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WO2009005704A1 - Procédés d'augmentation de la production d'isoprénoïde ou de précurseur d'isoprénoïde - Google Patents

Procédés d'augmentation de la production d'isoprénoïde ou de précurseur d'isoprénoïde Download PDF

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Publication number
WO2009005704A1
WO2009005704A1 PCT/US2008/007990 US2008007990W WO2009005704A1 WO 2009005704 A1 WO2009005704 A1 WO 2009005704A1 US 2008007990 W US2008007990 W US 2008007990W WO 2009005704 A1 WO2009005704 A1 WO 2009005704A1
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isoprenoid
cells
pathway
heterologous
culture medium
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Jeffery Lance Kizer
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
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
    • 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes

Definitions

  • Microbes have been designed to efficiently produce a wide range of industrially relevant compounds via heterologous pathway engineering including isoprenoids, bioplastics, polyketides, amino acids, and polymer precursors. It can be exceedingly difficult, in such complexly engineered systems, to predict what aspect of heterologous host metabolism limits final product titers.
  • the unregulated consumption of cellular resources, metabolic burden of heterologous protein production or the accumulation of pathway intermediates/products that are inhibitory or toxic to the heterologous host are all significant issues that arise from novel pathway engineering.
  • the present invention provides methods of producing an isoprenoid or isoprenoid precursor in an isoprenoid-producing host cell, generally involving culturing isoprenoid-producing cells in a defined culture medium that includes serine.
  • Figure 1 is a schematic depiction of mevalonate and 1 -deoxy-D-xylulose 5-diphosphate (DXP) pathways.
  • Figure 2 is a schematic depiction of synthesis of various isoprenoid compounds from the precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate.
  • Figure 3 is a schematic depiction of serine, glycine and single-carbon unit pathways.
  • Figure 4 depicts a heterologous amorpha-4-1 1-diene production pathway.
  • Figure 5 depicts specific amorpha-4-1 1-diene production (mg/L-OD) and total amorpha-4-11- diene production in various media formulations.
  • isoprenoid isoprenoid compound
  • terpene compound terpene compound
  • terpenoid compound refers to any compound that is capable of being derived from isopentenyl pyrophosphate (IPP).
  • IPP isopentenyl pyrophosphate
  • the number of C-atoms present in the isoprenoids is typically evenly divisible by five (e.g., C5, ClO, C15, C20, C25, C30 and C40).
  • Isoprenoid compounds include, but are not limited to, hemiterpenes, monoterpenes, diterpenes, triterpenes, sesquiterpenes, and polyterpenes.
  • Isoprenoid compounds include, e.g., C 5 -Ci 0 isoprenoids, C 5 -C] 5 isoprenoids, C 5 -C 2 O isoprenoids, C 1 0-C 2 0 isoprenoids, CiO-C 25 isoprenoids, Ci 0 -C 30 isoprenoids, Ci 0 -C 40 isoprenoids, and the like.
  • 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 (BPP) and its isomer dimethylallyl pyrophosphate (DMAPP).
  • pene synthase refers to any enzyme that enzymatically modifies
  • IPP IPP
  • DMAPP dimethyl methacrylate
  • polyprenyl pyrophosphate such that a terpene or a terpenoid precursor compound is produced.
  • pyrophosphate is used interchangeably herein with “diphosphate.”
  • prenyl diphosphate and “prenyl pyrophosphate” are interchangeable;
  • isopentenyl pyrophosphate” and “isopentenyl diphosphate” are interchangeable;
  • mevalonate pathway or "MEV pathway” is used herein to refer to the biosynthetic pathway that converts acetyl-coenzyme A (acetyl-CoA) to D?P.
  • the mevalonate pathway comprises enzymes that catalyze the following steps: (a) condensing two molecules of acetyl-CoA to acetoacetyl- CoA; (b) condensing acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-CoA (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.
  • the mevalonate pathway is illustrated schematically in Figure 1. The "
  • DXP pathway 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 1.
  • 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.
  • standard amino acid refers to the twenty encoded amino acids including phenylalanine, leucine, isoleucine, methionine, valine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartic acid, glutamic acid, cysteine, tryptophan, arginine, serine, and glycine.
  • 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.
  • 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 (e.g., the nucleic acid encodes a polypeptide that is heterologous to the host cell, e.g., the encoded polypeptide is not normally produced by the 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
  • 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.
  • 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.
  • 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 can be 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 prokaryotic host cell is a genetically modified prokaryotic host cell (e.g., a bacterium), 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 eukaryotic host cell is a genetically modified eukaryotic host cell, 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 suitable prokaryotic host cell e.g., a bacterium
  • a heterologous nucleic acid
  • a host cell includes a plurality of host cells and reference to “the culture medium” includes reference to one or more culture media and equivalents thereof known to those skilled in the art, and so forth.
  • the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
  • the present invention provides methods of producing an isoprenoid or isoprenoid precursor in vitro in a cell that is capable of producing the isoprenoid or isoprenoid compound, generally involving culturing the cell in a defined culture medium.
  • the defined culture medium includes at least one supplement (e.g., an amino acid) that feeds into and increases single-carbon metabolism in the isoprenoid-producing cell, e.g., where the amino acid provides a single-carbon unit, such as a methyl group, that is essential to a metabolic pathway in the cell.
  • Isoprenoid compounds are synthesized from a universal five carbon precursor, isopentenyl pyrophosphate (IPP). IPP is synthesized via two different pathways: the mevalonate (MEV) pathway and the l-deoxyxylulose-5-phosphate (DXP) or non-mevalonate pathway.
  • MEV mevalonate
  • DXP l-deoxyxylulose-5-phosphate
  • the MEV pathway and the DXP pathway are depicted schematically in Figure 1.
  • 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.
  • the DXP pathway produces BPP and DMAPP from pyruvate and glyceraldehyde-3 -phosphate, as depicted schematically in Figure 1.
  • the pathway begins with the formation of l-deoxy-D-xylulose-5- phosphate (DXP) from pyruvate and glyceraldehyde-3 -phosphate by DXP synthase (Dxs).
  • DXP is then isomerized and reduced to 2-C-methyl-D-erythritol-4-phosphate (MEP), the first committed step of the non-mevalonate pathway, by DXP reductoisomerase (IspC or Dxr).
  • DXP reductoisomerase IspC or Dxr
  • a cytidylic acid moiety is added to MEP by the action 2-C-methylerythritol-4-phosphate cytidyltransferase (IspD) to produce 4-diphosphocytidyl-2C-methyl-D-erythritol.
  • IspD 2-C-methylerythritol-4-phosphate cytidyltransferase
  • 4-diphosphocytidyl-2C-methyl-D-erythritol is then phosphorylated by 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE) and further converted to l-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate by the sequential action of 2-C-methylerythritol- 2,4-cyclodiphosphate synthase (IspF) and l-hydroxy-2-methyl-2-(E)-butenyI-4-diphosphate synthase (IspG).
  • the terminal enzyme of the DXP pathway in E. coli has recently been identified as the product of ispH (formerly lytB), and has been shown to convert l-hydroxy-2-methyl-2-(E)-butenyl 4- diphosphate to both IPP and DMAPP at a 5: 1 ratio.
  • DMAPP acts as a primer for the sequential additions of
  • IPP by the isoprenyl pyrophosphate synthases (also known as the prenyl transferases) to form C ]0 geranyl pyrophosphate (GPP), C )5 farnesyl pyrophosphate (FPP), C 2 o geranylgeranyl pyrophosphate (GGPP), and larger isoprenyl pyrophosphates.
  • the isoprenyl pyrophosphates are then cyclized by the terpene cyclases (synthases) to form the various terpene classes.
  • Carotenoids are synthesized by a series of enzymatic reactions beginning with the condensation of two GGPP molecules.
  • An isoprenoid compound (and/or an isoprenoid precursor such as mevalonate, IPP, a DXP pathway intermediate, and a polyprenyl diphosphate) can be produced in vitro in cells that are capable of producing the isoprenoid or isoprenoid precursor (also referred to herein as "isoprenoid-producing cells"). It has now been found that the composition of the culture medium in which the cells are cultured can be modified in specific ways to increase the amount of isoprenoid or isoprenoid precursor recoverable from the cells and/or the culture medium.
  • the present invention provides methods for producing an isoprenoid or an isoprenoid precursor, the methods generally involving culturing in vitro a plurality of isoprenoid-producing cells in a defined culture medium comprising at least one supplement that feeds into and increases single- carbon metabolism in the genetically modified host cell.
  • the cells have a biosynthetic pathway for converting a substrate(s) (e.g., acetoacetyl-CoA; glyceraldehyde-3 -phosphate and pyruvate; mevalonate) to EPP (also referred to herein as an "IPP biosynthetic pathway").
  • a substrate(s) e.g., acetoacetyl-CoA; glyceraldehyde-3 -phosphate and pyruvate; mevalonate
  • EPP also referred to herein as an "IPP biosynthetic pathway”
  • Illustrative examples include: 1) cells that have an endogenous EPP biosynthetic pathway (e.g., cells that have an endogenous mevalonate pathway, such as eukaryotic cells such as yeast cells, fungal cells, etc.; and cells that have an endogenous DXP pathway, e.g., a prokaryotic cell that normally produces EPP via a DXP pathway), and that synthesize the isoprenoid or isoprenoid precursor compound via the endogenous EPP biosynthetic pathway; 2) cells that have been genetically modified with one or more exogenous nucleic acids comprising nucleotide sequences encoding one or more heterologous EPP biosynthetic pathway enzymes (thus having an exogenous EPP biosynthetic pathway); and 3) cells that have a modified endogenous EPP biosynthetic pathway, e.g., where the endogenous pathway is genetically modified, where the cells synthesize the isoprenoid or isoprenoid
  • Culturing of the isoprenoid-producing cells in vitro in the defined culture medium provides for production of the isoprenoid or isoprenoid precursor by the cells in a recoverable amount.
  • the isoprenoid or isoprenoid precursor can then be recovered (e.g., isolated) from the cells, the culture medium, or both.
  • the present invention provides methods for producing an isoprenoid or an isoprenoid precursor.
  • the methods generally involve culturing a plurality of cells in vitro in a defined culture medium comprising a supplement (e.g., serine) that is a substrate for a single-carbon metabolic pathway.
  • the cells are capable of synthesizing the isoprenoid or isoprenoid precursor.
  • the cells synthesize the isoprenoid or isoprenoid precursor via an endogenous EPP biosynthetic pathway.
  • the cells synthesize the isoprenoid or isoprenoid precursor via an exogenous IPP biosynthetic pathway.
  • the cells synthesize the isoprenoid or isoprenoid precursor via a modified endogenous IPP biosynthetic pathway.
  • a cell that is capable of synthesizing an isoprenoid or isoprenoid precursor is cultured in a defined culture medium comprising a supplement (e.g., serine) that is a substrate for a single-carbon metabolic pathway, e.g., a metabolic pathway that requires one-carbon units (e.g., methyl groups).
  • a supplement e.g., serine
  • Such metabolic pathways include, e.g., metabolic pathways that depend on serine, e.g., where serine or a serine metabolite is a substrate for the pathway; and metabolic pathways that depend on methionine, e.g., where methionine is a substrate for the pathway.
  • serine is an important source of single carbon units in Escherichia coli during growth.
  • Serine is converted to glycine by action of serine hydroxymethyltransferase (SHMT, encoded by the glyA gene), in a reaction that transfers a methyl group to the Cl carrier molecule, tetrahydrofolate.
  • SHMT serine hydroxymethyltransferase
  • the conversion of serine to glycine provides the majority of single-carbon units required during growth.
  • methionine is a key precursor to the primary methyl- carrier cofactor in E. coli, S-adenosyl-methionine (SAM).
  • a subject method comprises culturing in vitro a cell that is capable of synthesizing an isoprenoid or isoprenoid precursor in a culture medium comprising: a) serine; and b) a subset of amino acids selected from the group consisting of alanine, glutamine, glutamic acid, isoleucine, leucine, valine, and methionine, where the subset consists of zero to seven amino acids, and where the medium does not comprise one or more amino acids that are not part of the subset.
  • the subset consists of zero amino acid.
  • the subset consists of one amino acid.
  • the subset consists of two amino acids.
  • the subset consists of three amino acids. In other embodiments, the subset consists of four amino acids. In other embodiments, the subset consists of five amino acids. In other embodiments, the subset consists of six amino acids. In still other embodiments, the subset consists of seven amino acids. In certain embodiments, the medium does not additionally comprise an amino acid (e.g., a standard amino acid) other than serine and the one or more amino acids that are in the subset.
  • an amino acid e.g., a standard amino acid
  • the culture medium comprises: a) serine; and b) a subset of amino acids consisting of glutamine, glutamic acid, isoleucine, leucine, valine, and methionine; where the culture medium does not comprise alanine.
  • the culture medium comprises: a) serine; and b) a subset of amino acids consisting of glutamic acid, isoleucine, leucine, valine, and methionine; where the culture medium does not comprise alanine and glutamine.
  • the culture medium comprises: a) serine; and b) a subset of amino acids consisting of isoleucine, leucine, valine, and methionine; where the culture medium does not comprise alanine, glutamine, and glutamic acid.
  • the culture medium comprises: a) serine; and b) a subset of amino acids consisting of leucine, valine, and methionine; where the culture medium does not comprise alanine, glutamine, glutamic acid, and isoleucine.
  • the culture medium comprises: a) serine; and b) a subset of amino acids consisting of alanine, glutamic acid, and glutamine; where the culture medium does not comprise isoleucine, leucine, valine, and methionine.
  • the culture medium comprises: a) serine; and b) a subset of amino acids consisting of valine and methionine; where the culture medium does not comprise alanine, glutamine, glutamic acid, isoleucine, and leucine.
  • the culture medium comprises: a) serine; and b) a subset of amino acids consisting of alanine and methionine; where the culture medium does not comprise glutamic acid, glutamine, isoleucine, leucine, and valine.
  • the culture medium comprises: a) serine; and b) methionine; and does not include any of alanine, glutamine, glutamic acid, isoleucine, leucine, and valine.
  • the culture medium comprises serine; does not include any of alanine, glutamine, glutamic acid, isoleucine, leucine, valine, and methionine.
  • the culture medium does not comprise one or more of the other standard amino acids selected from the group consisting of: arginine, cysteine, glycine, histidine, lysine, phenylalanine, proline, threonine, tryptophan, tyrosine, aspartic acid, and asparagine.
  • the defined culture medium does not comprise one or more of alanine, glutamine, glutamic acid, isoleucine, leucine, valine, arginine, cysteine, glycine, histidine, lysine, phenylalanine, proline, threonine, tryptophan, tyrosine, aspartic acid, and asparagine.
  • the defined culture medium comprises serine, alanine, glutamine, glutamic acid, isoleucine, leucine, methionine, and valine
  • the defined culture medium does not comprise one or more of arginine, cysteine, glycine, histidine, lysine, phenylalanine, proline, threonine, tryptophan, tyrosine, aspartic acid, and asparagine.
  • the defined culture medium does not additionally comprise an amino acid (e.g., a standard amino acid) other than serine and the one or more amino acids that are in the subset.
  • the defined culture medium does not comprise any of alanine, glutamine, glutamic acid, isoleucine, leucine, valine, arginine, cysteine, glycine, histidine, lysine, phenylalanine, proline, threonine, tryptophan, tyrosine, aspartic acid, and asparagine.
  • the defined culture medium comprises serine, alanine, glutamine, glutamic acid, isoleucine, leucine, methionine, and valine
  • the defined culture medium does not comprise any of arginine, cysteine, glycine, histidine, lysine, phenylalanine, proline, threonine, tryptophan, tyrosine, aspartic acid, and asparagine.
  • the culture medium further comprises a purine.
  • the purine is adenine.
  • the purine is guanine.
  • the purine is both adenine and guanine.
  • a subject method comprises culturing in vitro a cell that is capable of synthesizing an isoprenoid or isoprenoid precursor in a culture medium comprising: a) serine; and b) a subset of amino acids selected from the group consisting of glycine, alanine, glutamine, glutamic acid, isoleucine, leucine, valine, and methionine, where the subset consists of zero to eight amino acids, and where the medium does not comprise one or more amino acids that are not part of the subset.
  • the subset consists of zero amino acid.
  • the subset consists of one amino acid.
  • the subset consists of two amino acids.
  • the subset consists of three amino acids. In other embodiments, the subset consists of four amino acids. In other embodiments, the subset consists of five amino acids, hi other embodiments, the subset consists of six amino acids. In still other embodiments, the subset consists of seven amino acids. In still other embodiments, the subset consists of eight amino acids. In certain of these embodiments, the culture medium does not comprise one or more of the other standard amino acids selected from the group consisting of: arginine, cysteine, glycine, histidine, lysine, phenylalanine, proline, threonine, tryptophan, tyrosine, aspartic acid, and asparagine.
  • the defined culture medium comprises serine and a subset of amino acids, where the subset is glycine and methionine
  • the defined culture medium does not additionally comprise one or more of alanine, glutamine, glutamic acid, isoleucine, leucine, valine, arginine, cysteine, histidine, lysine, phenylalanine, proline, threonine, tryptophan, tyrosine, aspartic acid, and asparagine.
  • the defined culture medium does not additionally comprise an amino acid other than serine and the one or more amino acids that are in the subset.
  • the defined culture medium comprises serine and a subset of amino acids, where the subset is glycine and methionine
  • the defined culture medium does not additionally comprise an amino acid other than serine, glycine, and methionine, e.g., the defined culture medium does not comprise any of alanine, glutamine, glutamic acid, isoleucine, leucine, valine, arginine, cysteine, histidine, lysine, phenylalanine, proline, threonine, tryptophan, tyrosine, aspartic acid, and asparagine.
  • a subject method comprises culturing in vitro a cell that is capable of synthesizing an isoprenoid or isoprenoid precursor in a culture medium comprising: a) serine; and b) methionine and/or glycine; and c) a purine wherein the culture medium does not include any amino acid other than serine, and methionine and/or glycine.
  • the purine is adenine.
  • the purine is guanine.
  • the purine is both adenine and guanine.
  • a subject method comprises culturing in vitro a cell that is capable of synthesizing an isoprenoid or isoprenoid precursor in a culture medium comprising: a) serine; b) methionine; (c) glycine; and d) a purine wherein the culture medium does not include any amino acid other than serine, methionine, and glycine.
  • the purine is adenine.
  • the purine is guanine.
  • the purine is both adenine and guanine.
  • Serine can be present in the defined culture medium in a concentration range of from about 5 mM to about 20 mM, e.g., from about 5 mM to about 10 mM, from about 10 mM to about 15 mM, or from about 15 mM to about 20 mM; and in some embodiments is present in the defined culture medium at a concentration of 10 mM.
  • methionine can be present in the defined culture medium in a concentration range of from about 0.05 mM to about 0.5 mM, e.g., from about 0.05 mM to about 0.1 mM, from about 0.1 mM to about 0.15 mM, from about 0.15 mM to about 0.20 mM, from about 0.20 mM to about 0.30 mM, from about 0.30 mM to about 0.40 mM, or from about 0.40 mM to about 0.50 mM; and in some embodiments is present in the defined culture medium at a concentration of 0.2 mM.
  • adenine and/or guanine can each be present in the defined culture medium in a concentration range of from about 0.05 mM to about 0.4 mM, e.g., from about 0.05 mM to about 0.1 mM, from about 0.1 mM to about 0.15 mM, from about 0.15 mM to about 0.2 mM, from about 0.2 mM to about 0.3 mM, or from about 0.3 mM to about 0.4 mM; and in some embodiments are present in the defined culture medium at a concentration of 0.2 mM each.
  • each of these amino acids can each be present in the defined medium in a concentration range of from about 0.2 mM to about 1.2 mM, e.g., from about 0.2 mM to about 0.4 mM, from about 0.4 mM to about 0.6 mM, from about 0.6 mM to about 0.8 mM, from about 0.8 mM to about 1.0 mM, or from about 1.0 mM to about 1.2 mM; and in some embodiments are present in the defined medium at a concentration of 0.8 mM (alanine and leucine), 0.6 mM (glutamine, glutamic acid, and valine), and 0.4 mM (isoleucine).
  • the culture medium can further comprise glycine.
  • Glycine can be present in the defined culture medium in a concentration range of from about 0.4 mM to about 1.5 mM, e.g., from about 0.4 mM to about 0.5 mM, from about 0.5 mM to about 0.6 mM, from about 0.6 mM to about 0.8 mM, from about 0.8 mM to about 1.0 mM, from about 1.0 mM to about 1.25 mM, or from about 1.25 mM to about 1.5 mM; and in some embodiments is present in the defined culture medium at a concentration of 0.8 mM.
  • the culture medium further includes an aromatic amino acid, e.g., one or more of tryptophan, tyrosine, and phenylalanine.
  • the culture medium further includes para-amino benzoic acid.
  • the culture medium further includes para- hydroxy benzoic acid.
  • the culture medium further includes 2,3- dihydroxybenzoic acid.
  • the culture medium further includes tryptophan, tyrosine, phenylalanine, para-amino benzoic acid, para-hydroxy benzoic acid, and 2,3- dihydroxybenzoic acid.
  • the culture medium includes an aromatic amino acid (e.g., one or more of tryptophan, tyrosine, and phenylalanine)
  • the aromatic amino acid can be present in the defined medium in a concentration range of from about 0.05 mM to about 0.6 mM, e.g., from about 0.05 mM to about 0.1 mM, from about 0.1 mM to about 0.2 mM, from about 0.2 mM to about 0.4 mM, or from about 0.4 mM to about 0.6 mM; and in some embodiments are present in the defined culture medium at a concentration of 0.1 mM (tryptophan), 0.2 mM (tyrosine), and 0.4 mM (phenylalanine).
  • Para-amino benzoic acid, para-hydroxy benzoic acid, and 2,3-dihydroxybenzoic acid can each be present in the defined medium in a concentration range of from about 0.005 mM to about 0.1 mM, e.g., from about 0.005 mM to about 0.01 mM, from about 0.01 mM to about 0.05 mM, or from about 0.05 mM to about 0.1 mM; and in some embodiments are present in the defined culture medium at a concentration of 0.01 mM each.
  • the defined culture medium further comprises a C 12 -C 22 fatty acid.
  • the genetically modified host cell includes an endogenous type II fatty acid biosynthetic pathway.
  • the C 12 -C 22 fatty acid is a Ci 2 saturated fatty acid.
  • the Ci 2 -C 22 fatty acid is a Ci 4 saturated fatty acid.
  • the Cj 2 - C 22 fatty acid is a Q 6 saturated fatty acid.
  • the Ci 2 -C 22 fatty acid is a Ci 8 saturated fatty acid.
  • the Ci 2 -C 22 fatty acid is a C 20 saturated fatty acid.
  • the Ci 2 -C 22 fatty acid is a C 22 saturated fatty acid.
  • the fatty acid is palmitic acid.
  • Suitable saturated fatty acids include, but are not limited to, Ci 2 -C 22 saturated fatty acids, e.g.,
  • Suitable saturated fatty acids include, but are not limited to, myristic acid (tetradecanoic acid), pentadecanoic acid, palmitic acid (hexadecanoic acid), stearic acid (octadecanoic acid), arachidic acid (eicosanoic acid), docsosanoic acid, and tetracosanoic acid.
  • Suitable salts include, but are not limited to, lithium salts, potassium salts, sodium salts, and the like.
  • Suitable unsaturated fatty acids include, but are not limited to, oleic acid, vaccenic acid, linoleic acid, palmitelaidic acid, and arachidonic acid. Also suitable for use are salts of an unsaturated fatty acid, derivatives of an unsaturated fatty acid, and salts of a derivative of an unsaturated fatty acid. Suitable salts include, but are not limited to, lithium salts, potassium salts, sodium salts, and the like.
  • the fatty acid (or compound that yields a fatty acid) will in some embodiments be present in the culture medium in an amount or a concentration that is effective to reduce HMG-CoA accumulation- induced growth inhibition of the cell.
  • the culture medium comprises a fatty acid in a concentration range of from about 0.10 mM to about 0.50 mM, e.g., from about 0.1 mM to about 0.15 mM, from about 0.15 mM to about 0.2 mM, from about 0.2 mM to about 0.25 mM, from about 0.25 mM to about 0.3 mM, from about 0.3 mM to about 0.35 mM, from about 0.3 M to about 0.4 mM, from about 0.35 mM to about 0.4 mM, from about 0.35 mM to about 0.45 mM, or from about 0.45 mM to about 0.5 mM.
  • Ci 2 -C 22 fatty acid the genetically modified host cell has an endogenous type II fatty acid biosynthetic pathway.
  • Type II fatty acid biosynthetic pathway enzymes include, but are not limited to, malonyl- CoA:ACP transacylase, ⁇ -ketoacyl-ACP synthase I, ⁇ -ketoacyl-ACP synthase II, ⁇ -ketoacyl-ACP synthase III, acetyl-CoA:ACP transacylase, malonyl-ACP decarboxylase, ⁇ -ketoacyl-ACP reductase, ⁇ - hydroxyacyl-ACP dehydratase, ⁇ -hydroxydecanoyl-ACP dehydrase, trans-2-decenoyl-ACP isomerase, and enoyl-ACP reductase.
  • ACP is "acetyl carrier protein.”
  • the culture medium further comprises a supplement that increases the osmolarity compared to the ⁇ smolarity of the control culture medium.
  • the supplement is a salt in a concentration range of from about 50 mM to about 500 mM.
  • An agent(s) that increases osmolarity is present in the culture medium at a concentration that increases the osmolarity of the culture medium by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about75%, at least about 100% (or 2-fold), at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, at least about 10 fold, at least about 25 fold, at least about 50 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, compared to the osmolarity of a control culture medium not containing the agent (e.g., compared to the defined medium without the agent, or, where the agent is a salt, with the salt but at a concentration below 50 mM).
  • the osmolarity of control culture medium is from about 50 mOsM to about 100 mOsM; and a supplemented culture medium comprising one or more agents that increase osmolarity has an osmolarity of from about 100 mOsM to about 500 mOsM, e.g., from about 100 mOsM to about 1 10 mOsM , from about 100 mOsM to about 125 mOsM, from about 125 mOsM to about 150 mOsM, from about 150 mOsM to about 200 mOsM, from about 200 mOsM to about 250 mOsM, from about 250 mOsM to about 300 mOsM, from about 300 mOsM to about 350 mOsM, from about 350 mOsM to about 400 mOsM, from about 400 mOsM to about 450 mOsM, or from about
  • Agents that increase osmolarity include, but are not limited to, salts, sugars (e.g. monosaccharides such as glucose; disaccharides; etc.), sugar alcohols, starches, polysaccharides, glycerol, and the like.
  • Suitable salts include, but are not limited to, NaCl, sodium citrate, Na 2 HPO 4 , CaCl 2 , KCl, KH 2 PO 4 , K 2 HPO 4 , NH 4 Cl, MgSO 4 , and the like.
  • Suitable sugars and sugar alcohols include sorbitol, trehalose, and the like.
  • the culture medium comprises a salt, a sugar, or a sugar alcohol in a concentration range of from about 50 mM to about 500 mM, e.g., from about 50 mM to about 75 mM, from about 75 mM to about 100 mM, from about 100 mM to about 125 mM, from about 125 mM to about 150 mM, from about 150 mM to about 175 mM, from about 175 mM to about 200 mM, from about 200 mM to about 250 mM, from about 250 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, or from about 450 mM to about 500 mM.
  • a salt e.g., from about 50 mM to about 75 mM, from about 75 mM to about 100 mM, from about 100 mM to about 125 m
  • the defined culture medium comprises a minimal medium that is supplemented with serine and a subset of amino acids, as described above.
  • the defined culture medium comprises: a) a minimal culture medium; b) serine; and c) a subset of amino acids selected from the group consisting of alanine, glutamine, glutamic acid, isoleucine, leucine, valine, and methionine, where the subset consists of zero to seven amino acids, and where the medium does not comprise one or more amino acids that are not part of the subset.
  • a "minimal culture medium” is a culture medium that provides the minimum nutrients possible for bacterial growth; for example, a minimal culture medium includes a carbon source (e.g., citrate, succinate, glucose), magnesium, nitrogen, phosphorus, and sulfur, which components allow the bacterium to synthesize proteins and nucleic acids.
  • a minimal culture medium does not include amino acids or nucleotides.
  • Minimal culture media suitable for in vitro culture are known in the art, and any minimal culture medium can be used to generate a defined culture medium suitable for use in a subject method.
  • a suitable minimal culture medium comprises: a buffer that is compatible with the isoprenoid-producing cells (e.g., is not substantially toxic or growth- inhibiting to the isoprenoid-producing cells; e.g., is not substantially bacteriostatic or bactericidal to the isoprenoid-producing cells at the concentration used in the minimal culture medium); salts that provide sources of magnesium, nitrogen, phosphorus, and sulfur (e.g., a potassium salt, a sodium salt, an ammonium salt, a magnesium salt); and micronutrients (e.g., zinc, manganese, copper, cobalt, boron, and molybdenum).
  • Suitable buffers include, e.g., morpholinopropane sulfonate (MOPS), phosphat
  • Minimal media are known in the art; and any known minimal medium can be used.
  • M9 minimal medium is described in, e.g., Miller (1972) Experiments in molecular genetics, p. 431-432, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and Sambrook, J., E. F. Fritsch, and T. Maniatis (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • An exemplary, non-limiting minimal culture medium suitable for use is a culture medium comprising MOPS, tricine, a carbon source, nitrogen, phosphorus, sulfur, and micronutrients.
  • a suitable minimal culture medium can include MOPS (e.g., in a concentration range of from about 20 mM to about 80 mM), potassium phosphate (e.g., in a concentration range of from about 0.5 mM to about 2 mM), ammonium chloride (e.g., in a concentration range of from about 5 mM to about 15 mM), magnesium chloride (e.g., in a concentration range of from about 0.2 mM to about 1 mM), potassium sulfate (e.g., in a concentration range of from about 0.1 mM to about 1 mM), iron sulfate (e.g., in a concentration range of from about 0.005 mM to about 0.1 mM), calcium chloride (e.g., in a concentration range of from about 10 "4 mM to about 10 '3 mM), sodium chloride (e.g., in a concentration range of from about 10 mM to about 100 mM), N
  • One example of a suitable minimal medium is the MOPS minimal medium described in
  • MOPS minimal medium includes potassium phosphate (1.32 mM); ammonium chloride (9.52 mM); MgCl 2 (0.523 mM); potassium sulfate (0.276 mM); iron sulfate (0.010 mM); CaCl 2 (5 X lO "4 mM); NaCl (50 mM); MOPS (40 mM); Tricine (4 mM); and micronutrients (ammonium molybdate, 2 x 10 "6 mM; borate, 4 x 10 ⁇ mM; cobalt chloride, 2 x 10 '5 mM; copper sulfate, 10 "5 mM; manganese chloride, 8 x 10 "5 mM; and zinc sulfate, 10 "5 mM). Isoprenoid-producing cells
  • Suitable cells include: 1) cells that have an endogenous mevalonate pathway, e.g., eukaryotic cells such as yeast cells, fungal cells, etc., and that synthesize the isoprenoid or isoprenoid precursor compound at least in part via the endogenous mevalonate pathway; 2) cells that have an endogenous DXP pathway, e.g., eukaryotic cells such as a prokaryotic cell that normally produces IPP via a DXP pathway, and that synthesize the isoprenoid or isoprenoid precursor compound at least in part via the endogenous DXP pathway; 3) cells that have been genetically modified with one or more exogenous nucleic acids comprising nucleotide sequences encoding one or more heterologous mevalonate pathway enzymes, and that synthesize the isoprenoid or isoprenoid precursor at least in part via the exogenous mevalonate pathway, e.g., a prokaryotic cell that does
  • Isoprenoid-producing cells can be unicellular organisms, or can be cells that grown in culture as single cells.
  • the isoprenoid-producing 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, Pichiapastoris, Pichia finlandica, 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, Klicyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramine
  • the isoprenoid-producing 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) J. Immunol. 148: 1176-1 181 ; 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.
  • Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae.
  • the laboratory strain is one that is nonpathogenic.
  • 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. Genetically modified host cells
  • an isoprenoid-producing cell can be a cell that produces an isoprenoid or isoprenoid precursor via a genetically modified (heterologous) IPP biosynthetic pathway.
  • An isoprenoid-producing cell that produces an isoprenoid or isoprenoid precursor via a genetically modified (heterologous) IPP biosynthetic pathway is referred to as a "genetically modified isoprenoid-producing host cell” or simply a “genetically modified host cell.”
  • Suitable genetically modified host cells include: 1) cells that have been genetically modified with one or more exogenous nucleic acids comprising nucleotide sequences encoding one or more heterologous mevalonate pathway enzymes, and that synthesize the isoprenoid or isoprenoid precursor at least in part via the exogenous mevalonate pathway, e.g., a prokaryotic cell that does not normally produce IPP via a mevalonate pathway, and that has been genetically modified with one or more nucleic acids comprising nucleotide sequences encoding one or more heterologous mevalonate pathway enzymes; 2) cells that have been genetically modified with one or more exogenous nu
  • a genetically modified isoprenoid-producing host cell comprises one or more nucleic acids comprising nucleotide sequences encoding one or more heterologous MEV pathway enzymes; and may further comprise nucleotide sequences encoding one or more of a heterologous IPP isomerase, a heterologous prenyl transferase, and a heterologous terpene synthase.
  • a genetically modified isoprenoid-producing host cell comprises one or more nucleic acids comprising nucleotide sequences encoding one or more heterologous DXP pathway enzymes; and may further comprise nucleotide sequences encoding one or more of a heterologous DPP isomerase, a heterologous prenyl transferase, and a heterologous terpene synthase.
  • 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.
  • 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.
  • 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-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.
  • the mevalonate pathway enzymes required for production of IPP vary, depending on the culture conditions.
  • a genetically modified host cell is one that has been genetically modified with one or more heterologous nucleic acids comprising nucleotide sequences encoding mevalonate kinase (MK), phosphomevalonate kinase (PMK), and mevalonate pyrophosphate decarboxylase (MPD).
  • MK mevalonate kinase
  • PMK phosphomevalonate kinase
  • MPD mevalonate pyrophosphate decarboxylase
  • a genetically modified host cell is one that has been genetically modified with one or more heterologous nucleic acids comprising nucleotide sequences encoding MK, PMK, MPD, and isopentenyl pyrophosphate isomerase (IDI).
  • IDI isopentenyl pyrophosphate isomerase
  • a genetically modified host cell is one that has been genetically modified with one or more heterologous nucleic acids comprising nucleotide sequences encoding MK, PMK, MPD, EDI, and a prenyltransferase.
  • a genetically modified host cell is one that has been genetically modified with one or more heterologous nucleic acids comprising nucleotide sequences encoding acetoacetyl- CoA thiolase, hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR) HMGS, HMGR, MK, PMK, and MPD.
  • HMGS hydroxymethylglutaryl-CoA synthase
  • HMGR hydroxymethylglutaryl-CoA reductase
  • a genetically modified host cell is one that has been genetically modified with one or more heterologous nucleic acids comprising nucleotide sequences encoding acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, and IDI.
  • a genetically modified host cell is one that has been genetically modified with one or more heterologous nucleic acids comprising nucleotide sequences encoding acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, DDI, and a prenyl transferase.
  • Suitable prokaryotic host 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) J. 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 sonnei, 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 nucleic acid with which the host cell is genetically modified such that it produces IPP and/or mevalonate via a mevalonate pathway is an expression vector that includes a nucleic acid comprising a nucleotide sequence that encodes a mevalonate pathway enzyme(s).
  • 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).
  • Pl -based artificial chromosomes based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, and the like
  • Pl -based artificial chromosomes Pl -based artificial chromosomes
  • yeast plasmids yeast artificial chromosomes
  • any other vectors specific for specific hosts of interest such as E. coli and yeast.
  • a nucleic acid encoding a mevalonate pathway gene product(s) is included in any one of a variety of expression vectors for expressing the mevalonate pathway gene product(s
  • 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 pRIT2T (Pharmacia).
  • any other plasmid or other vector may be used so long as it is compatible with the host cell.
  • a mevalonate pathway enzyme-encoding nucleotide sequence is inserted into an expression vector.
  • the mevalonate pathway enzyme-encoding nucleotide sequence in the expression vector is operably linked to an appropriate expression control sequence(s) (e.g., a promoter) to direct synthesis of the encoded gene product.
  • an expression vector comprising nucleotide sequences encoding a mevalonate pathway enzyme will be used.
  • the mevalonate pathway enzyme coding sequences are operably linked to appropriate expression control sequence(s) to direct synthesis of the encoded gene product.
  • appropriate expression control sequence(s) to direct synthesis of the encoded gene product.
  • 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; a salicylate promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S.
  • Patent Publication No. 20040131637 discloses 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 (Harborne 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.
  • the expression vectors will in many embodiments contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as antibiotic resistance (e.g., tetracycline or ampicillin resistance) in prokaryotic host cells such as E. coli.
  • antibiotic resistance e.g., tetracycline or ampicillin resistance
  • prokaryotic host cells such as E. coli.
  • a mevalonate pathway enzyme-encoding nucleotide sequence is operably linked to an inducible promoter.
  • the genetically modified host cell is further genetically modified with one or more additional heterologous nucleic acid(s) comprising nucleotide sequences encoding enzymes other than mevalonate pathway enzymes
  • the nucleotide sequences can be 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 (DPTG)-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. (1995) J. Bacteriol.
  • 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
  • the nucleotide sequence encoding a mevalonate 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.
  • nucleotide sequences encoding the two or more enzymes will in some embodiments each be contained on separate expression vectors.
  • nucleotide sequences encoding the one or more mevalonate pathway enzymes will in some embodiments be contained in a single expression vector.
  • nucleotide sequences encoding the one or more mevalonate 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 mevalonate pathway enzyme-encoding nucleotide sequences on the single expression vector.
  • a common control element e.g., a promoter
  • nucleotide sequences encoding the mevalonate 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 mevalonate pathway enzyme-encoding nucleotide sequences separately on a single expression vector.
  • control elements e.g., a promoters
  • Nucleotide sequences encoding 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.
  • nucleotide sequences encoding acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, and IDI are known in the art.
  • the following are non-limiting examples of known nucleotide sequences encoding MEV pathway gene products, with GenBank Accession numbers and organism following each MEV pathway enzyme, in parentheses: acetoacetyl- CoA thiolase: (NC_000913 REGION: 2324131..2325315; E.
  • HMGS (NC OOl 145. complement 19061..20536; Saccharomyces cerevisiae), (X96617; Saccharomyces cerevisiae), (X83882; Arabidopsis thalian ⁇ ), (AB037907; Kitasatospora griseola), and (BT007302; Homo sapiens); 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_001 145: complement (1 15734..1 18898; Saccharomyces cerevisiae)); MK: (L77688; Arabidopsis thaliana), and (X55875; Saccharomyces cerevisiae); PMK: (AF429385; Hevea brasiliensis), (NM 006556; Homo sapiens), (NC OOl 145.
  • mevalonate pathway enzymes of gram positive bacteria e.g., as described in Wilding et al. (2000) J. Bacteriol. 182:4319-4327.
  • Various mevalonate pathway enzyme- encoding nucleotide sequences are known in the art; and any known mevalonate pathway enzyme- encoding nucleotide sequence, or a functional variant thereof, can be used. See, e.g., Streptococcus pneumoniae MK, MPD, and PMK, GenBank Accession No. AF290099; Streptococcus pneumoniae HMGS and HMGR, GenBank Accession No.
  • AF290098 Enterococcus faecium MK, PMK, and MPD, GenBank Accession No. AF290095; Enterococcus faecium HMGS, acetyl-CoA acetyltransferases, and HMGR, GenBank Accession No. AF290094; Enterococcus faecalis MK, MPD, and PMK, GenBank Accession No. AF290093; Enterococcus faecalis HMGS, acetyl-CoA acetyltransferases, and HMGR, GenBank Accession No.
  • AF290092 Staphylococcus aureus MK, MPD, and PMK, GenBank Accession No. AF290087; Staphylococcus aureus HMGS and HMGR, GenBank Accession No. AF290086; Streptococcus pyogenes MK, MPD, and PMK, GenBank Accession No. AF290097; and Streptococcus pyogenes HMGS and HMGR, GenBank Accession No. AF290096.
  • a non-limiting example of 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 ED NO: 13 of U.S. Patent No.
  • tHMGR 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. 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-erythritol
  • a genetically modified host cell is one that has been genetically modified with one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more 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.
  • 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.
  • AF282878 Pseudomonas aeruginosa dxs
  • GenBank Accession No. NM 121176 Alignidopsis thaliana dxs
  • GenBank Accession No. AB026631 GenBank Accession No. 078328 (Capsicum annum). See also Figure 5 of U.S. Patent Publication No. 2003/0219798 for nucleotide sequences encoding dxs.
  • 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.
  • 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 l 05070 Arabidopsis thaliana
  • GenBank Accession No. AEO 14073.1 nucleotides 2838621-283841; Shigella flexneri.
  • GenBank Accession No. CP000034.1 nucleotides 2505082 to 2503964; Shigella dysenteriae IspG
  • GenBank Accession No. NM_180902 Arabidopsis thaliana
  • GenBank Accession No. AE008814.1 nucleotides 15609-14491; Salmonella typhimurium IsgG
  • GenBank Accession No. AE014613.1 nucleotides 383225-384343; Salmonella enterica GcpE
  • AEO 17220.1 (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 EPP 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.
  • the nucleic acid with which the host cell is genetically modified such that it produces IPP via a DXP pathway is an expression vector that includes a nucleic acid comprising a nucleotide sequence that encodes a DXP pathway enzyme(s).
  • 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.
  • 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).
  • a nucleotide sequence encoding a DXP pathway enzyme is operably linked to a promoter.
  • 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 Pichi ⁇ ). 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.
  • an expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., 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.
  • the promoter can be constitutive or inducible.
  • 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, IRL 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. 1 1, A Practical Approach, Ed. DM Glover, 1986, IRL Press, Wash., D.C.).
  • vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • a host cell is genetically modified to include one or more nucleic acids comprising a nucleotide sequence(s) encoding one or more mevalonate pathway enzymes or one or more DXP 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 IPP 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
  • Suitable prenyltransferases include, but are not limited to, an £-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.
  • 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 nucleotide sequence encoding a prenyltransferase can be operably linked to a promoter.
  • Suitable promoters are as described above, and include, e.g., constitutive promoters and inducible promoters.
  • Terpene synthases are as described above, and include, e.g., constitutive promoters and inducible promoters.
  • a host cell is genetically modified to include one or more nucleic acids comprising a nucleotide sequence(s) encoding one or more mevalonate pathway enzymes or one or more DXP pathway enzymes, as described above; a nucleic acid comprising a nucleotide sequence that encodes a prenyl transferase; and a nucleic acid comprising a nucleotide sequence encoding a terpene synthase.
  • the terpene synthase is one that modifies FPP to generate a sesquiterpene. In other embodiments, the terpene synthase is one that modifies GPP to generate a monoterpene. In other embodiments, the terpene synthase is one that modifies GGPP to generate a diterpene.
  • the terpene synthase acts on a polyprenyl diphosphate substrate, modifying the polyprenyl diphosphate substrate by cyclizing, rearranging, or coupling the substrate, yielding an isoprenoid or isoprenoid precursor.
  • Suitable terpene synthases include, but are not limited to, an abietadiene synthase, an amorphadiene synthase, a camphene synthase, a carene synthase, an ⁇ -farnesene synthase, a ⁇ - farnesene synthase, a geraniol synthase, a germacrene synthase, a humulene synthase, a linalool synthase, a limonene synthase, a myrcene synthase, an ocimene synthase, a patchoulol synthase, an ⁇ -pinene synthase, a ⁇ -pinene synthase, a selinene synthase, a sabinine synthase, a ⁇ -terpinene synthase, a terpino
  • Nucleotide sequences encoding terpene synthases are known in the art, and any known terpene synthase-encoding nucleotide sequence can be used to genetically modify a host cell.
  • any known terpene synthase-encoding nucleotide sequence can be used to genetically modify a host cell.
  • the following terpene 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,E- alpha-farnesene synthase mRNA (AY640154; Cucumis sativus); 1,8-cineole synthase mRNA (AY691947; Arabidopsis thaliana); terpene synthase 5 (TPS5) mRNA (AY518314; Zea mays); terpene 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 (AY 195609; Antirrhinum majus);
  • a nucleotide sequence encoding a terpene synthase can be operably linked to a promoter.
  • Suitable promoters are as described above, and include, e.g., constitutive promoters and inducible promoters.
  • a nucleic acid comprising a nucleotide sequence encoding a mevalonate pathway enzyme, a DXP pathway enzyme, a prenyltransferase, a terpene synthase, or other enzyme mentioned above, is present as an extrachromosomal element in the host cell.
  • a nucleic acid comprising a nucleotide sequence encoding a mevalonate pathway enzyme, a DXP pathway enzyme, a prenyltransferase, a terpene synthase, or other enzyme mentioned above, is integrated into the host cell's genome. Codon usage
  • nucleotide sequence encoding a mevalonate pathway enzyme a mevalonate pathway enzyme
  • the DXP pathway enzyme a prenyltransferase, a terpene synthase, or other enzyme mentioned above, is modified such that the nucleotide sequence reflects the codon preference for the particular host cell.
  • the nucleotide sequence can be modified for E. coli codon preference. See, e.g., Gouy and Gautier (1982) Nucleic Acids Res. 10(22):7055-7074; Eyre- Walker (1996) MoI. Biol. Evol. 13(6):864-872. See also Nakamura et al. (2000) Nucleic Acids Res. 28(1):292.
  • the nucleotide sequence will in some embodiments be modified for yeast codon preference. See, e.g., Bennetzen and Hall (1982) J. Biol. Chem. 257(6): 3026-3031. Additional genetic modifications
  • a genetically modified host cell is one that is genetically modified to include one or more nucleic acids comprising a nucleotide sequence(s) that encode heterologous IPP biosynthetic pathway enzymes; and that is further genetically modified to achieve enhanced production of an isoprenoid or isoprenoid precursor, and/or that is further genetically modified such that an endogenous isoprenoid biosynthetic pathway gene (e.g., an endogenous DXP pathway gene; and endogenous mevalonate pathway gene) is functionally disabled.
  • an endogenous isoprenoid biosynthetic pathway gene e.g., an endogenous DXP pathway gene; and endogenous mevalonate pathway gene
  • the term "functionally disabled,” as used herein, refers to a genetic modification of a nucleic acid, which modification results in production of a gene product encoded by the nucleic acid that is produced at below normal levels, and/or is nonfunctional.
  • an isoprenoid-producing cell is cultured in vitro in a defined medium as described above.
  • the isoprenoid-producing cell is cultured in vitro under suitable conditions and for such a time that IPP biosynthetic pathway enzymes are produced by the cell; and the IPP biosynthetic pathway enzymes catalyze the production of an isoprenoid or isoprenoid precursor.
  • a subject method is useful for production of a variety of isoprenoid or isoprenoid precursor compounds.
  • the temperature at which isoprenoid-producing cell is cultured is generally from about 18°C to about 40 0 C, e.g., from about 18°C to about 20 0 C, from about 20 0 C to about 25°C, from about 25°C to about 30 0 C, from about 30 0 C to about 35°C, or from about 35°C to about 4O 0 C (e.g., at about 37°C).
  • a subject method provides for production of an isoprenoid or isoprenoid precursor in a recoverable amount of from about 1 mg/L to about 50 g/L, e.g., from about 1 mg/L to about 5 mg/L, from about 5 mg/L to about 10 mg/L, from about 10 mg/L to about 25 mg/L, from about 25 mg/L to about 50 mg/L, from about 50 mg/L to about 100 mg/L, from about 100 mg/L to about 250 mg/L, from about 250 mg/L to about 500 mg/L, from about 500 mg/L to about 1 g/L, from about 1 g/L to about 5 g/L, from about 5 g/L to about 10 g/L, from about 10 g/L to about 15 g/L, from about 15 g/L to about 20 g/L, from about 20 g/L to about 25 g/L, from about 25 g/L to about 30 g/L, from about 30
  • Isoprenoid and/or isoprenoid precursor production is readily determined using well-known methods, e.g., gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, ion chromatography-mass spectrometry, pulsed amperometric detection, uv-vis spectrometry, and the like.
  • the amount of isoprenoid or isoprenoid precursor that is produced using a subject method can also be expressed as an amount per gram of dry cell weight.
  • a subject method provides for production of an isoprenoid or isoprenoid precursor in a recoverable amount of at least 1 mg isoprenoid compound per gram of dry cell weight (mg/g), at least 2 mg/g, at least 5 mg/g, at least 10 mg/g, at least 15 mg/g, at least 20 mg/g, at least 25 mg/g, at least 30 mg/g, at least 35 mg/g, at least 40 mg/g, at least 45 mg/g, at least 50 mg/g, or more than 50 mg/g.
  • Isoprenoid precursors that can be produced using the method of the invention include, but are not limited to, EPP, DMAPP, an intermediate in a mevalonate pathway (e.g., mevalonate), and an intermediate in a DXP pathway.
  • Isoprenoids that can be produced using a subject method include, e.g., C 5 -C 10 isoprenoids, C 5 -
  • Isoprenoids that can be produced using ta subject method include, but are not limited to, hemiterpenes, monoterpenes, diterpenes, triterpenes, and polyterpenes.
  • isoprenoids that can be produced using a subject method 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, farnesol, gossypol, sanonin, periplanone, santatol, 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,
  • 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, and coenzyme Q- 10.
  • an isoprenoid that can be produced using a subject method is selected from abietadiene, amorphadiene, carene, ⁇ -farnesene, ⁇ -farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, ⁇ - pinene, sabinene, ⁇ -terpinene, terpinolene, and valencene.
  • the isoprenoid or isoprenoid precursor is produced at a level that is at least 2-fold higher than the level of the isoprenoid or isoprenoid precursor that is produced when the isoprenoid-producing cell is cultured in minimal medium (e.g., minimal medium without the supplement(s)).
  • minimal medium e.g., minimal medium without the supplement(s)
  • the total production of an isoprenoid or isoprenoid precursor is increased, compared to the total production of the isoprenoid or isoprenoid precursor when the isoprenoid-producing cell is grown in minimal medium (e.g., compared to the total production of the isoprenoid or isoprenoid precursor when the isoprenoid-producing cell is grown in MOPS minimal medium).
  • the total production can be expressed as mg/L-OD, where the OD (e.g., OD 6 oo is a measure of the number of cells.
  • the total production (in mg/L-optical density, or mg/L-OD) of an isoprenoid or isoprenoid precursor produced by an isoprenoid-producing cell that is cultured in vitro in a defined culture medium as described above is at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-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, higher than the total production of the isoprenoid or isoprenoid compound when the isopre
  • the total production of an isoprenoid or isoprenoid precursor produced by an isoprenoid-producing cell cultured in vitro in a defined medium is from about 2-fold to about 2.5-fold, from about 2.5-fold to about 3-fold, from about 3-fold to about 3.5- fold, from about 3.5-fold to about 4-fold, from about 4-fold to about 4.5-fold, from about 4.5-fold to about 5-fold, from about 5-fold to about 7-fold, or from about 7-fold to about 10-fold, from about 10- fold to about 50-fold, from about 50-fold to about 100-fold, from about 100-fold to about 200-fold, from about 200-fold to about 300-fold, from about 300-fold to about 400-fold, or from about 400-fold to about 500-fold, or more, higher than the total production of the isoprenoid or isoprenoid compound when the cell is cultured in the minimal medium.
  • a subject method of producing an isoprenoid or isoprenoid precursor compound comprises culturing an isoprenoid-producing cell, as described above; and further comprises recovering the isoprenoid or isoprenoid precursor compound.
  • An isoprenoid or isoprenoid produced by the isoprenoid-producing cell can be recovered (e.g., isolated, purified) from a cell lysate, from a cell supernatant (e.g., from the culture medium), or both cell lysate and cell supernatant.
  • an isoprenoid or isoprenoid precursor compound from cell lysate and from cell supernatant (e.g., from cell culture medium) are known in the art.
  • the isoprenoid-producing cell can be sonicated, subjected to detergent lysis, or subjected to another method for releasing the contents of the cytosol.
  • An isoprenoid or isoprenoid precursor compound can be recovered from the cell culture medium and/or a cell lysate using any of a variety of methods, including, but not limited to, high performance liquid chromatography (HPLC), size exclusion chromatography, and the like.
  • HPLC high performance liquid chromatography
  • an isoprenoid or isoprenoid precursor compound is secreted from the isoprenoid- producing cell, and is captured in an organic solvent which overlays the cell culture medium; in these embodiments, the isoprenoid or isoprenoid precursor compound can be recovered from the organic solvent.
  • isoprenoid-producing cells are cultured in a defined medium as described above, optionally supplemented with one or more additional agents, such as an inducer (e.g., where the isoprenoid-producing cell is a prokaryotic cell that is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding one or more mevalonate pathway enzymes, and where the nucleotide sequence is under the control of an inducible promoter, or where a nucleotide sequence encoding an enzyme that is not directly in the mevalonate pathway but that generates a precursor that feeds into the mevalonate pathway, or that modifies a product of the mevalonate pathway, is under the control of an inducible promoter, etc.); and the culture medium is overlaid with an organic solvent, e.g.
  • an inducer e.g., where the isoprenoid-producing cell is a prokaryotic cell that is genetically modified with one or more nucleic acids comprising
  • the isoprenoid compound produced by the isoprenoid-producing cell partitions into the organic layer, from which it can be purified.
  • 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 or isoprenoid precursor 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.
  • the isoprenoid or isoprenoid precursor compound that is recovered 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 or isoprenoid precursor compounds, macromolecules, contaminants, etc.
  • Example 1 Varying culture medium composition to increase isoprenoid production
  • the base media formulation (referred to as "minimal-MOPS" medium hereafter) contained MOPS, tricine, 1% glucose, FesO 4 , NH 4 Cl, K 2 SO 4 , CaCl 2 , MgCl 2 , NaCl, K 2 HPO 4 , micronutrients (ammonium molybdate, boric acid, cobalt chloride, cupric sulfate, manganese chloride, zinc sulfate), and antibiotics (Neidhardt et al. (1974) supra) [125].
  • the variants of this medium contained the following supplements at concentrations as per Neidhardt et al. (Neidhardt et al.
  • E. coli DHl was used as the cloning and expression host in this study (see Table 1 for strains used in this study).
  • the amorpha-4-1 1 -diene-producing strain used in this study contained a three plasmid system encoding the E. coli genes atoB, idi, ispA; the yeast genes HMGS, HMGR, MK, PMK, MPD; and a synthetic ADS as previously reported. Martin et al. (2003) Nat. Biotech. 21:796-802. Table 1. Strains and plasmids used in this study.
  • Plasmids pMevT, pMBIS, and pADS are described in, e.g., e.g., U.S. Patent No. 7, 183,089;
  • strain DHl is described in, e.g., Meselson Yuan (1968) Nature 217(134): p. 1 110-4. Plasmids pMevT-C159A, pMBI*S*, and pADS-mutl are described below.
  • pMevT(C159A) also referred to as pBad33MevT(C159A), (SEQ ID NO: 11, U.S. Patent No. 7,183,089).
  • the inactivated pMBIS plasmid was produced by point mutations in the idi and ispA genes.
  • the IDIEl 16V mutant was generated from pBAD24-IDI using the standard QuikChange (Stratagene, La Jolla, CA) procedure with the idi-f and idi-R primers. IDIEl 16V was then amplified using the primers idi-f and idi-r and ligated into the Xma I site of pMevB.
  • the IspAR96Q mutant was generated from pBAD24-IspA using the standard QuikChange (Stratagene, La Jolla, CA) procedure with the ispa-f and ispa-r primers. IspAR96Q was then amplified using the primers ispa-f and ispa-r and ligated into the SacII-SacI site of the pMB-BEl 16V plasmid. The final plasmid, pMB-IEl 16V-SR96Q, was verified by sequencing.
  • QuikChange Stratagene, La Jolla, CA
  • DHlOB was co-transformed with pADS and pMevB, pMBIS, or pMB-IEl 16V-SR96Q.
  • Fifty (50) mL of 2YT containing carbenicillin (50 ⁇ g/L) and tetracycline (5 ⁇ g/L) in a 250-mL baffled shake flask was inoculated with 1 mL of overnight LB culture of each of the freshly transformed strains. The cultures were grown at 37°C at 200 rpm to OD 600 0.2-0.3 before inducing with IPTG (1 mM). At this time, mevalonate (final concentration of 5 mM) and dodecane (5 mL) were added to the culture. After 48 h of growth, the dodecane layer was sampled and analyzed by GC-MS. The resulting plasmid was designated pMBI*S*.
  • E. coli DHl was transformed with plasmids encoding the amorpha-4-1 1-diene synthesis pathway (pMevT, pMBIS and pADS) or the inactive pathway control (pMevT-C159A, pMBI*S* and pADS-mutl) and grown on LB agar plates. Single colonies were chosen for 10-ml, overnight culture in Luria Broth with Millers modification. Overnight LB cultures were used to inoculate a second overnight culture in M9-glucose medium supplemented with micronutrients. The M9-glucose overnight cultures were then used as 1% inoculum for a 200-ml starter culture (M9-glucose).
  • the 200- ml starter cultures were grown overnight and then used to inoculate a 10-L batch reactor (M9-glucose) to a starting OD 60O of -0.05.
  • the batch reactor was operated with a 1 volume/initial volume house air supply, 700 RPM stirring, pH controlled with 5% NaOH at 6.4-6.9 pH, and an operating temperature of 3O 0 C.
  • the exhaust air was routed through a gas trap containing 200-ml of dodecane to capture any amorpha-4-1 1-diene the culture produced.
  • the exhaust from the gas trap was then analyzed with an inline mass selective detector for CO 2 concentration.
  • the cDNA was base hydrolyzed in 100 mM NaOH/10 mM EDTA at 65 0 C for ten minutes and then neutralized using 7.0 pH HEPES at a final concentration of 500 mM.
  • the Tris remaining in the cDNA suspension was removed by three buffer exchange spins using Micron YM-30 columns (Millipore) and eluted in a final volume of ⁇ 15 Dl water.
  • the cDNA was then labeled using either Alexa 555 or Alexa 647 (Invitrogen, Carslbad, CA) following the manufacturers protocol.
  • the hybridization program included a pre-hybridization (5x SSC/0.2% SDS/1% BSA, 42 0 C, 60 minutes), a 17-hour hybridization (Ambion Hyb Solution #3, 41 0 C, medium agitation), two low stringency washes (Ix SSC/0.2%SDS, 41 0 C, 2 minutes each), two high stringency washes (O.lx SSC/0.2%SDS, 25 0 C, 2 minutes each), and two final washes (O.lx SSC, 25°C, 2 minutes each). Following hybridization, the slides were scanned with an Axon 4500 with at 60% laser power and PMT gain adjusted to balance the total intensity histogram. Transcriptional profile data analysis
  • SNOMAD's loess normalization to correct for any hybridization artifacts Colduoni et al., SNOMAD (Standardization and Normalization of MicroArray Data): web-accessible gene expression data analysis. Bioinformatics, 2002. 18(1 1): p. 1540-1). Local Z-scores generated by SNOMAD and the serial analysis for microarray (SAM) software (Tusher et al. (2001) Proc Natl Acad Sci U S A, 98(9): p. 51 16-21) were used as guides to determine biologically significant expression changes.
  • SAM serial analysis for microarray
  • Cluster 3.0 Hierarchal clustering
  • k-means clustering was also used to search the data set for temporal patterns in gene expression (Cluster 3.0).
  • amorpha-4-1 1-diene producing strain (APS) was grown in 50-ml shake flask cultures using variations of Neidhart's MOPS-buffered minimal medium listed above.
  • E. coli DHl was transformed with pMevT, pMBIS and pADS and plated onto minimal agar plates. Single colonies were chosen and grown in a 10-ml overnight culture in minimal medium supplemented with 1% glucose media. This overnight culture was used to inoculate 50-ml shake flasks containing the various media formulations and a 20% v/v dodecane overlay in order to capture the volatile amorpha-4-11-diene.
  • the cultures were grown to ⁇ 0.3 OD 60O and induced with 50 mM IPTG and then sampled approximately every 6-12 hours for 48-76 hours for amorpha-4-1 1-diene production.
  • Amo ⁇ ha-4,1 1-diene production levels from E. coli strains expressing the full amorpha-4-11- diene production pathway as well as the inactivated production pathway were assayed under a variety of conditions.
  • the amorpha-4-1 1-diene concentration in the dodecane capture fluid was assayed at multiple time points by diluting 10- ⁇ L of the dodecane overlay into 990- ⁇ L of ethyl acetate spiked with 5 ⁇ g/mL trans,trans-caryophyllene (both Sigma) as an internal standard.
  • the resolved samples were analyzed by a Hewlett-Packard model 5973 mass selective detector that monitored ions 189 and 204 m/z.
  • a standard curve for amorpha-4-11- diene was determined, based on a pure standard.
  • the amorpha-4, 1 1-diene concentration is based on the relative abundance of 189 and 204 m/z ions to the abundance of the total ions in the mass spectra.
  • Mevalonate (mevalonic acid) concentration in cultures of engineered E. coli was determined by
  • E. coli culture (560 ⁇ L) was mixed with 140 ⁇ L of 500 mM HCl in a glass GC vial to convert mevalonate from mevalonic acid to mevalonic acid lactone.
  • Ethyl acetate 700 ⁇ L
  • 500 ⁇ g/ml (-)-trans-caryophyllene 500 ⁇ 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 (Fisher Scientific) for 3-5 minutes.
  • the ethyl acetate extract of acidified culture was diluted 1 : 100 with fresh ethyl acetate in a clean GC vial before analysis.
  • the oven cycle for each sample and the ions monitored were modified from published methods (Woollen (2001) J Chromatogr B Biomed Sci Appl, 760(1): p. 179-84).
  • the column temperature profile was 7O 0 C for 2 minutes; ramped at 15°C/min to 185°C; ramped at 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. Retention time, mass spectrum and concentration of extracted mevalonic acid lactone were confirmed using commercial DL-mevalonic acid lactone (Sigma). Intracellular metabolite extraction and analysis
  • acyl-CoAs and adenylate pool constituents were determined by LC-MS analysis of trichloroacetic acid (TCA) culture extracts taken during the exponential phase of growth.
  • TCA trichloroacetic acid
  • isolate E. coli cells from the growth medium and extract metabolites cells were centrifuged through a layer of silicone oil into a denser solution of TCA by method similar to that of Shimazu et al (Shimazu (2004) Anal Biochem, 328(1): p. 51-9).
  • the spent medium was carefully removed by aspiration and the TCA extract layer was transferred to a 2-mL centrifuge tube using a small gauge needle and syringe.
  • 1 mL of ice cold 0.5 M tri-n-octylamine in 1 , 1 ,2-trichloro- 1 ,2,2-trifluoroethane (both Sigma) was added, tubes were vortexed for 1 minute and then centrifuged at max speed for 2 minutes to separate the layers. The aqueous layer was removed for analysis by LC-MS.
  • the neutralized TCA extract was analyzed using a Hewlett-Packard 1 100 series LC-MS using electrospray ionization.
  • a 50- ⁇ L sample was separated on a C- 18 reverse phase HPLC column (250 x 2.1 mm Inertsil 3-um ODS-3 by Varian) using a two solvent gradient system adapted from J.J. Dal luge et al (Dalluge (2002) Anal Bioanal Chem, 374(5): p. 835-40).
  • 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 12 minutes.
  • the elutant program was: 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 26 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
  • heterologous pathway proteins due solely to the expression of heterologous pathway proteins was evaluated by comparing post-induction to pre-induction profiles in the inactivated pathway strain (designated IAPS).
  • IAPS inactivated pathway strain
  • the combined burdens of heterologous protein expression and active flux through the engineered pathway were profiled in the amorpha-4-1 1 -diene-producing strain (designated APS) by comparing post-induction biomass samples with pre-induction samples.
  • the specific burden on the heterologous host associated solely with active biochemical flux through the amorpha-4-11-diene production pathway was profiled using direct comparison of biomass from each strain at each point over a time course.
  • the expression profiles reveal that the amorpha-4-11-diene producing strain (APS) is experiencing a high serine turn over which is straining the single carbon metabolism. This strain is reflected in the time course expression profile of the LRP regulon as well as the genes in the methionine, serine, glycine and histidine biosynthetic pathways. Single-carbon metabolism
  • Serine is subsequently converted into glycine by serine hydroxymethyltransferase (SHMT) in a reaction that transfers a methyl group to the Cl carrier molecule, tetrahydrofolate (THF).
  • SHMT serine hydroxymethyltransferase
  • THF tetrahydrofolate
  • This reaction provides all of the estimated 902 ⁇ mol/g-biomass of single carbon units required during growth in minimal medium, and as such the expression level of the gene encoding SHMT, glyA, is an important indicator of the state of single carbon metabolism.
  • the expression of glyA was up-regulated 2.8-fold at T3 in the APS (Table T). Expression of this gene was also up-regulated 2.8-fold in the IAPS control though this was observed at T2, after which expression of this gene returned to pre-induction levels at T3.
  • glyA expression was significantly higher at all time points in the APS relative to the control strain.
  • Methionine is the least utilized amino acid for protein synthesis in E. coli, but it is the key precursor to the primary methyl-carrier cofactor in E. coli, S-adenosyl-methionine (SAM). SAM acts as a transcriptional co-repressor, along with MetJ, of all the methionine biosynthetic genes except for metH.
  • SAM acts as a transcriptional co-repressor, along with MetJ, of all the methionine biosynthetic genes except for metH.
  • MetR another transcriptional regulator
  • methionine biosynthesis plays a central role in single carbon metabolism and the transcriptional profile of the met genes provides a biomarker for the state of single carbon metabolism during growth in minimal media.
  • the expression of all methionine biosynthetic genes except for metH were strongly up-regulated in the APS at T3, matching the profile observed for glyA.
  • Expression of the met genes also matched glyA expression in the LAPS, with an up-regulation at T2 followed by a down-regulation at T3.
  • the two strains are compared directly, the combined pattern is seen in the methionine biosynthetic genes: expression is higher at T2 in the control strain and higher at T3 in the amorpha-4- 1 1-diene-producing strain.
  • the leucine-responsive regulatory protein plays an important role in coordinating amino acid metabolism with the nutritional quality of the growth medium by regulating the transcription of several genes in E. coli. While there were no significant differences observed in the Lrp levels in either strain over the time course (Table 2), there were changes observed in the expression of leucine biosynthetic genes which likely had a significant impact on both strain's metabolism. Lrp's affinity for certain promoters is modulated by the intracellular leucine concentration, and the activity of this transcriptional regulator is reported to be 4-10 fold higher in minimal media (Calvo and Matthews (1994) Microbiol Rev, 58(3): p. 466-90).
  • Lrp is a transcriptional regulator
  • any significant changes in leucine availability would be observed as modulation of gene expression in the Lrp regulon in the microarray data. Indeed, significant transcriptional changes were observed in the Lrp regulon for both strains though determining whether changes in leucine biosynthesis contributed to this response was complicated by the fact that most transcriptional units in this regulon are controlled by additional regulatory signals and the effect of leucine on LRP affinity varies for each promoter.
  • HvKHMGF which encodes a branched-chain amino acid transporter and is repressed by Lrp but only in the presence of excess intracellular leucine
  • the time course expression profile of this transcriptional unit exhibited a post-induction up-regulation at T2 in the APS followed by a significant down-regulation at T3 while the LAPS control had a minor down-regulation at T2.
  • Expression of the Hv operon was observed to increase in the APS relative to the LAPS control until T2, after which expression was strongly down-regulated at T3.
  • the dipeptide transporter encoded by the oppABCDF is repressed by Lrp alone but activated by Lrp bound to leucine (Andrews and Short (1986) J Bacteriol, 165(2): p. 434-42).
  • There was a transient down-regulation of the opp genes observed in the APS which reached a minimum at T2, after which expression returned to pre-induction levels at T3.
  • expression of oppABCDF remained stable in the first two post-induction samples but was up-regulated by T3.
  • the strain-to-strain comparison of the opp expression profile indicates that expression of these genes was significantly higher in the APS prior to LPTG induction.
  • Histidine starts with ATP and 5-phosphoribosyl-a-l -pyrophosphate (PRPP) and requires ten biochemical reactions utilizing eight enzymes and 41 molecules of ATP. Histidine biosynthesis is also a consumer of single-carbon units because it starts with an adenine molecule (in the form of ATP), and thus requires two single-carbon units, one of which is recycled to the purine pool as 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR).
  • AICAR 5-aminoimidazole-4-carboxamide ribonucleotide
  • the his operon is regulated by the alarmone ppGpp, where increasing concentrations of the nucleotide Jead to higher operon expression.
  • the second main regulatory feature is transcription attenuation in the leader section of the his operon, which coordinates expression based on the amount of His-tRNA Hls .
  • the attenuator regulation of this operon is not as sensitive as would be theoretically possible — even when 77-88% of the tRNA's are charged there is still significant basal readthrough. Charged tRNA levels need to drop to ⁇ 12% for there to be a significant increase in operon transcription due to attenuator regulation.
  • the his operon's full length transcript has an in vivo half life of ⁇ 3 minutes while a secondary transcript of the 5 distal genes (hisBHAFI) is much more stable and has a ⁇ 15 minute half life in the cell.
  • the processing of the primary transcript into several secondary transcripts is modulated by the concentrations of various pathway intermediates. Inhibition of the first enzymatic step in the pathway leads to an increase in the primary transcript levels, while inhibitors that block steps down stream of AICAR lead to increases in both the primary transcript and the stable secondary transcript.
  • the addition of an inhibitor that lowers the over all formylation level in the cell causes a decrease in primary transcript and an increase in the stable secondary transcript (Alifano (1994) Gene, 146(1): p. 15-21). Because AICAR must be formylated in order to return to the purine pool, the organism must coordinate the generation of AICAR by histidine biosynthesis with the overall formylating capacity of the cell.
  • Serine and single carbon units are utilized by several biochemical pathways branching from chorismate.
  • the biosynthesis of tryptophan and enterobactin directly consume one and three molecules of serine, respectively, while quinone biosynthesis utilizes SAM in three sequential methylation reactions.
  • Many of the genes in the highly branched chorismate pathway are expressed constitutively, but several are transcriptionally regulated in response to aromatic amino acid and iron levels.
  • E. coli releases the catecholate siderophore enterobactin into its growth medium to bind Fe(III).
  • Enterobactin is synthesized from the shikimate pathway intermediate chorismate via a pathway consisting of six enzymes encoded in three operons (entABCE, entD and entF). These operons were up-regulated in both the LAPS and the APS, though the post-induction response was far stronger in the APS. Transcription of the ent operon is controlled by the ferric uptake regulation protein (Fur); thus, an up-regulation of this pathway suggests a more significant iron limitation in the APS than in the LAPS. Since the Fur regulon was observed to be up- regulated in both strains relative to pre-induction levels the strain-to-strain comparison provided insight into the relative activation of this regulon.
  • the iron-sulfur cluster repair genes encoded by the sufABCSDE operon, the succinate dehydrogenase/2 -ketoglutarate dehydrogenase complex encoded by sdhABCDE-sucABCD, and portions of a ribonucleoside-diphosphate reductase complex encoded by nrdHEIF have all been reported to be Fur regulated, and each exhibited a temporal pattern consistent with de-repression of this regulon in the APS.
  • active biochemical flux through the amorpha-4- 1 1-diene production pathway activates the Fur regulon.
  • E. coli As a colonizer of the mammalian gut, E. coli has an optimal growth temperature around 37°C but can grow in a fairly wide range of temperatures (at least 25 0 C to 42 0 C).
  • the heat shock regulon is actually the cell's response to misfolded protein in the cytoplasm or in the periplasm and can be induced by a wide range of conditions including growth at high temperature ( ⁇ 42°C or higher), exposure to solvents, protein over expression, viral infection and alterations in Cl metabolism. While this regulon is most often identified as a stress regulon, this is really a misnomer since this group of proteins is required for growth at all temperatures.
  • the genes in this regulon consist mostly of chaperones and proteases and, as such, are critical for the normal functioning of E. co//'s translational machinery.
  • Glucose and nitrogen uptake were higher in the amorpha-4- 1 1 -diene producing strain (APS) in the first two cell-doublings following LPTG induction of heterologous protein expression. Additionally, the APS accumulated acetyl-CoA in the post-induction samples and excreted more acetate over the entire time course than the LAPS. Both strains up-regulated expression of the acetate-evolving ackA-pta pathway 2-3-fold at later time points, though by an equal amount, since the strain-to-strain transcriptomic comparisons show no differential expression for these genes. Since both strains grew in a condition of carbon excess, the higher glucose uptake, acetate excretion and CO 2 evolution in the APS suggest there was higher glycolytic flux in this strain compared to the LAPS.
  • HMG-CoA There was no significant accumulation of HMG-CoA observed, while acetoacetyl-CoA and mevalonate concentrations increased over time. Malonyl-CoA is an important biomarker of a growth inhibition associated with HMG-CoA accumulation (Pitera et al. (2007) Metab Eng 9(2): 193-207), but there was no significant accumulation of this acyl-CoA observed in the APS, indicating that the enzymatic activity in the engineered pathway was balanced, and HMG-CoA levels remained below growth-inhibiting concentrations.
  • Mevalonate accumulation to approximately 400 nM/OD 6 oo was observed at later time points in the APS, indicating the rate of biochemical flux through the later stages of the amorpha-4-11-diene pathway limited production.
  • the only heterologous proteins that were not detected by proteomic analysis were mevalonate kinase and mevalonate phosphokinase, which implies these proteins were not highly expressed and the conversion of mevalonate into prenyl phosphates may limit overall production titer.
  • Media supplementation improves amorpha-4-11-diene titer
  • the APS transcriptional and proteomic profiles suggested a possible iron and single carbon unit limitation was associated with amorpha-4-1 1-diene production in E. coli.
  • the APS was grown in M9-glucose medium with and without additional FeSO 4 ; there was no significant difference in amorpha-4-1 1-diene production or growth (data not shown).
  • MOPS-minimal medium supplemented with the aromatic amino acids as well as precursors to tetrahydrofolate, quinone and enterobactin (shikimate media) increased specific production to 64 mg/L-OD.
  • the MOPS-minimal, MGL and shikimate media all had a low total production titer, most likely because they did not support growth to as high a final cell density as the MOPS-defined rich, 8-AA or Cl media.
  • the MOPS-defined rich growth medium which contains all supplements, provided both the high final titer (402 mg/L) and specific production (69 mg/L-OD) that would be expected.
  • the 8-AA medium has been reported to increase recombinant protein production by supplementing eight amino acids (alanine, glutamine, glutamic acid, isoleucine, leucine, methionine, serine and valine).
  • the performance of this growth medium matched MOPS-defined rich medium very closely, both in final cell density and amorpha-4- 11 -diene production.
  • the Cl medium which is MOPS minimal medium supplemented with serine, glycine, methionine, adenine, and guanine, provided the highest final production titer of 499 mg/L and specific production at 92 mg/L-OD. This represented a nearly 5-fold improvement in total amorpha-4-11-diene production and a 2-fold improvement in specific production.

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Abstract

La présente invention concerne des procédés de fabrication d'un isoprénoïde ou d'un précurseur d'isoprénoïde dans une cellule hôte productrice d'isoprénoïde, qui comprennent généralement la culture de cellules productrices d'isoprénoïde dans un milieu de culture défini qui comprend de la sérine.
PCT/US2008/007990 2007-07-03 2008-06-27 Procédés d'augmentation de la production d'isoprénoïde ou de précurseur d'isoprénoïde Ceased WO2009005704A1 (fr)

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