzyme. This can be achieved by the presence of a synthetic IGR, as described herein.
  • Isoprenoid compounds are synthesized from a universal five carbon precursor, isopentenyl pyrophosphate (D?P).
  • D?P isopentenyl pyrophosphate
  • Mevalonate pathway enzymes are depicted in Figure 9.
  • the mevalonate pathway comprises the following enzymatic reactions: (a) condensing two molecules of acetyl-CoA to acetoacetyl-CoA; (b) condensing acetoacetyl-CoA with acetyl-CoA to form HMG-CoA; (c) converting HMG-CoA to mevalonate; (d) phosphorylating mevalonate to mevalonate 5-phosphate; (e) converting mevalonate 5- phosphate to mevalonate 5-pyrophosphate; and (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate.
  • Enzymes that carry out these reactions include acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), and mevalonate pyrophosphate decarboxylase (MPD).
  • HMGS hydroxymethylglutaryl-CoA synthase
  • HMGR hydroxymethylglutaryl-CoA reductase
  • MK mevalonate kinase
  • PMK phosphomevalonate kinase
  • MPD mevalonate pyrophosphate decarboxylase
  • FIG. 10 depicts schematically the DXP pathway, in which pyruvate and D-glyceraldehyde-3- phosphate are converted via a series of reactions to IPP and DMAPP.
  • the pathway involves action of the following enzymes: l-deoxy-D-xylulose-5-phosphate synthase (Dxs), 1 -deoxy-D-xylulose-5- phosphate reductoisomerase (IspC), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD), 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), l-hydroxy-2-methyl-2-(£)-butenyl 4-diphosphate synthase (IspG), and
  • Eukaryotic cells other than plant cells use the mevalonate pathway exclusively to convert acetyl- CoA to IPP, which is subsequently isomerized to DMAPP. Plants use both the mevalonae and the DXP pathways for isoprenoid synthesis. Prokaryotes, with some exceptions, use the DXP pathway to produce IPP and DMAPP separately through a branch point.
  • the IPP produced by the mevalonate pathway can be isomerized to produce DMAPP.
  • the IPP and/or the DMAPP can be acted on by prenyltransferases to produce polyprenyl pyrophosphates.
  • prenyltransferases to produce polyprenyl pyrophosphates.
  • IPP or DMAPP can be modified by prenyl transferases to generate the polyprenyl diphosphates geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP).
  • GPP and FPP are further modified by terpene synthases to generate monoterpenes and sesquiterpenes, respectively; and GGPP is further modified by terpene synthases to generate diterpenes and carotenoids.
  • IPP and DMAPP are generated by one of two pathways: the mevalonate (MEV) pathway and the l-deoxy-D-xylulose-5-phosphate (DXP) pathway.
  • MEV mevalonate
  • DXP l-deoxy-D-xylulose-5-phosphate
  • the present invention provides methods of producing an isoprenoid or an isoprenoid precursor in a host cell that comprises a biosynthetic pathway that converts a substrate to IPP (an "IPP biosynthetic pathway”), where the IPP biosynthetic pathway is modified to include a synthetic IGR between at least two coding regions encoding enzymes in the biosynthetic pathway.
  • An IPP biosynthetic pathway that is modified to include a synthetic IGR between at least two coding regions encoding enzymes in the IPP biosynthetic pathway is also referred to herein as an "IGR-modified EPP biosynthetic pathway.”
  • a subject method generally involves culturing a host cell in vitro in a suitable medium, wherein the host cell comprises a biosynthetic pathway that converts a substrate to IPP.
  • the IPP biosynthetic pathway is modified to include at least one synthetic IGR, where at least one synthetic IGR is disposed between a set of two coding regions encoding two enzymes in the biosynthetic pathway.
  • the host cell is "genetically modified,” as it includes a biosynthetic pathway modified to include a synthetic IGR.
  • Illustrative examples of an IPP biosynthetic pathway include: 1) an IPP biosynthetic pathway that is endogenous to a cell (e.g., an endogenous mevalonate pathway present in a eukaryotic cell that normally produces IPP via a mevalonate pathway, where such cells include, e.g., a yeast cell, a fungal cell, etc.; and an endogenous DXP pathway present in a prokaryotic cell that normally produces IPP via a DXP pathway); 2) an IPP biosynthetic pathway that is exogenous to a cell (e.g., where the cell has been genetically modified with one or more exogenous nucleic acids comprising nucleotide sequences encoding one or more IPP biosynthetic pathway enzymes heterologous to the cell (e.g., an exogenous mevalonate pathway in a prokaryotic cell that does not normally produce IPP via a mevalonate pathway; e.g.
  • the biosynthetic pathway that converts a substrate to IPP includes a single synthetic IGR disposed between a first coding region and a second coding region, where the first coding region comprises a nucleotide sequence encoding a first enzyme in the biosynthetic pathway and the second coding region comprises a nucleotide sequence encoding a second enzyme in the biosynthetic pathway.
  • the second enzyme is one that acts on a product of the first enzyme.
  • the biosynthetic pathway that converts a substrate to IPP includes two or more synthetic IGR, each disposed between two coding regions encoding enzymes in the pathway, where the coding regions encode at least three different enzymes in the pathway.
  • the biosynthetic pathway that converts a substrate to IPP includes a first synthetic IGR disposed between a first coding region and a second coding region; and a second synthetic IGR disposed between the second coding region and a third coding region, where each of the first, second, and third coding regions comprises nucleotide sequences encoding different enzymes in the biosynthetic pathway.
  • the second enzyme is one that acts on a product of the first enzyme; and the third enzyme acts on a product of the second enzyme.
  • the biosynthetic pathway that converts a substrate to IPP includes two or more synthetic IGR, each disposed between two coding regions encoding enzymes in the pathway, where the coding regions encode at least four different enzymes in the pathway.
  • the biosynthetic pathway that converts a substrate to EPP includes a first synthetic IGR disposed between a first coding region and a second coding region; and a second synthetic IGR disposed between a third coding region and a fourth coding region, where each of the first, second, third, and fourth coding regions comprises nucleotide sequences encoding different enzymes in the biosynthetic pathway.
  • a synthetic IGR is disposed between a first coding region encoding a gene product A (where gene product A is an mRNA encoding an enzyme in the IPP biosynthetic pathway) and a second coding region encoding a gene product B (where gene product B is an mRNA encoding another enzyme in the IPP biosynthetic pathway).
  • the presence of a synthetic IGR between a first coding region and a second coding region reduces the level of gene product A relative to gene product B, and thus provides for a reduced activity level of enzyme A relative to enzyme B.
  • the presence of a synthetic IGR reduces the activity level of enzyme A by from about 5% to about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50%, or more than 50%, compared to the activity level of enzyme B.
  • the presence of a synthetic IGR between a first coding region and a second coding region reduces the level of gene product B relative to gene product A, and thus provides for a reduced activity level of enzyme B relative to enzyme A.
  • the presence of a synthetic IGR reduces the activity level of enzyme B by from about 5% to about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50%, or more than 50%, compared to the activity level of enzyme A.
  • the presence of a synthetic IGR between a first coding region and a second coding region reduces the stability of gene product B relative to gene product A, or reduces the stability of gene product A relative to gene product B, such that in either case, the activity levels of enzyme A and enzyme B are substantially the same, e.g., the activity levels of the first and second enzymes differ from one another by less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1%.
  • the presence of a synthetic IGR between a first coding region and a second coding region reduces the stability of gene product A relative to gene product B, and thus " provides for a reduced activity level of enzyme A relative to enzyme B.
  • the presence of a synthetic IGR reduces the activity level of enzyme A by from about 5% to about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50%, or more than 50%, compared to the activity level of enzyme B.
  • the presence of a synthetic IGR between a first coding region and a second coding region reduces the stability of gene product B relative to gene product A, and thus provides for a reduced activity level of enzyme B relative to enzyme A.
  • the presence of a synthetic IGR reduces the activity level of enzyme B by from about 5% to about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50%, or more than 50%, compared to the activity level of enzyme A.
  • the presence of a synthetic IGR between a first coding region and a second coding region modulates reduces stability of gene product B relative to gene product A, or reduces the stability of gene product A relative to gene product B, such that in either case, the activity levels of enzyme A and enzyme B are substantially the same, e.g., the activity levels of the first and second enzymes differ from one another by less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1%
  • the presence of the at least one synthetic IGR provides for production of one or more intermediates in the biosynthetic pathway at a level that is non-toxic to the host cell, e.g., at a level that does not substantially reduce growth of the cell.
  • the presence of the at least one synthetic IGR provides for production of one or more intermediates in the biosynthetic pathway at a level that inhibits growth of the cell by less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5% or less than about 2%, compared to inhibition of cell growth in the absence of the synthetic IGR(s).
  • a synthetic IGR can have a length in a range of from about 15 nucleotides (nt) to about 500 nt, e.g., from about 15 nt to about 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about 50 nt, from about 50 nt to about 75 nt, from about 75 nt to about 100 nt, from about 100 nt to about 125 nt, from about 125 nt to about 150 nt, from about 150 nt to about 175 nt, from about 175 nt to about 200 nt, from about 200 nt to about 225 nt, from about 225 nt to about 250 nt, from about 250 nt to about 275 nt, from about 275 nt to about 300 nt, from about 300 nt to about 350 nt, from about 350 nt to about 400 nt, from about 400 nt to about 400 nt
  • a synthetic IGR can have a length in a range of from about 75 nt to about 275 nt. In other embodiments, a synthetic IGR can have a length in a range of from about 25 nt to about 135 nt.
  • a synthetic IGR is a nucleotide sequence having a length of from about
  • a synthetic IGR is a nucleotide sequence having a length of from about 15 nt to about 500 nt and comprising a nucleotide sequence that is a riboendonuclease recognition site.
  • a synthetic IGR is a nucleotide sequence having a length of from about 15 nt to about 500 nt, wherein the synthetic IGR comprises both a nucleotide sequence that forms a hairpin and a nucleotide sequence that is a riboendonuclease recognition site.
  • the term "hairpin” refers to a three-dimensional structure formed by a first complementary nucleotide sequence and a second complementary nucleotide sequence in the same nucleic acid sequence, where the nucleic acid comprising the hairpin-forming nucleotide sequence folds back on itself, such that the first and second complementary nucleotide sequences form hydrogen bonds with one another.
  • the first and second complementary nucleotide sequences can be immediately adjacent one another, or can be separated by from one to 200 nucleotides (e.g., from two to about 100, from six to about 50, from about 10 to about 20, etc.).
  • the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex.
  • the loop can vary in length. In some embodiments the loop is from about 5 nt to about 10 nt, from about 10 nt to about 100 nt, from about 15 nt to about 50 nt, from about 20 nt to about 30 nt nucleotides in length, and the like.
  • the first and second complementary sequences may be 100% complementary, or may include one or more mismatches.
  • the first and second complementary sequences form at least one base pair, and can form from one to 20 or more base pairs, where the base pairs can be contiguous or separated by one or more nucleotides.
  • a hairpin can be a short hairpin or a long hairpin.
  • a hairpin can be included in a secondary structure such as a stem-loop structure, an internal loop, a bulge loop, a branched structure, or a pseudoknot, multiple stem loop structures, cloverleaf type structures or any three dimensional structure that includes a hairpin.
  • a synthetic IGR can include a single hairpin, or more than one hairpin, e.g., two, three, four, or more hairpins.
  • a riboendonuclease recognition site includes an RNAse III recognition site, an RNAseE recognition site, and the like.
  • RNAse recognition sequences are known in the art.
  • the riboendonuclease recognition sequence is a recognition sequence for RNAse HI.
  • the riboendonuclease recognition sequence is a recognition sequence for RNAseE.
  • the synthetic IGR comprises, in order from 5'-3', a first hairpin-forming nucleotide sequence; an RNAse recognition sequence; and a second hairpin-forming nucleotide sequence.
  • Suitable synthetic IGR nucleotide sequences include, but are not limited to, a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% nucleotide sequence identity to a nucleotide sequence set forth in one of SEQ ID NOs:7, 8, 11, 12, 15, 16, 19, 20, and 62-77.
  • a synthetic IGR comprises a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% nucleotide sequence identity to a nucleotide sequence set forth in any one of SEQ ID NOs:5, 6, 9, 10, 13, 14, 17, or 18.
  • a synthetic IGR comprises a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% nucleotide sequence identity to a nucleotide sequence set forth in any one of SEQ ID NOs:7, 8, 11 , 12, 15, 16, 19, or 20.
  • a synthetic IGR comprises a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% nucleotide sequence identity to a nucleotide sequence set forth in any one of SEQ ID NOs:62-77.
  • the host cell produces EPP and/or mevalonate via the mevalonate pathway.
  • the host cell comprises a mevalonate pathway that comprises at least one synthetic IGR, where the at least one synthetic IGR is disposed between two coding regions encoding two enzymes in the mevalonate pathway.
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences encoding a mevalonate pathway, where the one or more nucleic acids comprises a single synthetic IGR, where the single synthetic IGR is between a coding region comprising a nucleotide sequence encoding acetoacetyl-CoA thiolase and a coding region comprising a nucleotide sequence encoding HMGS.
  • the host cell comprises a mevalonate pathway that comprises a single synthetic IGR, where the single synthetic IGR is between a coding region comprising a nucleotide sequence encoding HMGS, and a coding region comprising a nucleotide sequence encoding HMGR.
  • the single synthetic IGR is between a coding region comprising a nucleotide sequence encoding HMGR and a coding region comprising a nucleotide sequence encoding MK; between a coding region comprising a nucleotide sequence encoding MK and a coding region comprising a nucleotide sequence encoding PMK; or between a coding region comprising a nucleotide sequence encoding PMK and a coding region comprising a nucleotide sequence encoding MPD.
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences encoding a mevalonate pathway, where the one or more nucleic acids comprises two or more synthetic IGRs, where the two or more synthetic IGRs are each disposed between two coding regions encoding enzymes in the pathway.
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences encoding a mevalonate pathway, where the one or more nucleic acids comprises a first synthetic IGR between a coding region comprising a nucleotide sequence encoding acetoacetyl CoA thiolase and a coding region comprising a nucleotide sequence encoding HMGS; and a second synthetic IGR between a coding region comprising a nucleotide sequence encoding HMGS and HMGR, where the first and second IGR can be the same or different (e.g., have the same nucleotide sequence or two different nucleotide sequences).
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences r encoding a mevalonate pathway, where the one or more nucleic acids comprises a first synthetic IGR between a coding region comprising a nucleotide sequence encoding HMGS and a coding region comprising a nucleotide sequence encoding HMGR; and a second synthetic IGR between a coding region comprising a nucleotide sequence encoding HMGR and a coding region comprising a nucleotide sequence encoding MK.
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences encoding a mevalonate pathway, where the one or more nucleic acids comprises a first synthetic IGR between a coding region comprising a nucleotide sequence encoding acetoacetyl-CoA thiolase and a coding region comprising a nucleotide sequence encoding HMGS; and a second synthetic IGR between a coding region comprising a nucleotide sequence encoding HMGR and a coding region comprising a nucleotide sequence encoding MK.
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences encoding a mevalonate pathway, where the one or more nucleic acids comprises three, four, or five synthetic IGRs, where the three, four, or five synthetic IGRs are each disposed between two coding regions encoding enzymes in the pathway.
  • a host cell that comprises one or more nucleic acids comprising nucleotide sequences encoding a mevalonate pathway is a host cell that normally produces IPP via a mevalonate pathway.
  • a host cell that comprises one or more nucleic acids comprising nucleotide sequences encoding a mevalonate pathway is a host cell that does not normally produces IPP via a mevalonate pathway.
  • the host cell produces IPP via a DXP pathway.
  • the host cell comprises a mevalonate pathway that comprises at least one synthetic IGR, where the at least one synthetic IGR is disposed between two coding regions encoding two enzymes in the DXP pathway.
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences encoding a DXP pathway, where the one or more nucleic acids comprises a single synthetic IGR, where the single synthetic IGR is between a coding region comprising a nucleotide sequence encoding Dxs and a coding region comprising a nucleotide sequence encoding Dxr.
  • the host cell comprises a DXP pathway that comprises a single synthetic IGR, where the single synthetic IGR is between a coding region comprising a nucleotide sequence encoding Dxr, and a coding region comprising a nucleotide sequence encoding IspD.
  • the single synthetic IGR is between a coding region comprising a nucleotide sequence encoding IspD and a coding region comprising a nucleotide sequence encoding IspF; between a coding region comprising a nucleotide sequence encoding IspF and a coding region comprising a nucleotide sequence encoding IspG; or between a coding region comprising a nucleotide sequence encoding IspG and a coding region comprising a nucleotide sequence encoding IspH.
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences encoding a DXP pathway, where the one or more nucleic acids comprises two or more synthetic IGRs, where the two or more synthetic IGRs are each disposed between two coding regions encoding enzymes in the pathway.
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences encoding a DXP pathway, where the one or more nucleic acids comprises a first synthetic IGR between a coding region comprising a nucleotide sequence encoding Dxs and a coding region comprising a nucleotide sequence encoding Dxr; and a second synthetic IGR between a coding region comprising a nucleotide sequence encoding Dxr and IspD, where the first and second IGR can be the same or different (e.g., have the same nucleotide sequence or two different nucleotide sequences).
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences encoding a DXP pathway, where the one or more nucleic acids comprises a first synthetic IGR between a coding region comprising a nucleotide sequence encoding Dxr and a coding region comprising a nucleotide sequence encoding IspD; and a second synthetic IGR between a coding region comprising a nucleotide sequence encoding IspD and a coding region comprising a nucleotide sequence encoding IspE.
  • the one or more nucleic acids comprises a first synthetic IGR between a coding region comprising a nucleotide sequence encoding Dxr and a coding region comprising a nucleotide sequence encoding IspD; and a second synthetic IGR between a coding region comprising a nucleotide sequence encoding IspD and a coding region comprising a nucleotide
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences encoding a DXP pathway, where the one or more nucleic acids comprises a first synthetic IGR between a coding region comprising a nucleotide sequence encoding Dxs and a coding region comprising a nucleotide sequence encoding Dxr; and a second synthetic IGR between a coding region comprising a nucleotide sequence encoding IspD and a coding region comprising a nucleotide sequence encoding IspE.
  • the host cell comprises one or more nucleic acids comprising nucleotide sequences encoding a DXP pathway, where the one or more nucleic acids comprises three, four, or five synthetic IGRs, where the three, four, or five synthetic IGRs are each disposed between two coding regions encoding enzymes in the pathway.
  • a host cell that comprises one or more nucleic acids comprising nucleotide sequences encoding a DXP pathway is a host cell that normally produces DPP via a DXP pathway. In other embodiments, a host cell that comprises one or more nucleic acids comprising nucleotide sequences encoding a DXP pathway is a host cell that does not normally produces IPP via a DXP pathway.
  • the host cell that produces an isoprenoid or isoprenoid precursor is genetically modified such that it produces the isoprenoid or isoprenoid precursor via an IPP biosynthetic pathway that has been modified to include one or more synthetic IGRs.
  • the genetically modified host cell is a genetically modified version of a parent host cell.
  • the EPP biosynthetic pathway is in some embodiments substantially the same as an endogenous pathway but for the inclusion of the one or more synthetic IGRs, e.g., the IPP biosynthetic pathway comprises nucleotide sequence encoding enzymes that are endogenous to the host cell.
  • the host cell is a prokaryotic cell that normally produces DPP via an endogenous DXP pathway
  • the IGR-modified IPP biosynthetic pathway comprises the endogenous DXP pathway, modified to include one or more synthetic IGRs.
  • the host cell is a eukaryotic cell (e.g., a yeast cell) that normally produces D?P via an endogenous mevalonate pathway
  • the IGR-modified IPP biosynthetic pathway comprises the endogenous mevalonate pathway, modified to include one or more synthetic IGRs.
  • the IGR-modified IPP biosynthetic pathway comprises both synthetic
  • the host cell is a prokaryotic cell that does not normally synthesize IPP via a mevalonate pathway; and the host cell is genetically modified with an exogenous mevalonate pathway that includes one or more synthetic IGRs and nucleotide sequences encoding mevalonate pathway enzymes that are heterologous to the cell.
  • the host cell is a eukaryotic cell (e.g., a yeast cell) that does not normally synthesize DPP via a DXP pathway; and the host cell is genetically modified with an exogenous DXP pathway that includes one or more synthetic IGRs and nucleotide sequences encoding DXP pathway enzymes that are heterologous to the cell.
  • Production of an isoprenoid or an isoprenoid precursor is increased in the genetically modified host cell, compared to a control, parent cell.
  • production of an isoprenoid or isoprenoid precursor is increased by at least about 10%, at least about 20%, at least about 50%, at least about 2- fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100- fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold, or more, in the genetically modified host cell, compared to the control host cell.
  • the level of mevalonate produced in a subject genetically modified host cell is at least about 10%, at least about 20%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30- fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold, or more, greater than the level of mevalonate produced in a control cell.
  • the level of mevalonate produced in a subject genetically modified host cell is greater than about 275 mM, e.g., from about 280 mM to about 290 mM, from about 290 mM to about 300 mM, from about 300 mM to about 350 mM, from about 350 mM to about 400 mM, from about 400 mM to about 450 mM, from about 450 mM to about 500 mM, from about 500 mM to about 550 mM, or from about 550 mM to about 600 mM, or greater, at 24 hours in culture, where the concentrations are normalized to OD (e.g., OD 600 ).
  • OD 600 e.g., OD 600
  • the level of IPP produced in a subject genetically modified host cell is at least about 10%, at least about 20%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30- fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold, or more, greater than the level of IPP produced in a control cell.
  • the level of an isoprenoid compound produced in a subject genetically modified host cell is at least about 10%, at least about 20%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20- fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold, or more, greater than the level of the isoprenoid compound produced in a control cell.
  • the growth rate of a subject genetically modified host cell is greater than the growth rate of a control cell.
  • a subject genetically modified host cell grows at a rate that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or 2- fold, at least about 5-fold, at least about 10-fold, or more, higher than the growth rate of a control cell.
  • Cell growth o is readily determined using well-known methods, e.g., optical density (OD) measurement at about 600 nm (OD 600 ) of liquid cultures of bacteria; colony size; growth rate; and the like.
  • the presence of a synthetic IGR between a first coding region and a second coding region provides for altered levels of a gene product encoded by the first coding region relative to a gene product encoded by the second coding region.
  • the level of HMGS mRNA in a genetically modified host cell is less than the level of a mevalonate pathway mRNA other than HMGS mRNA in the genetically modified host cell.
  • the level of HMGS mRNA is less than the level of acetoacetyl-CoA thiolase mRNA in the genetically modified host cell.
  • the level of HMGS protein in a genetically modified host cell is less than the level of a mevalonate pathway protein other than HMGS protein in the genetically modified host cell.
  • the level of HMGS protein in a genetically modified host cell is less than the level of acetoacetyl-CoA thiolase protein in the genetically modified host cell.
  • the level of HMGS mRNA in a subject genetically modified host cell is from about 10% less to about 15% less, from about 15% less to about 20% less, from about 20% less to about 30% less, from about 30% less to about 40% less, from about 40% less to about 50% less, or from about 50% less to about 60% less, than the level of acetoacetyl-CoA thiolase mRNA in the genetically modified host cell.
  • the level of HMGS protein in a subject genetically modified host cell is from about 10% less to about 15% less, from about 15% less to about 20% less, from about 20% less to about 30% less, from about 30% less to about 40% less, from about 40% less to about 50% less, from about 50% less to about 60% less, or from about 60% less to about 70% less, than the level of acetoacetyl-CoA thiolase protein in the genetically modified host cell.
  • the level of both HMGS mRNA and HMGR mRNA in a genetically modified host cell is less than the level of a mevalonate pathway mRNA other than HMGS mRNA and HMGR mRNA in the genetically modified host cell.
  • the level of both HMGS mRNA and HMGR mRNA is less than the level of acetoacetyl-CoA thiolase mRNA in the genetically modified host cell.
  • the level of both HMGS protein and HMGR protein in a genetically modified host cell is less than the level of a mevalonate pathway protein other than HMGS and HMGR proteins in the genetically modified host cell.
  • the level of both HMGS protein and HMGR protein in a genetically modified host cell is less than the level of acetoacetyl-CoA thiolase protein in the genetically modified host cell.
  • the level of both HMGS mRNA and HMGR mRNA in a subject genetically modified host cell is from about 10% less to about 15% less, from about 15% less to about 20% less, from about 20% less to about 30% less, from about 30% less to about 40% less, from about 40% less to about 50% less, or from about 50% less to about 60% less, than the level of acetoacetyl- CoA thiolase mRNA in the genetically modified host cell.
  • the level of both HMGS protein and HMGR protein in a subject genetically modified host cell is from about 10% less to about 15% less, from about 15% less to about 20% less, from about 20% less to about 30% less, from about 30% less to about 40% less, from about 40% less to about 50% less, from about 50% less to about 60% less, or from about 60% less to about 70% less, than the level of acetoacetyl-CoA thiolase protein in the genetically modified host cell.
  • a subject method of increasing production of an isoprenoid compound, or an isoprenoid precursor compound, in a host cell comprises decreasing the level of HMGS activity in the cell and/or decreasing the level of HMGR activity in the cell, compared to the levels in a control host cell.
  • Decreasing the level of HMGS activity in a cell includes decreasing the total amount of HMGS polypeptide within the cell; and decreasing the specific activity of HMGS polypeptide within the cell.
  • the level of HMGS activity in a cell is decreased by decreasing the total amount of HMGS in the cell.
  • the level of HMGS activity in a cell is decreased by decreasing the specific activity of HMGS in the cell.
  • decreasing the level of HMGR activity in a cell includes decreasing the total amount of HMGR polypeptide within the cell; and decreasing the specific activity of HMGR polypeptide within the cell.
  • the level of HMGR activity in a cell is decreased by decreasing the total amount of HMGR in the cell. In other embodiments, the level of HMGR activity in a cell is decreased by decreasing the specific activity of HMGR in the cell.
  • Isoprenoids that can be produced using the method of the invention include, but are not limited to, monoterpenes, including but not limited to, limonene, citranellol, geraniol, menthol, perillyl alcohol, linalool, thujone; sesquiterpenes, including but not limited to, periplanone B, gingkolide B, amorphadiene, artemisinin, artemisinic acid, valencene, nootkatone, epi-cedrol, epi-aristolochene, famesol, gossypol, sanonin, periplanone, and forskolin; diterpenes, including but not limited to, casbene, eleutherobin, paclitaxel, prostratin, and pseudopterosin; triterpenes, including but not limited to, arbrusideE, bruceantin, testosterone, progesterone, cortisone, digitoxin.
  • Isoprenoids also include, but are not limited to, carotenoids such as lycopene, ⁇ - and ⁇ -carotene, ⁇ - and ⁇ -cryptoxanthin, bixin, zeaxanthin, astaxanthin, and lutein.
  • Isoprenoids also include, but are not limited to, triterpenes, steroid compounds, and compounds that are composed of isoprenoids modified by other chemical groups, such as mixed terpene-alkaloids, menaq ⁇ inones (e.g., vitamin K-2), and coenzyme Q-10.
  • HETEROLOGOUS NUCLEIC ACIDS HETEROLOGOUS NUCLEIC ACIDS
  • the present invention provides nucleic acids that are useful in generating a genetically modified host cell, for use in producing an isoprenoid or isoprenoid precursor, e.g., in a subject method.
  • a subject nucleic acid comprises nucleotide sequences encoding one or more synthetic IGRs.
  • a subject nucleic acid comprises nucleotide sequences encoding one or more synthetic IGRs; and nucleotide sequences encoding one or more enzymes in an IPP biosynthetic pathway (e.g., a mevalonate pathway; or a DXP pathway), where a synthetic IGR is located 5' to at least one nucleotide sequence encoding an enzyme in the IPP biosynthetic pathway.
  • a subject nucleic acid is a synthetic nucleic acid.
  • a subject nucleic acid is a recombinant nucleic acid.
  • a subject nucleic acid is an expression construct (an "expression vector").
  • a subject nucleic acid comprises nucleotide sequences encoding one or more synthetic IGRs, where synthetic IGRs are as described above; and nucleotide sequences encoding one or more enzymes in an EPP biosynthetic pathway (e.g., a mevalonate pathway; or a DXP pathway), where a synthetic IGR is located 5' to at least one nucleotide sequence encoding an enzyme in the IPP biosynthetic pathway.
  • EPP biosynthetic pathway e.g., a mevalonate pathway; or a DXP pathway
  • a subject nucleic acid comprises nucleotide sequences encoding two or more enzymes in an IPP biosynthetic pathway, and a single synthetic IGR disposed between two coding regions comprising nucleotide sequences encoding the two of the two or more enzymes in the IPP biosynthetic pathway, or a single synthetic IGR at the 5' end of the pathway.
  • a subject nucleic acid comprises a nucleotide sequence encoding a single synthetic IGR, and nucleotide sequences comprising a first coding region and a second coding region, where the single IGR is disposed between a first coding region and a second coding region, where the first coding region comprises a nucleotide sequence encoding a first enzyme in the biosynthetic pathway and the second coding region comprises a nucleotide sequence encoding a second enzyme in the biosynthetic pathway.
  • the second enzyme is one that acts on a product of the first enzyme.
  • a subject nucleic acid comprises nucleotide sequences encoding three or more enzymes in an IPP biosynthetic pathway; and nucleotide sequences encoding two or more synthetic IGRs, where each of the two or more synthetic IGRs is disposed between two coding regions encoding two of the three or more enzymes in the pathway.
  • a subject nucleic acid comprises a nucleotide sequence encoding an IGR-modified IPP biosynthetic pathway, and includes a first synthetic IGR disposed between a first coding region and a second coding region; and a second synthetic IGR disposed between the second coding region and a third coding region, where each of the first, second, and third coding regions comprises nucleotide sequences encoding different enzymes in the IPP biosynthetic pathway.
  • the second enzyme is one that acts on a product of the first enzyme; and the third enzyme acts on a product of the second enzyme.
  • a subject nucleic acid comprises nucleotide sequences encoding an IGR- modified IPP biosynthetic pathway that includes two or more synthetic IGR, each disposed between two coding regions encoding enzymes in the pathway, where the coding regions encode at least four different enzymes in the pathway.
  • a subject nucleic acid comprises nucleotide sequences encoding an IGR-modified IPP biosynthetic pathway that includes a first synthetic IGR disposed between a first coding region and a second coding region; and a second synthetic IGR disposed between a third coding region and a fourth coding region, where each of the first, second, third, and fourth coding regions comprises nucleotide sequences encoding different enzymes in the biosynthetic pathway.
  • the mevalonate pathway comprises: (a) condensing two molecules of acetyl-CoA to acetoacetyl-CoA; (b) condensing acetoacetyl-CoA with acetyl-CoA to form HMG-CoA; (c) converting HMG-CoAto mevalonate; (d) phosphorylating mevalonate to mevalonate 5 -phosphate; (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate; and (f) converting mevalonate 5- pyrophosphate to isopentenyl pyrophosphate.
  • the mevalonate pathway enzymes required for production of IPP vary, depending on the culture conditions.
  • a subject nucleic acid comprises nucleotide sequence encoding one or more synthetic IGRs; and nucleotide sequences encoding acetoacetyl-CoA thiolase, HMGS, and HMGR, where the one or more synthetic IGRs are disposed between two coding regions encoding the enzymes.
  • a subject nucleic acid comprises nucleotide sequence encoding one or more synthetic IGRs; and nucleotide sequences encoding acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, and MPD (and optionally also IPP isomerase), where each of the one or more synthetic IGRs are disposed between two coding regions encoding the enzymes.
  • a subject nucleic acid comprises nucleotide sequence encoding one or more synthetic IGRs; and nucleotide sequences encoding acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, IPP isomerase, and a prenyl transferase, where each of the one or more synthetic IGRs are disposed between two coding regions encoding the enzymes.
  • a subject nucleic acid comprises nucleotide sequence encoding one or more synthetic IGRs; and nucleotide sequences encoding acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, IPP isomerase, a prenyl transferase, and a terpene synthase, where each of the one or more synthetic IGRs are disposed between two coding regions encoding the enzymes.
  • Nucleotide sequences encoding mevalonate (MEV) pathway gene products are known in the art, and any known MEV pathway gene product-encoding nucleotide sequence can used to generate a subject genetically modified host cell.
  • MEV pathway gene product-encoding nucleotide sequence can be used to generate a subject genetically modified host cell.
  • nucleotide sequences encoding acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, and IDI are known in the art.
  • acetoacetyl-CoA thiolase (NC_000913 REGION: 2324131..232531S; ⁇ . coli), (D49362; Paracoccus denitrificans), and (L20428; Saccharomyces cerevisiae); HMGS: (NC_001145.
  • HMGR (NM 206548; Drosophila melanogaster), (NM_204485; Gallus gallus), (ABOl 5627; Streptomyces sp.
  • KO-3988 (AF542543; Nicotiana attenuata), (AB037907; Kitasatospora griseola), (AX128213, providing the sequence encoding a truncated HMGR; Saccharomyces cerevisiae), and (NC OOl 145: complement (115734..1 18898; Saccharomyces cerevisiae)); MK: (L77688; Arabidopsis thalian ⁇ ), and (X55875; Saccharomyces cerevisiae); PMK: (AF429385; Hevea brasiliensis), (NM 006556; Homo sapiens), (NCJ)Ol 145.
  • nucleotide sequences encoding aceoacetyl-CoA thiolase, HMGS, and HMGR is set forth in Figures 13A-C (SEQ ID NO:1) of U.S. Patent No. 7,183,089.
  • a non-limiting example of nucleotide sequences encoding MK, PMK, MPD, and isopentenyl diphosphate isomerase (IDI) is set forth in Figures 16A-D of U.S. Patent No. 7,183,089.
  • the HMGR coding region is set forth in SEQ ID NO: 13 of U. S . Patent
  • HMGR truncated form of HMGR
  • the transmembrane domain of HMGR contains the regulatory portions of the enzyme and has no catalytic activity.
  • the coding sequence of any known MEV pathway enzyme may be altered in various ways known in the art to generate targeted changes in the amino acid sequence of the encoded enzyme.
  • the amino acid of a variant MEV pathway enzyme will usually be substantially similar to the amino acid sequence of any known MEV pathway enzyme, i.e. will differ by at least one amino acid, and may differ by at least two, at least 5, at least 10, or at least 20 amino acids, but typically not more than about fifty amino acids.
  • the sequence changes may be substitutions, insertions or deletions.
  • the nucleotide sequence can be altered for the codon bias of a particular host cell.
  • one or more nucleotide sequence differences can be introduced that result in conservative amino acid changes in the encoded protein.
  • DXP pathway enzymes DXP pathway enzymes
  • the DXP pathway comprises: l-deoxy-D-xylulose-5 -phosphate synthase (Dxs), 1-deoxy-D- xylulose-5 -phosphate reductoisomerase (IspC), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2C-methyl-D-erythritol 2,4- cyclodiphosphate synthase (IspF), and l-hydroxy-2-methyl-2-(£)-butenyl 4-diphosphate synthase (IspG).
  • Dxs 1-deoxy-D- xylulose-5 -phosphate reductoisomerase
  • IspD 4-diphosphocytidyl-2-C-methyl-D-erythr
  • a subject nucleic acid comprises nucleotide sequence encoding one or more synthetic IGRs; and nucleotide sequences encoding two, three, four, five, six, or seven of the DXP pathway enzymes, where each of the one or more synthetic IGRs are disposed between two coding regions encoding the enzymes.
  • a subject nucleic acid comprises nucleotide sequence encoding one or more synthetic IGRs; and nucleotide sequences encoding two, three, four, five, six, or seven of the DXP pathway enzymes, and a prenyl transferase, where each of the one or more synthetic IGRs are disposed between two coding regions encoding the enzymes.
  • a subject nucleic acid comprises nucleotide sequence encoding one or more synthetic IGRs; and nucleotide sequences encoding two, three, four, five, six, or seven of the DXP pathway enzymes, a prenyl transferase, and a terpene synthase, where each of the one or more synthetic IGRs are disposed between two coding regions encoding the enzymes.
  • Nucleotide sequences encoding DXP pathway enzymes are nucleotide sequence encoding DXP pathway enzymes
  • Nucleotide sequences encoding DXP pathway enzymes are known in the art, and can be used in a subject method. Variants of any known nucleotide sequence encoding a DXP pathway enzyme can be used, where the encoded enzyme retains enzymatic activity.
  • Variants of any known nucleotide sequence encoding a DXP pathway enzyme selected from l-deoxy-D-xylulose-5-phosphate synthase (dxs); l-deoxy-D-xylulose-5-phosphate reductoisomerase (IspC; dxr), 4-diphosphocytidyl-2-C-methyl- D-erythritol synthase (IspD; YbgP), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE; YchB), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF; YbgB), l-hydroxy-2-methyl-2-(£)- butenyl 4-diphosphate synthase (IspG), and isopentenyl diphosphate isomerase can be used, where a variant
  • the coding sequence of any known DXP pathway enzyme may be altered in various ways known in the art to generate targeted changes in the amino acid sequence of the encoded enzyme.
  • the amino acid of a variant DXP pathway enzyme will in some embodiments be substantially similar to the amino acid sequence of any known DXP pathway enzyme, i.e. will differ by at least one amino acid, and may differ by at least two, at least 5, at least 10, or at least 20 amino acids, but typically not more than about fifty amino acids.
  • the sequence changes may be substitutions, insertions or deletions.
  • the nucleotide sequence can be altered for the codon bias of a particular host cell.
  • one or more nucleotide sequence differences can be introduced that result in conservative amino acid changes in the encoded protein.
  • Nucleotide sequences encoding l-deoxy-D-xylulose-5-phosphate synthase are known in the art. See, e.g., GenBank Accession No. DQ768815 ⁇ Yersinia pestis dxs); GenBank Accession No. AF143812 ⁇ Lycopersicon esculentum dxs); GenBank Accession No. Y18874 ⁇ Synechococcus PCC6301 dxs); GenBank Accession No. AF035440; E. coli dxs); GenBank Accession No.
  • Nucleotide sequences encoding l-deoxy-D-xylulose-5-phosphate reductoisomerase are known in the art. See, e.g., GenBank Accession No. AF282879 ⁇ Pseudomonas aeruginosa dxr); GenBank Accession No. AY081453 ⁇ Arabidopsis thaliana dxr); and GenBank Accession No. AJ297566 ⁇ Zea mays dxr). See also Figure 31 of U.S. Patent Publication No. 2003/0219798 for nucleotide sequences encoding dxr. [00117] Nucleotide sequences encoding 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD;
  • GenBank Accession No. AF230737 (Arabidopsis thaliana); GenBank Accession No. CP000034.1 (nucleotides 2725605-2724895; Shigella dysenteriae); and GenBank Accession No. CP000036.1 (nucleotides 2780789 to 2781448; Shigella boydii). See also SEQ ID NO:5 of U.S. Patent No. 6,660,507 (Methylomonas IspD).
  • Nucleotide sequences encoding 4-diphosphocytidyl-2-C-methyl-D-erythritol (IspE; YchB) kinase are known in the art. See, e.g., GenBank Accession No. CP000036.1 (nucleotides 1839782- 1840633; Shigella boydii); GenBank Accession No. AF288615 (Arabidopsis thaliana) and GenBank Accession No. CP000266.1 (nucleotides 1272480-1271629; Shigella flexneri). See also, SEQ ID NO.7 of U.S. Patent No. 6,660,507 (Methylomonas 16a IspE).
  • GenBank Accession No. AEOl 7220.1 nucleotides 3025667- 3025216; Salmonella enterica IspF
  • GenBank Accession No. NM_105070 Arabidopsis thaliana
  • GenBank Accession No. AE014073.1 nucleotides 2838621-283841; Shigella flexneri.
  • GenBank Accession No. CP000034.1 nucleotides 2505082 to 2503964; Shigella dysenteriae IspG
  • GenBank Accession No. NM l 80902 Arabidopsis thaliana
  • GenBank Accession No. AE008814.1 nucleotides 15609-14491; Salmonella typhimurium IsgG
  • GenBank Accession No. AE014613.1 nucleotides 383225-384343; Salmonella enterica GcpE
  • AEOl 7220.1 (nucleotides 2678054-2676936; Salmonella enterica GcpE; and GenBank Accession No. BX95085.1 (nucleotides 3604460-3603539; Erwinia carotova GcpE).
  • IspH genes are known in the art. See, e.g., GenBank Accession No. AYl 68881 (Arabidopsis thaliana).
  • Nucleotide sequences encoding IPP isomerase are known in the art. See, e.g., (J05090;
  • nucleotide sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or higher, nucleotide sequence identity to a known nucleotide sequence encoding a DXP pathway enzyme are also suitable for use, where the nucleotide sequence encodes a functional DXP pathway enzyme.
  • Prenyl transferases having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or higher, nucleotide sequence identity to a known nucleotide sequence encoding a DXP pathway enzyme are also suitable for use, where the nucleotide sequence encodes a functional DXP pathway enzyme.
  • a subject genetically modified host cell is genetically modified to include one or more nucleic acids comprising a nucleotide sequence(s) encoding one or more mevalonate pathway enzymes, as described above; and a nucleic acid comprising a nucleotide sequence that encodes a prenyl transferase.
  • Prenyltransferases constitute a broad group of enzymes catalyzing the consecutive condensation of IPP resulting in the formation of prenyl diphosphates of various chain lengths.
  • Suitable prenyltransferases include enzymes that catalyze the condensation of BPP with allylic primer substrates to form isoprenoid compounds with from about 2 isoprene units to about 6000 isoprene units or more, e.g., 2 isoprene units (Geranyl Pyrophosphate synthase), 3 isoprene units (Farnesyl pyrophosphate synthase), 4 isoprene units (geranylgeranyl pyrophosphate synthase), 5 isoprene units, 6 isoprene units (hexadecylpyrophosphate synthase), 7 isoprene units, 8 isoprene units (phytoene synthase, octaprenyl pyrophosphate synthase), 9 isoprene units (nonaprenyl pyrophosphate synthase, 10 isoprene units (decaprenyl pyrophosphate synth
  • Suitable prenyltransferases include, but are not limited to, an is-isoprenyl diphosphate synthase, including, but not limited to, geranyl diphosphate (GPP) synthase, farnesyl diphosphate (FPP) synthase, geranylgeranyl diphosphate (GGPP) synthase, hexaprenyl diphosphate (HexPP) synthase, heptaprenyl diphosphate (HepPP) synthase, octaprenyl (OPP) diphosphate synthase, solanesyl diphosphate (SPP) synthase, decaprenyl diphosphate (DPP) synthase, chicle synthase, and gutta-percha synthase; and a Z- isoprenyl diphosphate synthase, including, but not limited to, nonaprenyl diphosphate (NPP) synthase, unde
  • nucleotide sequences of a numerous prenyl transferases from a variety of species are known, and can be used or modified for use in generating a subject genetically modified host cell.
  • Nucleotide sequences encoding prenyl transferases are known in the art. See, e.g., Human farnesyl pyrophosphate synthetase mRNA (GenBank Accession No. J05262; Homo sapiens); farnesyl diphosphate synthetase (FPP) gene (GenBank Accession No.
  • J05091 Saccharomyces cerevisiae
  • isopentenyl diphosphate:dimethylallyl diphosphate isomerase gene J05090; Saccharomyces cerevisiae
  • Wang and Ohnuma (2000) Biochim. Biophys. Acta 1529:33-48; U.S. Patent No. 6,645,747; Arabidopsis thaliana farnesyl pyrophosphate synthetase 2 (FPS2) / FPP synthetase 2 / famesyl diphosphate synthase 2 (At4gl7190) mRNA (GenBank Accession No.
  • NM_202836 Ginkgo biloba geranylgeranyl diphosphate synthase (ggpps) mRNA (GenBank Accession No. AY371321); Arabidopsis thaliana geranylgeranyl pyrophosphate synthase (GGPSl) / GGPP synthetase / farnesyltranstransferase (At4g36810) mRNA (GenBank Accession No.
  • a nucleic acid comprising a nucleotide sequence encoding any known terpene synthase can be used.
  • Suitable terpene synthases include, but are not limited to, amorpha-4,11-diene synthase (ADS), beta-caryophyllene synthase, germacrene A synthase, 8-epicedrol synthase, valencene synthase, (+)- delta-cadinene synthase, germacrene C synthase, (E)-beta- farnesene synthase, Casbene synthase, vetispiradiene synthase, 5-epi-aristolochene synthase, Aristolchene synthase, beta-caryophyllene, alpha- humulene, (E,E)-al ⁇ ha-farnesene synthase, (-)-beta-pinene synthase, Gam
  • Nucleotide sequences encoding te ⁇ ene synthases are known in the art, and any known te ⁇ ene synthase-encoding nucleotide sequence can used to genetically modify a host cell.
  • any known te ⁇ ene synthase-encoding nucleotide sequence can be used to genetically modify a host cell.
  • the following te ⁇ ene synthase-encoding nucleotide sequences, followed by their GenBank accession numbers and the organisms in which they were identified are known and can be used: (-)-germacrene D synthase mRNA (AY438099; Populus balsamifera subsp.
  • E 1 E- alpha-famesene synthase mRNA (AY640154; Cucumis sativus); 1,8-cineole synthase mRNA (AY691947; Arabidopsis thaliana); te ⁇ ene synthase 5 (TPS5) mRNA (AY518314; Zea mays); te ⁇ ene synthase 4 (TPS4) mRNA (AY518312; Zea mays); myrcene/ocimene synthase (TPSlO) (At2g24210) mRNA (NM_127982; Arabidopsis thaliana); geraniol synthase (GES) mRNA (AY362553; Ocimum basilicum); pinene synthase mRNA (AY237645; Picea sitchensis); myrcene synthase le20 mRNA (AY195609; Anti
  • a subject nucleic acid comprises an expression construct, e.g., in some embodiments, a subject nucleic acid comprises a nucleotide sequence encoding at least one synthetic IGR, and nucleotide sequences encoding one or more enzymes in an IPP biosynthetic pathway, where the at least one synthetic IGR is 5 ' of a coding region for one of the enzymes, and can be disposed between two coding regions encoding two or more enzymes in the IPP biosynthetic pathway.
  • Suitable expression vectors include, but are not limited to, baculovirus vectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral vectors (e.g. viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, and the like), Pl -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as E. coli and yeast).
  • Suitable vectors include chromosomal, nonchromosomal and synthetic DNA sequences.
  • Suitable expression vectors are known to those of skill in the art, and many are commercially available.
  • the following vectors are provided by way of example; for bacterial host cells: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, lambda-ZAP vectors (Stratagene); pTrc99a, pKK223-3, pDR540, and pRTT2T (Pharmacia); for eukaryotic host cells: pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).
  • any other plasmid or other vector may be used so long as it is compatible with the host cell.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).
  • Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S. Patent Publication No.
  • apagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93; Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter (Harbome et al. (1992) MoI. Micro. 6:2805-2813), and the like ⁇ see, e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141 ; McKelvie et al. (2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.
  • sigma70 promoter e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, an spv promoter, and the like; a promoter derived from the pathogenicity island SPI-2 (see, e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al. (2002) Infect. Immun.
  • rpsM promoter see, e.g., Valdivia and Falkow (1996). MoI. Microbiol. 22:367-378
  • a tet promoter see, e.g., Hillen,W. and Wissmann,A. (1989) In Saenger.W. and Heinemann,U. (eds), Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162
  • SP6 promoter see, e.g., Melton et al. (1984) Nucl. Acids Res. 12:7035-7056; and the like.
  • Non-limiting examples of suitable eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.
  • Suitable promoters for expression in yeast include, but are not limited to, CYCl, HIS3, GALl, GALlO, ADHl, PGK, PHO5, GAPDH, ADCl, TRPl, URA3, LEU2, ENO, and TPl ; and, e.g., AOXl (e.g., for use in Pichia). Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector may also include appropriate sequences for amplifying expression.
  • the expression vectors include one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in prokaryotic host cells such as E. coli.
  • selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in prokaryotic host cells such as E. coli.
  • an expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli, the S. cerevisiae TRPl gene, etc.; and a promoter derived from a highly-expressed gene to direct transcription of the coding sequence.
  • Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ⁇ -factor, acid phosphatase, or heat shock proteins, among others.
  • PGK 3-phosphoglycerate kinase
  • ⁇ -factor acid phosphatase
  • heat shock proteins among others.
  • a nucleotide sequence encoding an IPP biosynthetic pathway enzyme is operably linked to an inducible promoter.
  • Inducible promoters are well known in the art. Suitable inducible promoters include, but are not limited to, the pL of bacteriophage ⁇ ; Plac; Ptrp; Ptac (Ptrp-lac hybrid promoter); an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter, e.g., a lacZ promoter; a tetracycline-inducible promoter; an arabinose inducible promoter, e.g., P BAD (see, e.g., Guzman et al.
  • a xylose-inducible promoter e.g., Pxyl (see, e.g., Kim et al. (1996) Gene 181:71-76); a GALl promoter; a tryptophan promoter; a lac promoter; an alcohol-inducible promoter, e.g., a methanol-inducible promoter, an ethanol-inducible promoter; a raffinose-inducible promoter; a heat-inducible promoter, e.g., heat inducible lambda P L promoter, a promoter controlled by a heat-sensitive repressor (e.g., CI857-repressed lambda-based expression vectors; see, e.g., Hoffmann et al. (1999) FEMS Microbiol Lett. 177(2):327-34); and the like.
  • a heat-sensitive repressor e.g., CI857-repressed lambda-
  • a nucleotide sequence encoding an IPP biosynthetic pathway enzyme is operably linked to a constitutive promoter.
  • Suitable constitutive promoters for use in prokaryotic cells include, but are not limited to, a sigma70 promoter, e.g., a consensus sigma70 promoter.
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • Current Protocols in Molecular Biology Vol. 2, 1988, Ed. Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant, et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol. II, ERL Press, Wash., D.C., Ch.
  • yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. DM Glover, 1986, ERL Press, Wash., D.C.).
  • vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • a subject nucleic acid comprises nucleotide sequences encoding two or more EPP biosynthetic pathway enzymes, where the nucleotide sequences encoding the two or more enzymes will in some embodiments each be contained on separate expression vectors. In other embodiments, nucleotide sequences encoding one or more EPP biosynthetic pathway enzymes are contained in a single expression vector.
  • nucleotide sequences encoding one or more EPP biosynthetic pathway enzymes are contained in a single expression vector
  • the nucleotide sequences will be operably linked to a common control element (e.g., a promoter), e.g., the common control element controls expression of all of the BPP biosynthetic pathway enzyme-encoding nucleotide sequences on the single expression vector.
  • a common control element e.g., a promoter
  • nucleotide sequences encoding the IPP biosynthetic pathway enzyme(s) are contained in a single expression vector, in some embodiments, the nucleotide sequences will be operably linked to different control elements (e.g., a promoters), e.g., the different control elements control expression of each of the IPP biosynthetic pathway enzyme-encoding nucleotide sequences separately on a single expression vector.
  • control elements e.g., a promoters
  • the present invention provides genetically modified host cells; and compositions comprising the genetically modified host cells.
  • the genetically modified host cells are useful for producing an isoprenoid compound or an isoprenoid precursor compound, as discussed above.
  • a subject method for producing an isoprenoid or isoprenoid precursor generally involves culturing a genetically modified host cell in a suitable medium.
  • the genetically modified host cell is one that has been genetically modified with one or more heterologous nucleic acids comprising nucleotide sequence(s) encoding one or more synthetic IGRs, where the one or more synthetic IGRs are each disposed between two coding regions comprising nucleotide sequences encoding IPP biosynthetic enzymes.
  • the genetically modified host cell is one that has been genetically modified with one or more heterologous nucleic acids comprising nucleotide sequencers) encoding one or more synthetic IGRs, and two or more coding regions comprising nucleotide sequences encoding enzymes in an B?P biosynthetic pathway, where the one or more synthetic IGRs are each disposed between two of the two or more coding regions.
  • a subject genetically modified host cell comprises a subject nucleic acid, e.g., is genetically modified with a subject nucleic acid.
  • a host cell that produces an isoprenoid or isoprenoid precursor is genetically modified such that it produces the isoprenoid or isoprenoid precursor via an IGR-modified BPP biosynthetic pathway.
  • the genetically modified host cell is a genetically modified version of a parent host cell.
  • the IGR-modified IPP biosynthetic pathway is in some embodiments substantially the same as an endogenous pathway but for the inclusion of the one or more synthetic IGRs, e.g., the IGR-modified DPP biosynthetic pathway comprises nucleotide sequence encoding enzymes that are endogenous to the host cell.
  • the host cell is a prokaryotic cell that normally produces LPP via an endogenous DXP pathway
  • the IGR-modified BPP biosynthetic pathway comprises the endogenous DXP pathway, modified to include one or more synthetic IGRs.
  • the host cell is a eukaryotic cell (e.g., a yeast cell) that normally produces IPP via an endogenous mevalonate pathway
  • the IGR-modified IPP biosynthetic pathway comprises the endogenous mevalonate pathway, modified to include one or more synthetic IGRs.
  • the IGR-modified BPP biosynthetic pathway comprises both synthetic
  • the host cell is a prokaryotic cell that does not normally synthesize IPP via a mevalonate pathway; and the host cell is genetically modified with an IGR-modified mevalonate pathway that includes one or more synthetic IGRs and nucleotide sequences encoding mevalonate pathway enzymes heterologous to the host cell.
  • the host cell is a eukaryotic cell (e.g., a yeast cell) that does not normally synthesize IPP via a DXP pathway; and the host cell is genetically modified with an IGR-modified DXP pathway that includes one or more synthetic IGRs and nucleotide sequences encoding DXP pathway enzymes heterologous to the host cell.
  • a eukaryotic cell e.g., a yeast cell
  • IGR-modified DXP pathway that includes one or more synthetic IGRs and nucleotide sequences encoding DXP pathway enzymes heterologous to the host cell.
  • a subject nucleic acid is introduced stably or transiently into a parent host cell, using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, DEAE-dextran mediated transfection, liposome- mediated transfection, and the like.
  • a nucleic acid will generally further include a selectable marker, e.g., any of several well-known selectable markers such as neomycin resistance, ampicillin resistance, tetracycline resistance, chloramphenicol resistance, kanamycin resistance, and the like.
  • Host cells can be unicellular organisms, or are grown in culture as single cells.
  • the host cell is a eukaryotic cell.
  • Suitable eukaryotic host cells include, but are not limited to, yeast cells, insect cells, plant cells, fungal cells, and algal cells.
  • Suitable eukaryotic host cells include, but are not limited to, Pichia pastoris, Pichia ⁇ nlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramine
  • the host cell is a prokaryotic cell.
  • Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus sp., Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al. (1992) 7. Immunol. 148:1176-1181; U.S. Patent No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302.
  • Salmonella strains which can be employed in the present invention include, but are not limited to, Salmonella typhi and S. typhimurium.
  • Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnet, and Shigella disenteriae.
  • the laboratory strain is one that is non-pathogenic.
  • suitable bacteria include, but are not limited to, Bacillus subtilis, Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and the like.
  • the host cell is Escherichia coli.
  • the present invention provides a method of producing an isoprenoid compound or an isoprenoid precursor compound.
  • the methods generally involve culturing a genetically modified host cell in a suitable medium.
  • the genetically modified host cell comprises an IGR-modified IPP biosynthetic pathway comprising at least one synthetic intergenic region (IGR) that comprises a nucleotide sequence that forms a hairpin structure; and the at least one synthetic IGR is disposed between a set of two coding regions encoding two enzymes in the biosynthetic pathway.
  • the methods involve culturing a subject genetically modified host cell in a suitable medium.
  • the genetically modified host cell produces the isoprenoid or isoprenoid precursor in a recoverable amount.
  • the methods further involve recovering the isoprenoid or isoprenoid from the genetically modified host cell, from the culture medium, or both the genetically modified host cell and the culture medium.
  • a subject genetically modified host cell is cultured in a suitable medium
  • the culture medium is overlaid with an organic solvent, e.g. dodecane, forming an organic layer.
  • an organic solvent e.g. dodecane
  • an inducer is added to the culture medium; and, after a suitable time, the isoprenoid compound is isolated from the organic layer overlaid on the culture medium.
  • the isoprenoid compound will be separated from other products which may be present in the organic layer. Separation of the isoprenoid compound from other products that may be present in the organic layer is readily achieved using, e.g., standard chromatographic techniques.
  • an isoprenoid compound synthesized by a subject method is further chemically modified in a cell-free reaction.
  • artemisinic acid is isolated from culture medium and/or a cell lysate, and the artemisinic acid is further chemically modified in a cell-free reaction to generate artemisinin.
  • the isoprenoid compound is pure, e.g., at least about 40% pure, at least about 50% pure, at least about 60% pure, at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98%, or more than 98% pure, where "pure" in the context of an isoprenoid compound refers to an isoprenoid compound that is free from other isoprenoid compounds, macromolecules, contaminants, etc.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base ⁇ air(s); nt, nucleotide(s); i.m., intramuscularQy); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like.
  • Example 1 Combinatorial engineering of intergenic regions of operons MATERIALS AND METHODS
  • Oligonucleotides are depicted in Table 1 of U.S. Provisional Patent Application No. 60/819,706; and features of the oligonucleotides are depicted in Table 1 , below. The strains and plasmids used in this study are listed in Table 2, below. Table 1
  • A7 RNase III site 1 AATGTAAGCCCTCTCAGACATCTGCATAGTCTG (SEQ ID NO:27)
  • D6 match C6 TGCATGTCTTGAGCGGATAAGG (SEQ ID NO: 57) GGATACAGTATCTGCGGTACCCTAGATGCGTTCCGAG
  • DPS DHlOB, ispA ispA : : PLAC-(MK, PMK, MPD, idi, ispA) ; This work ispA, ⁇ ispC; E. coli Strain auxotrophic for mevalonate p701g pBad24 based reporter plasmid (amp r , pBR origin) (Smolke carrying lacZ and gfpuy. and
  • Keasling 2002 pBadRFP ⁇ c pBad24 based reporter plasmid (amp r , pBR origin) This work carrying E. coli optimized DsRed. p70rg p701g with rfp EC replacing lacZ. This work p70rg 1-15 p70rg with various IGR sequences This work p70gr p70rg with rfp E c and gfpuv in reverse order This work pGEM-4Z In-vitro transcript cloning vector carrying SP6 and T7 Promega promoter. pCSOl pGEM-4Z with gfp (Smolke, Carrier et al.
  • pBAD24MevT (SEQ ID NO: 1); pBAD33MevT (SEQ ID NO:2); pMevT (SEQ ID NO:3); pMBIS (SEQ ID NO:4); pADS (SEQ ED NO:5); pAtoB (SEQ ID NO:6); pHMGS (SEQ ED NO:7); pHMGR (SEQ ID NO:8; pBAD18HMGR (SEQ DD NO:9); pHMGSR (SEQ ID NO: 10); pMevT(C159A), also referred to as pBAD33MevT(C159A) (SEQ ID NO:11); pHMGS(C159A) (SEQ ID NO:12); and tHMGR (SEQ ID NO: 13).
  • TIGR libraries Assembly of TIGR libraries. Cloning of the initial reporter vectors and mevalonate pathway constructs are described in the supplementary material. TIGRs were synthesized using PCR to assemble oligonucleotides into chimeric DNA sequences. Four-hundred (400) picomoles of an equimolar oligonucleotide mixture were added to a mixture containing 2.5 units of AmpliTaq Gold® polymerase (Applied Biosystems, Foster City, CA). The assembly was conducted over 35 rounds of 15 sec at 95°C, 30 sec at 72°C, and 20 + 5 seconds/cycle at 72°C.
  • the resulting assembly products were purified with a nucleotide removal column (Qiagen) and amplified using end specific primers containing BgHl and Asp! 18 restriction sites.
  • the amplified libraries were subcloned into p70rg, the ligation products were electroporated into E. coli DHlOB, and the resulting transformants were plated on LB agar with carbenecillin.
  • the OD 6O o arid GFP and DsRed fluorescence were measured using a Tecan Safire (Maennedorf, Switzerland) plate reader. GFP and DsRed were measured at excitation / emission wavelengths of 400nm / 510nm and 558 run / 583 nm, respectively. Each fluorescence value was normalized to the number of cells by dividing by the OD 600 .
  • RNA methods Messenger RNA analysis was performed by dot-blot hybridization, Northern blot hybridization, and real-time PCR. Details for these methods are found in the supplementary materials. Briefly, total RNA was isolated using a RiboPureTM-Bacteria kit (Ambion, Austin, TX) and quantified on a Bioanalyzer Total RNA Nanochip (Agilent Technologies, Palo Alto, CA). Dot and Northern blots were generated according to standard protocols 29 . The construction of probe templates is described in the supplementary materials.
  • Probes were synthesized by in vitro transcription from these gel extracted templates with SP6 RNA polymerase (Promega) in the presence of [ 32 P]-labeled ⁇ - CTP (PerkinElmer, Wellesley, MA) and unlabeled nucleotide triphosphates (Promega) according to the manufacturer's instructions. All probes are specific for their own genes and did not generate any cross reactivity to the other genes.
  • Biosensor screening of mevalonate producing libraries Colonies containing functional operons were transferred into 96-well plates and grown overnight in C-medium with chloramphenicol and 0.1 % glucose. Cultures were back-diluted 1 :100 into fresh C-medium with chloramphenicol and 0.2 % arabinose. After 24 h, the cells were pelleted and the spent media collected. A culture of the biosensor cells was grown overnight in C-medium with 50 ⁇ g/mL kanamycin and 1 mM mevalonate and back-diluted to an OD ⁇ oo of 0.02.
  • One-hundred ninety (190) ⁇ L of this culture was combined with either 10 ⁇ L of the spent media or 10 ⁇ L of a 1:10 dilution of the spent media in separate screening plates.
  • Two wells per plate were run in triplicate as internal controls. Mevalonate controls between 100 ⁇ M to 2 mM and the highest mevalonate producer were run on each plate.
  • the biosensor plates were grown for 48 h, during which the GFP fluorescence was periodically measured using a Typhoon laser scanner. Samples were clustered into groups based on their relative fluorescence compared to the best mevalonate producer and average standard deviation from the triplicate wells. The mevalonate producing cells corresponding to the highest fluorescent biosensor wells were subjected to further analysis.
  • MevT expression analysis ⁇ ii ⁇ i mevalonate producing library members were assayed for cell growth, mRNA levels, enzyme activity, intracellular acyl-coenzyme A levels, and mevalonate concentrations.
  • Cultures were inoculated to an OO 600 of 0.016 from glucose-repressed overnight cultures, grown to an OD ⁇ oo of 0.05, and induced with 0.2% arabinose.
  • Dot blots and Northern blots were prepared in triplicate from total RNA isolated at an OD 60O of 0.4. Blots were probed with appropriate radiolabeled probes generated as described in the supplementary material.
  • tHMGR protein levels were assayed enzymatically by monitoring the disappearance of NADPH (tHMGR cofactor) by measuring the absorbance at 340 ran 30 .
  • Mevalonate levels were determined by GC-MS as described below.
  • Acyl-CoA levels were determined by LC-MS analysis of cell extracts described below.
  • TIGR libraries Construction of TIGR libraries.
  • the reporter vector, p70rg, used for TIGR screening was constructed by replacing the lacZ gene in p701g ! with the previously described rfp EC gene 2 . Transcription of this reporter operon was controlled using the arabinose-inducible promoter, P BAD - In addition, the operon incorporated a 126-nucleotide (nt) IGR containing a protective hairpin 3' o ⁇ rfp E c and the endoribonuclease (RNase E) site from the Pap operon 3 of E. coli as well as identical RBSs (ACGAGG) upstream of each coding region.
  • nt 126-nucleotide
  • RNase E endoribonuclease
  • the rfp ⁇ c gene was amplified using primers RG-Fwd and RG-Rev in a mixture containing PFU Turbo DNA polymerase (Stratagene, La Jolla, CA).
  • the PCR product and p701g were digested with JVAeI and BgIQ. and subsequently ligated together. Ligation mixtures were transformed by electroporation into E. coli DHlOB (Invitrogen) and resulting transformants were plated on LB agar with carbenecillin.
  • the reporter construct, p70gr with the genes in the reverse order was similarly constructed from p70gl" and a PCR product derived from primers, GR_Fwd, GR_Rev.
  • RNA probe template synthesis was constructed by gel extracting the
  • the rfp EC probe template was constructed by inserting the Ncol-EcoRl fragment of pDsRed-Express into pGEM-4Z (Promega).
  • the resulting plasmid, pBP3 2 was digested with BgH and the 874 bp fragment was isolated by gel extraction.
  • the probes for atoB, HMGS, and XHMGR were constructed by PCR using the following primer pairs: atoB- Apro_Fwd, Apro_Rev; HMGS- SproJFwd, Spro_Rev; XHMGR- Rpro_Fwd, RproJRev.
  • the amplified gene fragments were cloned into pGEM-4Z with either Aspl ⁇ 8 and Pstl or Xm ⁇ l and Pstl.
  • the resulting plasmids, pPAtoB, pPHMGS, and pPHMGR were digested with PvuU and Pstl.
  • the approximately 750 bp fragments were purified by gel extraction and used for in vitro transcription described in RNA methods.
  • RNA samples were transferred to the membrane and RNA was immobilized by baking at 80 0 C for 1 h. Specific transcript levels were measured by hybridizing appropriate radiolabeled probes. The probe intensity was quantified using a Typhoon phosphorimager (Amersham Biosciences, Piscataway, NJ).
  • Real-time PCR was performed in an ICycler (BioRad, Hercules, CA) monitoring double-stranded DNA assayed continuously with SYBR®-Green (Invitrogen). Dilutions of cDNA and linearized plasmid (standard) were amplified in a PCR with AmpliTaq Gold® by gene-specific primer sets (Table 1 of U.S. Provisional Patent Application No. 60/819,706). The Ct values were determined for each reaction and the quantity of mRNA was determined from a standard curve prepared from linearized plasmid. Replicate reactions of three dilutions of cDNA were run for each sample.
  • denaturing agent 3.5 ⁇ L formaldehyde, 2 ⁇ L 5X MOPS running buffer, 10 ⁇ L formamide
  • RNA was transferred to a Nytran SuPerCharge membrane using a turboblotter transfer system (Schleicher & Schuell) with 20* SSC as the transfer buffer. The RNA was subsequently crosslinked to the membrane in a UV crosslinker and transcripts were identified and quantified as described above.
  • the rfp ⁇ c gene was cloned initially as junk DNA to simplify the gel extraction of cut vector prior to library cloning.
  • the rfp ⁇ c gene also served as a negative control for ligations of libraries, similar to traditional blue-white screening.
  • Mevalonate (mevalonic acid) concentration in cultures of engineered E. coli was determined by GC-MS analysis. 560 ⁇ L of E. coli culture was mixed with 140 ⁇ L of 500 mM HCl in a glass GC vial to convert mevalonate from mevalonic acid to mevalonic acid lactone. 700 ⁇ L of ethyl acetate, spiked with 50 ⁇ g/mL (-)-trans-caryophyllene as an internal standard, was added to each vial and then the samples were shaken at maximum speed on a Fisher Vortex Genie 2 mixer (Fischer Scientific) for three minutes.
  • the ethyl acetate extract of acidified culture was diluted 1 :10 with fresh ethyl acetate in a clean GC vial before analysis.Diluted ethyl acetate extracts were analyzed using an Agilent Technologies 6890 gas chromatograph with an Agilent Technologies model 5973 mass selective detector (GC-MS) operating in electron impact mode.
  • the GC column used was an Agilent Technologies DB-5ms (30 m x 250 ⁇ m x 0.25 ⁇ m).
  • Helium was used as the carrier gas at a constant flow of 1 rnL/min and 1 ⁇ L injections split 1:5 were preformed.
  • the injection port was maintained at 250 0 C, the MS source temperature was maintained at 230 0 C, and the MS quad temperature was held constant at 150 0 C.
  • the oven cycle for each sample and the ions monitored were modified from the method of B. H. Woollen et al. 7 .
  • the column temperature profile was 70 0 C for 2 minutes; 15°C/min to 185°C; 30°C/min to 300 0 C; and held at 300 0 C for 3 minutes.
  • the selected ions monitored were m/z 71 and 58 for mevalonic acid lactone, and m/z 189 and 204 for (-)-trans- caryophyllene.
  • Total mevalonate production was defined as the concentration of mevalonate in a culture sample.
  • Specific mevalonate production was defined as the total mevalonate concentration divided by the cell density (OD 60 o)-
  • LC-MS Analysis of Acyl-coenzyme A intermediates The concentrations of free coenzyme A, acetyl-CoA, acetoacetyl-CoA, and 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) were determined by LC-MS analysis of Trichloroacetic acid (TCA) culture extracts taken during the exponential phase of growth. To simultaneously and rapidly quench cellular metabolism, isolate E. coli cells from growth media and extract metabolites, cells were centrifuged through a layer of silicone oil into a denser solution of TCA by method similar to that of M. Shimazu et al 8 .
  • TCA Trichloroacetic acid
  • the neutralized TCA extract was analyzed using a Hewlett-Packard 1100 series LC-MS using electrospray ionization.
  • a 50 ⁇ L sample was separated on a C-18 reversed phase HPLC column (250 x 2.1 mm Inertsil 3 urn ODS-3 by Varian) using a two solvent gradient system adapted from J.J. Dalluge et al 9 .
  • Solvent A was 100 mM Ammonium Acetate buffer at pH 6 and Solvent B was 70% Solvent A and 30% Acetonitrile.
  • the HPLC column was equilibrated each run with 8% Solvent B (92% Solvent A) for 10 minutes.
  • the eluent program was the following: 8% Solvent B at 0 min to 50% Solvent B at 5 min, gradient increase to 100% Solvent B at 13 min, isocratic at 100% Solvent B until 19 min, gradient returning to 8% Solvent B at 24 min.
  • the resolved metabolite samples were analyzed by electrospray ionization mass selective detector (ESI-MS) operated in positive mode.
  • ESI-MS electrospray ionization mass selective detector
  • TIGRs Tunable InterGenic Regions
  • the synthesis of natural or unnatural products in microorganisms usually involves the introduction of several genes encoding the enzymes of a metabolic pathway 1 ' 2 .
  • the genes need to be expressed in a coordinated fashion at appropriately balanced levels to avoid bottlenecks in the biosynthetic pathway that result in suboptimal yields and can lead to the accumulation of potentially toxic intermediates.
  • the introduction into a cell or manipulation of multi-subunit proteins usually involves coordinated expression of several genes to produce the subunits at the appropriate stoichiometrics 3 .
  • IGR intergenic region
  • Fig. Ia An operon reporter system (Fig. Ia) containing the genes encoding the red fluorescent protein DsRed (rfp ⁇ c) 20 ' 2I and the green fluorescent protein GFP igfp ⁇ v) was designed to facilitate high-throughput measurement of relative gene expression resulting from the libraries of TIGRs.
  • a large library of TIGR sequences (>10 4 ) was assembled combinatorially from four sets of oligonucleotides (Table 1 of U.S. Provisional Patent Application No. 60/819,706) using polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Each oligonucleotide contained two 15-nt sequences that hybridized to a corresponding sequence in the neighboring oligonucleotide, such that a series of chimeric DNA molecules containing oligonucleotides from each of the four sets was created after several rounds of PCR (Fig. Ib). Between the hybridization sequences at either end of each oligonucleotide was a variable sequence that provided the diversity of features designed into the library.
  • PCR amplification of this DNA pool with end-specific oligonucleotides enriched the population with full-length TIGRs containing a member from each set of oligonucleotides (Fig. Ib).
  • Specific restriction sites incorporated into the amplification primers were used to clone the TIGR library between the two reporter genes.
  • FIGS. IA-E Figures IA-E. TIGR assembly and reporter operon.
  • the reporter plasmid p70rg consists of the reporter genes rfp EC and gfp ⁇ v downstream of the P BAD promoter. The black scissors indicate the location of the cloning site used to insert the library of TIGRs.
  • (b) Layout of the TIGR assembly reaction. Members of each region (A-D) anneal to members of neighboring regions and are extended by PCR. Eventually full-length TIGRs containing members of each region are assembled and then amplified using end-specific primers containing the restriction sites for cloning,
  • the oligos used for TIGR construction were designed to make three separate regions.
  • A-B form 5' hairpins
  • B-C form the single-stranded region with RNase E sites
  • C-D form a 3' hairpin
  • TIGR sequence is designed to be processed at a cleavage site between two secondary structures. Cleavage results in two independent secondary transcripts whose stability is determined by the remaining TIGR sequence.
  • Table 3 provides Sequences of TIGRs from library samples. Below are the sequences and predicted secondary structures of the characterized samples from the fluorescent reporter library and the mevalonate library. They are written from the stop codon of the upstream (5') gene through the start codon of the downstream (3') gene.
  • the TIGR pool that resulted from the assembly of the oligonucleotides was designed to contain three regions, two variable hairpin sequences flanking a single-stranded region incorporating various RNase E sites 22 ' 23 (Fig. Ic and Id). When transcribed, those TIGR sequences that contained a strong endonuclease site would be cleaved generating two secondary transcripts whose stability could be individually modulated by the secondary structures flanking the RNase site 17 (Fig. Ie).
  • the TIGR sequences also incorporated mRNA secondary structures of various lengths, GC contents, asymmetries, and mismatched bulges.
  • Cells harboring the reporter libraries produced a wide range of fluorescence phenotypes.
  • cells were collected and sorted via FACS to isolate highly fluorescent cells. Cultures of the sorted cells were grown in 96-well plates to determine the fluorescence of each construct after 24 hours.
  • the relative fluorescence ratio, red to green varied by two orders-of- magnitude, from 45:1 DsRed:GFP to 3:1 GFP:DsRed (Fig. 2a), depending on the IGR.
  • the distributions of relative mRNA levels and fluorescence ratios were not identical indicating that TIGRs may also affect translation (Fig. 2b and c).
  • the TIGR sequences had a stronger influence on the expression of the gene 3' to the TIGR than on the gene 5' to the TIGR.
  • Figures 2A-C Expression from TIGR RG library. Colonies of fluorescent strains were imaged using a laser scanner detecting fluorescence at 526 nm and 580 nm. The image was an overlay of the two signals, (a) DsRed:GFP fluorescence ratios of the library after 24 hours of growth, (b) Fifteen clones were assayed for fluorescence during exponential growth. Shown are DsRed:GFP fluorescence ratios normalized to the fluorescence of p70rg. (c) The ratios o ⁇ rfp ⁇ c and gfp mRNA from exponentially growing cells were determined by real-time PCR and dot blot hybridization.
  • rfp E c'gfp mRNA ratios determined by real-time PCR. Note the similarity in the distributions of the two samples, indicating that the TIGR is altering mRNA levels which in turn alter fluorescence ratios.
  • RG refers to the original operon vector p70RG described in the text.
  • Cleavage in the TIGR would result in two independent secondary transcripts whose stability and ultimately the amount of protein produced from them would be dictated by the remaining TIGR sequences at the 3'- and 5'-ends of the separated transcripts. Differences in the intensities of the primary and secondary transcripts support differential transcript stability (Fig.3b and 3c).
  • Northern Blots revealed a large quantity of stable rfp ⁇ c transcript corresponding to the size of a single gene (Fig. 3f) and very little gfp transcript, either full-length or containing gfp alone (Fig. 3g).
  • the strong hairpins in this TIGR can serve two functions, premature transcription termination and protection from exoribonucleases 25 resulting in an increased ratio of the first gene product to the second gene product (red:green).
  • Ribosome binding site sequestration resulted in a number of samples showing a significant difference between the relative mRNA and protein levels (Fig. 2b and c).
  • the gfp RBS was part of a secondary structure, such that cis basepairing of the RBS may have prevented the ribosome from loading onto the transcript.
  • Secondary structures incorporating the RBS have been previously shown to reduce the rate of translation initiation 14"16 ' 26 .
  • the structure of sample 3 which best illustrated this mechanism (Fig. 3h) consisted of four hairpins, the last of which incorporated the RBS at the base of its stem (Fig. 3i).
  • TIGR samples Cleavage of the TIGR generates two secondary transcripts, (b) Northern blot of total RNA from exponentially growing cultures of numbered clones probed for rfp E c- (c) Northern blots of the same RNA in (b) probed for gfp. (d) Premature transcription termination mechanism of sample 1.
  • the large IGR structure prevents transcription of the gfp gene,
  • the predicted secondary structure of sample 1 's TIGR contains two large hairpins, (f) rfp ⁇ c probed Northern blot of total RNA harvested at various timepoints (bottom) after transcription was stopped by adding rifampicin.
  • TIGR libraries were used to generate a series of operons that were subsequently screened for increased mevalonate production.
  • a megaprimer PCR approach 27 was used to simultaneously place TIGR libraries between the first and second genes and between the second and third genes of the MevT operon (Fig.4c).
  • Functional operons from the libraries were selected by transforming the plasmid library into an E. coli strain engineered to be auxotrophic for mevalonate 28 .
  • the plasmids isolated from this enriched pool were recovered and subsequently transformed into a production strain (DHlOB) and screened for the highest-producing version using a mevalonate auxotroph transformed with a plasmid harboring a constitutively-expressed gfp as a biosensor for mevalonate 28 .
  • This fluorescence-based screen incorporates a GFP-producing sensor strain whose growth is dependent on the level of mevalonate secreted into the surrounding medium by the producer strain.
  • 18% were significantly (more than three standard deviations greater, calculated from two sets of triplicate controls on each plate) more fluorescent than the original operon (control without TIGRs).
  • Figures 4A-F Mevalonate pathway optimization using the TIGR method, (a) Biosyntheic pathway of heterologous artemisinin production. Pink box highlights the yeast mevalonate pathway converting Acetyl-CoA (AcCoA) to the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through mevalonate (MEV).
  • AcCoA Acetyl-CoA
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • Dashed box, blown up in (b), represents the first three genes in the mevalonate pathway, atoB (acetoacetyl-CoA thiolase), HMGS, (HMG-CoA synthase), and tHMGR (truncated HMG-CoA reductase). These three genes have been clustered into one operon to make the vector pBad33MevT (c).
  • (d) Growth of the top four producers and the sampling points for induction (green), RNA (red), acyl-CoAs, and mevalonate assays (orange). The data for each of the sampling points is shown in Table 4.
  • acyl-CoAs in the pathway were not significantly changed among the strains expressing mevalonate pathway genes.
  • the data suggest that the improved production was due primarily to increased growth correlated with the availability of acetyl-CoA.
  • TIGR sequence altered the relative expression levels of the three genes.
  • the HMGS sn ⁇ tHMGR transcripts were noticeably reduced in the library strains in comparison to the transcript expressed from pBad33MevT (Table 4).
  • the tHMGR enzyme activity was significantly reduced in library sample A compared to pBad33MevT (Table 4). The reduction in expression was not expected, but suggests that reduced expression of HMGS and tHMGR was responsible for the improved growth and increased mevalonate production.

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Abstract

La présente invention concerne des procédés de production d'un isoprénoïde ou d'un précurseur d'isoprénoïde dans une cellule hôte. Les procédés de production comportent une voie biosynthétique qui convertit un substrat en pyrophosphate d'isopentényle, la voie biosynthétique étant modifiée pour présenter une zone intergénique synthétique (IGR) entre au moins deux zones codantes codant pour des enzymes dans la voie biosynthétique. La présente invention concerne en outre des produits de construction d'acide nucléique recombinant, comprenant un IGR synthétique et des cellules hôtes génétiquement modifiées incluant un IGR synthétique.
PCT/US2007/015498 2006-07-07 2007-07-03 Procédés permettant d'améliorer la production de composés isoprénoïdes par des cellules hôtes Ceased WO2008008256A2 (fr)

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