# Patent

## Title
Compositions and methods for producing isoprene

## Abstract

The invention features methods for producing isoprene from cultured cells. The invention also provides compositions that include these cultured cells.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority benefit of U.S. Provisional patent application No. 61/013,574, filed on Dec. 13, 2007. The content of that application is hereby incorporated by reference herein in its entirety.


FIELD OF THE INVENTION
The present invention relates generally to methods for producing isoprene from cultured cells and compositions that include these cultured cells.
BACKGROUND OF THE INVENTION
Isoprene (2-methyl-1,3-butadiene) is the critical starting material for a variety of synthetic polymers, most notably synthetic rubbers. Isoprene is naturally produced by a variety of microbial, plant, and animal species. In particular, two pathways have been identified for the biosynthesis of isoprene: the mevalonate (MVA) pathway and the non-mevalonate (DXP) pathway (FIGS. 19A and 19B). However, the yield of isoprene from naturally-occurring organisms is commercially unattractive. About 800,000 tons per year of cis-polyisoprene are produced from the polymerization of isoprene; most of this polyisoprene is used in the tire and rubber industry. Isoprene is also copolymerized for use as a synthetic elastomer in other products such as footwear, mechanical products, medical products, sporting goods, and latex.
Currently, the tire and rubber industry is based on the use of natural and synthetic rubber. Natural rubber is obtained from the milky juice of rubber trees or plants found in the rainforests of Africa. Synthetic rubber is based primarily on butadiene polymers. For these polymers, butadiene is obtained as a co-product from ethylene and propylene manufacture.
While isoprene can be obtained by fractionating petroleum, the purification of this material is expensive and time-consuming. Petroleum cracking of the C5 stream of hydrocarbons produces only about 15% isoprene. Thus, more economical methods for producing isoprene are needed. In particular, methods that produce isoprene at rates, titers, and purity that are sufficient to meet the demands of a robust commercial process are desirable. Also desired are systems for producing isoprene from inexpensive starting materials.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the invention features cells in culture that produce isoprene. In some embodiments, the invention provides cells in culture that produce greater than about 400 nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/gwcm/hr) of isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate (e.g., xylose or glucose), acetate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source (e.g., a hydrolyzed biomass carbon source), polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing.
In some embodiments, the invention provides cells in culture that have an average volumetric productivity of isoprene greater than about 0.1 mg/Lbroth/hr. In some embodiments, the invention provides cells in culture that have a peak volumetric productivity of isoprene greater than about 0.5 mg/Lbroth/hr. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate (e.g., xylose or glucose), acetate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source (e.g., a hydrolyzed biomass carbon source), polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing.
In some embodiments, the invention provides cells in culture that convert more than about 0.002% of the carbon in a cell culture medium into isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate (e.g., xylose or glucose), acetate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source (e.g., a hydrolyzed biomass carbon source), polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing.
In some embodiments, the invention provides cells in culture that comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate (e.g., xylose or glucose), acetate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source (e.g., a hydrolyzed biomass carbon source), polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing.
In one aspect, the invention features methods of producing isoprene, such as methods of using any of the cells described herein to produce isoprene. In some embodiments, the method involves culturing cells under conditions sufficient to produce greater than about 400 nmole/gwcm/hr of isoprene. In some embodiments, the method also includes recovering isoprene produced by the cells. In some embodiments, the method includes purifying isoprene produced by the cells. In some embodiments, the method includes polymerizing the isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate (e.g., xylose or glucose), acetate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source (e.g., a hydrolyzed biomass carbon source), polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing.
In one aspect, the invention features methods of producing isoprene, such as methods of using any of the cells described herein to produce isoprene. In some embodiments, the method involves culturing cells under conditions resulting in an average volumetric productivity of isoprene greater than about 0.1 mg/Lbroth/hr. In some embodiments, the method involves culturing cells under conditions resulting in a peak volumetric productivity of isoprene greater than about 0.5 mg/Lbroth/hr. In some embodiments, the method also includes recovering isoprene produced by the cells. In some embodiments, the method includes purifying isoprene produced by the cells. In some embodiments, the method includes polymerizing the isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate (e.g., xylose or glucose), acetate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source (e.g., a hydrolyzed biomass carbon source), polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing.
In some embodiments, the method includes culturing cells under conditions sufficient to convert more than about 0.002% of the carbon in a cell culture medium into isoprene. In some embodiments, the method also includes recovering isoprene produced by the cells. In some embodiments, the method includes purifying isoprene produced by the cells. In some embodiments, the method includes polymerizing the isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate (e.g., xylose or glucose), acetate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source (e.g., a hydrolyzed biomass carbon source), polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing.
In some embodiments of any of the aspects of the invention, the cells in culture produce isoprene at greater than or about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 12,500, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000, 125,000, 150,000, 188,000, or more nmole/gwcm/hr isoprene. In some embodiments, the cells in culture have an average volumetric productivity of isoprene at greater than or about 0.1, 1.0, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100, 1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, or more mg of isoprene/L of broth/hr (mg/Lbroth/hr, wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments, the cells in culture have a peak volumetric productivity of isoprene at greater than or about 0.5, 1.0, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100, 1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, 5,000, 5,250, 5,500, 5,750, 6,000, 6,250, 6,500, 6,750, 7,000, 7,250, 7,500, 7,750, 8,000, 8,250, 8,500, 8,750, 9,000, 9,250, 9,500, 9,750, 10,000, 12,500, 15,000, or more mg of isoprene/L of broth/hr (mg/Lbroth/hr, wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments of any of the aspects of the invention, the cells in culture convert greater than or about 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 2.0, 2.2, 2.4, 2.6, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 23.2, 23.4, 23.6, 23.8, 24.0, 25.0, 30.0, 31.0, 32.0, 33.0, 35.0, 37.5, 40.0, 45.0, 47.5, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0 molar %, or more of the carbon in the cell culture medium into isoprene. In some embodiments of any of the aspects of the invention, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wet weight of the cells/hr (ng/gwcm/h). In some embodiments of any of the aspects of the invention, the cells in culture produce a cumulative titer (total amount) of isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth (mg/Lbroth, wherein the volume of broth includes the volume of the cells and the cell medium). Other exemplary rates of isoprene production and total amounts of isoprene production are disclosed herein.
In some embodiments of any of the aspects of the invention, the cells further comprise a heterologous nucleic acid encoding an IDI polypeptide. In some embodiments of any of the aspects of the invention, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding an IDI polypeptide. In some embodiments of any of the aspects of the invention, the cells further comprise a heterologous nucleic acid encoding a DXS polypeptide. In some embodiments of any of the aspects of the invention, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding a DXS polypeptide. In some embodiments of any of the aspects of the invention, the cells further comprise one or more nucleic acids encoding an IDI polypeptide and a DXS polypeptide. In some embodiments of any of the aspects of the invention, one nucleic acid encodes the isoprene synthase polypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments of any of the aspects of the invention, one vector encodes the isoprene synthase polypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments, the vector comprises a selective marker, such as an antibiotic resistance nucleic acid.
In some embodiments of any of the aspects of the invention, the heterologous isoprene synthase nucleic acid is operably linked to a T7 promoter, such as a T7 promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects of the invention, the heterologous isoprene synthase nucleic acid is operably linked to a Trc promoter, such as a Trc promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects of the invention, the heterologous isoprene synthase nucleic acid is operably linked to a Lac promoter, such as a Lac promoter contained in a low copy plasmid. In some embodiments of any of the aspects of the invention, the heterologous isoprene synthase nucleic acid is operably linked to an endogenous promoter, such as an endogenous alkaline serine protease promoter. In some embodiments, the heterologous isoprene synthase nucleic acid integrates into a chromosome of the cells without a selective marker.
In some embodiments of any of the aspects of the invention, at least a portion of the cells maintain the heterologous isoprene synthase nucleic acid for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects of the invention, the nucleic acid comprising the isoprene synthase, IDI, or DXS nucleic acid also comprises a selective marker, such as an antibiotic resistance nucleic acid.
In some embodiments of any of the aspects of the invention, the cells further comprise a heterologous nucleic acid encoding an MVA pathway polypeptide (such as an MVA pathway polypeptide from Saccharomyces cerevisi, Methanosarcina mazei, or Enterococcus faecalis). In some embodiments of any of the aspects of the invention, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding an MVA pathway polypeptide (such as an MVA pathway polypeptide from Saccharomyces cerevisia, Methanosarcina mazei, or Enterococcus faecalis). In some embodiments of any of the aspects of the invention, the cells comprise an isoprene synthase, DXS, and MVA pathway nucleic acid. In some embodiments of any of the aspects of the invention, the cells comprise an isoprene synthase nucleic acid, a DXS nucleic acid, an IDI nucleic acid, and a MVA pathway nucleic (in addition to the IDI nucleic acid).
In some embodiments of any of the aspects of the invention, the isoprene synthase polypeptide is a naturally-occurring polypeptide from a plant such as Pueraria (e.g., Pueraria montana) or Populus (e.g., Populus tremuloides, Populus alba (P. alba), Populus nigra, Populus trichocarpa, or the hybrid, Populus alba x Populus tremula).
In some embodiments of any of the aspects of the invention, the cells are bacterial cells, such as gram-positive bacterial cells (e.g., Bacillus cells such as Bacillus subtilis cells or Streptomyces cells such as Streptomyces lividans, Streptomyces coelicolor, Streptomyces coelicolor, Streptomyces albus, or Streptomyces griseus cells). In some embodiments of any of the aspects of the invention, the cells are gram-negative bacterial cells (e.g., Escherichia cells such as Escherichia coli cells or Pantoea cells such as Pantoea citrea cells). In some embodiments, the E. coli cells are E. coli FadR atoC mutant cells. In some embodiments, the E. coli cells express (such as constitutively express) ybhE (also known as pgl). In some embodiments of any of the aspects of the invention, the cells are fungal, cells such as filamentous funal cells (e.g., Trichoderma cells such as Trichoderma reesei cells or Aspergillus cells such as Aspergillus oryzae and Aspergillus niger) or yeast cells (e.g., Yarrowia cells such as Yarrowia lipolytica cells).
In some embodiments of any of the aspects of the invention, the microbial polypeptide carbon source includes one or more polypeptides from yeast or bacteria. In some embodiments of any of the aspects of the invention, the plant polypeptide carbon source includes one or more polypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.
In one aspect, the invention provides a tire comprising polyisoprene. In some embodiments, the polyisoprene is produced by (i) polymerizing isoprene from any of the compositions or methods described herein or (ii) polymerizing isoprene recovered from any of the compositions or methods described herein. In some embodiments, the polyisoprene comprises cis-1,4-polyisoprene.
In one aspect, the invention features a product (such as a tire) produced by any of the compositions or methods of the invention.
It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is the nucleotide sequence of a kudzu isoprene synthase gene codon-optimized for expression in E. coli (SEQ ID NO:1). The atg start codon is in italics, the stop codon is in bold and the added PstI site is underlined.
FIG. 2 is a map of pTrcKudzu.
FIG. 3 is the nucleotide sequence of pTrcKudzu (SEQ ID NO:2). The RBS is underlined, the kudzu isoprene synthase start codon is in bold capitol letters and the stop codon is in bold, capitol, italics letters. The vector backbone is pTrcHis2B.
FIG. 4 is a map of pETNHisKudzu.
FIG. 5 is the nucleotide sequence of pETNHisKudzu (SEQ ID NO:5).
FIG. 6 is a map of pCL-lac-Kudzu.
FIG. 7 is the nucleotide sequence of pCL-lac-Kudzu (SEQ ID NO:7).
FIG. 8A is a graph showing the production of isoprene in E. coli BL21 cells with no vector.
FIG. 8B is a graph showing the production of isoprene in E. coli BL21 cells with pCL-lac-Kudzu
FIG. 8C is a graph showing the production of isoprene in E. coli BL21 cells with pTrcKudzu.
FIG. 8D is a graph showing the production of isoprene in E. coli BL21 cells with pETN-HisKudzu.
FIG. 9A is a graph showing OD over time of fermentation of E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.
FIG. 9B is a graph showing isoprene production over time of fermentation of E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.
FIG. 10A is a graph showing the production of isoprene in Panteoa citrea. Control cells without recombinant kudzu isoprene synthase. Grey diamonds represent isoprene synthesis, black squares represent OD600.
FIG. 10B is a graph showing the production of isoprene in Panteoa citrea expressing pCL-lac Kudzu. Grey diamonds represent isoprene synthesis, black squares represent OD600.
FIG. 10C is a graph showing the production of isoprene in Panteoa citrea expressing pTrcKudzu. Grey diamonds represent isoprene synthesis, black squares represent OD600.
FIG. 11 is a graph showing the production of isoprene in Bacillus subtilis expressing recombinant isoprene synthase. BG3594comK is a B. subtilis strain without plasmid (native isoprene production). CF443-BG3594comK is a B. subtilis strain with pBSKudzu (recombinant isoprene production). IS on the y-axis indicates isoprene.
FIG. 12 is the nucleotide sequence of pBS Kudzu #2 (SEQ ID NO:57).
FIG. 13 is the nucleotide sequence of kudzu isoprene synthase codon-optimized for expression in Yarrowia (SEQ ID NO:8).
FIG. 14 is a map of pTrex3g comprising a kudzu isoprene synthase gene codon-optimized for expression in Yarrowia. 
FIG. 15 is the nucleotide sequence of vector pSPZ1(MAP29Spb) (SEQ ID NO:11).
FIG. 16 is the nucleotide sequence of the synthetic kudzu (Pueraria montana) isoprene gene codon-optimized for expression in Yarrowia (SEQ ID NO:12).
FIG. 17 is the nucleotide sequence of the synthetic hybrid poplar (Populus alba x Populus tremula) isoprene synthase gene (SEQ ID NO:13). The ATG start codon is in bold and the stop codon is underlined.
FIG. 18A shows a schematic outlining construction of vectors pYLA 1, pYL1 and pYL2.
FIG. 18B shows a schematic outlining construction of the vector pYLA(POP1).
FIG. 18C shows a schematic outlining construction of the vector pYLA(KZ1)
FIG. 18D shows a schematic outlining construction of the vector pYLI(KZ1)
FIG. 18E shows a schematic outlining construction of the vector pYLI(MAP29)
FIG. 18F shows a schematic outlining construction of the vector pYLA(MAP29)
FIG. 19A shows the MVA and DXP metabolic pathways for isoprene (based on F. Bouvier et al., Progress in Lipid Res. 44: 357-429, 2005). The following description includes alternative names for each polypeptide in the pathways and a reference that discloses an assay for measuring the activity of the indicated polypeptide (each of these references are each hereby incorporated by reference in their entireties, particularly with respect to assays for polypeptide activity for polypeptides in the MVA and DXP pathways). Mevalonate Pathway: AACT; Acetyl-CoA acetyltransferase, MvaE, EC 2.3.1.9. Assay: J. Bacteriol., 184: 2116-2122, 2002; HMGS; Hydroxymethylglutaryl-CoA synthase, MvaS, EC 2.3.3.10. Assay: J. Bacteriol., 184: 4065-4070, 2002; HMGR; 3-Hydroxy-3-methylglutaryl-CoA reductase, MvaE, EC 1.1.1.34. Assay: J. Bacteriol., 184: 2116-2122, 2002; MVK; Mevalonate kinase, ERG12, EC 2.7.1.36. Assay: Curr Genet. 19:9-14, 1991. PMK; Phosphomevalonate kinase, ERG8, EC 2.7.4.2, Assay: Mol Cell Biol., 11:620-631, 1991; DPMDC; Diphosphomevalonate decarboxylase, MVD1, EC 4.1.1.33. Assay: J. Biochemistry, 33:13355-13362, 1994; IDI; Isopentenyl-diphosphate delta-isomerase, IDI1, EC 5.3.3.2. Assay: J. Biol. Chem. 264:19169-19175, 1989. DXP Pathway: DXS; 1-Deoxyxylulose-5-phosphate synthase, dxs, EC 2.2.1.7. Assay: PNAS, 94:12857-62, 1997; DXR; 1-Deoxy-D-xylulose 5-phosphate reductoisomerase, dxr, EC 2.2.1.7. Assay: Eur. J. Biochem. 269:4446-4457, 2002; MCT; 4-Diphosphocytidyl-2C-methyl-D-erythritol synthase, IspD, EC 2.7.7.60. Assay: PNAS, 97: 6451-6456, 2000; CMK; 4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspE, EC 2.7.1.148. Assay: PNAS, 97:1062-1067, 2000; MCS; 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase, IspF, EC 4.6.1.12. Assay: PNAS, 96:11758-11763, 1999; HDS; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, ispG, EC 1.17.4.3. Assay: J. Org. Chem., 70:9168-9174, 2005; HDR; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, IspH, EC 1.17.1.2. Assay: JACS, 126:12847-12855, 2004.
FIG. 19B illustrates the classical and modified MVA pathways. 1, acetyl-CoA acetyltransferase (AACT); 2, HMG-CoA synthase (HMGS); 3, HMG-CoA reductase (HMGR); 4, mevalonate kinase (MVK); 5, phosphomevalonate kinase (PMK); 6, diphosphomevalonate decarboxylase (MVD or DPMDC); 7, isopentenyl diphosphate isomerase (IDI); 8, phosphomevalonate decarboxylase (PMDC); 9, isopentenyl phosphate kinase (IPK). The classical MVA pathway proceeds from reaction 1 through reaction 7 via reactions 5 and 6, while a modified MVA pathway goes through reactions 8 and 9. P and PP in the structural formula are phosphate and pyrophosphate, respectively. This figure was taken from Koga and Morii, Microbiology and Mol. Biology. Reviews, 71:97-120, 2007, which is incorporated by reference in its entirety, particularly with respect to nucleic acids and polypeptides of the modified MVA pathway. The modified MVA pathway is present, for example, in some archaeal organisms, such as Methanosarcina mazei. 
FIG. 20 shows graphs representing results of the GC-MS analysis of isoprene production by recombinant Y. lipolytica strains without (left) or with (right) a kudzu isoprene synthase gene. The arrows indicate the elution time of the authentic isoprene standard.
FIG. 21 is a map of pTrcKudzu yIDI DXS Kan.
FIG. 22 is the nucleotide sequence of pTrcKudzu yIDI DXS Kan (SEQ ID NO:20).
FIG. 23A is a graph showing production of isoprene from glucose in BL21/pTrcKudzukan. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).
FIG. 23B is a graph showing production of isoprene from glucose in BL21/pTrcKudzu yIDI kan. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).
FIG. 23C is a graph showing production of isoprene from glucose in BL21/pTrcKudzu DXS kan. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).
FIG. 23D is a graph showing production of isoprene from glucose in BL21/pTrcKudzu yIDI DXS kan. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).
FIG. 23E is a graph showing production of isoprene from glucose in BL21/pCL PtrcKudzu. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).
FIG. 23F is a graph showing production of isoprene from glucose in BL21/pCL PtrcKudzu yIDI. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).
FIG. 23G is a graph showing production of isoprene from glucose in BL21/pCL PtrcKudzu DXS. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).
FIG. 23H is a graph showing production of isoprene from glucose in BL21/pTrcKudzuIDIDXSkan. The arrow indicates the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Black diamonds represent OD600, black triangles represent isoprene productivity (μg/L) and white squares represent specific productivity of isoprene (μg/L/OD).
FIG. 24 is a map of pTrcKKDyIkIS kan.
FIG. 25 is a nucleotide sequence of pTrcKKDyIkIS kan (SEQ ID NO:33).
FIG. 26 is a map of pCL PtrcUpperPathway.
FIG. 27 is a nucleotide sequence of pCL PtrcUpperPathway (SEQ ID NO:46).
FIG. 28 shows a map of the cassette containing the lower MVA pathway and yeast idi for integration into the B. subtilis chromosome at the nprE locus. nprE upstream/downstream indicates 1 kb each of sequence from the nprE locus for integration. aprE promoter (alkaline serine protease promoter) indicates the promoter (−35, −10, +1 transcription start site, RBS) of the aprE gene. MVK1 indicates the yeast mevalonate kinase gene. RBS-PMK indicates the yeast phosphomevalonte kinase gene with a Bacillus RBS upstream of the start site. RBS-MPD indicates the yeast diphosphomevalonate decarboxylase gene with a Bacillus RBS upstream of the start site. RBS-IDI indicates the yeast idi gene with a Bacillus RBS upstream of the start site. Terminator indicates the terminator alkaline serine protease transcription terminator from B. amyliquefaciens. SpecR indicates the spectinomycin resistance marker. “nprE upstream repeat for amp.” indicates a direct repeat of the upstream region used for amplification.
FIG. 29 is a nucleotide sequence of cassette containing the lower MVA pathway and yeast idi for integration into the B. subtilis chromosome at the nprE locus (SEQ ID NO:47).
FIG. 30 is a map of p9796-poplar.
FIG. 31 is a nucleotide sequence of p9796-poplar (SEQ ID NO:48).
FIG. 32 is a map of pTrcPoplar.
FIG. 33 is a nucleotide sequence of pTrcPoplar (SEQ ID NO:49).
FIG. 34 is a map of pTrcKudzu yIDI Kan.
FIG. 35 is a nucleotide sequence of pTrcKudzu yIDI Kan (SEQ ID NO:50).
FIG. 36 is a map of pTrcKudzuDXS Kan.
FIG. 37 is a nucleotide sequence of pTrcKudzuDXS Kan (SEQ ID NO:51).
FIG. 38 is a map of pCL PtrcKudzu.
FIG. 39 is a nucleotide sequence of pCL PtrcKudzu (SEQ ID NO:52).
FIG. 40 is a map of pCL PtrcKudzu A3.
FIG. 41 is a nucleotide sequence of pCL PtrcKudzu A3 (SEQ ID NO:53).
FIG. 42 is a map of pCL PtrcKudzu yIDI.
FIG. 43 is a nucleotide sequence of pCL PtrcKudzu yIDI (SEQ ID NO:54).
FIG. 44 is a map of pCL PtrcKudzu DXS.
FIG. 45 is a nucleotide sequence of pCL PtrcKudzu DXS (SEQ ID NO:55).
FIG. 46 shows graphs representing isoprene production from biomass feedstocks. Panel A shows isoprene production from corn stover, Panel B shows isoprene production from bagasse, Panel C shows isoprene production from softwood pulp, Panel D shows isoprene production from glucose, and Panel E shows isoprene production from cells with no additional feedstock. Grey squares represent OD600 measurements of the cultures at the indicated times post-inoculation and black triangles represent isoprene production at the indicated times post-inoculation.
FIG. 47A shows a graph representing isoprene production by BL21 (λDE3) pTrcKudzu yIDI DXS (kan) in a culture with no glucose added. Squares represent OD600, and triangles represent isoprene produced (μg/ml).
FIG. 47B shows a graph representing isoprene production from 1% glucose feedstock invert sugar by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squares represent OD600, and triangles represent isoprene produced (μg/ml).
FIG. 47C shows a graph representing isoprene production from 1% invert sugar feedstock by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squares represent OD600, and triangles represent isoprene produced (μg/ml).
FIG. 47D shows a graph representing isoprene production from 1% AFEX corn stover feedstock by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squares represent OD600, and triangles represent isoprene produced (μg/ml).
FIG. 48 shows graphs demonstrating the effect of yeast extract of isoprene production. Panel A shows the time course of optical density within fermentors fed with varying amounts of yeast extract. Panel B shows the time course of isoprene titer within fermentors fed with varying amounts of yeast extract. The titer is defined as the amount of isoprene produced per liter of fermentation broth. Panel C shows the effect of yeast extract on isoprene production in E. coli grown in fed-batch culture.
FIG. 49 shows graphs demonstrating isoprene production from a 500 L bioreactor with E. coli cells containing the pTrcKudzu and yIDI and DXS plasmid. Panel A shows the time course of optical density within the 500-L bioreactor fed with glucose and yeast extract. Panel B shows the time course of isoprene titer within the 500-L bioreactor fed with glucose and yeast extract. The titer is defined as the amount of isoprene produced per liter of fermentation broth. Panel C shows the time course of total isoprene produced from the 500-L bioreactor fed with glucose and yeast extract.
FIG. 50 is a map of pJMupperpathway2.
FIG. 51 is the nucleotide sequence of pJMupperpathway2 (SEQ ID NO:56).
FIG. 52 is a map of pBS Kudzu #2.
FIG. 53A is a graph showing growth during fermentation time of Bacillus expressing recombinant kudzu isoprene synthase in 14 liter fed batch fermentation. Black diamonds represent a control strain (BG3594comK) without recombinant isoprene synthase (native isoprene production) and grey triangles represent Bacillus with pBSKudzu (recombinant isoprene production).
FIG. 53B is a graph showing isoprene production during fermentation time of Bacillus expressing recombinant kudzu isoprene synthase in 14 liter fed batch fermentation. Black diamonds represent a control strain (BG3594comK) without recombinant isoprene synthase (native isoprene production) and grey triangles represent Bacillus with pBSKudzu (recombinant isoprene production).
FIG. 54 is a map of plasmid pET24 P. alba HGS.
FIGS. 55A and 55B are the nucleotide sequence of plasmid pET24 P. alba HGS (SEQ ID NO:87).
FIG. 56 is a schematic diagram showing restriction sites used for endonuclease digestion to construct plasmid EWL230 and compatible cohesive ends between BspHI and NcoI sites.
FIG. 57 is a map of plasmid EWL230.
FIGS. 58A and 58B are the nucleotide sequence of plasmid EWL230 (SEQ ID NO:88).
FIG. 59 is a schematic diagram showing restriction sites used for endonuclease digestion to construct plasmid EWL244 and compatible cohesive ends between NsiI and PstI sites.
FIG. 60 is a map of EWL244.
FIGS. 61A and 61B are the nucleotide sequence of plasmid EWL244 (SEQ ID NO:89).
FIG. 62 is a map of plasmids MCM484-487.
FIGS. 63A-63C are the nucleotide sequence of plasmid MCM484 (SEQ ID NO:90).
FIGS. 64A-64C are the nucleotide sequence of plasmid MCM485 (SEQ ID NO:91).
FIGS. 65A-65C are the nucleotide sequence of plasmid MCM486 (SEQ ID NO:92).
FIGS. 66A-66C are the nucleotide sequence of plasmid MCM487 (SEQ ID NO:93).
FIGS. 67A-67D are graphs of isoprene production by E. coli strain (EWL256) expressing genes from the MVA pathway and grown in fed-batch culture at the 15-L scale without yeast extract feeding. FIG. 67A shows the time course of optical density within the 15-L bioreactor fed with glucose. FIG. 67B shows the time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.
FIG. 67C shows the time course of total isoprene produced from the 15-L bioreactor fed with glucose. FIG. 67D shows the total carbon dioxide evolution rate (TCER), or metabolic activity profile, within the 15-L bioreactor fed with glucose.
FIGS. 68A-68E are graphs of isoprene production by E. coli strain (EWL256) expressing genes from the MVA pathway and grown in fed-batch culture at the 15-L scale with yeast extract feeding. FIG. 68A shows the time course of optical density within the 15-L bioreactor fed with glucose. FIG. 68B shows the time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth. FIG. 68C shows the time course of total isoprene produced from the 15-L bioreactor fed with glucose. FIG. 68D shows the volumetric productivity within the 15-L bioreactor fed with glucose. An average value of 1.1 g/L/hr was maintained for a 40-hour period (23-63 hours) with yeast extract feeding. FIG. 68E shows the carbon dioxide evolution rate (CER), or metabolic activity profile, within the 15-L bioreactor fed with glucose.
FIGS. 69A-69D shows production of isoprene from different carbon sources via the MVA (pathway). FIG. 69A shows growth of E. coli EWL256, which contains both the MVA pathway and isoprene synthase, on either glucose, biomass hydrolysate, glycerol, or acetate as the only carbon source. The different carbon sources were added to a concentration of 1% in the media. A negative control with no added carbon source was included. Growth was measured as optical density at 600 nM. FIG. 69B shows specific productivity of isoprene from E. coli EWL256 containing both the MVA pathway and isoprene synthase when grown on either glucose, biomass hydrolysate, glycerol, or acetate as only carbon source. The different carbon sources were added to a concentration of 1% in the media. A negative control with no added carbon source was included. Samples were taken 190 minutes, 255 minutes and 317 minutes after inoculation and isoprene produced by the bacteria was measured using GC-MS. FIG. 69C shows growth of E. coli EWL256 on either glucose or xylose as the only carbon source. The different carbon sources were added to a concentration of 1% in the media. A negative control with no added carbon source was included. Growth was measured as optical density at 600 nM. FIG. 69D shows specific productivity of isoprene from E. coli EWL256 when grown on either glucose or xylose as only carbon source. The carbon sources were added to a concentration of 1% in the media. A negative control with no added carbon source was included. Samples were taken 260 minutes, 322 minutes and 383 minutes after inoculation and isoprene produced by the bacteria was measured using GC-MS.
FIGS. 70A and 70B show the production of isoprene by E. coli strains from glucose and from fatty acid, respectively. For FIG. 70A, eleven colonies from the transformation of WW4 with pMCM118, the plasmid bearing the lower mevalonic acid pathway, were picked to verify the presence of the lower pathway. Cell from the colonies were cultured in TM3 medium containing 0.1% yeast extract and 2% glucose. Aliquots of induced culture were assayed for isoprene production after 4 hours of induction. All colonies showed the production of isoprene. The inducer IPTG had a strong growth inhibitory effect as was evident from the 3 to 4.6-fold reduced cell density in going from 50 to 900 uM concentration of the inducer (data not shown). The graph shows that higher induction, yields a higher specific titer of isoprene. For FIG. 70B, the production culture was inoculated from a washed overnight culture at 1 to 10 dilution. The culture was grown for several hours and induced with 50 uM IPTG. The left bar shows isoprene assay results four hours after induction followed by a one hour isoprene accumulation assay. The middle bar shows the one hour normalized value for the same culture with the same induction period but analyzed by a 12 hour isoprene accumulation assay. The right bar shows the value for a one hour isoprene accumulation assay of the culture that was induced for 13 hours.
FIG. 71 is a map of the E. coli-Streptomyces shuttle vector pUWL201PW (6400 bp) used for cloning isoprene synthase from Kudzu. Tsr, thiostrepton resistance gene. Picture is taken from Doumith et al., Mol. Gen. Genet. 264: 477-485, 2000.
FIG. 72 shows isoprene formation by Streptomyces albus wild type strain (“wt”) and strains harboring plasmid pUWL201PW (negative control) or pUWL201_iso (encoding isoprene synthase from Kudzu).
FIG. 73A is a map of the M. mazei archaeal Lower Pathway operon.
FIGS. 73B and 73C are the nucleotide sequence of the M. mazei archaeal lower Pathway operon (SEQ ID NO:113).
FIG. 74A is a map of MCM376-MVK from M. mazei archaeal Lowerin pET200D.
FIGS. 74B and 74C are the nucleotide sequence of MCM376-MVK from M. mazei archaeal Lowerin pET200D (SEQ ID NO:114).
FIGS. 75A-75D show growth and specific productivity of isoprene production for EWL256 compared to RM11608-2. Growth (OD550) is represented by the white diamonds; specific productivity of isoprene is represented by the solid bars. The x-axis is time (hours) post-induction with either 200 (FIGS. 75A and 75B) or 400 (FIGS. 75C and 75D) uM IPTG. Y-1 axis is productivity of isoprene (ug/L/OD/hr) and Y-2 is arbitrary units of optical density at a wavelength of 550. These values for the OD550 must be multiplied by 6.66 to obtain the actual OD of the culture.
FIG. 76 is a map of plasmid pBBRCMPGI1.5-pgl.
FIGS. 77A and 77B are the nucleotide sequence of plasmid pBBRCMPGI1.5-pgl (SEQ ID NO:122).
FIGS. 78A-78F are graphs of isoprene production by E. coli strain expressing M. mazei mevalonate kinase, P. alba isoprene synthase, and pgl (RHM111608-2), and grown in fed-batch culture at the 15-L scale. FIG. 78A shows the time course of optical density within the 15-L bioreactor fed with glucose. FIG. 78B shows the time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth. Method for calculating isoprene: cumulative isoprene produced in 59 hrs, g/Fermentor volume at 59 hrs, L [=] g/L broth. FIG. 78C also shows the time course of isoprene titer within the 15-L bioreactor fed with glucose. Method for calculating isoprene: ∫ (Instantaneous isoprene production rate, g/L/hr)dt from t=0 to 59 hours [=] g/L broth. FIG. 78D shows the time course of total isoprene produced from the 15-L bioreactor fed with glucose. FIG. 78E shows volumetric productivity within the 15-L bioreactor fed with glucose. FIG. 78F shows carbon dioxide evolution rate (CER), or metabolic activity profile, within the 15-L bioreactor fed with glucose.
FIG. 79A is a map of plasmid pJ201:19813.
FIGS. 79B and 79C are the nucleotide sequence of pJ201:19813 (SEQ ID NO:123).



DETAILED DESCRIPTION OF THE INVENTION
The invention features compositions and methods for the production of increased amounts of isoprene. In particular, these compositions and methods increase the rate of isoprene production and increase the total amount of isoprene that is produced. For example, cell culture systems that generate 4.8×104 nmole/gwcm/hr of isoprene have been produced (Table 1). The efficiency of these systems is demonstrated by the conversion of  ̃23.6 molar % yield (10.7 weight % yield) of the carbon that the cells consume from a cell culture medium into isoprene (% carbon yield). As shown in the Examples and Table 2, approximately 60.5 g of isoprene per liter of broth was generated. Isoprene was produced at a peak specific rate of 1.88×105 nmol/OD/hr (1.88×105 nmole/gwcm/hr). If desired, even greater amounts of isoprene can be obtained using other conditions, such as those described herein. In some embodiments, a renewable carbon source is used for the production of isoprene. The compositions and methods of the present invention are desirable because they allow high isoprene yield per cell, high carbon yield, high isoprene purity, high productivity, low energy usage, low production cost and investment, and minimal side reactions. This efficient, large scale, biosynthetic process for isoprene production provides an isoprene source for synthetic isoprene-based rubber and provides a desirable, low-cost alternative to using natural rubber.
As discussed further below, the amount of isoprene produced by cells can be greatly increased by introducing a heterologous nucleic acid encoding an isoprene synthase polypeptide (e.g., a plant isoprene synthase polypeptide) into the cells. Isoprene synthase polypeptides convert dimethylallyl diphosphate (DMAPP) into isoprene. As shown in the Examples, a heterologous Pueraria Montana (kudzu) or Populus alba (Poplar) isoprene synthase polypeptide was expressed in a variety of host cells, such as Escherichia coli, Panteoa citrea, Bacillus subtilis, Yarrowia lipolytica, and Trichoderma reesei. As also shown in the Examples, a heterologous Methanosarcina mazei (M. mazei) mevalonate kinase (MVK) was expressed in host cells such as Escherichia coli to increase isoprene production. All of these cells produced more isoprene than the corresponding cells without the heterologous isoprene synthase polypeptide. As illustrated in Tables 1 and 2, large amounts of isoprene are produced using the methods described herein. For example, B. subtilis cells with a heterologous isoprene synthase nucleic acid produced approximately 10-fold more isoprene in a 14 liter fermentor than the corresponding control B. subtilis cells without the heterologous nucleic acid (Table 2). The production of 60.5 g of isoprene per liter of broth (mg/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells) by E. coli and 30 mg/L by B. subtilis in fermentors indicates that significant amounts of isoprene can be generated (Table 2). If desired, isoprene can be produced on an even larger scale or other conditions described herein can be used to further increase the amount of isoprene. The vectors listed in Tables 1 and 2 and the experimental conditions are described in further detail below and in the Examples section.







TABLE 1







Exemplary yields of isoprene from a shake flask using


the cell cultures and methods of the invention.









Isoprene Production in a Headspace vial*










Headspace
Specific Rate



concentration
μg/Lbroth/hr/OD


Strain
μg/Lgas
(nmol/gwcm/hr)












E. coli BL21/pTrcKudzu IS
1.40
53.2




(781.2) 


E. coli BL21/Pcl DXS yidi
7.61
289.1 


Kudzu IS

(4.25 × 103)


E. coli BL21/MCM127 with
23.0
874.1 


kudzu IS and entire MVA

(1.28 × 104)


pathway


E. coli BL21/Pet N-
1.49
56.6


HisKudzu IS

(831.1) 


Pantoea citrea/pTrcKudzu
0.66
25.1


IS

(368.6) 


E. coli w/Poplar IS
—
 5.6


[Miller (2001)]

(82.2)


Bacillis licheniformis Fall
—
 4.2


U.S. Pat. No. 5,849,970

(61.4)


Yarrowia lipolytica with
~0.05 μg/L
~2  


kudzu isoprene synthase

(~30)  


Trichoderma reesei with
~0.05 μg/L
~2  


kudzu isoprene synthase

(~30)  


E. coli BL21/
85.9
3.2 × 103


pTrcKKDyIkIS with kudzu

(4.8 × 104)


IS and lower MVA pathway





The assay for measuring isoprene production is described in Example I, part II. For this assay, a sample was removed at one or more time points from the shake flask and cultured for 30 minutes. The amount of isoprene produced in this sample was then measured. The headspace concentration and specific rate of isoprene production are listed in Table 1 and described further herein.


*Normalized to 1 mL of 1 OD600, cultured for 1 hour in a sealed headspace vial with a liquid to headspace volume ratio of 1:19.













TABLE 2







Exemplary yields of isoprene in a fermentor using


the cell cultures and methods of the invention.









Isoprene Production in Fermentors











Peak Headspace

Peak Specific rate



concentration**
Titer
μg/Lbroth/hr/OD


Strain
(ug/Lgas)
(mg/Lbroth)
(nmol/gwcm/hr)













E. coli BL21/
52
41.2
37 


pTrcKudzu with


(543.3)


Kudzu IS


E. coli
3
3.5
 21.4


FM5/pTrcKudzu IS


(308.1)


E. coli BL21/triple
285
300
240  


strain (DXS, yidi,


(3.52 × 103)


IS)


E. coli FM5/triple
50.8
29
180.8


strain (DXS, yidi,


(2.65 × 103)


IS)


E. coli/MCM127
1094
250
875  


with Kudzu IS and


(1.28 × 104)


entire MVA pathway


Bacillus subtilis
1.5
2.5
 0.8


wild-type


 (11.7)


Bacillus pBS Kudzu
16.6
~30
5


IS

(over 100
 (73.4)




hours)


Bacillus Marburg
2.04
0.61
 24.5


6051 [Wagner and


(359.8)


Fall (1999)]


Bacillus Marburg
0.7
0.15
 6.8


6051 Fall U.S.


(100)  


Pat. No. 5,849,970


E. coli
2.03 × 104
3.22 × 104
 5.9 × 103


BL21/pCLPtrcUpper


(8.66 × 104)


Pathway and


gil.2KKDyI and


pTrcAlba-mMVK


E. coli
3.22 × 104
6.05 × 104
1.28 × 104


BL21/pCLPtrcUpper


(1.88 × 105)


Pathway and


gi1.2KKDyI and


pTrcAlba-mMVK


plus


pBBRCMPGI1.5pgl





The assay for measuring isoprene production is described in Example I, part II. For this assay, a sample of the off-gas of the fermentor was taken and analyzed for the amount of isoprene. The peak headspace concentration (which is the highest headspace concentration during the fermentation), titer (which is the cumulative, total amount of isoprene produced per liter of broth), and peak specific rate of isoprene production (which is the highest specific rate during the fermentation) are listed in Table 2 and described further herein.


**Normalized to an off-gas flow rate of 1 vvm (1 volume off-gas per 1 Lbroth per minute).






Additionally, isoprene production by cells that contain a heterologous isoprene synthase nucleic acid can be enhanced by increasing the amount of a 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptide and/or an isopentenyl diphosphate isomerase (IDI) polypeptide expressed by the cells. For example, a DXS nucleic acid and/or an IDI nucleic acid can be introduced into the cells. The DXS nucleic acid may be a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid. Similarly, the IDI nucleic acid may be a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid. In some embodiments, the amount of DXS and/or IDI polypeptide is increased by replacing the endogenous DXS and/or IDI promoters or regulatory regions with other promoters and/or regulatory regions that result in greater transcription of the DXS and/or IDI nucleic acids. In some embodiments, the cells contain both a heterologous nucleic acid encoding an isoprene synthase polypeptide (e.g., a plant isoprene synthase nucleic acid) and a duplicate copy of an endogenous nucleic acid encoding an isoprene synthase polypeptide.
The encoded DXS and IDI polypeptides are part of the DXP pathway for the biosynthesis of isoprene (FIG. 19A). DXS polypeptides convert pyruvate and D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate. While not intending to be bound by any particular theory, it is believed that increasing the amount of DXS polypeptide increases the flow of carbon through the DXP pathway, leading to greater isoprene production. IDI polypeptides catalyze the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). While not intending to be bound by any particular theory, it is believed that increasing the amount of IDI polypeptide in cells increases the amount of IPP that is converted into DMAPP, which in turn is converted into isoprene.
For example, fermentation of E. coli cells with a kudzu isoprene synthase, S. cerevisia IDI, and E. coli DXS nucleic acids was used to produce isoprene. The levels of isoprene varied from 50 to 300 μg/L over a time period of 15 hours (Example 7, part VII). As another example, fermentation of E. coli with M. mazei mevalonate kinase (MVK), P. alba isoprene synthase, the upper MVA pathway, and the integrated lower MVA pathway was used to produce isoprene. The levels of isoprene varied from 32 to 35.6 g/L over a time period of 67 hours (Example 10, part III).
In yet another example, fermentation of E. coli with M. mazei mevalonate kinase (MVK), P. alba isoprene synthase, pgl over-expression (RHM111608-2), the upper MVA pathway, and the integrated lower MVA pathway were used to produce isoprene. The levels of isoprene vary from 33.2 g/L to 40.0 g/L over a time period of 40 hours or 48.6 g/L to 60.5 g/L over a time period of 59 hours (Example 13, part (ii)).
In some embodiments, the presence of heterologous or extra endogenous isoprene synthase, IDI, and DXS nucleic acids causes cells to grow more reproducibly or remain viable for longer compared to the corresponding cell with only one or two of these heterologous or extra endogenous nucleic acids. For example, cells containing heterologous isoprene synthase, IDI, and DXS nucleic acids grew better than cells with only heterologous isoprene synthase and DXS nucleic acids or with only a heterologous isoprene synthase nucleic acid. Also, heterologous isoprene synthase, IDI, and DXS nucleic acids were successfully operably linked to a strong promoter on a high copy plasmid that was maintained by E. coli cells, suggesting that large amounts of these polypeptides could be expressed in the cells without causing an excessive amount of toxicity to the cells. While not intending to be bound to a particular theory, it is believed that the presence of heterologous or extra endogenous isoprene synthase and IDI nucleic acids may reduce the amount of one or more potentially toxic intermediates that would otherwise accumulate if only a heterologous or extra endogenous DXS nucleic acid was present in the cells.
In some embodiments, the production of isoprene by cells that contain a heterologous isoprene synthase nucleic acid is augmented by increasing the amount of a MVA polypeptide expressed by the cells (FIGS. 19A and 19B). Exemplary MVA pathways polypeptides include any of the following polypeptides: acetyl-CoA acetyltransferase (AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK) polypeptides, phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonte decarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDI polypeptides, and polypeptides (e.g., fusion polypeptides) having an activity of two or more MVA pathway polypeptides. For example, one or more MVA pathway nucleic acids can be introduced into the cells. In some embodiments, the cells contain the upper MVA pathway, which includes AA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase nucleic acids. In some embodiments, the cells contain the lower MVA pathway, which includes MVK, PMK, MVD, and IDI nucleic acids. In some embodiments, the cells contain the entire MVA pathway, which includes AA-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, MVK, PMK, MVD, and IDI nucleic acids. In some embodiments, the cells contain an entire MVA pathway that includes AA-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, MVK, PMDC, IPK, and IDI nucleic acids. The MVA pathway nucleic acids may be heterologous nucleic acids or duplicate copies of endogenous nucleic acids. In some embodiments, the amount of one or more MVA pathway polypeptides is increased by replacing the endogenous promoters or regulatory regions for the MVA pathway nucleic acids with other promoters and/or regulatory regions that result in greater transcription of the MVA pathway nucleic acids. In some embodiments, the cells contain both a heterologous nucleic acid encoding an isoprene synthase polypeptide (e.g., a plant isoprene synthase nucleic acid) and a duplicate copy of an endogenous nucleic acid encoding an isoprene synthase polypeptide.
For example, E. coli cells containing a nucleic acid encoding a kudzu isoprene synthase polypeptide and nucleic acids encoding Saccharomyces cerevisia MVK, PMK, MVD, and IDI polypeptides generated isoprene at a rate of 6.67×104 nmol/Lbroth/OD600/hr (see Example 8). Additionally, a 14 liter fermentation of E. coli cells with nucleic acids encoding Enterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase polypeptides produced 22 grams of mevalonic acid (an intermediate of the MVA pathway). A shake flask of these cells produced 2-4 grams of mevalonic acid per liter. These results indicate that heterologous MVA pathways nucleic acids are active in E. coli. E. coli cells that contain nucleic acids for both the upper MVA pathway and the lower MVA pathway as well as a kudzu isoprene synthase (strain MCM 127) produced significantly more isoprene (874 μg/Lbroth/hr/OD) compared to E. coli cells with nucleic acids for only the lower MVA pathway and the kudzu isoprene synthase (strain MCM 131) (see Table 3 and Example 8, part VIII).
As another example, E. coli cells containing a nucleic acid encoding a P. alba isoprene synthase polypeptide and a nucleic acid encoding M. mazei MVK polypeptide generated 320.6 g (at a peak specific rate of 9.54×104 nmol/Lbroth/OD600/hr (i.e. 9.5×10−5 mol/Lbroth/OD600/hr)) of isoprene during a 67 hour fermentation in the absence of yeast extract feeding or 395.5 g (at a peak specific rate of 8.66×104 nmol/Lbroth/OD600/hr) during a 68 hour fermentation in the presence of yeast extract feeding (see Example 10).
In some embodiments, at least a portion of the cells maintain the heterologous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acid for at least about 5, 10, 20, 50, 75, 100, 200, 300, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects of the invention, the nucleic acid comprising the heterologous or duplicate copy of an endogenous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acid also comprises a selective marker, such as a kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol antibiotic resistance nucleic acid.
As indicated in Example 7, part VI, the amount of isoprene produced can be further increased by adding yeast extract to the cell culture medium using E. coli cells with kudzu isoprene synthase, S. cerevisia IDI, and E. coli DXS nucleic acids to produce isoprene. In particular, the amount of isoprene produced was linearly proportional to the amount of yeast extract in the cell medium for the concentrations tested (FIG. 48C). Additionally, approximately 0.11 grams of isoprene per liter of broth was produced from a cell medium with yeast extract and glucose (Example 7, part VIII). Increasing the amount of yeast extract in the presence of glucose resulted in more isoprene being produced than increasing the amount of glucose in the presence of yeast extract. Also, increasing the amount of yeast extract allowed the cells to produce a high level of isoprene for a longer length of time and improved the health of the cells.
Isoprene production was also demonstrated using three types of hydrolyzed biomass (bagasse, corn stover, and soft wood pulp) as the carbon source (FIGS. 46A-C and FIGS. 69A and 69B). E. coli cells with kudzu isoprene synthase, S. cerevisia IDI, and E. coli DXS nucleic acids produced as much isoprene from these hydrolyzed biomass carbon sources as from the equivalent amount of glucose (e.g., 1% glucose, w/v). E. coli cells expressing P. alba isoprene synthase and the MVA pathway produced isoprene at a higher initial growth rate from ammonia fiber expansion (AFEX) pretreated corn stover than from the equivalent amount of glucose. (FIGS. 69A and 69B). If desired, any other biomass carbon source can be used in the compositions and methods of the invention. Biomass carbon sources are desirable because they are cheaper than many conventional cell mediums, thereby facilitating the economical production of isoprene.
Additionally, invert sugar was shown to function as a carbon source for the generation of isoprene (FIG. 47D).
Additionally, xylose, acetate, and glycerol were also shown to function as a carbon source for the generation of isoprene (FIGS. 69A-69D). For example, E. coli cells with P. alba isoprene synthase and the MVA pathway grown on acetate as the only carbon source had a specific productivity of isoprene about twice as high as during growth on glucose (Example 10, Part IV; FIGS. 69A and 69B).
In some embodiments, an oil is included in the cell medium. For example, B. subtilis cells containing a kudzu isoprene synthase nucleic acid produced isoprene when cultured in a cell medium containing an oil and a source of glucose (Example 4, part III). As another example, E. coli fadR atoC mutant cells containing the upper and lower MVA pathway plus kudzu isoprene synthase produced isoprene when cultured in a cell medium containing palm oil and a source of glucose (Example 12, part II). In some embodiments, more than one oil (such as 2, 3, 4, 5, or more oils) is included in the cell medium. While not intending to be bound to any particular theory, it is believed that (i) the oil may increase the amount of carbon in the cells that is available for conversion to isoprene, (ii) the oil may increase the amount of acetyl-CoA in the cells, thereby increasing the carbon flow through the MVA pathway, and/or (ii) the oil may provide extra nutrients to the cells, which is desirable since a lot of the carbon in the cells is converted to isoprene rather than other products. In some embodiments, cells that are cultured in a cell medium containing oil naturally use the MVA pathway to produce isoprene or are genetically modified to contain nucleic acids for the entire MVA pathway. In some embodiments, the oil is partially or completely hydrolyzed before being added to the cell culture medium to facilitate the use of the oil by the host cells.
Exemplary Polypeptides and Nucleic Acids
Various isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids can be used in the compositions and methods of the invention.
As used herein, “polypeptides” includes polypeptides, proteins, peptides, fragments of polypeptides, and fusion polypeptides that include part or all of a first polypeptide (e.g., an isoprene synthase, DXS, IDI, or MVA pathway polypeptide) and part or all of a second polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag). In some embodiments, the fusion polypeptide has an activity of two or more MVA pathway polypeptides (such as AA-CoA thiolase and HMG-CoA reductase polypeptides). In some embodiments, the polypeptide is a naturally-occurring polypeptide (such as the polypeptide encoded by an Enterococcus faecalis mvaE nucleic acid) that has an activity of two or more MVA pathway polypeptides.
In various embodiments, a polypeptide has at least or about 50, 100, 150, 175, 200, 250, 300, 350, 400, or more amino acids. In some embodiments, the polypeptide fragment contains at least or about 25, 50, 75, 100, 150, 200, 300, or more contiguous amino acids from a full-length polypeptide and has at least or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of an activity of a corresponding full-length polypeptide. In particular embodiments, the polypeptide includes a segment of or the entire amino acid sequence of any naturally-occurring isoprene synthase, DXS, IDI, or MVA pathway polypeptide. In some embodiments, the polypeptide has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) isoprene synthase, DXS, IDI, or MVA pathway polypeptide.
In some embodiments, the polypeptide is an isolated polypeptide. As used herein, an “isolated polypeptide” is not part of a library of polypeptides, such as a library of 2, 5, 10, 20, 50 or more different polypeptides and is separated from at least one component with which it occurs in nature. An isolated polypeptide can be obtained, for example, by expression of a recombinant nucleic acid encoding the polypeptide.
In some embodiments, the polypeptide is a heterologous polypeptide. By “heterologous polypeptide” is meant a polypeptide whose amino acid sequence is not identical to that of another polypeptide naturally expressed in the same host cell.
As used herein, a “nucleic acid” refers to two or more deoxyribonucleotides and/or ribonucleotides in either single or double-stranded form. In some embodiments, the nucleic acid is a recombinant nucleic acid. By “recombinant nucleic acid” means a nucleic acid of interest that is free of one or more nucleic acids (e.g., genes) which, in the genome occurring in nature of the organism from which the nucleic acid of interest is derived, flank the nucleic acid of interest. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA, a genomic DNA fragment, or a cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In some embodiments, an isoprene synthase, DXS, IDI, or MVA pathway nucleic acid is operably linked to another nucleic acid encoding all or a portion of another polypeptide such that the recombinant nucleic acid encodes a fusion polypeptide that includes an isoprene synthase, DXS, IDI, or MVA pathway polypeptide and all or part of another polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag). In some embodiments, part or all of a recombinant nucleic acid is chemically synthesized.
In some embodiments, the nucleic acid is a heterologous nucleic acid. By “heterologous nucleic acid” is meant a nucleic acid whose nucleic acid sequence is not identical to that of another nucleic acid naturally found in the same host cell.
In particular embodiments, the nucleic acid includes a segment of or the entire nucleic acid sequence of any naturally-occurring isoprene synthase, DXS, IDI, or MVA pathway nucleic acid. In some embodiments, the nucleic acid includes at least or about 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, or more contiguous nucleotides from a naturally-occurring isoprene synthase nucleic acid DXS, IDI, or MVA pathway nucleic acid. In some embodiments, the nucleic acid has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) isoprene synthase, DXS, IDI, or MVA pathway nucleic acid. In some embodiments, the nucleic acid has one or more mutations (e.g., a silent mutation) that increase the transcription or translation of isoprene synthase, DXS, IDI, or MVA pathway nucleic acid. In some embodiments, the nucleic acid is a degenerate variant of any nucleic acid encoding an isoprene synthase, DXS, IDI, or MVA pathway polypeptide.
“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid for improved expression in a host cell, it is desirable in some embodiments to design the nucleic acid such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
The accession numbers of exemplary isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids are listed in Appendix 1 (the accession numbers of Appendix 1 and their corresponding sequences are herein incorporated by reference in their entireties, particularly with respect to the amino acid and nucleic acid sequences of isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids). The Kegg database also contains the amino acid and nucleic acid sequences of numerous exemplary isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids (see, for example, the world-wide web at “genome.jp/kegg/pathway/map/map00100.html” and the sequences therein, which are each hereby incorporated by reference in their entireties, particularly with respect to the amino acid and nucleic acid sequences of isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids). In some embodiments, one or more of the isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and/or nucleic acids have a sequence identical to a sequence publicly available on Dec. 12, 2007 or Dec. 11, 2008, such as any of the sequences that correspond to any of the accession numbers in Appendix 1 or any of the sequences present in the Kegg database. Additional exemplary isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids are described further below.
Exemplary Isoprene Synthase Polypeptides and Nucleic Acids
As noted above, isoprene synthase polypeptides convert dimethylallyl diphosphate (DMAPP) into isoprene. Exemplary isoprene synthase polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of an isoprene synthase polypeptide. Standard methods can be used to determine whether a polypeptide has isoprene synthase polypeptide activity by measuring the ability of the polypeptide to convert DMAPP into isoprene in vitro, in a cell extract, or in vivo. In an exemplary assay, cell extracts are prepared by growing a strain (e.g., the E. coli/pTrcKudzu strain described herein) in the shake flask method as described in Example 1. After induction is complete, approximately 10 mLs of cells are pelleted by centrifugation at 7000×g for 10 minutes and resuspended in 5 ml of PEB without glycerol. The cells are lysed using a French Pressure cell using standard procedures. Alternatively the cells are treated with lysozyme (Ready-Lyse lysozyme solution; EpiCentre) after a freeze/thaw at −80° C.
Isoprene synthase polypeptide activity in the cell extract can be measured, for example, as described in Silver et al., J. Biol. Chem. 270:13010-13016, 1995 and references therein, which are each hereby incorporated by reference in their entireties, particularly with respect to assays for isoprene synthase polypeptide activity. DMAPP (Sigma) is evaporated to dryness under a stream of nitrogen and rehydrated to a concentration of 100 mM in 100 mM potassium phosphate buffer pH 8.2 and stored at −20° C. To perform the assay, a solution of 5 μl of 1M MgCl2, 1 mM (250 μg/ml) DMAPP, 65 μl of Plant Extract Buffer (PEB) (50 mM Tris-HCl, pH 8.0, 20 mM MgCl2, 5% glycerol, and 2 mM DTT) is added to 25 μl of cell extract in a 20 ml Headspace vial with a metal screw cap and teflon coated silicon septum (Agilent Technologies) and cultured at 37° C. for 15 minutes with shaking. The reaction is quenched by adding 200 μl of 250 mM EDTA and quantified by GC/MS as described in Example 1, part II.
Exemplary isoprene synthase nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an isoprene synthase polypeptide. Exemplary isoprene synthase polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.
In some embodiments, the isoprene synthase polypeptide or nucleic acid is from the family Fabaceae, such as the Faboideae subfamily. In some embodiments, the isoprene synthase polypeptide or nucleic acid is a polypeptide or nucleic acid from Pueraria montana (kudzu) (Sharkey et al., Plant Physiology 137: 700-712, 2005), Pueraria lobata, poplar (such as Populus alba, Populus nigra, Populus trichocarpa, Populus alba x tremula (CAC35696), or Populus alba) (Sasaki et al., FEBS Letters 579(11): 2514-2518, 2005; Miller et al., Planta 213: 483-487, 2001), aspen (such as Populus tremuloides) (Silver et al., JBC 270(22): 13010-1316, 1995), or English Oak (Quercus robur) (Zimmer et al., WO 98/02550), which are each hereby incorporated by reference in their entireties, particularly with respect to isoprene synthase nucleic acids and the expression of isoprene synthase polypeptides. Suitable isoprene synthases include, but are not limited to, those identified by Genbank Accession Nos. AY341431, AY316691, AY279379, AJ457070, and AY182241, which are each hereby incorporated by reference in their entireties, particularly with respect to sequences of isoprene synthase nucleic acids and polypeptides. In some embodiments, the isoprene synthase polypeptide or nucleic acid is not a naturally-occurring polypeptide or nucleic acid from Quercus robur (i.e., the isoprene synthase polypeptide or nucleic acid is an isoprene synthase polypeptide or nucleic acid other than a naturally-occurring polypeptide or nucleic acid from Quercus robur). In some embodiments, the isoprene synthase nucleic acid or polypeptide is a naturally-occurring polypeptide or nucleic acid from poplar. In some embodiments, the isoprene synthase nucleic acid or polypeptide is not a naturally-occurring polypeptide or nucleic acid from poplar.
Exemplary DXS Polypeptides and Nucleic Acids
As noted above, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptides convert pyruvate and D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate. Exemplary DXS polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of a DXS polypeptide. Standard methods (such as those described herein) can be used to determine whether a polypeptide has DXS polypeptide activity by measuring the ability of the polypeptide to convert pyruvate and D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate in vitro, in a cell extract, or in vivo. Exemplary DXS nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of a DXS polypeptide. Exemplary DXS polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.
Exemplary IDI Polypeptides and Nucleic Acids
Isopentenyl diphosphate isomerase polypeptides (isopentenyl-diphosphate delta-isomerase or IDI) catalyses the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (e.g., converting IPP into DMAPP and/or converting DMAPP into IPP). Exemplary IDI polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of an IDI polypeptide. Standard methods (such as those described herein) can be used to determine whether a polypeptide has IDI polypeptide activity by measuring the ability of the polypeptide to interconvert IPP and DMAPP in vitro, in a cell extract, or in vivo. Exemplary IDI nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an IDI polypeptide. Exemplary IDI polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.
Exemplary MVA Pathway Polypeptides and Nucleic Acids
Exemplary MVA pathway polypeptides include acetyl-CoA acetyltransferase (AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK) polypeptides, phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonte decarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDI polypeptides, and polypeptides (e.g., fusion polypeptides) having an activity of two or more MVA pathway polypeptides. In particular, MVA pathway polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of an MVA pathway polypeptide. Exemplary MVA pathway nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an MVA pathway polypeptide. Exemplary MVA pathway polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.
In particular, acetyl-CoA acetyltransferase polypeptides (AA-CoA thiolase or AACT) convert two molecules of acetyl-CoA into acetoacetyl-CoA. Standard methods (such as those described herein) can be used to determine whether a polypeptide has AA-CoA thiolase polypeptide activity by measuring the ability of the polypeptide to convert two molecules of acetyl-CoA into acetoacetyl-CoA in vitro, in a cell extract, or in vivo.
3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase or HMGS) polypeptides convert acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA. Standard methods (such as those described herein) can be used to determine whether a polypeptide has HMG-CoA synthase polypeptide activity by measuring the ability of the polypeptide to convert acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA in vitro, in a cell extract, or in vivo.
3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase or HMGR) polypeptides convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate. Standard methods (such as those described herein) can be used to determine whether a polypeptide has HMG-CoA reductase polypeptide activity by measuring the ability of the polypeptide to convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate in vitro, in a cell extract, or in vivo.
Mevalonate kinase (MVK) polypeptide phosphorylates mevalonate to form mevalonate-5-phosphate. Standard methods (such as those described herein) can be used to determine whether a polypeptide has MVK polypeptide activity by measuring the ability of the polypeptide to convert mevalonate into mevalonate-5-phosphate in vitro, in a cell extract, or in vivo.
Phosphomevalonate kinase (PMK) polypeptides phosphorylates mevalonate-5-phosphate to form mevalonate-5-diphosphate. Standard methods (such as those described herein) can be used to determine whether a polypeptide has PMK polypeptide activity by measuring the ability of the polypeptide to convert mevalonate-5-phosphate into mevalonate-5-diphosphate in vitro, in a cell extract, or in vivo.
Diphosphomevalonte decarboxylase (MVD or DPMDC) polypeptides convert mevalonate-5-diphosphate into isopentenyl diphosphate (IPP). Standard methods (such as those described herein) can be used to determine whether a polypeptide has MVD polypeptide activity by measuring the ability of the polypeptide to convert mevalonate-5-diphosphate into IPP in vitro, in a cell extract, or in vivo.
Phosphomevalonate decarboxylase (PMDC) polypeptides convert mevalonate-5-phosphate into isopentenyl phosphate (IP). Standard methods (such as those described herein) can be used to determine whether a polypeptide has PMDC polypeptide activity by measuring the ability of the polypeptide to convert mevalonate-5-phosphate into IP in vitro, in a cell extract, or in vivo.
Isopentenyl phosphate kinase (IPK) polypeptides phosphorylate isopentyl phosphate (IP) to form isopentenyl diphosphate (IPP). Standard methods (such as those described herein) can be used to determine whether a polypeptide has IPK polypeptide activity by measuring the ability of the polypeptide to convert IP into IPP in vitro, in a cell extract, or in vivo.
Exemplary IDI polypeptides and nucleic acids are described above.
Exemplary Methods for Isolating Nucleic Acids
Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids can be isolated using standard methods. Methods of obtaining desired nucleic acids from a source organism of interest (such as a bacterial genome) are common and well known in the art of molecular biology (see, for example, WO 2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to the isolation of nucleic acids of interest). For example, if the sequence of the nucleic acid is known (such as any of the known nucleic acids described herein), suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired nucleic acid sequence. Once the sequence is isolated, the DNA may be amplified using standard primer directed amplification methods such as polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202, which is incorporated by reference in its entirety, particularly with respect to PCR methods) to obtain amounts of DNA suitable for transformation using appropriate vectors.
Alternatively, isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids (such as any isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids with a known nucleic acid sequence) can be chemically synthesized using standard methods.
Additional isoprene synthase, DXS, IDI, or MVA pathway polypeptides and nucleic acids which may be suitable for use in the compositions and methods described herein can be identified using standard methods. For example, cosmid libraries of the chromosomal DNA of organisms known to produce isoprene naturally can be constructed in organisms such as E. coli, and then screened for isoprene production. In particular, cosmid libraries may be created where large segments of genomic DNA (35-45 kb) are packaged into vectors and used to transform appropriate hosts. Cosmid vectors are unique in being able to accommodate large quantities of DNA. Generally cosmid vectors have at least one copy of the cos DNA sequence which is needed for packaging and subsequent circularization of the heterologous DNA. In addition to the cos sequence, these vectors also contain an origin of replication such as ColEI and drug resistance markers such as a nucleic acid resistant to ampicillin or neomycin. Methods of using cosmid vectors for the transformation of suitable bacterial hosts are well described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to transformation methods.
Typically to clone cosmids, heterologous DNA is isolated using the appropriate restriction endonucleases and ligated adjacent to the cos region of the cosmid vector using the appropriate ligases. Cosmid vectors containing the linearized heterologous DNA are then reacted with a DNA packaging vehicle such as bacteriophage. During the packaging process, the cos sites are cleaved and the heterologous DNA is packaged into the head portion of the bacterial viral particle. These particles are then used to transfect suitable host cells such as E. coli. Once injected into the cell, the heterologous DNA circularizes under the influence of the cos sticky ends. In this manner, large segments of heterologous DNA can be introduced and expressed in host cells.
Additional methods for obtaining isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids include screening a metagenomic library by assay (such as the headspace assay described herein) or by PCR using primers directed against nucleotides encoding for a length of conserved amino acids (for example, at least 3 conserved amino acids). Conserved amino acids can be identified by aligning amino acid sequences of known isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides. Conserved amino acids for isoprene synthase polypeptides can be identified based on aligned sequences of known isoprene synthase polypeptides. An organism found to produce isoprene naturally can be subjected to standard protein purification methods (which are well known in the art) and the resulting purified polypeptide can be sequenced using standard methods. Other methods are found in the literature (see, for example, Julsing et al., Applied. Microbiol. Biotechnol. 75: 1377-84, 2007; Withers et al., Appl Environ Microbiol. 73(19):6277-83, 2007, which are each hereby incorporated by reference in their entireties, particularly with respect to identification of nucleic acids involved in the synthesis of isoprene).
Additionally, standard sequence alignment and/or structure prediction programs can be used to identify additional DXS, IDI, or MVA pathway polypeptides and nucleic acids based on the similarity of their primary and/or predicted polypeptide secondary structure with that of known DXS, IDI, or MVA pathway polypeptides and nucleic acids. Standard databases such as the swissprot-trembl database (world-wide web at “expasy.org”, Swiss Institute of Bioinformatics Swiss-Prot group CMU-1 rue Michel Servet CH-1211 Geneva 4, Switzerland) can also be used to identify isoprene synthase, DXS, IDI, or MVA pathway polypeptides and nucleic acids. The secondary and/or tertiary structure of an isoprene synthase, DXS, IDI, or MVA pathway polypeptide can be predicted using the default settings of standard structure prediction programs, such as PredictProtein (630 West, 168 Street, BB217, New York, N.Y. 10032, USA). Alternatively, the actual secondary and/or tertiary structure of an isoprene synthase, DXS, IDI, or MVA pathway polypeptide can be determined using standard methods. Additional isoprene synthase, DXS, IDI, or MVA pathway nucleic acids can also be identified by hybridization to probes generated from known isoprene synthase, DXS, IDI, or MVA pathway nucleic acids.
Exemplary Promoters and Vectors
Any of the isoprene synthase, DXS, IDI, or MVA pathway nucleic acid described herein can be included in one or more vectors. Accordingly, the invention also features vectors with one more nucleic acids encoding any of the isoprene synthase, DXS, IDI, or MVA pathway polypeptides that are described herein. As used herein, a “vector” means a construct that is capable of delivering, and desirably expressing one or more nucleic acids of interest in a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, DNA or RNA expression vectors, cosmids, and phage vectors. In some embodiments, the vector contains a nucleic acid under the control of an expression control sequence.
As used herein, an “expression control sequence” means a nucleic acid sequence that directs transcription of a nucleic acid of interest. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. An “inducible promoter” is a promoter that is active under environmental or developmental regulation. The expression control sequence is operably linked to the nucleic acid segment to be transcribed.
In some embodiments, the vector contains a selective marker. The term “selective marker” refers to a nucleic acid capable of expression in a host cell that allows for ease of selection of those host cells containing an introduced nucleic acid or vector. Examples of selectable markers include, but are not limited to, antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/or nucleic acids that confer a metabolic advantage, such as a nutritional advantage on the host cell. Exemplary nutritional selective markers include those markers known in the art as amdS, argB, and pyr4. Markers useful in vector systems for transformation of Trichoderma are known in the art (see, e.g., Finkelstein, Chapter 6 in Biotechnology of Filamentous Fungi, Finkelstein et al., Eds. Butterworth-Heinemann, Boston, Mass., Chap. 6, 1992; and Kinghorn et al., Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, Chapman and Hall, London, 1992, which are each hereby incorporated by reference in their entireties, particularly with respect to selective markers). In some embodiments, the selective marker is the amdS nucleic acid, which encodes the enzyme acetamidase, allowing transformed cells to grow on acetamide as a nitrogen source. The use of an A. nidulans amdS nucleic acid as a selective marker is described in Kelley et al., EMBO J. 4:475-479, 1985 and Penttila et al., Gene 61:155-164, 1987 (which are each hereby incorporated by reference in their entireties, particularly with respect to selective markers). In some embodiments, an isoprene synthase, DXS, IDI, or MVA pathway nucleic acid integrates into a chromosome of the cells without a selective marker.
Suitable vectors are those which are compatible with the host cell employed. Suitable vectors can be derived, for example, from a bacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, a yeast, or a plant. Protocols for obtaining and using such vectors are known to those in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to the use of vectors).
Promoters are well known in the art. Any promoter that functions in the host cell can be used for expression of an isoprene synthase, DXS, IDI, or MVA pathway nucleic acid in the host cell. Initiation control regions or promoters, which are useful to drive expression of isoprene synthase, DXS, IDI, or MVA pathway nucleic acids in various host cells are numerous and familiar to those skilled in the art (see, for example, WO 2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors for the expression of nucleic acids of interest). Virtually any promoter capable of driving these nucleic acids is suitable for the present invention including, but not limited to, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, and TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, λPL, λPR, T7, tac, and trc (useful for expression in E. coli).
In some embodiments, a glucose isomerase promoter is used (see, for example, U.S. Pat. No. 7,132,527 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect promoters and plasmid systems for expressing polypeptides of interest). Reported glucose isomerase promoter mutants can be used to vary the level of expression of the polypeptide encoded by a nucleic acid operably linked to the glucose isomerase promoter (U.S. Pat. No. 7,132,527). In various embodiments, the glucose isomerase promoter is contained in a low, medium, or high copy plasmid (U.S. Pat. No. 7,132,527).
In various embodiments, an isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acid is contained in a low copy plasmid (e.g., a plasmid that is maintained at about 1 to about 4 copies per cell), medium copy plasmid (e.g., a plasmid that is maintained at about 10 to about 15 copies per cell), or high copy plasmid (e.g., a plasmid that is maintained at about 50 or more copies per cell). In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVA pathway nucleic acid is operably linked to a T7 promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVA pathway nucleic acid operably linked to a T7 promoter is contained in a medium or high copy plasmid. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVA pathway nucleic acid is operably linked to a Trc promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVA pathway nucleic acid operably linked to a Trc promoter is contained in a medium or high copy plasmid. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVA pathway nucleic acid is operably linked to a Lac promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVA pathway nucleic acid operably linked to a Lac promoter is contained in a low copy plasmid. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVA pathway nucleic acid is operably linked to an endogenous promoter, such as an endogenous Escherichia, Panteoa, Bacillus, Yarrowia, Streptomyces, or Trichoderma promoter or an endogenous alkaline serine protease, isoprene synthase, DXS, IDI, or MVA pathway promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVA pathway nucleic acid operably linked to an endogenous promoter is contained in a high copy plasmid. In some embodiments, the vector is a replicating plasmid that does not integrate into a chromosome in the cells. In some embodiments, part or all of the vector integrates into a chromosome in the cells.
In some embodiments, the vector is any vector which when introduced into a fungal host cell is integrated into the host cell genome and is replicated. Reference is made to the Fungal Genetics Stock Center Catalogue of Strains (FGSC, the world-wide web at “fgsc.net” and the references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors) for a list of vectors. Additional examples of suitable expression and/or integration vectors are provided in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds) 1987, Supplement 30, section 7.7.18); van den Hondel et al. in Bennett and Lasure (Eds.) More Gene Manipulations in Fungi, Academic Press pp. 396-428, 1991; and U.S. Pat. No. 5,874,276, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors. Particularly useful vectors include pFB6, pBR322, PUC18, pUC100, and pENTR/D.
In some embodiments, an isoprene synthase, DXS, IDI, or MVA pathway nucleic acid is operably linked to a suitable promoter that shows transcriptional activity in a fungal host cell. The promoter may be derived from one or more nucleic acids encoding a polypeptide that is either endogenous or heterologous to the host cell. In some embodiments, the promoter is useful in a Trichoderma host. Suitable non-limiting examples of promoters include cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2, xyn1, and amy. In some embodiments, the promoter is one that is native to the host cell. For example, in some embodiments when T. reesei is the host, the promoter is a native T. reesei promoter. In some embodiments, the promoter is T. reesei cbh1, which is an inducible promoter and has been deposited in GenBank under Accession No. D86235, which is incorporated by reference in its entirety, particularly with respect to promoters. In some embodiments, the promoter is one that is heterologous to the fungal host cell. Other examples of useful promoters include promoters from the genes of A. awamori and A. niger glucoamylase (glaA) (Nunberg et al., Mol. Cell. Biol. 4:2306-2315, 1984 and Boel et al., EMBO J. 3:1581-1585, 1984, which are each hereby incorporated by reference in their entireties, particularly with respect to promoters); Aspergillus niger alpha amylases, Aspergillus oryzae TAKA amylase, T. reesei xln1, and the T. reesei cellobiohydrolase 1 (EP 137280, which is incorporated by reference in its entirety, particularly with respect to promoters).
In some embodiments, the expression vector also includes a termination sequence. Termination control regions may also be derived from various genes native to the host cell. In some embodiments, the termination sequence and the promoter sequence are derived from the same source. In another embodiment, the termination sequence is endogenous to the host cell. A particularly suitable terminator sequence is cbh1 derived from a Trichoderma strain (such as T. reesei). Other useful fungal terminators include the terminator from an A. niger or A. awamori glucoamylase nucleic acid (Nunberg et al., Mol. Cell. Biol. 4:2306-2315, 1984 and Boel et al., EMBO J. 3:1581-1585, 1984; which are each hereby incorporated by reference in their entireties, particularly with respect to fungal terminators). Optionally, a termination site may be included. For effective expression of the polypeptides, DNA encoding the polypeptide are linked operably through initiation codons to selected expression control regions such that expression results in the formation of the appropriate messenger RNA.
In some embodiments, the promoter, coding, region, and terminator all originate from the isoprene synthase, DXS, IDI, or MVA pathway nucleic acid to be expressed. In some embodiments, the coding region for an isoprene synthase, DXS, IDI, or MVA pathway nucleic acid is inserted into a general-purpose expression vector such that it is under the transcriptional control of the expression construct promoter and terminator sequences. In some embodiments, genes or part thereof are inserted downstream of the strong cbh1 promoter.
An isoprene synthase, DXS, IDI, or MVA pathway nucleic acid can be incorporated into a vector, such as an expression vector, using standard techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is hereby incorporated by reference in its entirety, particularly with respect to the screening of appropriate DNA sequences and the construction of vectors). Methods used to ligate the DNA construct comprising a nucleic acid of interest (such as an isoprene synthase, DXS, IDI, or MVA pathway nucleic acid), a promoter, a terminator, and other sequences and to insert them into a suitable vector are well known in the art. For example, restriction enzymes can be used to cleave the isoprene synthase, DXS, IDI, or MVA pathway nucleic acid and the vector. Then, the compatible ends of the cleaved isoprene synthase, DXS, IDI, or MVA pathway nucleic acid and the cleaved vector can be ligated. Linking is generally accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide linkers are used in accordance with conventional practice (see, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, and Bennett and Lasure, More Gene Manipulations in Fungi, Academic Press, San Diego, pp 70-76, 1991, which are each hereby incorporated by reference in their entireties, particularly with respect to oligonucleotide linkers). Additionally, vectors can be constructed using known recombination techniques (e.g., Invitrogen Life Technologies, Gateway Technology).
In some embodiments, it may be desirable to over-express isoprene synthase, DXS, IDI, or MVA pathway nucleic acids at levels far higher than currently found in naturally-occurring cells. This result may be accomplished by the selective cloning of the nucleic acids encoding those polypeptides into multicopy plasmids or placing those nucleic acids under a strong inducible or constitutive promoter. Methods for over-expressing desired polypeptides are common and well known in the art of molecular biology and examples may be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to cloning techniques.
The following resources include descriptions of additional general methodology useful in accordance with the invention: Kreigler, Gene Transfer and Expression; A Laboratory Manual, 1990 and Ausubel et al., Eds. Current Protocols in Molecular Biology, 1994, which are each hereby incorporated by reference in their entireties, particularly with respect to molecular biology and cloning techniques.
Exemplary Source Organisms
Isoprene synthase, DXS, IDI, or MVA pathway nucleic acids (and their encoded polypeptides) can be obtained from any organism that naturally contains isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids. As noted above, isoprene is formed naturally by a variety of organisms, such as bacteria, yeast, plants, and animals. Organisms contain the MVA pathway, DXP pathway, or both the MVA and DXP pathways for producing isoprene (FIG. 19). Thus, DXS nucleic acids can be obtained, e.g., from any organism that contains the DXP pathway or contains both the MVA and DXP pathways. IDI and isoprene synthase nucleic acids can be obtained, e.g., from any organism that contains the MVA pathway, DXP pathway, or both the MVA and DXP pathways. MVA pathway nucleic acids can be obtained, e.g., from any organism that contains the MVA pathway or contains both the MVA and DXP pathways.
In some embodiments, the nucleic acid sequence of the isoprene synthase, DXS, IDI, or MVA pathway nucleic is identical to the sequence of a nucleic acid that is produced by any of the following organisms in nature. In some embodiments, the amino acid sequence of the isoprene synthase, DXS, IDI, or MVA pathway polypeptide is identical to the sequence of a polypeptide that is produced by any of the following organisms in nature. In some embodiments, the isoprene synthase, DXS, IDI, or MVA pathway nucleic acid or polypeptide is a mutant nucleic acid or polypeptide derived from any of the organisms described herein. As used herein, “derived from” refers to the source of the nucleic acid or polypeptide into which one or more mutations is introduced. For example, a polypeptide that is “derived from a plant polypeptide” refers to polypeptide of interest that results from introducing one or more mutations into the sequence of a wild-type (i.e., a sequence occurring in nature) plant polypeptide.
In some embodiments, the source organism is a fungus, examples of which are species of Aspergillus such as A. oryzae and A. niger, species of Saccharomyces such as S. cerevisiae, species of Schizosaccharomyces such as S. pombe, and species of Trichoderma such as T. reesei. In some embodiments, the source organism is a filamentous fungal cell. The term “filamentous fungi” refers to all filamentous forms of the subdivision Eumycotina (see, Alexopoulos, C. J. (1962), Introductory Mycology, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligatory aerobic. The filamentous fungal parent cell may be a cell of a species of, but not limited to, Trichoderma, (e.g., Trichoderma reesei, the asexual morph of Hypocrea jecorina, previously classified as T. longibrachiatum, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol. Biotechnol 20: 46-53, 1984; ATCC No. 56765 and ATCC No. 26921); Penicillium sp., Humicola sp. (e.g., H. insolens, H. lanuginose, or H. grisea); Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp., Aspergillus sp. (e.g., A. oryzae, A. niger, A. sojae, A. japonicus, A. nidulans, or A. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993 and Goedegebuur et al., Genet. 41: 89-98, 2002), Fusarium sp., (e.g., F. roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum), Neurospora sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., M. miehei), Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci. 228: 21-26, 1985). The term “Trichoderma” or “Trichoderma sp.” or “Trichoderma spp.” refer to any fungal genus previously or currently classified as Trichoderma. 
In some embodiments, the fungus is A. nidulans, A. awamori, A. oryzae, A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F. oxysporum, or F. solani, Aspergillus strains are disclosed in Ward et al., Appl. Microbiol. Biotechnol. 39:738-743, 1993 and Goedegebuur et al., Curr Gene 41:89-98, 2002, which are each hereby incorporated by reference in their entireties, particularly with respect to fungi. In particular embodiments, the fungus is a strain of Trichoderma, such as a strain of T. reesei. Strains of T. reesei are known and non-limiting examples include ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCC No. 56765, ATCC No. 56767, and NRRL 15709, which are each hereby incorporated by reference in their entireties, particularly with respect to strains of T. reesei. In some embodiments, the host strain is a derivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al., Appl. Microbiol. Biotechnology 20:46-53, 1984, which is hereby incorporated by reference in its entirety, particularly with respect to strains of T. reesei. 
In some embodiments, the source organism is a yeast, such as Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp.
In some embodiments, the source organism is a bacterium, such as strains of Bacillus such as B. lichenformis or B. subtilis, strains of Pantoea such as P. citrea, strains of Pseudomonas such as P. alcaligenes, strains of Streptomyces such as S. albus, S. lividans, or S. rubiginosus, or strains of Escherichia such as E. coli. 
As used herein, “the genus Bacillus” includes all species within the genus “Bacillus,” as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “Geobacillus stearothermophilus.” The production of resistant endospores in the presence of oxygen is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus. 
In some embodiments, the source organism is a gram-positive bacterium. Non-limiting examples include strains of Streptomyces (e.g., S. albus, S. lividans, S. coelicolor, or S. griseus) and Bacillus. In some embodiments, the source organism is a gram-negative bacterium, such as E. coli or Pseudomonas sp.
In some embodiments, the source organism is a plant, such as a plant from the family Fabaceae, such as the Faboideae subfamily. In some embodiments, the source organism is kudzu, poplar (such as Populus alba x tremula CAC35696 or Populus alba) (Sasaki et al., FEBS Letters 579(11): 2514-2518, 2005), aspen (such as Populus tremuloides), or Quercus robur. 
In some embodiments, the source organism is an algae, such as a green algae, red algae, glaucophytes, chlorarachniophytes, euglenids, chromista, or dinoflagellates.
In some embodiments, the source organism is a cyanobacteria, such as cyanobacteria classified into any of the following groups based on morphology: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales, or Stigonematales. 
Exemplary Host Cells
A variety of host cells can be used to express isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and to produce isoprene in the methods of the claimed invention. Exemplary host cells include cells from any of the organisms listed in the prior section under the heading “Exemplary Source Organisms.” The host cell may be a cell that naturally produces isoprene or a cell that does not naturally produce isoprene. In some embodiments, the host cell naturally produces isoprene using the DXP pathway, and an isoprene synthase, DXS, and/or IDI nucleic acid is added to enhance production of isoprene using this pathway. In some embodiments, the host cell naturally produces isoprene using the MVA pathway, and an isoprene synthase and/or one or more MVA pathway nucleic acids are added to enhance production of isoprene using this pathway. In some embodiments, the host cell naturally produces isoprene using the DXP pathway and one or more MVA pathway nucleic acids are added to produce isoprene using part or all of the MVA pathway as well as the DXP pathway. In some embodiments, the host cell naturally produces isoprene using both the DXP and MVA pathways and one or more isoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added to enhance production of isoprene by one or both of these pathways.
Exemplary Transformation Methods
Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids or vectors containing them can be inserted into a host cell (e.g., a plant cell, a fungal cell, a yeast cell, or a bacterial cell described herein) using standard techniques for expression of the encoded isoprene synthase, DXS, IDI, and/or MVA pathway polypeptide. Introduction of a DNA construct or vector into a host cell can be performed using techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated or DEAE-Dextrin mediated transfection or transfection using a recombinant phage virus), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. General transformation techniques are known in the art (see, e.g., Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds) Chapter 9, 1987; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989; and Campbell et al., Curr. Genet. 16:53-56, 1989, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods). The expression of heterologous polypeptide in Trichoderma is described in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; U.S. Pat. No. 7,262,041; WO 2005/001036; Harkki et al.; Enzyme Microb. Technol. 13:227-233, 1991; Harkki et al., Bio Technol. 7:596-603, 1989; EP 244,234; EP 215,594; and Nevalainen et al., “The Molecular Biology of Trichoderma and its Application to the Expression of Both Homologous and Heterologous Genes,” in Molecular Industrial Mycology, Eds. Leong and Berka, Marcel Dekker Inc., NY pp. 129-148, 1992, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation and expression methods). Reference is also made to Cao et al., (Sci. 9:991-1001, 2000; EP 238023; and Yelton et al., Proceedings. Natl. Acad. Sci. USA 81:1470-1474, 1984 (which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods) for transformation of Aspergillus strains. The introduced nucleic acids may be integrated into chromosomal DNA or maintained as extrachromosomal replicating sequences.
Any method known in the art may be used to select transformants. In one non-limiting example, stable transformants including an amdS marker are distinguished from unstable transformants by their faster growth rate and the formation of circular colonies with a smooth, rather than ragged outline on solid culture medium containing acetamide. Additionally, in some cases a further test of stability is conducted by growing the transformants on a solid non-selective medium (e.g., a medium that lacks acetamide), harvesting spores from this culture medium, and determining the percentage of these spores which subsequently germinate and grow on selective medium containing acetamide.
In some embodiments, fungal cells are transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a known manner. In one specific embodiment, the preparation of Trichoderma sp. for transformation involves the preparation of protoplasts from fungal mycelia (see, Campbell et al., Curr. Genet. 16:53-56, 1989, which is incorporated by reference in its entirety, particularly with respect to transformation methods). In some embodiments, the mycelia are obtained from germinated vegetative spores. The mycelia are treated with an enzyme that digests the cell wall resulting in protoplasts. The protoplasts are then protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Usually the concentration of these stabilizers varies between 0.8 M and 1.2 M. It is desirable to use about a 1.2 M solution of sorbitol in the suspension medium.
Uptake of DNA into the host Trichoderma sp. strain is dependent upon the calcium ion concentration. Generally, between about 10 mM CaCl2 and 50 mM CaCl2 is used in an uptake solution. In addition to the calcium ion in the uptake solution, other compounds generally included are a buffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethylene glycol (PEG). While not intending to be bound to any particular theory, it is believed that the polyethylene glycol acts to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoderma sp. strain and the plasmid DNA to be transferred to the nucleus. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.
Usually a suspension containing the Trichoderma sp. protoplasts or cells that have been subjected to a permeability treatment at a density of 105 to 107/mL (such as 2×106/mL) are used in the transformation. A volume of 100 μL of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaCl2) are mixed with the desired DNA. Generally, a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension. In some embodiments, about 0.25 volumes are added to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, and the like may also be added to the uptake solution and aid in transformation. Similar procedures are available for other fungal host cells (see, e.g., U.S. Pat. Nos. 6,022,725 and 6,268,328, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods).
Generally, the mixture is then cultured at approximately 0° C. for a period of between 10 to 30 minutes. Additional PEG is then added to the mixture to further enhance the uptake of the desired nucleic acid sequence. The 25% PEG 4000 is generally added in volumes of 5 to 15 times the volume of the transformation mixture; however, greater and lesser volumes may be suitable. The 25% PEG 4000 is desirably about 10 times the volume of the transformation mixture. After the PEG is added, the transformation mixture is then cultured either at room temperature or on ice before the addition of a sorbitol and CaCl2 solution. The protoplast suspension is then further added to molten aliquots of a growth medium. When the growth medium includes a growth selection (e.g., acetamide or an antibiotic) it permits the growth of transformants only.
The transformation of bacterial cells may be performed according to conventional methods, e.g., as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is hereby incorporated by reference in its entirety, particularly with respect to transformation methods.
Exemplary Cell Culture Media
The invention also includes a cell or a population of cells in culture that produce isoprene. By “cells in culture” is meant two or more cells in a solution (e.g., a cell medium) that allows the cells to undergo one or more cell divisions. “Cells in culture” do not include plant cells that are part of a living, multicellular plant containing cells that have differentiated into plant tissues. In various embodiments, the cell culture includes at least or about 10, 20, 50, 100, 200, 500, 1,000, 5,000, 10,000 or more cells.
Any carbon source can be used to cultivate the host cells. The term “carbon source” refers to one or more carbon-containing compounds capable of being metabolized by a host cell or organism. For example, the cell medium used to cultivate the host cells may include any carbon source suitable for maintaining the viability or growing the host cells.
In some embodiments, the carbon source is a carbohydrate (such as monosaccharide, disaccharide, oligosaccharide, or polysaccharides), invert sugar (e.g., enzymatically treated sucrose syrup), glycerol, glycerine (e.g., a glycerine byproduct of a biodiesel or soap-making process), dihydroxyacetone, one-carbon source, oil (e.g., a plant or vegetable oil such as corn, palm, or soybean oil), acetate, animal fat, animal oil, fatty acid (e.g., a saturated fatty acid, unsaturated fatty acid, or polyunsaturated fatty acid), lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, polypeptide (e.g., a microbial or plant protein or peptide), renewable carbon source (e.g., a biomass carbon source such as a hydrolyzed biomass carbon source), yeast extract, component from a yeast extract, polymer, acid, alcohol, aldehyde, ketone, amino acid, succinate, lactate, acetate, ethanol, or any combination of two or more of the foregoing. In some embodiments, the carbon source is a product of photosynthesis, including, but not limited to, glucose. In some embodiment, the carbohydrate is xylose or glucose.
Exemplary monosaccharides include glucose and fructose; exemplary oligosaccharides include lactose and sucrose, and exemplary polysaccharides include starch and cellulose. Exemplary carbohydrates include C6 sugars (e.g., fructose, mannose, galactose, or glucose) and C5 sugars (e.g., xylose or arabinose). In some embodiments, the cell medium includes a carbohydrate as well as a carbon source other than a carbohydrate (e.g., glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, or a component from a yeast extract). In some embodiments, the cell medium includes a carbohydrate as well as a polypeptide (e.g., a microbial or plant protein or peptide). In some embodiments, the microbial polypeptide is a polypeptide from yeast or bacteria. In some embodiments, the plant polypeptide is a polypeptide from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.
In some embodiments, the concentration of the carbohydrate is at least or about 5 grams per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such as at least or about 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400, or more g/L. In some embodiments, the concentration of the carbohydrate is between about 50 and about 400 g/L, such as between about 100 and about 360 g/L, between about 120 and about 360 g/L, or between about 200 and about 300 g/L. In some embodiments, this concentration of carbohydrate includes the total amount of carbohydrate that is added before and/or during the culturing of the host cells.
Exemplary lipids are any substance containing one or more fatty acids that are C4 and above fatty acids that are saturated, unsaturated, or branched.
Exemplary oils are lipids that are liquid at room temperature. In some embodiments, the lipid contains one or more C4 or above fatty acids (e.g., contains one or more saturated, unsaturated, or branched fatty acid with four or more carbons). In some embodiments, the oil is obtained from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, linseed, oleagineous microbial cells, Chinese tallow, or any combination of two or more of the foregoing.
Exemplary fatty acids include compounds of the formula RCOOH, where “R” is a hydrocarbon. Exemplary unsaturated fatty acids include compounds where “R” includes at least one carbon-carbon double bond. Exemplary unsaturated fatty acids include, but are not limited to, oleic acid, vaccenic acid, linoleic acid, palmitelaidic acid, and arachidonic acid. Exemplary polyunsaturated fatty acids include compounds where “R” includes a plurality of carbon-carbon double bonds. Exemplary saturated fatty acids include compounds where “R” is a saturated aliphatic group. In some embodiments, the carbon source includes one or more C12-C22 fatty acids, such as a C12 saturated fatty acid, a C14 saturated fatty acid, a C16 saturated fatty acid, a C1-8 saturated fatty acid, a C20 saturated fatty acid, or a C22 saturated fatty acid. In an exemplary embodiment, the fatty acid is palmitic acid. In some embodiments, the carbon source is a salt of a fatty acid (e.g., an unsaturated fatty acid), a derivative of a fatty acid (e.g., an unsaturated fatty acid), or a salt of a derivative of fatty acid (e.g., an unsaturated fatty acid). Suitable salts include, but are not limited to, lithium salts, potassium salts, sodium salts, and the like. Di- and triglycerols are fatty acid esters of glycerol.
In some embodiments, the concentration of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is at least or about 1 gram per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such as at least or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400, or more g/L. In some embodiments, the concentration of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is between about 10 and about 400 g/L, such as between about 25 and about 300 g/L, between about 60 and about 180 g/L, or between about 75 and about 150 g/L. In some embodiments, the concentration includes the total amount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride that is added before and/or during the culturing of the host cells. In some embodiments, the carbon source includes both (i) a lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride and (ii) a carbohydrate, such as glucose. In some embodiments, the ratio of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride to the carbohydrate is about 1:1 on a carbon basis (i.e., one carbon in the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride per carbohydrate carbon). In particular embodiments, the amount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is between about 60 and 180 g/L, and the amount of the carbohydrate is between about 120 and 360 g/L.
Exemplary microbial polypeptide carbon sources include one or more polypeptides from yeast or bacteria. Exemplary plant polypeptide carbon sources include one or more polypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.
Exemplary renewable carbon sources include cheese whey permeate, cornsteep liquor, sugar beet molasses, barley malt, and components from any of the foregoing. Exemplary renewable carbon sources also include acetate, glucose, hexose, pentose and xylose present in biomass, such as corn, switchgrass, sugar cane, cell waste of fermentation processes, and protein by-product from the milling of soy, corn, or wheat. In some embodiments, the biomass carbon source is a lignocellulosic, hemicellulosic, or cellulosic material such as, but are not limited to, a grass, wheat, wheat straw, bagasse, sugar cane bagasse, soft wood pulp, corn, corn cob or husk, corn kernel, fiber from corn kernels, corn stover, switch grass, rice hull product, or a by-product from wet or dry milling of grains (e.g., corn, sorghum, rye, triticate, barley, wheat, and/or distillers grains). Exemplary cellulosic materials include wood, paper and pulp waste, herbaceous plants, and fruit pulp. In some embodiments, the carbon source includes any plant part, such as stems, grains, roots, or tubers. In some embodiments, all or part of any of the following plants are used as a carbon source: corn, wheat, rye, sorghum, triticate, rice, millet, barley, cassaya, legumes, such as beans and peas, potatoes, sweet potatoes, bananas, sugarcane, and/or tapioca. In some embodiments, the carbon source is a biomass hydrolysate, such as a biomass hydrolysate that includes both xylose and glucose or that includes both sucrose and glucose.
In some embodiments, the renewable carbon source (such as biomass) is pretreated before it is added to the cell culture medium. In some embodiments, the pretreatment includes enzymatic pretreatment, chemical pretreatment, or a combination of both enzymatic and chemical pretreatment (see, for example, Farzaneh et al., Bioresource Technology 96 (18): 2014-2018, 2005; U.S. Pat. No. 6,176,176; U.S. Pat. No. 6,106,888; which are each hereby incorporated by reference in their entireties, particularly with respect to the pretreatment of renewable carbon sources). In some embodiments, the renewable carbon source is partially or completely hydrolyzed before it is added to the cell culture medium.
In some embodiments, the renewable carbon source (such as corn stover) undergoes ammonia fiber expansion (AFEX) pretreatment before it is added to the cell culture medium (see, for example, Farzaneh et al., Bioresource Technology 96 (18): 2014-2018, 2005). During AFEX pretreatment, a renewable carbon source is treated with liquid anhydrous ammonia at moderate temperatures (such as about 60 to about 100° C.) and high pressure (such as about 250 to about 300 psi) for about 5 minutes. Then, the pressure is rapidly released. In this process, the combined chemical and physical effects of lignin solubilization, hemicellulose hydrolysis, cellulose decrystallization, and increased surface area enables near complete enzymatic conversion of cellulose and hemicellulose to fermentable sugars. AFEX pretreatment has the advantage that nearly all of the ammonia can be recovered and reused, while the remaining serves as nitrogen source for microbes in downstream processes. Also, a wash stream is not required for AFEX pretreatment. Thus, dry matter recovery following the AFEX treatment is essentially 100%. AFEX is basically a dry to dry process. The treated renewable carbon source is stable for long periods and can be fed at very high solid loadings in enzymatic hydrolysis or fermentation processes. Cellulose and hemicellulose are well preserved in the AFEX process, with little or no degradation. There is no need for neutralization prior to the enzymatic hydrolysis of a renewable carbon source that has undergone AFEX pretreatment. Enzymatic hydrolysis of AFEX-treated carbon sources produces clean sugar streams for subsequent fermentation use.
In some embodiments, the concentration of the carbon source (e.g., a renewable carbon source) is equivalent to at least or about 0.1, 0.5, 1, 1.5 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50% glucose (w/v). The equivalent amount of glucose can be determined by using standard HPLC methods with glucose as a reference to measure the amount of glucose generated from the carbon source. In some embodiments, the concentration of the carbon source (e.g., a renewable carbon source) is equivalent to between about 0.1 and about 20% glucose, such as between about 0.1 and about 10% glucose, between about 0.5 and about 10% glucose, between about 1 and about 10% glucose, between about 1 and about 5% glucose, or between about 1 and about 2% glucose.
In some embodiments, the carbon source includes yeast extract or one or more components of yeast extract. In some embodiments, the concentration of yeast extract is at least 1 gram of yeast extract per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such at least or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, or more g/L. In some embodiments, the concentration of yeast extract is between about 1 and about 300 g/L, such as between about 1 and about 200 g/L, between about 5 and about 200 g/L, between about 5 and about 100 g/L, or between about 5 and about 60 g/L. In some embodiments, the concentration includes the total amount of yeast extract that is added before and/or during the culturing of the host cells. In some embodiments, the carbon source includes both yeast extract (or one or more components thereof) and another carbon source, such as glucose. In some embodiments, the ratio of yeast extract to the other carbon source is about 1:5, about 1:10, or about 1:20 (w/w).
Additionally the carbon source may also be one-carbon substrates such as carbon dioxide, or methanol. Glycerol production from single carbon sources (e.g., methanol, formaldehyde, or formate) has been reported in methylotrophic yeasts (Yamada et al., Agric. Biol. Chem., 53(2) 541-543, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources) and in bacteria (Hunter et. al., Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). These organisms can assimilate single carbon compounds, ranging in oxidation state from methane to formate, and produce glycerol. The pathway of carbon assimilation can be through ribulose monophosphate, through serine, or through xylulose-momophosphate (Gottschalk, Bacterial Metabolism, Second Edition, Springer-Verlag: New York, 1986, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). The ribulose monophosphate pathway involves the condensation of formate with ribulose-5-phosphate to form a six carbon sugar that becomes fructose and eventually the three carbon product glyceraldehyde-3-phosphate. Likewise, the serine pathway assimilates the one-carbon compound into the glycolytic pathway via methylenetetrahydrofolate.
In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7th ed., 415-32. Editors: Murrell et al., Publisher: Intercept, Andover, UK, 1993, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). Similarly, various species of Candida metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153(5), 485-9, 1990, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources).
In some embodiments, cells are cultured in a standard medium containing physiological salts and nutrients (see, e.g., Pourquie, J. et al., Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al., Academic Press, pp. 71-86, 1988 and Ilmen et al., Appl. Environ. Microbiol. 63:1298-1306, 1997, which are each hereby incorporated by reference in their entireties, particularly with respect to cell media). Exemplary growth media are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of particular host cells are known by someone skilled in the art of microbiology or fermentation science.
In addition to an appropriate carbon source, the cell medium desirably contains suitable minerals, salts, cofactors, buffers, and other components known to those skilled in the art suitable for the growth of the cultures or the enhancement of isoprene production (see, for example, WO 2004/033646 and references cited therein and WO 96/35796 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect cell medias and cell culture conditions). In some embodiments where an isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acid is under the control of an inducible promoter, the inducing agent (e.g., a sugar, metal salt or antimicrobial), is desirably added to the medium at a concentration effective to induce expression of an isoprene synthase, DXS, IDI, and/or MVA pathway polypeptide. In some embodiments, cell medium has an antibiotic (such as kanamycin) that corresponds to the antibiotic resistance nucleic acid (such as a kanamycin resistance nucleic acid) on a vector that has one or more DXS, IDI, or MVA pathway nucleic acids.
Exemplary Cell Culture Conditions
Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Exemplary techniques may be found in Manual of Methods for General Bacteriology Gerhardt et al., eds), American Society for Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., which are each hereby incorporated by reference in their entireties, particularly with respect to cell culture techniques. In some embodiments, the cells are cultured in a culture medium under conditions permitting the expression of one or more isoprene synthase, DXS, IDI, or MVA pathway polypeptides encoded by a nucleic acid inserted into the host cells.
Standard cell culture conditions can be used to culture the cells (see, for example, WO 2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to cell culture and fermentation conditions). Cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as at about 20 to about 37° C., at about 6% to about 84% CO2, and at a pH between about 5 to about 9). In some embodiments, cells are grown at 35° C. in an appropriate cell medium. In some embodiments, e.g., cultures are cultured at approximately 28° C. in appropriate medium in shake cultures or fermentors until desired amount of isoprene production is achieved. In some embodiments, the pH ranges for fermentation are between about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH 8.0 or about 6.5 to about 7.0). Reactions may be performed under aerobic, anoxic, or anaerobic conditions based on the requirements of the host cells. Exemplary culture conditions for a given filamentous fungus are known in the art and may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection and Fungal Genetics Stock Center.
In various embodiments, the cells are grown using any known mode of fermentation, such as batch, fed-batch, or continuous processes. In some embodiments, a batch method of fermentation is used. Classical batch fermentation is a closed system where the composition of the media is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the cell medium is inoculated with the desired host cells and fermentation is permitted to occur adding nothing to the system. Typically, however, “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly until the time the fermentation is stopped. Within batch cultures, cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. In some embodiments, cells in log phase are responsible for the bulk of the isoprene production. In some embodiments, cells in stationary phase produce isoprene.
In some embodiments, a variation on the standard batch system is used, such as the Fed-Batch system. Fed-Batch fermentation processes comprise a typical batch system with the exception that the carbon source is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of carbon source in the cell medium. Fed-batch fermentations may be performed with the carbon source (e.g., glucose) in a limited or excess amount. Measurement of the actual carbon source concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen, and the partial pressure of waste gases such as CO2. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., which is hereby incorporated by reference in its entirety, particularly with respect to cell culture and fermentation conditions.
In some embodiments, continuous fermentation methods are used. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or isoprene production. For example, one method maintains a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allows all other parameters to moderate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration (e.g., the concentration measured by media turbidity) is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, the cell loss due to media being drawn off is balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., which is hereby incorporated by reference in its entirety, particularly with respect to cell culture and fermentation conditions.
In some embodiments, cells are immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for isoprene production.
In some embodiments, bottles of liquid culture are placed in shakers in order to introduce oxygen to the liquid and maintain the uniformity of the culture. In some embodiments, an incubator is used to control the temperature, humidity, shake speed, and/or other conditions in which a culture is grown. The simplest incubators are insulated boxes with an adjustable heater, typically going up to  ̃65° C. More elaborate incubators can also include the ability to lower the temperature (via refrigeration), or the ability to control humidity or CO2 levels. Most incubators include a timer; some can also be programmed to cycle through different temperatures, humidity levels, etc. Incubators can vary in size from tabletop to units the size of small rooms.
If desired, a portion or all of the cell medium can be changed to replenish nutrients and/or avoid the build up of potentially harmful metabolic byproducts and dead cells. In the case of suspension cultures, cells can be separated from the media by centrifuging or filtering the suspension culture and then resuspending the cells in fresh media. In the case of adherent cultures, the media can be removed directly by aspiration and replaced. In some embodiments, the cell medium allows at least a portion of the cells to divide for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution).
In some embodiments, a constitutive or leaky promoter (such as a Trc promoter) is used and a compound (such as IPTG) is not added to induce expression of the isoprene synthase, DXS, IDI, or MVA pathway nucleic acid(s) operably linked to the promoter. In some embodiments, a compound (such as IPTG) is added to induce expression of the isoprene synthase, DXS, IDI, or MVA pathway nucleic acid(s) operably linked to the promoter.
Exemplary Production of Isoprene
In some embodiments, the cells are cultured in a culture medium under conditions permitting the production of isoprene by the cells. By “peak absolute productivity” is meant the maximum absolute amount of isoprene in the off-gas during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By “peak absolute productivity time point” is meant the time point during a fermentation run when the absolute amount of isoprene in the off-gas is at a maximum during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). In some embodiments, the isoprene amount is measured at the peak absolute productivity time point. In some embodiments, the peak absolute productivity for the cells is about any of the isoprene amounts disclosed herein.
By “peak specific productivity” is meant the maximum amount of isoprene produced per cell during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By “peak specific productivity time point” is meant the time point during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run) when the amount of isoprene produced per cell is at a maximum. The peak specific productivity is determined by dividing the total productivity by the amount of cells, as determined by optical density at 600 nm (OD600). In some embodiments, the isoprene amount is measured at the peak specific productivity time point. In some embodiments, the peak specific productivity for the cells is about any of the isoprene amounts per cell disclosed herein.
By “peak volumetric productivity” is meant the maximum amount of isoprene produced per volume of broth (including the volume of the cells and the cell medium) during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By “peak specific volumetric productivity time point” is meant the time point during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run) when the amount of isoprene produced per volume of broth is at a maximum. The peak specific volumetric productivity is determined by dividing the total productivity by the volume of broth and amount of time. In some embodiments, the isoprene amount is measured at the peak specific volumetric productivity time point. In some embodiments, the peak specific volumetric productivity for the cells is about any of the isoprene amounts per volume per time disclosed herein.
By “peak concentration” is meant the maximum amount of isoprene produced during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By “peak concentration time point” is meant the time point during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run) when the amount of isoprene produced per cell is at a maximum. In some embodiments, the isoprene amount is measured at the peak concentration time point. In some embodiments, the peak concentration for the cells is about any of the isoprene amounts disclosed herein.
By “average volumetric productivity” is meant the average amount of isoprene produced per volume of broth (including the volume of the cells and the cell medium) during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). The average volumetric productivity is determined by dividing the total productivity by the volume of broth and amount of time. In some embodiments, the average specific volumetric productivity for the cells is about any of the isoprene amounts per volume per time disclosed herein.
By “cumulative total productivity” is meant the cumulative, total amount of isoprene produced during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). In some embodiments, the cumulative, total amount of isoprene is measured. In some embodiments, the cumulative total productivity for the cells is about any of the isoprene amounts disclosed herein.
In some embodiments, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 12,500, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000, 125,000, 150,000, 188,000, or more nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/gwcm/hr). In some embodiments, the amount of isoprene is between about 2 to about 200,000 nmole/gwcm/hr, such as between about 2 to about 100 nmole/gwcm/hr, about 100 to about 500 nmole/gwcm/hr, about 150 to about 500 nmole/gwcm/hr, about 500 to about 1,000 nmole/gwcm/hr, about 1,000 to about 2,000 nmole/gwcm/hr, about 2,000 to about 5,000 nmole/gwcm/hr, about 5,000 to about 10,000 nmole/gwcm/hr, about 10,000 to about 50,000 nmole/gwcm/hr, about 50,000 to about 100,000 nmole/gwcm/hr, about 100,000 to about 150,000 nmole/gwcm/hr, or about 150,000 to about 200,000 nmole/gwcm/hr. In some embodiments, the amount of isoprene is between about 20 to about 200,000 nmole/gwcm/hr, about 100 to about 5,000 nmole/gwcm/hr, about 200 to about 2,000 nmole/gwcm/hr, about 200 to about 1,000 nmole/gwcm/hr, about 300 to about 1,000 nmole/gwcm/hr, about 400 to about 1,000 nmole/gwcm/hr, about 1,000 to about 5,000 nmole/gwcm/hr, about 2,000 to about 20,000 nmole/gwcm/hr, about 5,000 to about 50,000 nmole/gwcm/hr, about 10,000 to about 100,000 nmole/gwcm/hr, about 20,000 to about 150,000 nmole/gwcm/hr, or about 20,000 to about 200,000 nmole/gwcm/hr.
The amount of isoprene in units of nmole/gwcm/hr can be measured as disclosed in U.S. Pat. No. 5,849,970, which is hereby incorporated by reference in its entirety, particularly with respect to the measurement of isoprene production. For example, two mL of headspace (e.g., headspace from a culture such as 2 mL of culture cultured in sealed vials at 32° C. with shaking at 200 rpm for approximately 3 hours) are analyzed for isoprene using a standard gas chromatography system, such as a system operated isothermally (85° C.) with an n-octane/porasil C column (Alltech Associates, Inc., Deerfield, Ill.) and coupled to a RGD2 mercuric oxide reduction gas detector (Trace Analytical, Menlo Park, Calif.) (see, for example, Greenberg et al, Atmos. Environ. 27A: 2689-2692, 1993; Silver et al., Plant Physiol. 97:1588-1591, 1991, which are each hereby incorporated by reference in their entireties, particularly with respect to the measurement of isoprene production). The gas chromatography area units are converted to nmol isoprene via a standard isoprene concentration calibration curve. In some embodiments, the value for the grams of cells for the wet weight of the cells is calculated by obtaining the A600 value for a sample of the cell culture, and then converting the A600 value to grams of cells based on a calibration curve of wet weights for cell cultures with a known A600 value. In some embodiments, the grams of the cells is estimated by assuming that one liter of broth (including cell medium and cells) with an A600 value of 1 has a wet cell weight of 1 gram. The value is also divided by the number of hours the culture has been incubating for, such as three hours.
In some embodiments, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wet weight of the cells/hr (ng/gwcm/h). In some embodiments, the amount of isoprene is between about 2 to about 5,000 ng/gwcm/h, such as between about 2 to about 100 ng/gwcm/h, about 100 to about 500 ng/gwcm/h, about 500 to about 1,000 ng/gwcm/h, about 1,000 to about 2,000 ng/gwcm/h, or about 2,000 to about 5,000 ng/gwcm/h. In some embodiments, the amount of isoprene is between about 20 to about 5,000 ng/gwcm/h, about 100 to about 5,000 ng/gwcm/h, about 200 to about 2,000 ng/gwcm/h, about 200 to about 1,000 ng/gwcm/h, about 300 to about 1,000 ng/gwcm/h, or about 400 to about 1,000 ng/gwcm/h. The amount of isoprene in ng/gwcm/h can be calculated by multiplying the value for isoprene production in the units of nmole/gwcm/hr discussed above by 68.1 (as described in Equation 5 below).
In some embodiments, the cells in culture produce a cumulative titer (total amount) of isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth (mg/Lbroth, wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments, the amount of isoprene is between about 2 to about 5,000 mg/Lbroth, such as between about 2 to about 100 mg/Lbroth, about 100 to about 500 mg/Lbroth, about 500 to about 1,000 mg/Lbroth, about 1,000 to about 2,000 mg/Lbroth, or about 2,000 to about 5,000 mg/Lbroth. In some embodiments, the amount of isoprene is between about 20 to about 5,000 mg/Lbroth, about 100 to about 5,000 mg/Lbroth, about 200 to about 2,000 mg/Lbroth, about 200 to about 1,000 mg/Lbroth, about 300 to about 1,000 mg/Lbroth, or about 400 to about 1,000 mg/Lbroth.
The specific productivity of isoprene in mg of isoprene/L of headspace from shake flask or similar cultures can be measured by taking a 1 ml sample from the cell culture at an OD600 value of approximately 1.0, putting it in a 20 mL vial, incubating for 30 minutes, and then measuring the amount of isoprene in the headspace (as described, for example, in Example I, part II). If the OD600 value is not 1.0, then the measurement can be normalized to an OD600 value of 1.0 by dividing by the OD600 value. The value of mg isoprene/L headspace can be converted to mg/Lbroth/hr/OD600 of culture broth by multiplying by a factor of 38. The value in units of mg/Lbroth/hr/OD600 can be multiplied by the number of hours and the OD600 value to obtain the cumulative titer in units of mg of isoprene/L of broth.
In some embodiments, the cells in culture have an average volumetric productivity of isoprene at greater than or about 0.1, 1.0, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100, 1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, or more mg of isoprene/L of broth/hr (mg/Lbroth/hr, wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments, the average volumetric productivity of isoprene is between about 0.1 to about 3,500 mg/Lbroth/hr, such as between about 0.1 to about 100 mg/Lbroth/hr, about 100 to about 500 mg/Lbroth/hr, about 500 to about 1,000 mg/Lbroth/hr, about 1,000 to about 1,500 mg/Lbroth/hr, about 1,500 to about 2,000 mg/Lbroth/hr, about 2,000 to about 2,500 mg/Lbroth/hr, about 2,500 to about 3,000 mg/Lbroth/hr, or about 3,000 to about 3,500 mg/Lbroth/hr. In some embodiments, the average volumetric productivity of isoprene is between about 10 to about 3,500 mg/Lbroth/hr, about 100 to about 3,500 mg/Lbroth/hr, about 200 to about 1,000 mg/Lbroth/hr, about 200 to about 1,500 mg/Lbroth/hr, about 1,000 to about 3,000 mg/Lbroth/hr, or about 1,500 to about 3,000 mg/Lbroth/hr.
In some embodiments, the cells in culture have a peak volumetric productivity of isoprene at greater than or about 0.5, 1.0, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100, 1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, 5,000, 5,250, 5,500, 5,750, 6,000, 6,250, 6,500, 6,750, 7,000, 7,250, 7,500, 7,750, 8,000, 8,250, 8,500, 8,750, 9,000, 9,250, 9,500, 9,750, 10,000, 12,500, 15,000, or more mg of isoprene/L of broth/hr (mg/Lbroth/hr, wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments, the peak volumetric productivity of isoprene is between about 0.5 to about 15,000 mg/Lbroth/hr, such as between about 0.5 to about 10 mg/Lbroth/hr, about 1.0 to about 100 mg/Lbroth/hr, about 100 to about 500 mg/Lbroth/hr, about 500 to about 1,000 mg/Lbroth/hr, about 1,000 to about 1,500 mg/Lbroth/hr, about 1,500 to about 2,000 mg/Lbroth/hr, about 2,000 to about 2,500 mg/Lbroth/hr, about 2,500 to about 3,000 mg/Lbroth/hr, about 3,000 to about 3,500 mg/Lbroth/hr, about 3,500 to about 5,000 mg/Lbroth/hr, about 5,000 to about 7,500 mg/Lbroth/hr, about 7,500 to about 10,000 mg/Lbroth/hr, about 10,000 to about 12,500 mg/Lbroth/h, or about 12,500 to about 15,000 mg/Lbroth/hr. In some embodiments, the peak volumetric productivity of isoprene is between about 10 to about 15,000 mg/Lbroth/hr, about 100 to about 2,500 mg/Lbroth/hr, about 1,000 to about 5,000 mg/Lbroth/hr, about 2,500 to about 7,500 mg/Lbroth/hr, about 5,000 to about 10,000 mg/Lbroth/hr, about 7,500 to about 12,500 mg/Lbroth/hr, or about 10,000 to about 15,000 mg/Lbroth/hr.
The instantaneous isoprene production rate in mg/Lbroth/hr in a fermentor can be measured by taking a sample of the fermentor off-gas, analyzing it for the amount of isoprene (in units such as mg of isoprene per Lgas) as described, for example, in Example I, part II and multiplying this value by the rate at which off-gas is passed though each liter of broth (e.g., at 1 vvm (volume of air/volume of broth/minute) this is 60 Lgas per hour). Thus, an off-gas level of 1 mg/Lgas corresponds to an instantaneous production rate of 60 mg/Lbroth/hr at air flow of 1 vvm. If desired, the value in the units mg/Lbroth/hr can be divided by the OD600 value to obtain the specific rate in units of mg/Lbroth/hr/OD. The average value of mg isoprene/Lgas can be converted to the total product productivity (grams of isoprene per liter of fermentation broth, mg/Lbroth) by multiplying this average off-gas isoprene concentration by the total amount of off-gas sparged per liter of fermentation broth during the fermentation. Thus, an average off-gas isoprene concentration of 0.5 mg/Lbroth/hr over 10 hours at 1 vvm corresponds to a total product concentration of 300 mg isoprene/Lbroth.
In some embodiments, the cells in culture convert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 2.0, 2.2, 2.4, 2.6, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 23.2, 23.4, 23.6, 23.8, 24.0, 25.0, 30.0, 31.0, 32.0, 33.0, 35.0, 37.5, 40.0, 45.0, 47.5, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, or 90.0 molar % of the carbon in the cell culture medium into isoprene. In some embodiments, the percent conversion of carbon into isoprene is between about 0.002 to about 90.0 molar %, such as about 0.002 to about 0.005%, about 0.005 to about 0.01%, about 0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about 1.0%, about 1.0 to about 1.6%, about 1.6 to about 3.0%, about 3.0 to about 5.0%, about 5.0 to about 8.0%, about 8.0 to about 10.0%, about 10.0 to about 15.0%, about 15.0 to about 20.0%, about 20.0 to about 25.0%, about 25.0% to 30.0%, about 30.0% to 35.0%, about 35.0% to 40.0%, about 45.0% to 50.0%, about 50.0% to 55.0%, about 55.0% to 60.0%, about 60.0% to 65.0%, about 65.0% to 70.0%, about 75.0% to 80.0%, about 80.0% to 85.0%, or about 85.0% to 90.0%. In some embodiments, the percent conversion of carbon into isoprene is between about 0.002 to about 0.4 molar %, 0.002 to about 0.16 molar %, 0.04 to about 0.16 molar %, about 0.005 to about 0.3 molar %, about 0.01 to about 0.3 molar %, about 0.05 to about 0.3 molar %, about 0.1 to 0.3 molar %, about 0.3 to about 1.0 molar %, about 1.0 to about 5.0 molar %, about 2 to about 5.0 molar %, about 5.0 to about 10.0 molar %, about 7 to about 10.0 molar %, about 10.0 to about 20.0 molar %, about 12 to about 20.0 molar %, about 16 to about 20.0 molar %, about 18 to about 20.0 molar %, about 18 to 23.2 molar %, about 18 to 23.6 molar %, about 18 to about 23.8 molar %, about 18 to about 24.0 molar %, about 18 to about 25.0 molar %, about 20 to about 30.0 molar %, about 30 to about 40.0 molar %, about 30 to about 50.0 molar %, about 30 to about 60.0 molar %, about 30 to about 70.0 molar %, about 30 to about 80.0 molar %, or about 30 to about 90.0 molar %
The percent conversion of carbon into isoprene (also referred to as “% carbon yield”) can be measured by dividing the moles carbon in the isoprene produced by the moles carbon in the carbon source (such as the moles of carbon in batched and fed glucose and yeast extract). This number is multiplied by 100% to give a percentage value (as indicated in Equation 1).

% Carbon Yield=(moles carbon in isoprene produced)/(moles carbon in carbon source)*100  Equation 1

For this calculation, yeast extract can be assumed to contain 50% w/w carbon. As an example, for the 500 liter described in Example 7, part VIII, the percent conversion of carbon into isoprene can be calculated as shown in Equation 2.

% Carbon Yield=(39.1 g isoprene*1/68.1 mol/g*5 C/mol)/[(181221 g glucose*1/180 mol/g*6 C/mol)+(17780 g yeast extract*0.5*1/12 mol/g)]*100=0.042%  Equation 2

For the two 500 liter fermentations described herein (Example 7, parts VII and VIII), the percent conversion of carbon into isoprene was between 0.04-0.06%. A 0.11-0.16% carbon yield has been achieved using 14 liter systems as described herein.
One skilled in the art can readily convert the rates of isoprene production or amount of isoprene produced into any other units. Exemplary equations are listed below for interconverting between units.
Units for Rate of Isoprene Production (Total and Specific)

1 g isoprene/Lbroth/hr=14.7 mmol isoprene/Lbroth/hr(total volumetric rate)  Equation 3

1 nmol isoprene/gwcm/hr=1 nmol isoprene/Lbroth/hr/OD600 (This conversion assumes that one liter of broth with an OD600 value of 1 has a wet cell weight of 1 gram.)  Equation 4

1 nmol isoprene/gwcm/hr=68.1 ng isoprene/gwcm/hr(given the molecular weight of isoprene)  Equation 5

1 nmol isoprene/LgasO2/hr=90 nmol isoprene/Lbroth/hr(at an O2 flow rate of 90 L/hr per L of culture broth)  Equation 6

1 ug isoprene/Lgas isoprene in off-gas=60 ug isoprene/Lbroth/hr at a flow rate of 60 Lgas per Lbroth(1 vvm)  Equation 7

Units for Titer (Total and Specific)

1 nmol isoprene/mg cell protein=150 nmol isoprene/Lbroth/OD600 (This conversion assumes that one liter of broth with an OD600 value of 1 has a total cell protein of approximately 150 mg) (specific productivity)  Equation 8

1 g isoprene/Lbroth=14.7 mmol isoprene/Lbroth(total titer)  Equation 9

If desired, Equation 10 can be used to convert any of the units that include the wet weight of the cells into the corresponding units that include the dry weight of the cells.

Dry weight of cells=(wet weight of cells)/3.3  Equation 10

In some embodiments encompassed by the invention, a cell comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide produces an amount of isoprene that is at least or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of isoprene produced from a corresponding cell grown under essentially the same conditions without the heterologous nucleic acid encoding the isoprene synthase polypeptide.
In some embodiments encompassed by the invention, a cell comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide and one or more heterologous nucleic acids encoding a DXS, IDI, and/or MVA pathway polypeptide produces an amount of isoprene that is at least or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of isoprene produced from a corresponding cell grown under essentially the same conditions without the heterologous nucleic acids.
Exemplary Isoprene Purification Methods
In some embodiments, any of the methods described herein further include recovering the isoprene. For example, the isoprene produced using the compositions and methods of the invention can be recovered using standard techniques. such as gas stripping, fractionation, adsorption/desorption, pervaporation, thermal or vacuum desorption of isoprene from a solid phase, or extraction of isoprene immobilized or absorbed to a solid phase with a solvent (see, for example, U.S. Pat. Nos. 4,703,007 and 4,570,029, which are each hereby incorporated by reference in their entireties, particularly with respect to isoprene recovery and purification methods). In some embodiments, the recovery of isoprene involves the isolation of isoprene in a liquid form (such as a neat solution of isoprene or a solution of isoprene in a solvent). Gas stripping involves the removal of isoprene vapor from the fermentation off-gas stream in a continuous manner. Such removal can be achieved in several different ways including, but not limited to, adsorption to a solid phase, partition into a liquid phase, or direct condensation. In some embodiments, membrane enrichment of a dilute isoprene vapor stream above the dew point of the vapor resulting in the condensation of liquid isoprene.
The recovery of isoprene may involve one step or multiple steps. In some embodiments, the removal of isoprene vapor from the fermentation off-gas and the conversion of isoprene to a liquid phase are performed simultaneously. For example, isoprene can be directly condensed from the off-gas stream to form a liquid. In some embodiments, the removal of isoprene vapor from the fermentation off-gas and the conversion of isoprene to a liquid phase are performed sequentially. For example, isoprene may be adsorbed to a solid phase and then extracted from the solid phase with a solvent.
In some embodiments, any of the methods described herein further include purifying the isoprene. For example, the isoprene produced using the compositions and methods of the invention can be purified using standard techniques. Purification refers to a process through which isoprene is separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is obtained as a substantially pure liquid. Examples of purification methods include (i) distillation from a solution in a liquid extractant and (ii) chromatography. As used herein, “purified isoprene” means isoprene that has been separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is at least about 20%, by weight, free from other components that are present when the isoprene is produced. In various embodiments, the isoprene is at least or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%, by weight, pure. Purity can be assayed by any appropriate method, e.g., by column chromatography, HPLC analysis, or GC-MS analysis.
In some embodiments, any of the methods described herein further include polymerizing the isoprene. For example, standard methods can be used to polymerize the purified isoprene to form cis-polyisoprene or other down stream products using standard methods.
Additional methods and compositions are described in U.S. Provisional patent application No. 61/097,186, filed on Sep. 15, 2008, U.S. Provisional patent application No. 61/097,189, filed on Sep. 15, 2008, and U.S. Provisional patent application No. 61/097,163, filed on Sep. 15, 2008, all of which are incorporated by reference in their entireties, particular with respect to compositions and methods for producing isoprene.
EXAMPLES
The examples, which are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention discussed above. Unless indicated otherwise, temperature is in degrees Centigrade and pressure is at or near atmospheric. The foregoing examples and detailed description are offered by way of illustration and not by way of limitation. All publications, patent applications, and patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or patent were specifically and individually indicated to be incorporated by reference. In particular, all publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies which might be used in connection with the invention. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Example 1
Production of Isoprene in E. coli Expressing Recombinant Kudzu Isoprene Synthase
I. Construction of Vectors for Expression of the Kudzu Isoprene Synthase in E. coli
The protein sequence for the kudzu (Pueraria montana) isoprene synthase gene (IspS) was obtained from GenBank (AAQ84170). A kudzu isoprene synthase gene, optimized for E. coli codon usage, was purchased from DNA2.0 (SEQ ID NO:1). The isoprene synthase gene was removed from the supplied plasmid by restriction endonuclease digestion with BspLU111/PstI, gel-purified, and ligated into pTrcHis2B (Invitrogen) that had been digested with NcoI/PstI. The construct was designed such that the stop codon in the isoprene synthase gene 5′ to the PstI site. As a result, when the construct was expressed the His-Tag is not attached to the isoprene synthase protein. The resulting plasmid, pTrcKudzu, was verified by sequencing (FIGS. 2 and 3).
The isoprene synthase gene was also cloned into pET16b (Novagen). In this case, the isoprene synthase gene was inserted into pET16b such that the recombinant isoprene synthase protein contained the N-terminal His tag. The isoprene synthase gene was amplified from pTrcKudzu by PCR using the primer set pET-His-Kudzu-2F: 5′-CGTGAGATCATATGTGTGCGACCTCTTCTCAATTTAC (SEQ ID NO:3) and pET-His-Kudzu-R: 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:4). These primers added an NdeI site at the 5′-end and a BamHI site at the 3′ end of the gene respectively. The plasmid pTrcKudzu, described above, was used as template DNA, Herculase polymerase (Stratagene) was used according to manufacture's directions, and primers were added at a concentration of 10 pMols. The PCR was carried out in a total volume of 25 μl. The PCR product was digested with NdeI/BamHI and cloned into pET16b digested with the same enzymes. The ligation mix was transformed into E. coli Top10 (Invitrogen) and the correct clone selected by sequencing. The resulting plasmid, in which the kudzu isoprene synthase gene was expressed from the T7 promoter, was designated pETNHisKudzu (FIGS. 4 and 5).
The kudzu isoprene synthase gene was also cloned into the low copy number plasmid pCL1920. Primers were used to amplify the kudzu isoprene synthase gene from pTrcKudzu described above. The forward primer added a HindIII site and an E. coli consensus RBS to the 5′ end. The PstI cloning site was already present in pTrcKudzu just 3′ of the stop codon so the reverse primer was constructed such that the final PCR product includes the PstI site. The sequences of the primers were: HindIII-rbs-Kudzu F: 5′-CATATGAAAGCTTGTATCGATTAAATAAGGAGGAATAAACC (SEQ ID NO:6) and BamH1-Kudzu R:
5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:4). The PCR product was amplified using Herculase polymerase with primers at a concentration of 10 μmol and with 1 ng of template DNA (pTrcKudzu). The amplification protocol included 30 cycles of (95° C. for 1 minute, 60° C. for 1 minute, 72° C. for 2 minutes). The product was digested with HindIII and PstI and ligated into pCL1920 which had also been digested with HindIII and PstI. The ligation mix was transformed into E. coli Top10, Several transformants were checked by sequencing. The resulting plasmid was designated pCL-lac-Kudzu (FIGS. 6 and 7).
II. Determination of Isoprene Production
For the shake flask cultures, one ml of a culture was transferred from shake flasks to 20 ml CTC headspace vials (Agilent vial cat#5188 2753; cap cat#5188 2759). The cap was screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After 30 minutes the vials were removed from the incubator and analyzed as described below (see Table 1 for some experimental values from this assay).
In cases where isoprene production in fermentors was determined, samples were taken from the off-gas of the fermentor and analyzed directly as described below (see Table 2 for some experimental values from this assay).
The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μm film thickness) was used for separation of analytes. The sampler was set up to inject 500 μL of headspace gas. The GC/MS method utilized helium as the carrier gas at a flow of 1 ml/minutes The injection port was held at 250° C. with a split ratio of 50:1. The oven temperature was held at 37° C. for the 2 minute duration of the analysis. The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on m/z 67. The detector was switched off from 1.4 to 1.7 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. A calibration table was used to quantify the absolute amount of isoprene and was found to be linear from 1 μg/L to 200 μg/L. The limit of detection was estimated to be 50 to 100 ng/L using this method.
III. Production of Isoprene in Shake Flasks Containing E. coli Cells Expressing Recombinant Isoprene Synthase
The vectors described above were introduced to E. coli strain BL21 (Novagen) to produce strains BL21/ptrcKudzu, BL21/pCL-lac-Kudzu and BL21/pETHisKudzu. The strains were spread for isolation onto LA (Luria agar) and carbenicillin (50 μg/ml) and incubated overnight at 37° C. Single colonies were inoculated into 250 ml baffled shake flasks containing 20 ml Luria Bertani broth (LB) and carbenicillin (100 μg/ml). Cultures were grown overnight at 20° C. with shaking at 200 rpm. The OD600 of the overnight cultures were measured and the cultures were diluted into a 250 ml baffled shake flask containing 30 ml MagicMedia (Invitrogen) and carbenicillin (100 μg/ml) to an OD600 ̃0.05. The culture was incubated at 30° C. with shaking at 200 rpm. When the OD600 ̃0.5-0.8, 400 μM IPTG was added and the cells were incubated for a further 6 hours at 30° C. with shaking at 200 rpm. At 0, 2, 4 and 6 hours after induction with IPTG, 1 ml aliquots of the cultures were collected, the OD600 was determined and the amount of isoprene produced was measured as described above. Results are shown in FIG. 8.
IV. Production of Isoprene from BL21/ptrcKudzu in 14 Liter Fermentation
Large scale production of isoprene from E. coli containing the recombinant kudzu isoprene synthase gene was determined from a fed-batch culture. The recipe for the fermentation media (TM2) per liter of fermentation medium was as follows: K2HPO4 13.6 g, KH2PO4 13.6 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, (NH4)2SO4 3.2 g, yeast extract 5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH2O. The pH was adjusted to 6.8 with potassium hydroxide (KOH) and q.s. to volume. The final product was filter sterilized with 0.22μ filter (only, do not autoclave). The recipe for 1000× Modified Trace Metal Solution was as follows: Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a time in diH2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22μ filter.
This experiment was carried out in 14 L bioreactor to monitor isoprene formation from glucose at the desired fermentation, pH 6.7 and temperature 34° C. An inoculum of E. coli strain BL21/ptrcKudzu taken from a frozen vial was prepared in soytone-yeast extract-glucose medium. After the inoculum grew to OD550=0.6, two 600 ml flasks were centrifuged and the contents resuspended in 70 ml supernatant to transfer the cell pellet (70 ml of OD 3.1 material) to the bioreactor. At various times after inoculation, samples were removed and the amount of isoprene produced was determined as described above. Results are shown in FIG. 9.
Example 2
Production of Isoprene in E. coli Expressing Recombinant Poplar Isoprene Synthase
The protein sequence for the poplar (Populus alba x Populus tremula) isoprene synthase (Schnitzler, J-P, et al. (2005) Planta 222:777-786) was obtained from GenBank (CAC35696). A gene, codon optimized for E. coli, was purchased from DNA2.0 (p9796-poplar, FIGS. 30 and 31). The isoprene synthase gene was removed from the supplied plasmid by restriction endonuclease digestion with BspLU11I/PstI, gel-purified, and ligated into pTrcHis2B that had been digested with NcoI/PstI. The construct is cloned such that the stop codon in the insert is before the PstI site, which results in a construct in which the His-Tag is not attached to the isoprene synthase protein. The resulting plasmid pTrcPoplar (FIGS. 32 and 33), was verified by sequencing.
Example 3
Production of Isoprene in Panteoa citrea Expressing Recombinant Kudzu Isoprene Synthase
The pTrcKudzu and pCL-lac Kudzu plasmids described in Example 1 were electroporated into P. citrea (U.S. Pat. No. 7,241,587). Transformants were selected on LA containing carbenicillin (200 μg/ml) or spectinomycin (50 μg/ml) respectively. Production of isoprene from shake flasks and determination of the amount of isoprene produced was performed as described in Example 1 for E. coli strains expressing recombinant kudzu isoprene synthase. Results are shown in FIG. 10.
Example 4
Production of Isoprene in Bacillus subtilis Expressing Recombinant Kudzu Isoprene Synthase
I. Construction of a B. subtilis Replicating Plasmid for the Expression of Kudzu Isoprene Synthase
The kudzu isoprene synthase gene was expressed in Bacillus subtilis aprEnprE Pxyl-comK strain (BG3594comK) using a replicating plasmid (pBS19 with a chloramphenicol resistance cassette) under control of the aprE promoter. The isoprene synthase gene, the aprE promoter and the transcription terminator were amplified separately and fused using PCR. The construct was then cloned into pBS19 and transformed into B. subtilis. 
a) Amplification of the aprE Promoter
The aprE promoter was amplified from chromosomal DNA from Bacillus subtilis using the following primers:
CF 797 (+) Start aprE Promoter MfeI







(SEQ ID NO: 58)









5′-GACATCAATTGCTCCATTTTCTTCTGCTATC






CF 07-43 (−) Fuse aprE Promoter to Kudzu ispS








(SEQ ID NO: 59)









5′-ATTGAGAAGAGGTCGCACACACTCTTTACCCTCTCCTTTTA






b) Amplification of the Isoprene Synthase Gene

The kudzu isoprene synthase gene was amplified from plasmid pTrcKudzu (SEQ ID NO:2). The gene had been codon optimized for E. coli and synthesized by DNA 2.0. The following primers were used:
CF 07-42 (+) Fuse the aprE Promoter to Kudzu Isoprene Synthase Gene (GTG Start Codon)







(SEQ ID NO: 60)









5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT






CF 07-45 (−) Fuse the 3′ End of Kudzu Isoprene Synthase Gene to the Terminator








(SEQ ID NO: 61)









5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC






c) Amplification of the Transcription Terminator

The terminator from the alkaline serine protease of Bacillus amyliquefaciens was amplified from a previously sequenced plasmid pJHPms382 using the following primers:
CF 07-44 (+) Fuse the 3′ end of Kudzu Isoprene Synthase to the Terminator







(SEQ ID NO: 62)









5′-GATTAACCAGCTGATGTATGTCTAAAAAAAACCGGCCTTGG






CF 07-46 (−) End of B. amyliquefaciens Terminator (BamHI)








(SEQ ID NO: 63)









5′-GACATGACGGATCCGATTACGAATGCCGTCTC






The kudzu fragment was fused to the terminator fragment using PCR with the following primers:
CF 07-42 (+) Fuse the aprE Promoter to Kudzu Isoprene Synthase Gene (GTG Start Codon)







(SEQ ID NO: 61)









5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT






CF 07-46 (−) End of B. amyliquefaciens Terminator (BamHI)








(SEQ ID NO: 63)









5′-GACATGACGGATCCGATTACGAATGCCGTCTC






The kudzu-terminator fragment was fused to the promoter fragment using PCR with the following primers:
CF 797 (+) Start aprE Promoter MfeI







(SEQ ID NO: 64)









5′-GACATCAATTGCTCCATTTTCTTCTGCTATC






CF 07-46 (−) End of B. amyliquefaciens Terminator (BamHI)








(SEQ ID NO: 63)


5′-GACATGACGGATCCGATTACGAATGCCGTCTC






The fusion PCR fragment was purified using a Qiagen kit and digested with the restriction enzymes MfeI and BamHI. This digested DNA fragment was gel purified using a Qiagen kit and ligated to a vector known as pBS19, which had been digested with EcoRI and BamHI and gel purified.
The ligation mix was transformed into E. coli Top 10 cells and colonies were selected on LA and 50 carbenicillin plates. A total of six colonies were chosen and grown overnight in LB and 50 carbenicillin and then plasmids were isolated using a Qiagen kit. The plasmids were digested with EcoRI and BamHI to check for inserts and three of the correct plasmids were sent in for sequencing with the following primers:
CF 149 (+) EcoRI Start of aprE Promoter







(SEQ ID NO: 65)









5′-GACATGAATTCCTCCATTTTCTTCTGC






CF 847 (+) Sequence in pXX 049 (End of aprE Promoter)








(SEQ ID NO: 66)









5′-AGGAGAGGGTAAAGAGTGAG






CF 07-45 (−) Fuse the 3′ end of Kudzu Isoprene Synthase to the Terminator








(SEQ ID NO: 61)









5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC






CF 07-48 (+) Sequencing primer for Kudzu Isoprene Synthase








(SEQ ID NO: 67)









5′-CTTTTCCATCACCCACCTGAAG






CF 07-49 (+) Sequencing in Kudzu Isoprene Synthase








(SEQ ID NO: 68)









5′-GGCGAAATGGTCCAACAACAAAATTATC






The plasmid designated pBS Kudzu #2 (FIGS. 52 and 12) was correct by sequencing and was transformed into BG 3594 comK, a Bacillus subtilis host strain. Selection was done on LA and 5 chloramphenicol plates. A transformant was chosen and struck to single colonies on LA and 5 chloramphenicol, then grown in LB and 5 chloramphenicol until it reached an OD600 of 1.5. It was stored frozen in a vial at −80° C. in the presence of glycerol. The resulting strain was designated CF 443.
II. Production of Isoprene in Shake Flasks Containing B. subtilis Cells Expressing Recombinant Isoprene Synthase
Overnight cultures were inoculated with a single colony of CF 443 from a LA and Chloramphenicol (Cm, 25 μg/ml). Cultures were grown in LB and Cm at 37° C. with shaking at 200 rpm. These overnight cultures (1 ml) were used to inoculate 250 ml baffled shake flasks containing 25 ml Grants II media and chloramphenicol at a final concentration of 25 μg/ml. Grants II Media recipe was 10 g soytone, 3 ml 1M K2HPO4, 75 g glucose, 3.6 g urea, 100 ml 10×MOPS, q.s. to 1 L with H2O, pH 7.2; 10×MOPS recipe was 83.72 g MOPS, 7.17 g tricine, 12 g KOH pellets, 10 ml 0.276M K2SO4 solution, 10 ml 0.528M MgCl2 solution, 29.22 g NaCl, 100 ml 100× micronutrients, q.s. to 1 L with H2O; and 100× micronutrients recipe was 1.47 g CaCl2*2H2O, 0.4 g FeSO4*7H2O, 0.1 g MnSO4*H2O, 0.1 g ZnSO4*H2O, 0.05 g CUCl2*2H2O, 0.1 g COCl2*6H2O, 0.1 g Na2MOO4*2H2O, q.s. to 1 L with H2O. Shake flasks were incubated at 37° C. and samples were taken at 18, 24, and 44 hours. At 18 hours the headspaces of CF443 and the control strain were sampled. This represented 18 hours of accumulation of isoprene. The amount of isoprene was determined by gas chromatography as described in Example 1. Production of isoprene was enhanced significantly by expressing recombinant isoprene synthase (FIG. 11).
III. Production of Isoprene by CF443 in 14 L Fermentation
Large scale production of isoprene from B. subtilis containing the recombinant kudzu isoprene synthase gene on a replication plasmid was determined from a fed-batch culture. Bacillus strain CF 443, expressing a kudzu isoprene synthase gene, or control stain which does not express a kudzu isoprene synthase gene were cultivated by conventional fed-batch fermentation in a nutrient medium containing soy meal (Cargill), sodium and potassium phosphate, magnesium sulfate and a solution of citric acid, ferric chloride and manganese chloride. Prior to fermentation the media is macerated for 90 minutes using a mixture of enzymes including cellulases, hemicellulases and pectinases (see, WO95/04134). 14-L batch fermentations are fed with 60% wt/wt glucose (Cargill DE99 dextrose, ADM Versadex greens or Danisco invert sugar) and 99% wt/wt oil (Western Family soy oil, where the 99% wt/wt is the concentration of oil before it was added to the cell culture medium). Feed was started when glucose in the batch was non-detectable. The feed rate was ramped over several hours and was adjusted to add oil on an equal carbon basis. The pH was controlled at 6.8-7.4 using 28% w/v ammonium hydroxide. In case of foaming, antifoam agent was added to the media. The fermentation temperature was controlled at 37° C. and the fermentation culture was agitated at 750 rpm. Various other parameters such as pH, DO %, airflow, and pressure were monitored throughout the entire process. The DO % is maintained above 20. Samples were taken over the time course of 36 hours and analyzed for cell growth (OD550) and isoprene production. Results of these experiments are presented in FIGS. 53A and 53B.
IV. Integration of the Kudzu Isoprene Synthase (ispS) in B. subtilis
The kudzu isoprene synthase gene was cloned in an integrating plasmid (pJH101-cmpR) under the control of the aprE promoter. Under the conditions tested, no isoprene was detected.
Example 5
Production of Isoprene in Trichoderma
I. Construction of Vectors for Expression of the Kudzu Isoprene Synthase in Trichoderma reesei
The Yarrowia lipolytica codon-optimized kudzu IS gene was synthesized by DNA 2.0 (SEQ ID NO:8) (FIG. 13). This plasmid served as the template for the following PCR amplification reaction: 1 μl plasmid template (20 ng/ul), 1 μl Primer EL-945 (10 uM) 5′-GCTTATGGATCCTCTAGACTATTACACGTACATCAATTGG (SEQ ID NO:9), 1 μl Primer EL-965 (10 uM) 5′-CACCATGTGTGCAACCTCCTCCCAGTTTAC (SEQ ID NO:10), 1 μl dNTP (10 mM), 5 μl 10× PfuUltra II Fusion HS DNA Polymerase Buffer, 1 μl PfuUltra II Fusion HS DNA Polymerase, 40 μl water in a total reaction volume of 50 μl. The forward primer contained an additional 4 nucleotides at the 5′-end that did not correspond to the Y. lipolytica codon-optimized kudzu isoprene synthase gene, but was required for cloning into the pENTR/D-TOPO vector. The reverse primer contained an additional 21 nucleotides at the 5′-end that did not correspond to the Y. lipolytica codon-optimized kudzu isoprene synthase gene, but were inserted for cloning into other vector backbones. Using the MJ Research PTC-200 Thermocycler, the PCR reaction was performed as follows: 95° C. for 2 minutes (first cycle only), 95° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 30 seconds (repeat for 27 cycles), 72° C. for 1 minute after the last cycle. The PCR product was analyzed on a 1.2% E-gel to confirm successful amplification of the Y. lipolytica codon-optimized kudzu isoprene synthase gene.
The PCR product was then cloned using the TOPO pENTR/D-TOPO Cloning Kit following manufacturer's protocol: 1 μl PCR reaction, 1 μl Salt solution, 1 μl TOPO pENTR/D-TOPO vector and 3 μl water in a total reaction volume of 6 μl. The reaction was incubated at room temperature for 5 minutes. One microliter of TOPO reaction was transformed into TOP10 chemically competent E. coli cells. The transformants were selected on LA and 50 μg/ml kanamycin plates. Several colonies were picked and each was inoculated into a 5 ml tube containing LB and 50 μg/ml kanamycin and the cultures grown overnight at 37° C. with shaking at 200 rpm. Plasmids were isolated from the overnight culture tubes using QIAprep Spin Miniprep Kit, following manufacturer's protocol. Several plasmids were sequenced to verify that the DNA sequence was correct.
A single pENTR/D-TOPO plasmid, encoding a Y. lipolytica codon-optimized kudzu isoprene synthase gene, was used for Gateway Cloning into a custom-made pTrex3g vector. Construction of pTrex3g is described in WO 2005/001036 A2. The reaction was performed following manufacturer's protocol for the Gateway LR Clonase II Enzyme Mix Kit (Invitrogen): 1 μl Y. lipolytica codon-optimized kudzu isoprene synthase gene pENTR/D-TOPO donor vector, 1 μl pTrex3g destination vector, 6 μl TE buffer, pH 8.0 in a total reaction volume of 8 μl. The reaction was incubated at room temperature for 1 hour and then 1 μl proteinase K solution was added and the incubation continued at 37° C. for 10 minutes. Then 1 μl of reaction was transformed into TOP10 chemically competent E. coli cells. The transformants were selected on LA and 50 μg/ml carbenicillin plates. Several colonies were picked and each was inoculated into a 5 ml tube containing LB and 50 μg/ml carbenicillin and the cultures were grown overnight at 37° C. with shaking at 200 rpm. Plasmids were isolated from the overnight culture tubes using QIAprep Spin Miniprep Kit (Qiagen, Inc.), following manufacturer's protocol. Several plasmids were sequenced to verify that the DNA sequence was correct.
Biolistic transformation of Y. lipolytica codon-optimized kudzu isoprene synthase pTrex3g plasmid (FIG. 14) into a quad delete Trichoderma reesei strain was performed using the Biolistic PDS-1000/HE Particle Delivery System (see WO 2005/001036 A2). Isolation of stable transformants and shake flask evaluation was performed using protocol listed in Example 11 of patent publication WO 2005/001036 A2.
II. Production of Isoprene in Recombinant Strains of T. reesei
One ml of 15 and 36 hour old cultures of isoprene synthase transformants described above were transferred to head space vials. The vials were sealed and incubated for 5 hours at 30° C. Head space gas was measured and isoprene was identified by the method described in Example 1. Two of the transformants showed traces of isoprene. The amount of isoprene could be increased by a 14 hour incubation. The two positive samples showed isoprene at levels of about 0.5 μg/L for the 14 hour incubation. The untransformed control showed no detectable levels of isoprene. This experiment shows that T. reesei is capable of producing isoprene from endogenous precursor when supplied with an exogenous isoprene synthase.
Example 6
Production of Isoprene in Yarrowia
I. Construction of Vectors for Expression of the Kudzu Isoprene Synthase in Yarrowia lipolytica.
The starting point for the construction of vectors for the expression of the kudzu isoprene synthase gene in Yarrowia lipolytica was the vector pSPZ1(MAP29Spb). The complete sequence of this vector (SEQ ID No:11) is shown in FIG. 15.
The following fragments were amplified by PCR using chromosomal DNA of a Y. lipolytica strain GICC 120285 as the template: a promotorless form of the URA3 gene, a fragment of 18S ribosomal RNA gene, a transcription terminator of the Y. lipolytica XPR2 gene and two DNA fragments containing the promoters of XPR2 and ICL1 genes. The following PCR primers were used:







ICL1 3


(SEQ ID NO: 69)


5′-GGTGAATTCAGTCTACTGGGGATTCCCAAATCTATATATACT


GCAGGTGAC


 


ICL1 5


(SEQ ID NO: 70)


5′-GCAGGTGGGAAACTATGCACTCC


 


XPR3


(SEQ ID NO: 71)


5′-CCTGAATTCTGTTGGATTGGAGGATTGGATAGTGGG


 


(SEQ ID NO: 72)


XPR5


5′-GGTGTCGACGTACGGTCGAGCTTATTGACC


 


(SEQ ID NO: 73)


XPRT3


5′-GGTGGGCCCGCATTTTGCCACCTACAAGCCAG


 


(SEQ ID NO: 74)


XPRT 5


5′-GGTGAATTCTAGAGGATCCCAACGCTGTTGCCTACAACGG


 


(SEQ ID NO: 75)


Y18S3


5′-GGTGCGGCCGCTGTCTGGACCTGGTGAGTTTCCCCG


 


(SEQ ID NO: 76)


Y18S 5


5′-GGTGGGCCCATTAAATCAGTTATCGTTTATTTGATAG


 


(SEQ ID NO: 77)


YURA3


5′-GGTGACCAGCAAGTCCATGGGTGGTTTGATCATGG


 


(SEQ ID NO: 78)


YURA 50


5′-GGTGCGGCCGCCTTTGGAGTACGACTCCAACTATG


 


(SEQ ID NO: 79)


YURA 51


5′-GCGGCCGCAGACTAAATTTATTTCAGTCTCC






For PCR amplification the PfuUltraII polymerase (Stratagene), supplier-provided buffer and dNTPs, 2.5 μM primers and the indicated template DNA were used as per the manufacturer's instructions. The amplification was done using the following cycle: 95° C. for 1 min; 34× (95° C. for 30 sec; 55° C. for 30 sec; 72° C. for 3 min) and 10 min at 72° C. followed by a 4° C. incubation.
Synthetic DNA molecules encoding the kudzu isoprene synthase gene, codon-optimized for expression in Yarrowia, was obtained from DNA 2.0 (FIG. 16; SEQ ID NO:12). Full detail of the construction scheme of the plasmids pYLA(KZ1) and pYLI(KZ1) carrying the synthetic kudzu isoprene synthase gene under control of XPR2 and ICL1 promoters respectively is presented in FIG. 18. Control plasmids in which a mating factor gene (MAP29) is inserted in place of an isoprene synthase gene were also constructed (FIGS. 18E and 18F).
A similar cloning procedure can be used to express a poplar (Populus alba x Populus tremula) isoprene synthase gene. The sequence of the poplar isoprene is described in Miller B. et al. (2001) Planta 213, 483-487 and shown in FIG. 17 (SEQ ID NO:13). A construction scheme for the generation the plasmids pYLA(POP1) and pYLI(POP1) carrying synthetic poplar isoprene synthase gene under control of XPR2 and ICL1 promoters respectively is presented in FIGS. 18A and B.
II. Production of Isoprene by Recombinant Strains of Y. lipolytica.
Vectors pYLA(KZ1), pYLI(KZ1), pYLA(MAP29) and pYLI(MAP29) were digested with SacII and used to transform the strain Y. lipolytica CLIB 122 by a standard lithium acetate/polyethylene glycol procedure to uridine prototrophy. Briefly, the yeast cells grown in YEPD (1% yeast extract, 2% peptone, 2% glucose) overnight, were collected by centrifugation (4000 rpm, 10 min), washed once with sterile water and suspended in 0.1 M lithium acetate, pH 6.0. Two hundred μl aliquots of the cell suspension were mixed with linearized plasmid DNA solution (10-20 μg), incubated for 10 minutes at room temperature and mixed with 1 ml of 50% PEG 4000 in the same buffer. The suspensions were further incubated for 1 hour at room temperature followed by a 2 minutes heat shock at 42° C. Cells were then plated on SC his leu plates (0.67% yeast nitrogen base, 2% glucose, 100 mg/L each of leucine and histidine). Transformants appeared after 3-4 days of incubation at 30° C.
Three isolates from the pYLA(KZ1) transformation, three isolates from the pYLI(KZ1) transformation, two isolates from the pYLA(MAP29) transformation and two isolates from the pYLI(MAP29) transformation were grown for 24 hours in YEP7 medium (1% yeast extract, 2% peptone, pH 7.0) at 30° C. with shaking. Cells from 10 ml of culture were collected by centrifugation, resuspended in 3 ml of fresh YEP7 and placed into 15 ml screw cap vials. The vials were incubated overnight at room temperature with gentle (60 rpm) shaking. Isoprene content in the headspace of these vials was analyzed by gas chromatography using mass-spectrometric detector as described in Example 1. All transformants obtained with pYLA(KZ1) and pYLI(KZ1) produced readily detectable amounts of isoprene (0.5 μg/L to 1 μg/L, FIG. 20). No isoprene was detected in the headspace of the control strains carrying phytase gene instead of an isoprene synthase gene.
Example 7
Production of Isoprene in E. coli Expressing Kudzu Isoprene Synthase and idi, or dxs, or idi and dxs
I. Construction of Vectors Encoding Kudzu Isoprene Synthase and idi, or dxs, or idi and dxs for the Production of Isoprene in E. coli
i) Construction of pTrcKudzuKan
The bla gene of pTrcKudzu (described in Example 1) was replaced with the gene conferring kanamycin resistance. To remove the bla gene, pTrcKudzu was digested with BspHI, treated with Shrimp Alkaline Phosphatase (SAP), heat killed at 65° C., then end-filled with Klenow fragment and dNTPs. The 5 kbp large fragment was purified from an agarose gel and ligated to the kanr gene which had been PCR amplified from pCR-Blunt-II-TOPO using primers MCM22 5′-GATCAAGCTTAACCGGAATTGCCAGCTG (SEQ ID NO:14) and MCM23 5′-GATCCGATCGTCAGAAGAACTCGTCAAGAAGGC (SEQ ID NO:15), digested with HindIII and PvuI, and end-filled. A transformant carrying a plasmid conferring kanamycin resistance (pTrcKudzuKan) was selected on LA containing kanamycin 50 μg/ml.
ii) Construction of pTrcKudzu yIDI Kan
pTrcKudzuKan was digested with PstI, treated with SAP, heat killed and gel purified. It was ligated to a PCR product encoding idi from S. cerevisiae with a synthetic RBS. The primers for PCR were NsiI-YIDI 1 F 5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC (SEQ ID NO:16) and PstI-YIDI 1 R 5′-CCTTCTGCAGGACGCGTTGTTATAGC (SEQ ID NO:17); and the template was S. cerevisiae genomic DNA. The PCR product was digested with NsiI and PstI and gel purified prior to ligation. The ligation mixture was transformed into chemically competent TOP10 cells and selected on LA containing 50 μg/ml kanamycin. Several transformants were isolated and sequenced and the resulting plasmid was called pTrcKudzu-yIDI(kan) (FIGS. 34 and 35).
iii) Construction of pTrcKudzu DXS Kan
Plasmid pTrcKudzuKan was digested with PstI, treated with SAP, heat killed and gel purified. It was ligated to a PCR product encoding dxs from E. coli with a synthetic RBS. The primers for PCR were MCM13 5′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ ID NO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19); and the template was E. coli genomic DNA. The PCR product was digested with NsiI and PstI and gel purified prior to ligation. The resulting transformation reaction was transformed into TOP10 cells and selected on LA with kanamycin 50 μg/ml. Several transformants were isolated and sequenced and the resulting plasmid was called pTrcKudzu-DXS(kan) (FIGS. 36 and 37).
iv) Construction of pTrcKudzu-yIDI-dxs (kan)
pTrcKudzu-yIDI(kan) was digested with PstI, treated with SAP, heat killed and gel purified. It was ligated to a PCR product encoding E. coli dxs with a synthetic RBS (primers MCM13 5′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ ID NO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19); template TOP10 cells) which had been digested with NsiI and PstI and gel purified. The final plasmid was called pTrcKudzu-yIDI-dxs (kan) (FIGS. 21 and 22).
v) Construction of pCL PtrcKudzu
A fragment of DNA containing the promoter, structural gene and terminator from Example 1 above was digested from pTrcKudzu using SspI and gel purified. It was ligated to pCL1920 which had been digested with PvuII, treated with SAP and heat killed. The resulting ligation mixture was transformed into TOP10 cells and selected in LA containing spectinomycin 50 μg/ml. Several clones were isolated and sequenced and two were selected. pCL PtrcKudzu and pCL PtrcKudzu (A3) have the insert in opposite orientations (FIGS. 38-41).
vi) Construction of pCL PtrcKudzu yIDI
The NsiI-PstI digested, gel purified, IDI PCR amplicon from (ii) above was ligated into pCL PtrcKudzu which had been digested with PstI, treated with SAP, and heat killed. The ligation mixture was transformed into TOP10 cells and selected in LA containing spectinomycin 50 μg/ml. Several clones were isolated and sequenced and the resulting plasmid is called pCL PtrcKudzu yIDI (FIGS. 42 and 43).
vii) Construction of pCL PtrcKudzu DXS
The NsiI-PstI digested, gel purified, DXS PCR amplicon from (iii) above was ligated into pCL PtrcKudzu (A3) which had been digested with PstI, treated with SAP, and heat killed. The ligation mixture was transformed into TOP10 cells and selected in LA containing spectinomycin 50 μg/ml. Several clones were isolated and sequenced and the resulting plasmid is called pCL PtrcKudzu DXS (FIGS. 44 and 45).
II. Measurement of Isoprene in Headspace from Cultures Expressing Kudzu Isoprene Synthase, idi, and/or dxs at Different Copy Numbers.
Cultures of E. coli BL21(λDE3) previously transformed with plasmids pTrcKudzu(kan) (A), pTrcKudzu-yIDI kan (B), pTrcKudzu-DXS kan (C), pTrcKudzu-yIDI-DXS kan (D) were grown in LB kanamycin 50 μg/mL. Cultures of pCL PtrcKudzu (E), pCL PtrcKudzu, pCL PtrcKudzu-yIDI (F) and pCL PtrcKudzu-DXS (G) were grown in LB spectinomycin 50 μg/mL. Cultures were induced with 400 μM IPTG at time 0 (OD600 approximately 0.5) and samples taken for isoprene headspace measurement (see Example 1). Results are shown in FIG. 23A-23G.
Plasmid pTrcKudzu-yIDI-dxs (kan) was introduced into E. coli strain BL21 by transformation. The resulting strain BL21/pTrc Kudzu IDI DXS was grown overnight in LB containing kanamycin (50 μg/ml) at 20° C. and used to inoculate shake flasks of TM3 (13.6 g K2PO4, 13.6 g KH2PO4, 2.0 g MgSO4.7H2O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH4)2SO4, 0.2 g yeast extract, 1.0 ml 1000× Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H2O, and filter sterilized) containing 1% glucose. Flasks were incubated at 30° C. until an OD600 of 0.8 was reached, and then induced with 400 μM IPTG. Samples were taken at various times after induction and the amount of isoprene in the head space was measured as described in Example 1. Results are shown in FIG. 23H.
III. Production of Isoprene from Biomass in E. coli/pTrcKudzu yIDI DXS
The strain BL21 pTrcKudzuIDIDXS was tested for the ability to generate isoprene from three types of biomass; bagasse, corn stover and soft wood pulp with glucose as a control. Hydrolysates of the biomass were prepared by enzymatic hydrolysis (Brown, L. and Torget, R., 1996, NREL standard assay method Lap-009 “Enzymatic Saccharification of Lignocellulosic Biomass”) and used at a dilution based upon glucose equivalents. In this example, glucose equivalents were equal to 1% glucose. A single colony from a plate freshly transformed cells of BL21 (DE3) pTrcKudzu yIDI DXS (kan) was used to inoculate 5 ml of LB plus kanamycin (50 μg/ml). The culture was incubated overnight at 25° C. with shaking. The following day the overnight culture was diluted to an OD600 of 0.05 in 25 ml of TM3 and 0.2% YE and 1% feedstock. The feedstock was corn stover, bagasse, or softwood pulp. Glucose was used as a positive control and no glucose was used as a negative control. Cultures were incubated at 30° C. with shaking at 180 rpm. The culture was monitored for OD600 and when it reached an OD600 of  ̃0.8, cultures were analyzed at 1 and 3 hours for isoprene production as described in Example 1. Cultures are not induced. All cultures containing added feedstock produce isoprene equivalent to those of the glucose positive control. Experiments were done in duplicate and are shown in FIG. 46.
IV. Production of Isoprene from Invert Sugar in E. coli/pTrcKudzuIDIDXS
A single colony from a plate freshly transformed cells of BL21 (λDE3)/pTrcKudzu yIDI DXS (kan) was used to inoculate 5 mL of LB and kanamycin (50 μg/ml). The culture was incubated overnight at 25° C. with shaking. The following day the overnight culture was diluted to an OD600 of 0.05 in 25 ml of TM3 and 0.2% YE and 1% feedstock. Feedstock was glucose, inverted glucose or corn stover. The invert sugar feedstock (Danisco Invert Sugar) was prepared by enzymatically treating sucrose syrup. AFEX corn stover was prepared as described below (Part V). The cells were grown at 30° C. and the first sample was measured when the cultures reached an OD600  ̃0.8-1.0 (0 hour). The cultures were analyzed for growth as measured by OD600 and for isoprene production as in Example 1 at 0, 1 and 3 hours. Results are shown in FIG. 47.
V. Preparation of Hydrolysate from AFEX Pretreated Corn Stover
AFEX pretreated corn stover was obtained from Michigan Biotechnology Institute. The pretreatment conditions were 60% moisture, 1:1 ammonia loading, and 90° C. for 30 minutes, then air dried. The moisture content in the AFEX pretreated corn stover was 21.27%. The contents of glucan and xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (dry basis), respectively. The saccharification process was as follows; 20 g of AFEX pretreated corn stover was added into a 500 ml flask with 5 ml of 1 M sodium citrate buffer pH 4.8, 2.25 ml of Accellerase 1000, 0.1 ml of Grindamyl H121 (Danisco xylanase product from Aspergillus niger for bread-making industry), and 72.65 ml of DI water. The flask was put in an orbital shaker and incubated at 50° C. for 96 hours. One sample was taken from the shaker and analyzed using HPLC. The hydrolysate contained 38.5 g/l of glucose, 21.8 g/l of xylose, and 10.3 g/l of oligomers of glucose and/or xylose.
VI. The Effect of Yeast Extract on Isoprene Production in E. coli Grown in Fed-Batch Culture
Fermentation was performed at the 14-L scale as previously described with E. coli cells containing the pTrcKudzu yIDI DXS plasmid described above. Yeast extract (Bio Springer, Montreal, Quebec, Canada) was fed at an exponential rate. The total amount of yeast extract delivered to the fermentor was varied between 70-830 g during the 40 hour fermentation. Optical density of the fermentation broth was measured at a wavelength of 550 nm. The final optical density within the fermentors was proportional to the amount of yeast extract added (FIG. 48A). The isoprene level in the off-gas from the fermentor was determined as previously described. The isoprene titer increased over the course of the fermentation (FIG. 48B). The amount of isoprene produced was linearly proportional to the amount of fed yeast extract (FIG. 48C).
VII. Production of Isoprene in 500 L Fermentation of pTrcKudzu DXS yIDI
A 500 liter fermentation of E. coli cells with a kudzu isoprene synthase, S. cerevisiae IDI, and E. coli DXS nucleic acids (E. coli BL21 (λDE3) pTrc Kudzu dxs yidi) was used to produce isoprene. The levels of isoprene varied from 50 to 300 μg/L over a time period of 15 hours. On the basis of the average isoprene concentrations, the average flow through the device and the extent of isoprene breakthrough, the amount of isoprene collected was calculated to be approximately 17 g.
VIII. Production of Isoprene in 500 L Fermentation of E. coli Grown in Fed-Batch Culture
Medium Recipe (per Liter Fermentation Medium):
K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium gas (NH3) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotic were added after sterilization and pH adjustment.
1000× Modified Trace Metal Solution:
Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO4*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component is dissolved one at a time in DI H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with 0.22 micron filter.
Fermentation was performed in a 500-L bioreactor with E. coli cells containing the pTrcKudzu yIDI DXS plasmid. This experiment was carried out to monitor isoprene formation from glucose and yeast extract at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was prepared in soytone-yeast extract-glucose medium. After the inoculum grew to OD 0.15, measured at 550 nm, 20 ml was used to inoculate a bioreactor containing 2.5-L soytone-yeast extract-glucose medium. The 2.5-L bioreactor was grown at 30° C. to OD 1.0 and 2.0-L was transferred to the 500-L bioreactor.
Yeast extract (Bio Springer, Montreal, Quebec, Canada) and glucose were fed at exponential rates. The total amount of glucose and yeast extract delivered to the bioreactor during the 50 hour fermentation was 181.2 kg and 17.6 kg, respectively. The optical density within the bioreactor over time is shown in FIG. 49A. The isoprene level in the off-gas from the bioreactor was determined as previously described. The isoprene titer increased over the course of the fermentation (FIG. 49B). The total amount of isoprene produced during the 50 hour fermentation was 55.1 g and the time course of production is shown in FIG. 49C.
Example 8
Production of Isoprene in E. coli Expressing Kudzu Isoprene Synthase and Recombinant Mevalonic Acid Pathway Genes
I. Cloning the Lower MVA Pathway
The strategy for cloning the lower mevalonic pathway was as follows. Four genes of the mevalonic acid biosynthesis pathway; mevalonate kinase (MVK), phosphomevalonate kinase (PMK), diphosphomevalonte decarboxylase (MVD) and isopentenyl diphosphate isomerase genes were amplified by PCR from S. cerevisiae chromosomal DNA and cloned individually into the pCR BluntII TOPO plasmid (Invitrogen). In some cases, the idi gene was amplified from E. coli chromosomal DNA. The primers were designed such that an E. coli consensus RBS (AGGAGGT (SEQ ID NO:80) or AAGGAGG (SEQ ID NO:81)) was inserted at the 5′ end, 8 bp upstream of the start codon and a PstI site was added at the 3′ end. The genes were then cloned one by one into the pTrcHis2B vector until the entire pathway was assembled.
Chromosomal DNA from S. cerevisiae S288C was obtained from ATCC (ATCC 204508D). The MVK gene was amplified from the chromosome of S. cerevisiae using primers MVKF (5′-AGGAGGTAAAAAAACATGTCATTACCGTTCTTAACTTCTGC, SEQ ID NO:21) and MVK-Pst1-R (5′-ATGGCTGCAGGCCTATCGCAAATTAGCTTATGAAGTCCATGGTAAATTCGTG, SEQ ID NO:22) using PfuTurbo as per manufacturer's instructions. The correct sized PCR product (1370 bp) was identified by electrophoresis through a 1.2% E-gel (Invitrogen) and cloned into pZeroBLUNT TOPO. The resulting plasmid was designated pMVK1. The plasmid pMVK1 was digested with SacI and Taq1 restriction endonucleases and the fragment was gel purified and ligated into pTrcHis2B digested with SacI and BstBI. The resulting plasmid was named pTrcMVK1.
The second gene in the mevalonic acid biosynthesis pathway, PMK, was amplified by PCR using primers: PstI-PMK1 R (5′-GAATTCGCCCTTCTGCAGCTACC, SEQ ID NO:23) and BsiHKA1-PMK1 F (5′-CGACTGGTGCACCCTTAAGGAGGAAAAAAACATGTCAG, SEQ ID NO:24). The PCR reaction was performed using Pfu Turbo polymerase (Stratagene) as per manufacturer's instructions. The correct sized product (1387 bp) was digested with PstI and BsiHKI and ligated into pTrcMVK1 digested with PstI. The resulting plasmid was named pTrcKK. The MVD and the idi genes were cloned in the same manner. PCR was carried out using the primer pairs PstI-MVD 1 R (5′-GTGCTGGAATTCGCCCTTCTGCAGC, SEQ ID NO:25) and NsiI-MVD 1 F (5′-GTAGATGCATGCAGAATTCGCCCTTAAGGAGG, SEQ ID NO:26) to amplify the MVD gene and PstI-YIDI 1 R (5′-CCTTCTGCAGGACGCGTTGTTATAGC, SEQ ID NO:27) and NsiI-YIDI 1 F (5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC, SEQ ID NO:28) to amplify the yIDI gene. In some cases the IPP isomerase gene, idi from E. coli was used. To amplify idi from E. coli chromosomal DNA, the following primer set was used: PstI-CIDI 1 R (5′-GTGTGATGGATATCTGCAGAATTCG, SEQ ID NO:29) and NsiI-CIDI 1 F (5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAACATG, SEQ ID NO:30). Template DNA was chromosomal DNA isolated by standard methods from E. coli FM5 (WO 96/35796 and WO 2004/033646, which are each hereby incorporated by reference in their entireties, particularly with respect to isolation of nucleic acids). The final plasmids were named pKKDIy for the construct encoding the yeast idi gene or pKKDIc for the construct encoding the E. coli idi gene. The plasmids were transformed into E. coli hosts BL21 for subsequent analysis. In some cases the isoprene synthase from kudzu was cloned into pKKDIy yielding plasmid pKKDIyIS.
The lower MVA pathway was also cloned into pTrc containing a kanamycin antibiotic resistance marker. The plasmid pTrcKKDIy was digested with restriction endonucleases ApaI and PstI, the 5930 bp fragment was separated on a 1.2% agarose E-gel and purified using the Qiagen Gel Purification kit according to the manufacturer's instructions. The plasmid pTrcKudzuKan, described in Example 7, was digested with restriction endonucleases ApaI and PstI, and the 3338 bp fragment containing the vector was purified from a 1.2% E-gel using the Qiagen Gel Purification kit. The 3338 bp vector fragment and the 5930 bp lower MVA pathway fragment were ligated using the Roche Quick Ligation kit. The ligation mix was transformed into E. coli TOP10 cells and transformants were grown at 37° C. overnight with selection on LA containing kanamycin (50 μg/ml). The transformants were verified by restriction enzyme digestion and one was frozen as a stock. The plasmid was designated pTrcKanKKDIy.
II. Cloning a Kudzu Isoprene Synthase Gene into pTrcKanKKDIy
The kudzu isoprene synthase gene was amplified by PCR from pTrcKudzu, described in Example 1, using primers MCM50 5′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGTGTGCGACCTCTTCTCAATTTAC T (SEQ ID NO:31) and MCM53 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:32). The resulting PCR fragment was cloned into pCR2.1 and transformed into E. coli TOP10. This fragment contains the coding sequence for kudzu isoprene synthase and an upstream region containing a RBS from E. coli. Transformants were incubated overnight at 37° C. with selection on LA containing carbenicillin (50 μg/ml). The correct insertion of the fragment was verified by sequencing and this strain was designated MCM93.
The plasmid from strain MCM93 was digested with restriction endonucleases NsiI and PstI to liberate a 1724 bp insert containing the RBS and kudzu isoprene synthase. The 1724 bp fragment was separated on a 1.2% agarose E-gel and purified using the Qiagen Gel Purification kit according to the manufacturer's instructions. Plasmid pTrcKanKKDIy was digested with the restriction endonuclease PstI, treated with SAP for 30 minutes at 37° C. and purified using the Qiagen PCR cleanup kit. The plasmid and kudzu isoprene synthase encoding DNA fragment were ligated using the Roche Quick Ligation kit. The ligation mix was transformed into E. coli TOP10 cells and transformants were grown overnight at 37° C. with selection on LA containing Kanamycin at 50 μg/ml. The correct transformant was verified by restriction digestion and the plasmid was designated pTrcKKDyIkISKan (FIGS. 24 and 25). This plasmid was transformed into BL21(λDE3) cells (Invitrogen).
III. Isoprene Production from Mevalonate in E. coli Expressing the Recombinant Lower Mevalonate Pathway and Isoprene Synthase from Kudzu.
Strain BL21/pTrcKKDyIkISKan was cultured in MOPS medium (Neidhardt et al., (1974) J. Bacteriology 119:736-747) adjusted to pH 7.1 and supplemented with 0.5% glucose and 0.5% mevalonic acid. A control culture was also set up using identical conditions but without the addition of 0.5% mevalonic acid. The culture was started from an overnight seed culture with a 1% inoculum and induced with 500 μM IPTG when the culture had reached an OD600 of 0.3 to 0.5. The cultures were grown at 30° C. with shaking at 250 rpm. The production of isoprene was analyzed 3 hours after induction by using the head space assay described in Example 1. Maximum production of isoprene was 6.67×104 nmol/Lbroth/OD600/hr where Lbroth is the volume of broth and includes both the volume of the cell medium and the volume of the cells. The control culture not supplemented with mevalonic acid did not produce measurable isoprene.
IV. Cloning the Upper MVA Pathway
The upper mevalonate biosynthetic pathway, comprising two genes encoding three enzymatic activities, was cloned from Enterococcus faecalis. The mvaE gene encodes a protein with the enzymatic activities of both acetyl-CoA acetyltransferase and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the first and third proteins in the pathway, and the mvaS gene encodes second enzyme in the pathway, HMG-CoA synthase. The mvaE gene was amplified from E. faecalis genomic DNA (ATCC 700802D-5) with an E. coli ribosome binding site and a spacer in front using the following primers:
CF 07-60 (+) Start of mvaE w/RBS+ATG Start Codon SacI







(SEQ ID NO: 34)









5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAG



TTATTATTG






CF 07-62 (−) Fuse mvaE to mvaS with RBS in Between








(SEQ ID NO: 35)


5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTG


 


TTTTCTTAAATC






The mvaS gene was amplified from E. faecalis genomic DNA (ATCC 700802D-5) with a RBS and spacer from E. coli in front using the following primers:
CF 07-61 (+) Fuse mvaE to mvaS with RBS in Between







(SEQ ID NO: 36)


5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATT


 


GGGATTGATAAA






CF 07-102 (−) End of mvaS Gene Bg/II








(SEQ ID NO: 37)









5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT






The PCR fragments were fused together with PCR using the following primers:
CF 07-60 (+) Start of mvaE w/RBS+ATG Start Codon SacI







(SEQ ID NO: 34)


5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTT


 


ATTATTG






CF 07-102 (−) End of mvaS Gene Bg/II








(SEQ ID NO: 37)









5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT






The fusion PCR fragment was purified using a Qiagen kit and digested with the restriction enzymes SacI and Bg/II. This digested DNA fragment was gel purified using a Qiagen kit and ligated into the commercially available vector pTrcHis2A, which had been digested with SacI and Bg/II and gel purified.
The ligation mix was transformed into E. coli Top 10 cells and colonies were selected on LA and 50 μg/ml carbenicillin plates. A total of six colonies were chosen and grown overnight in LB and 50 μg/ml carbenicillin and plasmids were isolated using a Qiagen kit. The plasmids were digested with SacI and Bg/II to check for inserts and one correct plasmid was sequenced with the following primers:
CF 07-58 (+) Start of mvaE Gene







(SEQ ID NO: 38)









5′-ATGAAAACAGTAGTTATTATTGATGC






CF 07-59 (−) End of mvaE Gene








(SEQ ID NO: 39)









5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGC






CF 07-82 (+) Start of mvaS Gene








(SEQ ID NO: 40)









5′-ATGACAATTGGGATTGATAAAATTAG






CF 07-83 (−) End of mvaS Gene








(SEQ ID NO: 41)









5′-TTAGTTTCGATAAGAACGAACGGT






CF 07-86 (+) Sequence in mvaE








(SEQ ID NO: 42)









5′-GAAATAGCCCCATTAGAAGTATC






CF 07-87 (+) Sequence in mvaE








(SEQ ID NO: 43)









5′-TTGCCAATCATATGATTGAAAATC






CF 07-88 (+) Sequence in mvaE








(SEQ ID NO: 44)









5′-GCTATGCTTCATTAGATCCTTATCG






CF 07-89 (+) Sequence mvaS








(SEQ ID NO: 45)









5′-GAAACCTACATCCAATCTTTTGCCC






The plasmid called pTrcHis2AUpperPathway#1 was correct by sequencing and was transformed into the commercially available E. coli strain BL21. Selection was done on LA and 50 μg/ml carbenicillin. Two transformants were chosen and grown in LB and 50 μg/ml carbenicillin until they reached an OD600 of 1.5. Both strains were frozen in a vial at −80° C. in the presence of glycerol. Strains were designated CF 449 for pTrcHis2AUpperPathway#1 in BL21, isolate #1 and CF 450 for pTrcHis2AUpperPathway#1 in BL21, isolate #2. Both clones were found to behave identically when analyzed.
V. Cloning of UpperMVA Pathway into pCL1920
The plasmid pTrcHis2AUpperPathway was digested with the restriction endonuclease SspI to release a fragment containing pTrc-mvaE-mvaS-(His tag)-terminator. In this fragment, the his-tag was not translated. This blunt ended 4.5 kbp fragment was purified from a 1.2% E-gel using the Qiagen Gel Purification kit. A dephosphorylated, blunt ended 4.2 kbp fragment from pCL1920 was prepared by digesting the vector with the restriction endonuclease PvuII, treating with SAP and gel purifying from a 1.2% E-gel using the Qiagen Gel Purification kit. The two fragments were ligated using the Roche Quick Ligation Kit and transformed into TOP10 chemically competent cells. Transformants were selected on LA containing spectinomycin (50 μg/ml). A correct colony was identified by screening for the presence of the insert by PCR. The plasmid was designated pCL PtrcUpperPathway (FIGS. 26 and 27).
VI. Strains Expressing the Combined Upper and Lower Mevalonic Acid Pathways
To obtain a strain with a complete mevalonic acid pathway plus kudzu isoprene synthase, plasmids pTrcKKDyIkISkan and pCLpTrcUpperPathway were both transformed into BL21(λDE3) competent cells (Invitrogen) and transformants were selected on LA containing kanamycin (50 μg/ml) and Spectinomycin (50 μg/ml). The transformants were checked by plasmid prep to ensure that both plasmids were retained in the host. The strain was designated MCM127.
VII. Production of Mevalonic Acid from Glucose in E. coli/pUpperpathway
Single colonies of the BL21/pTrcHis2A-mvaE/mvaS or FM5/p pTrcHis2A-mvaE/mvaS are inoculated into LB and carbenicillin (100 μg/ml) and are grown overnight at 37° C. with shaking at 200 rpm. These cultures were diluted into 50 ml medium in 250 ml baffled flasks to an OD600 of 0.1. The medium was TM3, 1 or 2% glucose, carbenicillin (100 ug/ml) or TM3, 1% glucose. hydrolyzed soy oil, and carbenicillin (100 ug/ml) or TM3 and biomass (prepared bagasse, corn stover or switchgrass). Cultures were grown at 30° C. with shaking at 200 rpm for approximately 2-3 hours until an OD600 of 0.4 was reached. At this point the expression from the mvaE mvaS construct was induced by the addition of IPTG (400 μM). Cultures were incubated for a further 20 or 40 hours with samples taken at 2 hour intervals to 6 hour post induction and then at 24, 36 and 48 hours as needed. Sampling was done by removing 1 ml of culture, measuring the OD600, pelleting the cells in a microfuge, removing the supernatant and analyzing it for mevalonic acid.
A 14 liter fermentation of E. coli cells with nucleic acids encoding Enterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase polypeptides produced 22 grams of mevalonic acid with TM3 medium and 2% glucose as the cell medium. A shake flask of these cells produced 2-4 grams of mevalonic acid per liter with LB medium and 1% glucose as the cell culture medium. The production of mevalonic acid in these strains indicated that the MVA pathway was functional in E. coli. 
VIII. Production of Isoprene from E. coli BL21 Containing the Upper and Lower MVA Pathway Plus Kudzu Isoprene Synthase.
The following strains were created by transforming in various combinations of plasmids containing the upper and lower MVA pathway and the kudzu isoprene synthase gene as described above and the plasmids containing the idi, dxs, and dxr and isoprene synthase genes described in Example 7. The host cells used were chemically competent BL21(λDE3) and the transformations were done by standard methods. Transformants were selected on L agar containing kanamycin (50 μg/ml) or kanamycin plus spectinomycin (both at a concentration of 50 μg/ml). Plates were grown at 37° C. The resulting strains were designated as follows:
Grown on Kanamycin Plus Spectinomycin (50 μg/ml Each)
MCM127—pCL Upper MVA and pTrcKKDyIkIS (kan) in BL21(λDE3)
MCM131—pCL1920 and pTrcKKDyIkIS (kan) in BL21(λDE3)
MCM125—pCL Upper MVA and pTrcHis2B (kan) in BL21(λDE3)
Grown on Kanamycin (50 μg/ml)
MCM64—pTrcKudzu yIDI DXS (kan) in BL21(λDE3)
MCM50—pTrcKudzu (kan) in BL21(λDE3)
MCM123—pTrcKudzu yIDI DXS DXR (kan) in BL21(λDE3)
The above strains were streaked from freezer stocks to LA and appropriate antibiotic and grown overnight at 37° C. A single colony from each plate was used to inoculate shake flasks (25 ml LB and the appropriate antibiotic). The flasks were incubated at 22° C. overnight with shaking at 200 rpm. The next morning the flasks were transferred to a 37° C. incubator and grown for a further 4.5 hours with shaking at 200 rpm. The 25 ml cultures were centrifuged to pellet the cells and the cells were resuspended in 5 ml LB and the appropriate antibiotic. The cultures were then diluted into 25 ml LB, % glucose, and the appropriate antibiotic to an OD600 of 0.1. Two flasks for each strain were set up, one set for induction with IPTG (800 μM) the second set was not induced. The cultures were incubated at 37° C. with shaking at 250 rpm. One set of the cultures were induced after 1.50 hours (immediately following sampling time point 1). At each sampling time point, the OD600 was measured and the amount of isoprene determined as described in Example 1. Results are presented in Table 3. The amount of isoprene made is presented as the amount at the peak production for the particular strain.







TABLE 3







Production of isoprene in E. coli strains










Strain
Isoprene μg/Lbroth/hr/OD














MCM50
23.8



MCM64
289



MCM125
ND



MCM131
Trace



MCM127
874







ND: not detected



Trace: peak present but not integrable.






IX. Analysis of Mevalonic Acid

Mevalonolactone (1.0 g, 7.7 mmol) (CAS#503-48-0) was supplied from Sigma-Aldrich (WI, USA) as a syrup that was dissolved in water (7.7 mL) and was treated with potassium hydroxide (7.7 mmol) in order to generate the potassium salt of mevalonic acid. The conversion to mevalonic acid was confirmed by 1H NMR analysis. Samples for HPLC analysis were prepared by centrifugation at 14,000 rpm for 5 minutes to remove cells, followed by the addition of a 300 μl aliquot of supernatant to 900 μl of H2O. Perchloric acid (36 μl of a 70% solution) was then added followed by mixing and cooling on ice for 5 minutes. The samples were then centrifuged again (14,000 rpm for 5 min) and the supernatant transferred to HPLC. Mevalonic acid standards (20, 10, 5, 1 and 0.5 g/L) were prepared in the same fashion. Analysis of mevalonic acid (20 uL injection volume) was performed by HPLC using a BioRad Aminex 87-H+ column (300 mm by 7.0 mm) eluted with 5 mM sulfuric acid at 0.6 mL/min with refractive index (R1) detection. Under these conditions mevalonic acid eluted as the lactone form at 18.5 minutes.
Example 9
Construction of the Upper and Lower MVA Pathway for Integration into Bacillus subtilis
I. Construction of the Upper MVA Pathway in Bacillus subtilis
The upper pathway from Enterococcus faecalis is integrated into B. subtilis under control of the aprE promoter. The upper pathway consists of two genes; mvaE, which encodes for AACT and HMGR, and mvaS, which encodes for HMGS. The two genes are fused together with a stop codon in between, an RBS site in front of mvaS, and are under the control of the aprE promoter. A terminator is situated after the mvaE gene. The chloramphenicol resistance marker is cloned after the mvaE gene and the construct is integrated at the aprE locus by double cross over using flanking regions of homology.
Four DNA fragments are amplified by PCR such that they contain overhangs that will allow them to be fused together by a PCR reaction. PCR amplifications are carried out using Herculase polymerase according to manufacturer's instructions.
1: PaprE
CF 07-134 (+) Start of aprE Promoter PstI







(SEQ ID NO: 82)









5′-GACATCTGCAGCTCCATTTTCTTCTGC






CF 07-94 (−) Fuse PaprE to mvaE








(SEQ ID NO: 83)


5′-CAATAATAACTACTGTTTTCACTCTTTACCCTCTCCTTTTAA






Template: Bacillus subtilis Chromosomal DNA

2: mvaE

CF 07-93 (+) Fuse mvaE to the aprE Promoter (GTG Start Codon)








(SEQ ID NO: 84)


5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTG






CF 07-62 (−) Fuse mvaE to mvaS with RBS in Between








(SEQ ID NO: 35)


5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGT


 


TTTCTTAAATC






Template: Enterococcus faecalis Chromosomal DNA (from ATCC)

3. mvaS

CF 07-61 (+) Fuse mvaE to mvaS with RBS in Between








(SEQ ID NO: 36)


5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTG


 


GGATTGATAAA






CF 07-124 (−) Fuse the End of mvaS to the Terminator








(SEQ ID NO: 85)


5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGT






Template: Enterococcus faecalis Chromosomal DNA

4. B. amyliquefaciens Alkaline Serine Protease Terminator 

CF 07-123 (+) Fuse the End of mvaS to the Terminator








(SEQ TD NO: 86)


5′-ACCGTTCGTTCTTATCGAAACTAAAAAAAACCGGCCTTGGCCCCG






CF 07-46 (−) End of B. amyliquefaciens Terminator BamHI








(SEQ ID NO: 63)









5′-GACATGACGGATCCGATTACGAATGCCGTCTC






Template: Bacillus amyliquefaciens Chromosomal DNA

PCR Fusion Reactions

5. Fuse mvaE to mvaS

CF 07-93 (+) Fuse mvaE to the aprE Promoter (GTG Start Codon)








(SEQ ID NO: 84)


5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTG






CF 07-124 (−) Fuse the End of mvaS to the Terminator








(SEQ ID NO: 85)


5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGT






Template: #2 and 3 from Above

6. Fuse mvaE-mvaS to aprE Promoter

CF 07-134 (+) Start of aprE Promoter PstI








(SEQ ID NO: 82)









5′-GACATCTGCAGCTCCATTTTCTTCTGC






CF 07-124 (−) Fuse the End of mvaS to the Terminator








(SEQ ID NO: 85)


5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGT






Template #1 and #4 from Above

7. Fuse PaprE-mvaE-mvaS to Terminator

CF 07-134 (+) Start of aprE Promoter PstI








(SEQ ID NO: 82)


5′-GACATCTGCAGCTCCATTTTCTTCTGC






CF 07-46 (−) End of B. amyliquefaciens Terminator BamHI








(SEQ ID NO: 63)


5′-GACATGACGGATCCGATTACGAATGCCGTCTC






Template: #4 and #6

The product is digested with restriction endonucleases PstI/BamHI and ligated to pJM102 (Perego, M. 1993. Integrational vectors for genetic manipulation in Bacillus subtilis, p. 615-624. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington, D.C.) which is digested with PstI/BamHI. The ligation is transformed into E. coli TOP 10 chemically competent cells and transformants are selected on LA containing carbenicillin (50 μg/ml). The correct plasmid is identified by sequencing and is designated pJMUpperpathway2 (FIGS. 50 and 51). Purified plasmid DNA is transformed into Bacillus subtilis aprEnprE Pxyl-comK and transformants are selected on L agar containing chloramphenicol (5 μg/ml). A correct colony is selected and is plated sequentially on L agar containing chloramphenicol 10, 15 and 25 μg/ml to amplify the number of copies of the cassette containing the upper pathway.
The resulting strain is tested for mevalonic acid production by growing in LB containing 1% glucose and 1%. Cultures are analyzed by GC for the production of mevalonic acid.
This strain is used subsequently as a host for the integration of the lower mevalonic acid pathway.
The following primers are used to sequence the various constructs above.
Sequencing Primers:
CF 07-134 (+) Start of aprE Promoter PstI







(SEQ ID NO: 82)


5′-GACATCTGCAGCTCCATTTTCTTCTGC






CF 07-58 (+) Start of mvaE Gene








(SEQ ID NO: 38)


5′-ATGAAAACAGTAGTTATTATTGATGC






CF 07-59 (−) End of mvaE Gene








(SEQ ID NO: 39)


5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGC






CF 07-82 (+) Start of mvaS Gene








(SEQ ID NO: 40)


5′-ATGACAATTGGGATTGATAAAATTAG






CF 07-83 (−) End of mvaS Gene








 (SEQ ID NO: 41)


5′-TTAGTTTCGATAAGAACGAACGGT






CF 07-86 (+) Sequence in mvaE








(SEQ ID NO: 42)


5′-GAAATAGCCCCATTAGAAGTATC 






CF 07-87 (+) Sequence in mvaE








(SEQ ID NO: 43)


5′-TTGCCAATCATATGATTGAAAATC 






CF 07-88 (+) Sequence in mvaE








(SEQ ID NO: 44)


5′-GCTATGCTTCATTAGATCCTTATCG 






CF 07-89 (+) Sequence mvaS








(SEQ ID NO: 45)


5′-GAAACCTACATCCAATCTTTTGCCC 






Transformants are selected on LA containing chloramphenicol at a concentration of 5 μg/ml. One colony is confirmed to have the correct integration by sequencing and is plated on LA containing increasing concentrations of chloramphenicol over several days, to a final level of 25 μg/ml. This results in amplification of the cassette containing the genes of interest. The resulting strain is designated CF 455: pJMupperpathway#1 X Bacillus subtilis aprEnprE Pxyl comK (amplified to grow on LA containing chloramphenicol 25 μg/ml).
II. Construction of the Lower MVA Pathway in Bacillus subtilis
The lower MVA pathway, consisting of the genes mvk1, pmk, mpd and idi are combined in a cassette consisting of flanking DNA regions from the nprE region of the B. subtilis chromosome (site of integration), the aprE promoter, and the spectinomycin resistance marker (see FIGS. 28 and 29). This cassette is synthesized by DNA2.0 and is integrated into the chromosome of B. subtilis containing the upper MVA pathway integrated at the aprE locus. The kudzu isoprene synthase gene is expressed from the replicating plasmid described in Example 4 and is transformed into the strain with both upper and lower pathways integrated.
Example 10
Production of Isoprene in E. coli Expressing M. mazei Mevalonate Kinase and P. alba Isoprene Synthase
I. Construction of Vectors and Strains Encoding M. mazei Mevalonate Kinase (MVK) and P. alba Isoprene Synthase
(i) Construction of Strain EWL201 (BL21, Cm-GI1.2-KKDyI)
E. coli BL21 (Novagen brand, EMD Biosciences, Inc.) was a recipient strain, transduced with MCM331 P1 lysate (lysate prepared according to the method described in Ausubel, et al., Current Protocols in Molecular Biology. John Wiley and Sons, Inc.). Transductants were selected for by spreading cells onto L Agar and 20 μg/μl chloramphenicol. The plates were incubated overnight at 30° C. Analysis of transductants showed no colonies on control plates (water+cells control plate for reversion and water and P1 lysate control plate for lysate contamination.
Four transductants were picked and used to inoculate 5 mL L Broth and 20 μg/μl chloramphenicol. The cultures were grown overnight at 30° C. with shaking at 200 rpm. To make genomic DNA preps of each transductant for PCR analysis, 1.5 mL of overnight cell culture were centrifuged. The cell pellet was resuspended with 400 μl Resuspension Buffer (20 mM Tris, 1 mM EDTA, 50 mM NaCl, pH 7.5) and 4 μl RNase, DNase-free (Roche) was added. The tubes were incubated at 37° C. for 30 minutes followed by the addition of 4 μl 10% SDS and 4 μl of 10 mg/ml Proteinase K stock solution (Sigma-Aldrich). The tubes were incubated at 37° C. for 1 hour. The cell lysate was transferred into 2 ml Phase Lock Light Gel tubes (Eppendorf) and 200 μl each of saturated phenol pH7.9 (Ambion Inc.) and chloroform were added. The tubes were mixed well and microcentrifuged for 5 minutes. A second extraction was done with 400 μl chloroform and the aqueous layer was transferred to a new eppendorf tube. The genomic DNA was precipitated by the addition of 1 ml of 100% ethanol and centrifugation for 5 minutes. The genomic DNA pellet was washed with 1 ml 70% ethanol. The ethanol was removed and the genomic DNA pellet was allowed to air dry briefly. The genomic DNA pellet was resuspended with 200 μl TE.
Using Pfu Ultra II DNA polymerase (Stratagene) and 200 ng/μl of genomic DNA as template, 2 different sets of PCR reaction tubes were prepared according to manufacturer's protocol. For set 1, primers MCM130 and GB Cm-Rev (Table 4) were used to ensure transductants were successfully integrated into the attTn7 locus. PCR parameters for set 1 were 95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 25 seconds, 72° C. for 25 seconds (repeat steps 2-4 for 28 cycles), 72° C. for 1 minute. For set 2, primers MVD For and MVD Rev (Table 4) were used to ensure that the gi1.2-KKDyI operon integrated properly. PCR parameters for set 2 were 95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 25 seconds, 72° C. for 10 seconds (repeat steps 2-4 for 28 cycles), 72° C. for 1 minute. Analysis of PCR amplicons on a 1.2% E-gel (Invitrogen Corp.) showed that all 4 transductant clones were correct (picked one and designated as strain EWL201).
ii) Construction of Strain EWL204 (BL21, Loopout-GI1.2-KKDyI)
The chloramphenicol marker was looped out of strain EWL201 using plasmid pCP20 as described by Datsenko and Wanner (2000) (Datsenko et al., Proc Natl. Acad. Sci. USA 97:6640-6645, 2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. (Datsenko et al., PNAS, 97: 6640-6645, 2000). EWL201 cells were grown in L Broth to midlog phase and then washed three times in ice-cold, sterile water. An aliquot of 50 μl of cell suspension was mixed with 1 μl of pCP20 and the cell suspension mixture was electroporated in a 2 mm cuvette (Invitrogen Corp.) at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator (Bio-Rad Inc.). 1 ml of LB was immediately added to the cells, then transferred to a 14 ml polypropylene tube (Sarstedt) with a metal cap. Cells were allowed to recover by growing for 1 hour at 30° C. Transformants were selected on L Agar and 20 μg/μl chloramphenicol and 50 μg/μl carbenicillin and incubated at 30° C. overnight. The next day, a single clone was grown in 10 ml L Broth and 50 μg/μl carbenicillin at 30° C. until early log phase. The temperature of the growing culture was then shifted to 42° C. for 2 hours. Serial dilutions were made, the cells were then spread onto LA plates (no antibiotic selection), and incubated overnight at 30° C. The next day, 20 colonies were picked and patched onto L Agar (no antibiotics) and LA and 20 μg/μl chloramphenicol plates. Plates were then incubated overnight at 30° C. Cells able to grow on LA plates, but not LA and 20 μg/μl chloramphenicol plates, were deemed to have the chloramphenicol marker looped out (picked one and designated as strain EWL204).
iii) Construction of Plasmid pEWL230 (pTrc P. alba)
Generation of a synthetic gene encoding Populus alba isoprene synthase (P. alba HGS) was outsourced to DNA2.0 Inc. (Menlo Park, Calif.) based on their codon optimization method for E. coli expression. The synthetic gene was custom cloned into plasmid pET24a (Novagen brand, EMD Biosciences, Inc.) and delivered lyophilized (FIGS. 54, 55A and 55B).
A PCR reaction was performed to amplify the P. alba isoprene synthase (P. alba HGS) gene using pET24 P. alba HGS as the template, primers MCM182 and MCM192, and Herculase II Fusion DNA polymerase (Stratagene) according to manufacturer's protocol. PCR conditions were as follows: 95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 20 seconds, 72° C. for 1 minute, repeat for 25 cycles, with final extension at 72° C. for 3 minutes. The P. alba isoprene synthase PCR product was purified using QIAquick PCR Purification Kit (Qiagen Inc.).
P. alba isoprene synthase PCR product was then digested in a 20 μl reaction containing 1 μl BspHI endonuclease (New England Biolabs) with 2 μl 10×NEB Buffer 4. The reaction was incubated for 2 hours at 37° C. The digested PCR fragment was then purified using the QIAquick PCR Purification Kit. A secondary restriction digest was performed in a 20 μl reaction containing 1 μl PstI endonuclease (Roche) with 2 μl 10× Buffer H. The reaction was incubated for 2 hours at 37° C. The digested PCR fragment was then purified using the QIAquick PCR Purification Kit. Plasmid pTrcHis2B (Invitrogen Corp.) was digested in a 20 μl reaction containing 1 μl NcoI endonuclease (Roche), 1 μl PstI endonuclease, and 2 μl 10× Buffer H. The reaction was incubated for 2 hours at 37° C. The digested pTrcHis2B vector was gel purified using a 1.2% E-gel (Invitrogen Corp.) and extracted using the QIAquick Gel Extraction Kit (Qiagen) (FIG. 56). Using the compatible cohesive ends of BspHI and NcoI sites, a 20 μl ligation reaction was prepared containing 5 μl P. alba isoprene synthase insert, 2 μl pTrc vector, 1 μl T4 DNA ligase (New England Biolabs), 2 μl 10× ligase buffer, and 10 μl ddH2O. The ligation mixture was incubated at room temperature for 40 minutes. The ligation mixture was desalted by floating a 0.025 cm nitrocellulose membrane filter (Millipore) in a petri dish of ddH2O and applying the ligation mixture gently on top of the nitrocellulose membrane filter for 30 minutes at room temperature. MCM446 cells (See section II) were grown in LB to midlog phase and then washed three times in ice-cold, sterile water. An aliquot of 50 μl of cell suspension was mixed with 5 μl of desalted pTrc P. alba HGS ligation mix. The cell suspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 ml of LB is immediately added to the cells, then transferred to a 14 ml polypropylene tube (Sarstedt) with a metal cap. Cells were allowed to recover by growing for 2 hour at 30° C. Transformants were selected on L Agar and 50 μg/μl carbenicillin and 10 mM mevalonic acid and incubated at 30° C. The next day, 6 transformants were picked and grown in 5 ml L Broth and 50 μg/μl carbenicillin tubes overnight at 30° C. Plasmid preps were performed on the overnight cultures using QIAquick Spin Miniprep Kit (Qiagen). Due to the use of BL21 cells for propagating plasmids, a modification of washing the spin columns with PB Buffer 5× and PE Buffer 3× was incorporated to the standard manufacturer's protocol for achieving high quality plasmid DNA. Plasmids were digested with PstI in a 20 μl reaction to ensure the correct sized linear fragment. All 6 plasmids were the correct size and shipped to Quintara Biosciences (Berkeley, Calif.) for sequencing with primers MCM65, MCM66, EL1000 (Table 4). DNA sequencing results showed all 6 plasmids were correct. Picked one and designated plasmid as EWL230 (FIGS. 57, 58A and 58B).
iv) Construction of Plasmid pEWL244 (pTrc P. alba-mMVK)
A PCR reaction was performed to amplify the Methanosarcina mazei (M. mazei) MVK gene using MCM376 as the template (see section v), primers MCM165 and MCM177 (see Table 4), and Pfu Ultra II Fusion DNA polymerase (Stratagene) according to manufacturer's protocol. PCR conditions were as follows: 95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 25 seconds, 72° C. for 18 seconds, repeat for 28 cycles, with final extension at 72° C. for 1 minute. The M. mazei MVK PCR product was purified using QIAquick PCR Purification Kit (Qiagen Inc.)
The M. mazei MVK PCR product was then digested in a 4001 reaction containing 8 μl PCR product, 2 μl PmeI endonuclease (New England Biolabs), 4 μl, 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 22 μl of ddH2O. The reaction was incubated for 3 hours at 37° C. The digested PCR fragment was then purified using the QIAquick PCR Purification Kit. A secondary restriction digest was performed in a 47 μl reaction containing 2 μl NsiI endonuclease (Roche), 4.7 μl 10× Buffer H, and 40 μl of PmeI digested M. mazei MVK fragment. The reaction was incubated for 3 hours at 37° C. The digested PCR fragment was then gel purified using a 1.2% E-gel and extracted using the QIAquick Gel Extraction Kit. Plasmid EWL230 was digested in a 40 μl reaction containing 10 μl plasmid, 2 μl PmeI endonuclease, 4 μl 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 20 μl of ddH2O. The reaction was incubated for 3 hours at 37° C. The digested PCR fragment was then purified using the QIAquick PCR Purification Kit. A secondary restriction digest was performed in a 47 μl reaction containing 2 μl PstI endonuclease, 4.7 μl 10× Buffer H, and 40 μl of PmeI digested EWL230 linear fragment. The reaction was incubated for 3 hours at 37° C. The digested PCR fragment was then gel purified using a 1.2% E-gel and extracted using the QIAquick Gel Extraction Kit (FIG. 59). Using the compatible cohesive ends of NsiI and PstI sites, a 20 μl ligation reaction was prepared containing 8 μl M. mazei MVK insert, 3 μl EWL230 plasmid, 1 μl T4 DNA ligase, 2 μl 10× ligase buffer, and 6 μl ddH2O. The ligation mixture was incubated at overnight at 16° C. The next day, the ligation mixture was desalted by floating a 0.025 μm nitrocellulose membrane filter in a petri dish of ddH2O and applying the ligation mixture gently on top of the nitrocellulose membrane filter for 30 minutes at room temperature. MCM446 cells were grown in LB to midlog phase and then washed three times in ice-cold, sterile water. An aliquot of 50 μl of cell suspension was mixed with 5 μl of desalted pTrc P. alba-mMVK ligation mix. The cell suspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 ml of LB is immediately added to the cells, then the cells are transferred to a 14 ml polypropylene tube with a metal cap. Cells were allowed to recover by growing for 2 hour at 30° C. Transformants were selected on LA and 50 μg/μl carbenicillin and 5 mM mevalonic acid plates and incubated at 30° C. The next day, 6 transformants were picked and grown in 5 ml LB and 50 μg/μl carbenicillin tubes overnight at 30° C. Plasmid preps were performed on the overnight cultures using QIAquick Spin Miniprep Kit. Due to the use of BL21 cells for propagating plasmids, a modification of washing the spin columns with PB Buffer 5× and PE Buffer 3× was incorporated to the standard manufacturer's protocol for achieving high quality plasmid DNA. Plasmids were digested with PstI in a 20 μl reaction to ensure the correct sized linear fragment. Three of the 6 plasmids were the correct size and shipped to Quintara Biosciences for sequencing with primers MCM65, MCM66, EL1000, EL1003, and EL1006 (Table 4). DNA sequencing results showed all 3 plasmids were correct. Picked one and designated plasmid as EWL244 (FIGS. 60 and 61A-B).
v) Construction of Plasmid MCM376-MVK from M. mazei Archaeal Lower in pET200D.
The MVK ORF from the M. mazei archaeal Lower Pathway operon (FIGS. 73A-C) was PCR amplified using primers MCM161 and MCM162 (Table 4) using the Invitrogen Platinum HiFi PCR mix. 45 uL of PCR mix was combined with 1 uL template, 1 uL of each primer at 10 uM, and 2 uL water. The reaction was cycled as follows: 94° C. for 2:00 minutes; 30 cycles of 94° C. for 0:30 minutes, 55° C. for 0:30 minutes and 68° C. for 1:15 minutes; and then 72° C. for 7:00 minutes, and 4° C. until cool. 3 uL of this PCR reaction was ligated to Invitrogen pET200D plasmid according to the manufacturer's protocol. 3 uL of this ligation was introduced into Invitrogen TOP10 cells, and transformants were selected on LA/kan50. A plasmid from a transformant was isolated and the insert sequenced, resulting in MCM376 (FIGS. 74A-C).
vi) Construction of Strain EWL251 (BL21(DE3), Cm-GI1.2-KKDyI, pTrc P. alba-mMVK)
MCM331 cells (which contain chromosomal construct gi1.2KKDyI encoding S. cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, and IPP isomerase) were grown in LB to midlog phase and then washed three times in ice-cold, sterile water. Mixed 50 μl of cell suspension with 1 μl of plasmid EWL244. The cell suspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 ml of LB is immediately added to the cells, then the cells were transferred to a 14 ml polypropylene tube with a metal cap. Cells were allowed to recover by growing for 2 hours at 30° C. Transformants were selected on LA and 50 μg/μl carbenicillin and 5 mM mevalonic acid plates and incubated at 37° C. One colony was selected and designated as strain EWL251.
vii) Construction of Strain EWL256 (BL21(DE3), Cm-GI1.2-KKDyI, pTrc P. alba-mMVK, pCL Upper MVA)
EWL251 cells were grown in LB to midlog phase and then washed three times in ice-cold, sterile water. Mixed 50 μl of cell suspension with 1 μl of plasmid MCM82 (which is pCL PtrcUpperPathway encoding E. faecalis mvaE and mvaS). The cell suspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 ml of LB was immediately added to the cells, then transferred to a 14 ml polypropylene tube with a metal cap. Cells were allowed to recover by growing for 2 hour at 30° C. Transformants were selected on LA and 50 μg/μl carbenicillin and 50 μg/μl spectinomycin plates and incubated at 37° C. Picked one colony and designated as strain EWL256.







TABLE 4







Primer Sequences








Primer



name
Primer sequence





MCM130
ACCAATTGCACCCGGCAGA



(SEQ ID NO: 94)


 


GB Cm
GCTAAAGCGCATGCTCCAGAC


Rev
(SEQ ID NO: 95)


 


MVD
GACTGGCCTCAGATGAAAGC


For
(SEQ ID NO: 96)


 


MVD
CAAACATGTGGCATGGAAAG


Rev
(SEQ ID NO: 97)


 


MCM182
GGGCCCGTTTAAACTTTAACTAGACTCTGCAGTTAGCGTTCAAA



CGGCAGAA



(SEQ ID NO: 98)


 


MCM192
CGCATGCATGTCATGAGATGTAGCGTGTCCACCGAAAA



(SEQ ID NO: 99


 


MCM65
ACAATTTCACACAGGAAACAGC



(SEQ ID NO: 100)


 


MCM66
CCAGGCAAATTCTGTTTTATCAG



(SEQ ID NO: 101)


 


EL1000
GCACTGTCTTTCCGTCTGCTGC



(SEQ ID NO: 102)


 


MCM165
GCGAACGATGCATAAAGGAGGTAAAAAAACATGGTATCCTGTTC



TGCGCCGGGTAAGATTTACCTG



(SEQ ID NO: 103)


 


MCM177
GGGCCCGTTTAAACTTTAACTAGACTTTAATCTACTTTCAGACC



TTGC



(SEQ ID NO: 104)


 


EL1003
GATAGTAACGGCTGCGCTGCTACC



(SEQ ID NO: 105)


 


EL1006
GACAGCTTATCATCGACTGCACG



(SEQ ID NO: 106)


 


MCM161
CACCATGGTATCCTGTTCTGCG



(SEQ ID NO: 107)


 


MCM162
TTAATCTACTTTCAGACCTTGC



(SEQ ID NO: 108)









II. Construction of MCM442-449: BL21 and BL21(DE3) with FRT-cmR-FRT-gi1.x-mKKDyI

i) Construction of Template for Recombination

FRT-based recombination cassettes, and plasmids for Red/ET-mediated integration and antibiotic marker loopout were obtained from Gene Bridges GmbH (Germany). Procedures using these materials were carried out according to Gene Bridges protocols. Primers MCM193 and MCM195 were used to amplify the resistance cassette from the FRT-gb2-Cm-FRT template using Stratagene Herculase II Fusion kit according to the manufacturer's protocol. The 50 uL reaction was cycled as follows: 95° C., 2 minutes; (95° C., 20 seconds, 55° C., 20 seconds, 72° C., 1 minute)×5, (95° C., 20 seconds, 60° C., 20 seconds, 72° C., 1 minute)×25; 72° C., 3 minutes; 4° C. until cool. The amplicon was purified by a Qiagen PCR column according to the manufacturer's protocol and eluted in 30 uL EB (Elution Buffer). DNA was digested with NdeI and PciI in a 20 uL reaction with 1× Roche H buffer and 0.5 uL BSA. Plasmid MCM376 was digested in a 10 uL reaction containing 1 uL each of NdeI, NcoI, and Roche H buffer. Reactions proceeded overnight at 37° C., and then cut DNA was purified on Qiagen PCR columns and eluted in 30 uL EB. The PCR product was ligated into MCM376 in a reaction containing 1 uL vector, 3 uL PCR product, 1 uL Roche Quick Ligase Buffer 2, 5 uL Buffer1, and 1 uL Ligase. The reaction proceeded at room temperature for 3 hours and then 5 uL was transformed into Invitrogen TOP10 cells according to the manufacturer's protocol. Transformants were selected on L agar (LA) and chloramphenicol (10 ug/mLO) at 37° C. overnight.
Transformant colonies were patched onto LA containing chloramphenicol (30 ug/mL) and kanamycin (50 ug/ml) for storage and sent to Quintara (Berkeley, Calif.) for sequencing. Four clones, one each with the four different nucleotides at the “N” in primer MCM 195, were found to have the correct sequence for the inserted promoter. Clones were grown in 5 mL LB containing chloramphenicol (30 ug/mL) and kanamycin (50 ug/mL) and used for the preparation of plasmid DNA. This plasmid was retransformed into TOP10 cells and strains were frozen as:







TABLE 5





MCM484-487
















MCM484
cmR-gi1.6-MVK(mazei) in pET (clone A1-3, variable nt A)


MCM485
cmR-gi1.0-MVK(mazei) in pET (clone B4-6, variable nt C)


MCM486
cmR-gi1.2-MVK(mazei) in pET (clone C1-5, variable nt G)


MCM487
cmR-gi1.5-MVK(mazei) in pET (clone C3-3, variable nt T)









ii) Creation of Recombination Target Strains MCM349 and MCM441

The chloramphenicol resistance (cmR) marker was looped out of strain MCM331 using plasmid pGB706 (GeneBridges) according to Manufacturer's instructions. MCM331 cells were grown to mid-log in LB and washed three times in iced, sterile water. A 1 uL aliquot of pGB706 DNA was added to 50 uL of cell suspension and this mixture was electroporated in a 2 mm cuvette at 2.5 volts, 25 uFd followed immediately by recovery in 500 uL LB for one hour at 30° C. Transformants were selected on LB containing tetracycline (5 ug/ml) at 30° C. The following day, a clone was grown up at 30° C. in LB containing tetracycline (5 ug/ml) until visibly turbid (OD600 ̃0.5-0.8). This culture was streaked onto LB and grown overnight at 37° C. A clone that was unable to grow on LB containing chloramphenicol (10 ug/mL) or LB containing tetracycline (5 ug/mL) was frozen as MCM348. Plasmid MCM356 (pRedET carbencillin; GeneBridges) was electroporated in as described above and transformants were selected on LB containing carbenicillin (50 ug/mL) at 30° C. A clone was grown in LB carbenicillin (50 ug/mL) at 30° C. and frozen as MCM349.
Strain MCM441 was created by electrotransforming plasmid MCM356 into EWL204 as above.
iii) Recombination of FRT-cmR-FRT-gi1.x-mMVK into MCM349 and MCM441
Plasmids MCM484-487 were used as template for PCR amplification with primers MCM120 and MCM196 and Herculase II Fusion kit, according to the manufacturer's protocol. Three reactions per template were carried out, with 0, 1, or 3 uL DMSO. The 50 uL reactions were cycled as follows: 95° C., 2 minutes; (95° C., 20 seconds; 55° C. 20 seconds; 72° C., 1.5 minutes) for five cycles; (95° C., 20 seconds; 60° C. 20 seconds; 72° C., 1.5 minutes) for 25 cycles; 72° C. for 3 minutes; 4° C., overnight.] The three reactions from a given template were pooled and purified on Qiagen PCR columns and eluted with 30 uL EB at 60° C. 5 uL DNA was digested with 1 uL DpnI in 1× Roche Buffer A for 3 hours at 37° C. This DNA was then microdialyzed against excess water for 30 minutes.
Strains were grown in 5 mL LB containing carbenicillin (50 ug/mL) from fresh streaks at 30° C. to an OD600 of  ̃0.5. 40 mM L-arabinose was added and cultures were incubated at 37° C. for 1.5 hours. Cells were harvested and electroporated with 3 uL dialyzed amplicons above, and then recovered in 500 uL SOC at 37° C. for 1.5-3 hours. Transformants were selected on LA plates containing chloramphenicol (5 ug/mL) at 37° C.
Kanamycin sensitive clones were screened by PCR for insertion of the amplicon. PCR products from positive clones were sequenced to verify the sequence of inserted DNA. Amplicons were consistent with the FRT-gi1.2-yKKDyI at attTn7 in MCM441 and 348 being replaced by FRT-cmR-FRT-gi1.x-mKKDyI (The yK and mK designations refer to the mevalonate kinase from Saccharomyces cerevisiae and Methanosarcina mazei respectively).







TABLE 6A







The following strains were grown in LB containing


chloramphenicol (5 ug/mL) and frozen.













Recombi-





nation


Strain


Amplicon


ID
Name
Parent
Template





MCM442
BL21(DE3) cmR-gi1.6mKKDyI A1,
MCM349
MCM484



clone37 (A)


MCM443
BL21(DE3) cmR-gi1.0mKKDyI B4,
MCM349
MCM485



clone27 (C)


MCM444
BL21(DE3) cmR-gi1.2mKKDyI C1,
MCM349
MCM486



clone16 (G)


MCM445
BL21(DE3) cmR-gi1.5mKKDyI C3,
MCM349
MCM487



clone7 (T)


MCM446
BL21 cmR-gi1.6mKKDyI A1-3 (A)
MCM441
MCM484


MCM447
BL21 cmR-gi1.0mKKDyI B4-6 (C)
MCM441
MCM485


MCM448
BL21 cmR-gi1.2mKKDyI C1-5 (G)
MCM441
MCM486


MCM449
BL21 cmR-gi1.5mKKDyI C3-3 (T)
MCM441
MCM487
















TABLE 6B





Primers
















MCM120
AAAGTAGCCGAAGATGACGGTTTGTCACATGGAGTTGGCAGGAT



GTTTGATTAAAAGCAATTAACCCTCACTAAAGGGCGG



(SEQ ID NO: 109)


 


MCM193
GATATACATATGAATTAACCCTCACTAAAGG



(SEQ ID NO: 110)


 


MCM195
GCATGCATGACATGTTTTTTTACCTCCTTTGTTATCCGCTCACA



ATTAGTGGTTGAATTATTTGCTCAGGATGTGGCATNGTCAAGGG



CGCGGCCGCGATCTAATACGACTCACTATAGGGCTCG



(SEQ ID NO: 111)


 


MCM196
AGGCTCTCAACTCTGACATGTTTTTTTCCTCCTTAAGGGTGCAG



GCCTATCGCAAATTAGCTTAATCTACTTTCAGACCTTGCTCGG



(SEQ ID NO: 112)









III. The Effect of Yeast Extract on Isoprene Production in E. coli Expressing Genes from the Mevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (per Liter Fermentation Medium):
K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.
1000× Modified Trace Metal Solution:
Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO4*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component is dissolved one at a time in Di H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micron filter.
Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the upper mevalonic acid (MVA) pathway (pCL Upper), the integrated lower MVA pathway (gi1.2KKDyI), and high expression of mevalonate kinase from M. mazei and isoprene synthase from P. alba (pTrcAlba-mMVK). This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30° C. A frozen vial of the E. coli strain was thawed and inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 15-L bioreactor bringing the initial volume to 5-L.
i) Production of Isoprene in E. coli Cells (EL256) Grown in Fed-Batch Culture without Yeast Extract Feeding
Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 67 hour fermentation was 3.9 kg. Induction was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The IPTG concentration was brought to 102 uM when the optical density at 550 nm (OD550) reached a value of 9. The IPTG concentration was raised to 192 uM when OD550 reached 140. The OD550 profile within the bioreactor over time is shown in FIG. 67A. The isoprene level in the off gas from the bioreactor was determined using a Hiden mass spectrometer. The isoprene titer increased over the course of the fermentation to a final value of 35.6 g/L (FIG. 67B). The total amount of isoprene produced during the 67 hour fermentation was 320.6 g and the time course of production is shown in FIG. 67C. The metabolic activity profile, as measured by TCER, is shown in FIG. 67D. The molar yield of utilized carbon that went into producing isoprene during fermentation was 17.9%. The weight percent yield of isoprene from glucose was 8.1%.
Production of Isoprene in E. coli Cells (EL256) Grown in Fed-Batch Culture with Yeast Extract Feeding
Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 68 hour fermentation was 7.1 kg. A total of 1.06 kg of yeast extract was also fed during the fermentation. Induction was achieved by adding IPTG. The IPTG concentration was brought to 208 uM when the optical density at 550 nm (OD550) reached a value of 7. The IPTG concentration was raised to 193 uM when OD550 reached 180. The OD550 profile within the bioreactor over time is shown in FIG. 68A. The isoprene level in the off gas from the bioreactor was determined using a Hiden mass spectrometer. The isoprene titer increased over the course of the fermentation to a maximum value of 32.2 g/L (FIG. 68B). The total amount of isoprene produced during the 68 hour fermentation was 395.5 g and the time course of production is shown in FIG. 68C. The time course of volumetric productivity is shown in FIG. 68D and shows that an average rate of 1.1 g/L/hr was maintained for between 23 and 63 hours. The metabolic activity profile, as measured by CER, is shown in FIG. 68E The molar yield of utilized carbon that went into producing isoprene during fermentation was 10.3%. The weight percent yield of isoprene from glucose was 5.2%.
IV. Production of Isoprene from Different Carbon Sources in E. coli Harboring the Mevalonic Acid (MVA) Pathway and Isoprene Synthase (EWL256)
Media Recipe (per Liter Fermentation Media):
K2HPO4 13.6 g, KH2PO4 13.6 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, (NH4)2SO4 3.2 g, yeast extract 0.2 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were dissolved sequentially in diH2O. The pH was adjusted to 6.8 with ammonium hydroxide (30%) and brought to volume. Media was filter sterilized with a 0.22 micron filter. Carbon source was added to a final concentration of 1%. Required antibiotics were added after sterilization and pH adjustment.
1000× Trace Metal Solution (per Liter Fermentation Media):
Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO4*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a time in diH2O, pH to 3.0 with HCl/NaOH, and then brought to volume and filter sterilized with a 0.22 micron filter.
i) Preparation of AFEX Biomass Hydrolysate
AFEX pretreated corn stover was hydrolyzed to prepare biomass hydrolysate containing both xylose, glucose and acetate.
AFEX pretreated corn stover, received from Michigan Biotechnology Institute, was used. The pretreatment conditions were, 60% moisture, 1:1 ammonia loading, and 90° C. for 30 minutes, then air dried. The moisture content in the AFEX pretreated corn stover was 21.27%. Content of glucan and xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (dry basis) respectively. The enzyme used was accellerase 1000, Grindamyl H121 (Danisco xylanase product from Aspergillus niger for bread-making industry).
For saccharification, 20 g of AFEX pretreated corn stover was added into a 500 ml flask, together with 5 ml of 1 M pH 4.8 sodium citrate buffer, 2.25 ml of Accellerase 1000, 0.1 ml of Grindamyl H121, and 72.65 ml of DI water. The flask was put in an orbital shaker, and incubated at 50° C. for 96 hours.
For analysis, one sample was taken from the shaker, and analyzed using HPLC. The hydrolysate contained 37.2 g/l of glucose and 24.3 g/L of xylose, and 7.6 g/L of oligomers of glucose and/or xylose. Additionally, the hydrolysate also contains 1.17 g/L acetate.
ii) Experimental Procedure
An inoculum of the E. coli strain EWL256 containing the MVA pathway and isoprene synthase was taken from a frozen vial and streaked onto an LB broth agar plate containing spectinomycin (50 ug/mL) and carbinicllin (50 ug/mL) and incubated at 30° C. overnight. A single colony was inoculated into TM3 media containing glucose, xylose, glycerol, acetate or biomass as only carbon source and grown overnight at 30° C. Cells grow on acetate reached a significantly lower optical density. Cells grown on glucose, glycerol, biomass hydrolysate or acetate were diluted into 20 mL of TM3 media containing the respective carbon sources to reach an optical density of between 0.1 measured at 600 nM. A negative control not containing any carbon source was prepared from the glucose overnight culture. A separate experiment was performed with glucose and xylose, where the cultures were diluted to an optical density of 0.05. All culture conditions (except for acetate and glycerol) were tested in duplicates and the presented results are averaged between these cultures. Production of isoprene was induced with 200 μM IPTG from the beginning of the experiment. The flasks were incubated at 30° C. in an orbital shaker (200 rpm) and growth was followed by measuring optical density. After the glucose fed cultures had reached an optical density of approximately 0.4, samples were analyzed for isoprene production from all the tested carbon sources every hour for three hours. Samples of 100 μL were transferred in duplicates to 2 mL glass vials, sealed and incubated for 30 min at 30° C. The bacteria were then heat killed by incubation at 80° C. for 8 minutes. The amount of produced isoprene was measured using GC-MS and specific productivity (μg/L*hr) was calculated.
iii) Results
Significant production of isoprene could be demonstrated during growth on all the tested carbon sources. These carbon sources are examples of common alcohols, organic acids, sugars containing 5 or 6 carbon units (C5 or C6), and biomass hydrolysate.
The initial growth rate on biomass hydrolysate was comparable to the growth rate on glucose (FIG. 69A). The initial specific productivity during growth on biomass hydrolysate was significantly higher than during growth on glucose. This demonstrates that biomass hydrolysate can be used as an efficient source of carbon for the production of isoprene. The specific productivity declined after 255 minutes of growth on biomass hydrolysate (FIG. 69B). The bacteria had a slower growth rate with xylose as only carbon source when compared to glucose (FIG. 69C), but a significant specific isoprene productivity was measured (FIG. 69D). This shows that both C5 and C6 sugars can be utilized for the production of isoprene via the mevalonate acid pathway.
Surprisingly, bacteria grown on acetate as the only carbon source had a specific productivity of isoprene approximately twice as high as during growth on glucose (FIG. 69A). The bacteria grew slower on acetate when compared to glucose (FIG. 69B), but the performed experiment demonstrates that acetate can also be used as a carbon source for the production of isoprene. Acetate was also present in the biomass hydrolysate as measured by HPLC.
The bacteria grew well with glycerol as only carbon source (FIG. 69A) and significant production of isoprene was demonstrated (FIG. 69B). This shows that common alcohols may also be used as carbon sources for production of isoprene via the mevalonate acid pathway.
Example 11
Expression of Isoprene-Synthase from Plant in Streptomyces sp
The gene for isoprene synthase Kudzu was obtained from plasmid pJ201:19813. Plasmid pJ201:19813 encodes isoprene synthase from Pueraia lobata (Kudzu plant) and was codon-optimized for Pseudomonas fluorescens, Pseudomonas putida, Rhodopseudomonas palustris and Corynebacterium (FIGS. 79A-79C (SEQ ID NO:123)). Digestion of plasmid pJ201:19813 with restriction enzymes NdeI and BamHI liberated gene iso19813 that was ligated into the Streptomyces-E. coli shuttle vector pUWL201PW (Doumith et al., Mol. Gen. Genet. 264: 477-485, 2000; FIG. 71) to generate pUWL201_iso. Successful cloning was verified by restriction analysis of pUWL201_iso. Expression of isoprene synthase iso19813 was under control of the erm-promoter which allows for constitutive expression in Streptomycetes species, but not for expression in E. coli. 
PUWL201PW (no insert) and pUWL201_iso were introduced in Streptomyces albus J1074 (Sanchez et al., Chem. Biol. 9:519-531, 2002) by transformation of protoplasts as described by Hopwood et al., The John innes foundation, Norwich, 1985.
A 200 μl aliquot of protoplast suspensions was transformed with 1.9 μg pUWL201PW or 2.9 μg pUWL201_iso. After incubation overnight at 28° C. on non-selective R5-agarplates, positive transformants were selected by further incubation for 4 days in R3-overlay agar containing thiostrepton (250 μg/ml). Thiostrepton resistant transformants were examined for presence of the pUWL-plasmids by plasmid preparation using Plasmid Mini Kit (Qiagen). Prepared plasmid DNA was reintroduced in E. coli DH50α to generate sufficient amounts of plasmid DNA to be analyzed by restriction analysis. Positive transformants were selected on ampicillin-containing L-agar plates and insert analysis was done by digestion of plasmid DNA with NdeI and BamHI endonucleases. Isoprene synthase was identified as a 1.7 kb fragment in positive pUWL201 iso clones while in the control strains (pUWL201PW) no such fragment was observed.
Wild type strain and transformants of S. albus containing control plasmid pUWL201PW or isoprene synthase encoding pUWL201_iso were analyzed for isoprene formation. Strains were cultivated in duplicate on solid media (tryptic soy broth agar, TSB; 2.5 ml) in presence or absence of thiostrepton (200 μg/ml) and incubated for 4 days at 28° C. in sealed head-space vials (total volume 20 ml). 500 μl head-space samples (end point measurements) were analyzed by GC-MS in SIM-mode and isoprene was identified according to reference retention times and molecular masses (67 m/z). Isoprene present in head-space samples was quantified by previously generated calibration curves. While wild-type S. albus and control strains harboring pUWL201PW produced isoprene in concentrations slightly higher than the detection limit (0.04-0.07 ppm), S. albus harboring pUWL201_iso produced isoprene in at least tenfold excess compared to controls (0.75 ppm; FIG. 72). The results demonstrate successful expression of plant-derived isoprene synthase in a prokaryotic organism of the Actinomycetes group.
Example 12
Production of Isoprene or Mevalonate from Fatty Acid or Palm Oil in E. coli fadR atoC LS5218 Containing the Upper or Upper and Lower Mevalonic Acid Pathway Plus Kudzu Isoprene Synthase
Escherichia coli fadR atoC strain LS5218 (#6966) was obtained from the Coli Genetic Stock Center. FadR encodes a transcription repressor that negatively regulates expression of the genes encoding fatty acid degradation enzymes (Campbell et al., J. Bacteriol. 183: 5982-5990, 2001). AtoC is a response regulator in a two-component regulatory system with AtoS, regulates acetolactate metabolism. The fadR atoC strain allows constitutive expression of the fatty acid degradation genes and incorporates long chain fatty acids into long-chain-length polyhydroxyalkanoates. When palm oil is used as a carbon source for either mevalonate or isoprene production, the palm oil was converted to glycerol plus fatty acid. Methods for this are well known in the art, and it can be done either enzymatically by incubation with a lipase (for example Porcine pancreatic lipase, Candida rugosa lipase, or other similar lipases) or chemically by saponification with a base such as sodium hydroxide.
i) E. coli fadR atoC Strain Expressing the Upper Mevalonic Acid Pathway
Strain WW4 was created by electroporating pCLPtrcUpperPathway into LS5218 using standard methods (Sambrooke et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989). Incorporation of the plasmid was demonstrated by the production of mevalonic acid (MVA) when cells were cultured in TM3 medium supplemented with either C12 fatty acid (FA) or palm oil as the carbon source. To demonstrate production of MVA by WW4 from fatty acid, cells from an overnight culture were diluted 1 to 100 into 5 mL of modified TM3 medium (TM3 without yeast extract) supplemented with 0.25% C12 FA (Sigma cat #L9755). The first sign of MVA production (24 mg/L) was apparent after overnight incubation at 30° C. of the IPTG induced culture. Production increased over three days with the final level of 194 mg/L of MVA produced. To demonstrate production of MVA by WW4 from oil, cells from an overnight culture were diluted 1 to 100 into modified TM3 medium supplemented with 200 mg of digested palm oil per 5 mL of TM3 medium. The first sign of MVA production (50 mg/L) was apparent after overnight incubation of the IPTG induced culture at 30° C. Production increased over three days with a final level of 500 mg/L of MVA produced.
ii) E. coli fadR atoC Strain Expressing the Upper and Lower MVA Pathway Plus Kudzu Isoprene Synthase
Escherichia coli strain WW4 (LS5218 fadR atoC pCLPtrcUpperPathway) was transformed with pMCM118 [pTrcKKDyIkIS] to yield WW10. The incorporation of the plasmid was demonstrated by evidence of production of isoprene when the strain was cultured in TM3 and glucose and induced with IPTG (100, 300, or 900 uM). The strain was relatively sensitive to IPTG and showed a significant growth defect even at 100 uM IPTG. These results are shown in FIG. 70A.
To test isoprene production from dodecanoic acid, WW10 was cultured overnight in L broth containing spectinomycin (50 ug/ml), and kanamycin (50 ug/ml) at 37° C. with shaking at 200 rpm. The cells were washed with modified TM3 medium by centrifugation and resuspension in their original culture volume with this medium. The washed and resuspended cells from this starter culture were diluted 1 to 100 and 1 to 10 into 5 mL of modified TM3 medium containing 0.125% C12 Fatty Acid (Sigma cat #L9755).
To demonstrate production of mevalonate from palm oil, the oil was predigested with lipase at 37° C. and 250 rpm for several days to release the fatty acids (evidence of hydrolysis was judged by the foam formed when tubes were shaken).
In addition, a culture was set up by diluting the washed cells at 1 to 10 into modified TM3 medium contained in test tubes with palm oil. A further tube was set up by the addition of 0.125% C12FA to the remainder (2.5 mL) of the washed cells without further dilution (bioconversion). After 3.75 hours of growth at 30° C. with shaking at 250 rpm all of the cultures were induced by the addition of 50 uM IPTG. Incubation was continued for 4 hours after which time 200 uL of each of the cultures was assayed for isoprene accumulation with a modified head space assay (1 hour accumulation at 30° C. with shaking at 500 rpm). An additional isoprene assay was conducted by a 12 hour incubation of the assay glass block prior to GCMS analysis. Incubation of the induced cultures was continued overnight and 200 uL aliquots were again assayed for isoprene production (1 hour, 30 deg, 500 rpm Shel-Lab shaker) the following morning. Analysis of these cultures showed the production of significant levels of isoprene. The highest levels of isoprene were observed in the culture which was seeded at 1/10 dilution from the overnight starter culture after it had been incubated and induced overnight. This result suggests that this culture continued to grow and increase in cell density. These results are shown in FIG. 70B. Cell density could not be measured directly because the fatty acid suspension had a turbid appearance. Cell density of this culture was therefore determined by plating an aliquot of the culture and showed 8×107 colony forming units. This corresponds approximately to an OD600 of 0.1. Nevertheless, this culture provided significant isoprene production; no isoprene is observed for similar strains without the pathway described in this example.
Example 13
Improvement of Isoprene Production by Constitutive Expression of ybhE in E. coli
This example shows production of isoprene in a strain constitutively expressing ybhE (pgl) compared to a control strain with wild type ybhE. The gene ybhE (pgl) encodes a 6-phosphogluconolactonase that suppresses posttranslational gluconylation of heterologously expressed proteins and improves product solubility and yield while also improving biomass yield and flux through the pentose phosphate pathway (Aon et al. Applied and Environmental Microbiology, 74(4): 950-958, 2008).
The BL21 strain of E. coli producing isoprene (EWL256) was constructed with constitutive expression of the ybhE gene on a replicating plasmid pBBR1MCS5(Gentamycin) (obtained from Dr. K. Peterson, Louisiana State University).
FRT-based recombination cassettes, and plasmids for Red/ET-mediated integration and antibiotic marker loopout were obtained from Gene Bridges GmbH (Germany). Procedures using these materials were carried out according to Gene Bridges protocols. Primers Pgl-F and PglGI1.5-R were used to amplify the resistance cassette from the FRT-gb2-Cm-FRT template using Stratagene Herculase II Fusion kit according to the manufacturer's protocol. The PCR reaction (50 uL final volume) contained: 5 uL buffer, 1 uL template DNA (FRT-gb2-Cm-F from Gene Bridges), 10 pmols of each primer, and 1.5 uL 25 mM dNTP mix, made to 50 uL with dH2O. The reaction was cycled as follows: 1×2 minutes, 95° C. then 30 cycles of (30 seconds at 95° C.; 30 seconds at 63° C.; 3 minutes at 72° C.).
The resulting PCR product was purified using the QiaQick PCR purification kit (Qiagen) and electroporated into electrocompetent MG1655 cells harboring the pRed-ET recombinase-containing plasmid as follows. Cells were prepared by growing in 5 mLs of L broth to and OD600 ̃0.6 at 30° C. The cells were induced for recombinase expression by the addition of 4% arabinose and allowed to grow for 30 minutes at 30° C. followed by 30 minutes of growth at 37° C. An aliquot of 1.5 mLs of the cells was washed 3-4 times in ice cold dH2O. The final cell pellet was resuspended in 40 uL of ice cold dH2O and 2-5 uL of the PCR product was added. The electroporation was carried out in 1-mm gap cuvettes, at 1.3 kV in a Gene Pulser Electroporator (Bio-Rad Inc.). Cells were recovered for 1-2 hours at 30° C. and plated on L agar containing chloramphenicol (5 ug/mL). Five transformants were analyzed by PCR and sequencing using primers flanking the integration site (2 primer sets: pgl and 49 rev and 3′ EcoRV-pgl stop; Bottom Pgb2 and Top GB's CMP (946)). A correct transformant was selected and this strain was designated MG1655 GI1.5-pgl::CMP.
The chromosomal DNA of MG1655 GI1.5-pgl::CMP was used as template to generate a PCR fragment containing the FRT-CMP-FRT-GI1.5-ybhE construct. This construct was cloned into pBBR1MCS5(Gentamycin) as follows. The fragment, here on referred to as CMP-GI1.5-pgl, was amplified using the 5′ primer Pglconfirm-F and 3′ primer 3′ EcoRV-pgl stop. The resulting fragment was cloned using the Invitrogen TOPO-Blunt cloning kit into the plasmid vector pCR-Blunt II-TOPO as suggested from the manufacturer. The NsiI fragment harboring the CMP-GI1.5-pgl fragment was cloned into the PstI site of pBBR1MCS5(Gentamycin). A 20 μl ligation reaction was prepared containing 5 μl CMP-GI1.5-pgl insert, 2 μl pBBR1MCS5(Gentamycin) vector, 1 μl T4 DNA ligase (New England Biolabs), 2 μl 10× ligase buffer, and 10 μl ddH2O. The ligation mixture was incubated at room temperature for 40 minutes then 2-4 uL were electroporated into electrocompetent Top10 cells (Invitrogen) using the parameters disclosed above. Transformants were selected on L agar containing 10 ug/ml chloramphenicol and 5 ug/ml Gentamycin. The sequence of the selected clone was determined using a number of the primers described above as well as with the in-house T3 and Reverse primers provided by Sequetech, CA. This plasmid was designated pBBRCMPGI1.5-pgl (FIGS. 77A-B and SEQ ID NO:122).
Plasmid pBBRCMPGI1.5-pgl was electroporated into EWL256, as described above in Example 10 and transformants were plated on L agar containing Chloramphenicol (10 ug/mL), Gentamycin (5 ug/mL), spectinomycin (50 ug/mL), and carbenicillin (50 ug/mL). One transformant was selected and designated RM11608-2.







Primers: 


Pg1-F


(SEQ ID NO: 115)


5′-ACCGCCAAAAGCGACTAATTTTAGCTGTTACAGTCAGTTGAATTAAC


 


CCTCACTAAAGGGCGGCCGC-3′


 


Pg1GI1.5-R


(SEQ ID NO: 116)


5′-GCTGGCGATATAAACTGTTTGCTTCATGAATGCTCCTTTGGGTTACC


 


TCCGGGAAACGCGGTTGATTTGTTTAGTGGTTGAATTATTTGCTCAGGAT


 


GTGGCATAGTCAAGGGCGTGACGGCTCGCTAATACGACTCACTATAGGGC


 


TCGAG-3′


 


3′EcoRV-pg1stop: 


(SEQ ID NO: 117)


5′-CTT GAT ATC TTA GTG TGC GTT AAC CAC CAC


 


pg1 + 49 rev: 


(SEQ ID NO: 118)


CGTGAATTTGCTGGCTCTCAG


 


Bottom Pgb2: 


(SEQ ID NO: 119)


GGTTTAGTTCCTCACCTTGTC


 


Top GB's CMP (946): 


(SEQ ID NO: 120)


ACTGAAACGTTTTCATCGCTC


 


Pg1confirm-F


(SEQ ID NO: 121)


5′-ACCGCCAAAAGCGACTAATTTTAGCT-3′






i) Small Scale Analysis

Media Recipe (per Liter Fermentation Media):
K2HPO4 13.6 g, KH2PO4 13.6 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, (NH4)2SO4 3.2 g, yeast extract 1 g, 1000× Trace Metals Solution 1 ml. All of the components were added together and dissolved in diH2O. The pH was adjusted to 6.8 with ammonium hydroxide (30%) and brought to volume. Media was filter-sterilized with a 0.22 micron filter. Glucose 5.0 g and antibiotics were added after sterilization and pH adjustment.
1000× Trace Metal Solution (per Liter Fermentation Media):
Citric Acid*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO4*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component is dissolved one at a time in diH2O. The pH is adjusted to 3.0 with HCl/NaOH, and then the solution is brought to volume and filter-sterilized with a 0.22 micron filter.
a) Experimental Procedure
Isoprene production was analyzed by growing the strains in a CelleratorTM from MicroReactor Technologies, Inc. The working volume in each of the 24 wells was 4.5 mL. The temperature was maintained at 30° C., the pH setpoint was 7.0, the oxygen flow setpoint was 20 sccm and the agitation rate was 800 rpm. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 30° C. A single colony was inoculated into media with antibiotics and grown overnight. The bacteria were diluted into 4.5 mL of media with antibiotics to reach an optical density of 0.05 measured at 550 nm.
Off-gas analysis of isoprene was performed using a gas chromatograph-mass spectrometer (GC-MS) (Agilent) headspace assay. Sample preparation was as follows: 100 μL of whole broth was placed in a sealed GC vial and incubated at 30° C. for a fixed time of 30 minutes. Following a heat kill step, consisting of incubation at 70° C. for 5 minutes, the sample was loaded on the GC.
Optical density (OD) at a wavelength of 550 nm was obtained using a microplate reader (Spectramax) during the course of the run. Specific productivity was obtained by dividing the isoprene concentration (μg/L) by the OD reading and the time (hour).
The two strains EWL256 and RM11608-2 were assessed at 200 and 400 uM IPTG induction levels. Samples were analyzed for isoprene production and cell growth (OD550) at 1, 2.5, 4.75, and 8 hours post-induction. Samples were done in duplicate.
b) Results
The experiment demonstrated that at 2 different concentrations of IPTG the strain expressing the ybhE (pgl) had a dramatic 2-3 fold increase in specific productivity of isoprene compared to the control strain.
ii) Isoprene Fermentation from E. coli Expressing M. mazei Mevalonate Kinase, P. alba Isoprene Synthase, and pgl Over-Expression (RHM111608-2) and Grown in Fed-Batch Culture at the 15-L Scale
Medium Recipe (per Liter Fermentation Medium)
K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.
1000× Modified Trace Metal Solution:
Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO4*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component is dissolved one at a time in Di H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micron filter
Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the upper mevalonic acid (MVA) pathway (pCL Upper), the integrated lower MVA pathway (gi1.2KKDyI), high expression of mevalonate kinase from M. mazei and isoprene synthase from P. alba (pTrcAlba-mMVK), and high expression of pgl (pBBR-pgl). This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 34° C. A frozen vial of the E. coli strain was thawed and inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 15-L bioreactor bringing the initial volume to 5-L.
Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 40 hour (59 hour) fermentation was 3.1 kg (4.2 kg at 59 hour). Induction was achieved by adding IPTG. The IPTG concentration was brought to 110 uM when the optical density at 550 nm (OD550) reached a value of 4. The IPTG concentration was raised to 192 uM when OD550 reached 150. The OD550 profile within the bioreactor over time is shown in FIG. 78A. The isoprene level in the off gas from the bioreactor was determined using a Hiden mass spectrometer. The isoprene titer increased over the course of the fermentation to a maximum value of 33.2 g/L at 40 hours (48.6 g/L at 59 hours) (FIG. 78B). The isoprene titer increased over the course of the fermentation to a maximum value of 40.0 g/L at 40 hours (60.5 g/L at 59 hours) (FIG. 78C). The total amount of isoprene produced during the 40-hour (59-hour) fermentation was 281.3 g (451.0 g at 59 hours) and the time course of production is shown in FIG. 78D. The time course of volumetric productivity is shown in FIG. 78E and shows that an average rate of 1.0 g/L/hr was maintained between 0 and 40 hours (1.4 g/L/hour between 19 and 59 hour). The metabolic activity profile, as measured by CER, is shown in FIG. 78F. The molar yield of utilized carbon that went into producing isoprene during fermentation was 19.6% at 40 hours (23.6% at 59 hours). The weight percent yield of isoprene from glucose was 8.9% at 40 hours (10.7% at 59 hours).
Unless defined otherwise, the meanings of all technical and scientific terms used herein are those commonly understood by one of skill in the art to which this invention belongs. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) provide one of skill with a general dictionary of many of the terms used in this invention. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. One of skill in the art will also appreciate that any methods and materials similar or equivalent to those described herein can also be used to practice or test the invention.
The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole.
For use herein, unless clearly indicated otherwise, use of the terms “a”, “an,” and the like refers to one or more.
Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.” Numeric ranges are inclusive of the numbers defining the range.
It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments.
APPENDIX 1
Exemplary 1-deoxy-D-xylulose-5-phosphate Synthase Nucleic Acids and Polypeptides


    ATH: AT3G21500(DXPS1) AT4G15560(CLA1) AT5G11380(DXPS3)
    OSA: 4338768 4340090 4342614
    CME: CMF089C
    PFA: MAL13P1.186
    TAN: TA20470
    TPV: TP01—0516
    ECO: b0420(dxs)
    ECJ: JW0410(dxs)
    ECE: Z0523(dxs)
    ECS: ECs0474
    ECC: c0531(dxs)
    ECI: UT189_C0443(dxs)
    ECP: ECP—0479
    ECV: APECO1—1590(dxs)
    ECW: EcE24377A—0451(dxs)
    ECX: EcHS_A0491
    STY: STY0461(dxs)
    STT: t2441(dxs)
    SPT: SPA2301(dxs)
    SEC: SC0463(dxs)
    STM: STM0422(dxs)
    YPE: YPO3177(dxs)
    YPK: y1008(dxs)
    YPM: YP—0754(dxs)
    YPA: YPA—2671
    YPN: YPN—0911
    YPP: YPDSF—2812
    YPS: YPTB0939(dxs)
    YPI: YpsIP31758—3112(dxs)
    SFL: SF0357(dxs)
    SFX: S0365(dxs)
    SFV: SFV—0385(dxs)
    SSN: SSON—0397(dxs)
    SBO: SBO—0314(dxs)
    SDY: SDY—0310(dxs)
    ECA: ECA1131(dxs)
    PLU: plu3887(dxs)
    BUC: BU464(dxs)
    BAS: BUsg448(dxs)
    WBR: WGLp144(dxs)
    SGL: SG0656
    KPN: KPN—00372(dxs)
    BFL: Bfl238(dxs)
    BPN: BPEN—244(dxs)
    HIN: HI1439(dxs)
    HIT: NTHI1691(dxs)
    HIP: CGSHiEE—04795
    HIQ: CGSHiGG—01080
    HDU: HD0441(dxs)
    HSO: HS—0905(dxs)
    PMU: PM0532(dxs)
    MSU: MS1059(dxs)
    APL: APL—0207(dxs)
    XFA: XF2249
    XFT: PD1293(dxs)
    XCC: XCC2434(dxs)
    XCB: XC—1678
    XCV: XCV2764(dxs)
    XAC: XAC2565(dxs)
    XOO: XOO2017(dxs)
    XOM: XOO—1900(XOO1900)
    VCH: VC0889
    VVU: VV1—0315
    VVY: VV0868
    VPA: VP0686
    VFI: VF0711
    PPR: PBPRA0805
    PAE: PA4044(dxs)
    PAU: PA14—11550(dxs)
    PAP: PSPA7—1057(dxs)
    PPU: PP—0527(dxs)
    PST: PSPTO—0698(dxs)
    PSB: Psyr—0604
    PSP: PSPPH—0599(dxs)
    PFL: PFL—5510(dxs)
    PFO: Pfl—5007
    PEN: PSEEN0600(dxs)
    PMY: Pmen—3844
    PAR: Psyc—0221(dxs)
    PCR: Pcryo—0245
    ACI: ACIAD3247(dxs)
    SON: SO—1525(dxs)
    SDN: Sden—2571
    SFR: Sfri—2790
    SAZ: Sama—2436
    SBL: Sba1—1357
    SLO: Shew—2771
    SHE: Shewmr4—2731
    SHM: Shewmr7—2804
    SHN: Shewana3—2901
    SHW: Sputw3181—2831
    ILO: IL2138(dxs)
    CPS: CPS—1088(dxs)
    PHA: PSHAa2366(dxs)
    PAT: Patl—1319
    SDE: Sde—3381
    PIN: Ping—2240
    MAQ: Maqu—2438
    MCA: MCA0817(dxs)
    FTU: FTT1018c(dxs)
    FTF: FTF1018c(dxs)
    FTW: FTW—0925(dxs)
    FTL: FTL—1072
    FTH: FTH—1047(dxs)
    FTA: FTA—1131(dxs)
    FTN: FTN—0896(dxs)
    NOC: Noc—1743
    AEH: Mlg—1381
    HCH: HCH—05866(dxs)
    CSA: Csa1—0099
    ABO: ABO—2166(dxs)
    AHA: AHA—3321(dxs)
    BCI: BCI—0275(dxs)
    RMA: Rmag—0386
    VOK: COSY—0360(dxs)
    NME: NMB1867
    NMA: NMA0589(dxs)
    NMC: NMC0352(dxs)
    NGO: NG00036
    CVI: CV—2692(dxs)
    RSO: RSc2221(dxs)
    REU: Reut_A0882
    REH: H16_A2732(dxs)
    RME: Rmet—2615
    BMA: BMAA0330(dxs)
    BMV: BMASAVP1—1512(dxs)
    BML: BMA10299—1706(dxs)
    BMN: BMA10247_A0364(dxs)
    BXE: Bxe_B2827
    BUR: Bcep18194_B2211
    BCN: Bcen—4486
    BCH: Bcen2424—3879
    BAM: Bamb—3250
    BPS: BPSS1762(dxs)
    BPM: BURPS1710b_A0842(dxs)
    BPL: BURPS1106A_A2392(dxs)
    BPD: BURPS668_A2534(dxs)
    BTE: BTH—110614(dxs)
    BPE: BP2798(dxs)
    BPA: BPP2464(dxs)
    BBR: BB1912(dxs)
    RFR: Rfer—2875
    POL: Bpro—1747
    PNA: Pnap—1501
    AJS: Ajs—1038
    MPT: Mpe_A2631
    HAR: HEAR0279(dxs)
    MMS: mma—0331
    NEU: NE1161(dxs)
    NET: Neut—1501
    NMU: Nmul_A0236
    EBA: ebA4439(dxs)
    AZO: azo1198(dxs)
    DAR: Daro—3061
    TBD: Tbd—0879
    MFA: Mfla—2133
    HPY: HPO354(dxs)
    HPJ: jhp0328(dxs)
    HPA: HPAG1—0349
    HHE: HH0608(dxs)
    HAC: Hac—0968(dxs)
    WSU: WS1996
    TDN: Tmden—0475
    CJE: Cj0321(dxs)
    CJR: CJE0366(dxs)
    CJJ: CJJ81176-0343(dxs)
    CJU: C8J=0298(dxs)
    CJD: JJD26997—1642(dxs)
    CFF: CFF8240—0264(dxs)
    CCV: CCV52592—1671(dxs) CCV52592—1722
    CHA: CHAB381—1297(dxs)
    CCO: CCC13826—1594(dxs)
    ABU: Abu—2139(dxs)
    NIS: NIS—0391(dxs)
    SUN: SUN—2055(dxs)
    GSU: GSU0686(dxs-1) GSU1764(dxs-2)
    GME: Gmet—1934 Gmet—2822
    PCA: Pcar—1667
    PPD: Ppro—1191 Ppro—2403
    DVU: DVU1350(dxs)
    DVL: Dvu1—1718
    DDE: Dde—2200
    LIP: L10408(dsx)
    DPS: DP2700
    ADE: Adeh—1097
    MXA: MXAN—4643(dxs)
    SAT: SYN—02456
    SFU: Sfum—1418
    PUB: SAR11—0611(dxs)
    MLO: m1r7474
    MES: Meso—0735
    SME: SMc00972(dxs)
    ATU: Atu0745(dxs)
    ATC: AGR_C—1351
    RET: RHE_CH00913(dxs)
    RLE: RL0973(dxs)
    BME: BMEI1498
    BMF: BAB1—0462(dxs)
    BMS: BR0436(dxs)
    BMB: BruAb1—0458(dxs)
    BOV: BOV—0443(dxs)
    BJA: bl12651(dxs)
    BRA: BRAD02161(dxs)
    BBT: BBta—2479(dxs)
    RPA: RPA0952(dxs)
    RPB: RPB—4460
    RPC: RPC—1149
    RPD: RPD—4305
    RPE: RPE—1067
    NWI: Nwi—0633
    NHA: Nham—0778
    BHE: BH04350(dxs)
    BQU: BQ03540(dxs)
    BBK: BARBAKC583—0400(dxs)
    CCR: CC—2068
    SIL: SPO0247(dxs)
    SIT: TM1040—2920
    RSP: RSP—0254(dxsA) RSP—1134(dxs)
    JAN: Jann—0088 Jann—0170
    RDE: RD1—0101(dxs) RD1—0548(dxs)
    MMR: Mmar10—0849
    HNE: HNE—1838(dxs)
    ZMO: ZMO1234(dxs) ZMO1598(dxs)
    NAR: Saro—0161
    SAL: Sala—2354
    ELI: ELI—12520
    GOX: GOX0252
    GBE: GbCGDNIH1—0221 GbCGDNIH1—2404
    RRU: Rru_A0054 Rru_A2619
    MAG: amb2904
    MGM: Mmc1—1048
    SUS: Acid—1783
    BSU: BG11715(dxs)
    BHA: BH2779
    BAN: BA4400(dxs)
    BAR: GBAA4400(dxs)
    BAA: BA—4853
    BAT: BAS4081
    BCE: BC4176(dxs)
    BCA: BCE—4249(dxs)
    BCZ: BCZK3930(dxs)
    BTK: BT9727—3919(dxs)
    BTL: BALH—3785(dxs)
    BLI: BL01523(dxs)
    BLD: BLi02598(dxs)
    BCL: ABC2462(dxs)
    BAY: RBAM—022600
    BPU: BPUM—2159
    GKA: GK2392
    GTN: GTNG—2322
    LMO: lmo1365(tktB)
    LMF: LMOf2365—1382(dxs)
    LIN: lin1402(tktB)
    LWE: lwe1380(tktB)
    LLA: L108911(dxsA) L123365(dxsB)
    LLC: LACR—1572 LACR—1843
    LLM: llmg—0749(dxsB)
    SAK: SAK—0263
    LPL: lp—2610(dxs)
    LJO: LJ0406
    LAC: LBA0356
    LSL: LSL—0209(dxs)
    LGA: LGAS—0350
    STH: STH1842
    CAC: CAC2077 CA_P0106(dxs)
    CPE: CPE1819
    CPF: CPF—2073(dxs)
    CPR: CPR—1787(dxs)
    CTC: CTC01575
    CNO: NTOICX—1983
    CTH: Cthe—0828
    CDF: CD1207(dxs)
    CBO: CB01881(dxs)
    CBA: CLB—1818(dxs)
    CBH: CLC—1825(dxs)
    CBF: C1-—1945(dxs)
    CKL: CKL—1231(dxs)
    CHY: CHY—1985(dxs)
    DSY: DSY2348
    DRM: Dred—1078
    PTH: PTH—1196(dxs)
    SWO: Swol—0582
    CSC: Csac—1853
    TTE: TTE1298(dxs)
    MTA: Moth—1511
    MPE: MYPE730
    MGA: MGA—1268(dxs)
    MTU: Rv2682c(dxs1) Rv3379c(dxs2)
    MTC: MT2756(dxs)
    MBO: Mb270c(dxs1) Mb3413c(dxs2)
    MLE: ML1038(dxs)
    MPA: MAP2803c(dxs)
    MAV: MAV—3577(dxs)
    MSM: MSMEG—2776(dxs)
    MMC: Mmcs—2208
    CGL: NCg11827(cg11902)
    CGB: cg2083(dxs)
    CEF: CE1796
    CDI: DIP1397(dxs)
    CJK: jk1078(dxs)
    NFA: nfa37410(dxs)
    RHA: RHA1_ro06843
    SCO: SCO6013(SC1C3.01) SCO6768(SC6A5.17)
    SMA: SAV1646(dxs1) SAV2244(dxs2)
    TWH: TWT484
    TWS: TW280(Dxs)
    LXX: Lxx10450(dxs)
    CMI: CMM—1660(dxsA)
    AAU: AAur—1790(dxs)
    PAC: PPA1062
    TFU: Tfu—1917
    FRA: Francci3—1326
    FAL: FRAAL2088(dxs)
    ACE: Acel—1393
    SEN: SACE—1815(dxs) SACE—4351
    BLO: BL1132(dxs)
    BAD: BAD—0513(dxs)
    FNU: FN1208 FN1464
    RBA: RB2143(dxs)
    CTR: CT331(dxs)
    CTA: CTA—0359(dxs)
    CMU: TC0608
    CPN: CPn1060(tktB—2)
    CPA: CP0790
    CPJ: CPj1060(tktB—2)
    CPT: CpB1102
    CCA: CCA00304(dxs)
    CAB: CAB301(dxs)
    CFE: CF0699(dxs)
    PCU: pc0619(dxs)
    TPA: TP0824
    TDE: TDE1910(dxs)
    LIL: LA3285(dxs)
    LIC: LIC10863(dxs)
    LBJ: LBJ=0917(dxs)
    LBL: LBL—0932(dxs)
    SYN: sll1945(dxs)
    SYW: SYNW1292(Dxs)
    SYC: syc1087_c(dxs)
    SYF: Synpcc7942—0430
    SYD: Syncc9605—1430
    SYE: Syncc9902—1069
    SYG: sync—1410(dxs)
    SYR: SynRCC307—1390(dxs)
    SYX: SynWH7803—1223(dxs)
    CYA: CYA—1701(dxs)
    CYB: CYB—1983(dxs)
    TEL: t110623
    GVI: g11094
    ANA: a1r0599
    AVA: Ava—4532
    PMA: Pro0928(dxs)
    PMM: PMM0907(Dxs)
    PMT: PMT0685(dxs)
    PMN: PMN2A—0300
    PMI: PMT9312—0893
    PMB: A9601—09541(dxs)
    PMC: P9515—09901(dxs)
    PMF: P9303—15371(dxs)
    PMG: P9301—09521(dxs)
    PMH: P9215—09851
    PMJ: P9211—08521
    PME: NATL1—09721(dxs)
    TER: Tery—3042
    BTH: BT—1403 BT—4099
    BFR: BF0873 BF4306
    BFS: BF0796(dxs) BF4114
    PGI: PG2217(dxs)
    CHU: CHU—3643(dxs)
    GFO: GFO—3470(dxs)
    FPS: FP0279(dxs)
    CTE: CT0337(dxs)
    CPH: Cpha266—0671
    PVI: Cvib—0498
    PLT: Plut—0450
    DET: DET0745(dxs)
    DEH: cbdb_A720(dxs)
    DRA: DR—1475
    DGE: Dgeo—0994
    TTH: TTC1614
    TTJ: TTHA0006
    AAE: aq—881
    TMA: TM1770
    PMO: Pmob—1001
    Exemplary Acetyl-CoA-Acetyltransferase Nucleic Acids and Polypeptides
    HSA: 38(ACAT1) 39(ACAT2)
    PTR: 451528(ACAT1)
    MCC: 707653(ACAT1) 708750(ACAT2)
    MMU: 110446(Acat1) 110460(Acat2)
    RNO: 25014(Acat1)
    CFA: 484063(ACAT2) 489-421(ACAT1)
    GGA: 418968(ACAT1) 421587(RCJMB04—34i5)
    XLA: 379569(MGC69098) 414622(MGC81403) 414639(MGC81256) 444-457(MGC83664)
    XTR: 394562(acat2)
    DRE: 30643(acat2)
    SPU: 759502(LOC759502)
    DME: Dme1_CG10932 Dme1_CG9149
    CEL: T02G5.4 T02G5.7 T02G5.8(kat-1)
    ATH: AT5G48230(ACAT2/EMB1276)
    OSA: 4326136 4346520
    CME: CMA042C CME087C
    SCE: YPL028W(ERG10)
    AGO: AGOS_ADR165c
    PIC: PICST—31707(ERG10)
    CAL: CaO19.1591(erg10)
    CGR: CAGLOL12364g
    SPO: SPBC215.09c
    MGR: MGG—01755 MGG—13499
    ANI: AN1409.2
    AFM: AFUA—6G14200 AFUA—8G04000
    AOR: AO090103000012 AO090103000406
    CNE: CNC05280
    UMA: UM03571.1
    DDI: DDB—0231621
    PFA: PF14—0484
    TET: TTHERM—00091590 TTHERM—00277470 TTHERM—00926980
    TCR: 511003.60
    ECO: b2224(atoB)
    ECJ: JW2218(atoB) JW5453(yqeF)
    ECE: Z4164(yqeF)
    ECS: ECs3701
    ECC: c2767(atoB) c3441(yqeF)
    ECI: UTI89_C2506(atoB) UTI89_C3247(yqeF)
    ECP: ECP—2268 ECP—2857
    ECV: APECO1—3662(yqeF) APECO1—4335(atoB) APECO1—43352(atoB)
    ECX: EcHS_A2365
    STY: STY3164(yqeF)
    STT: t2929(yqeF)
    SPT: SPA2886(yqeF)
    SEC: SC2958(yqeF)
    STM: STM3019(yqeF)
    SFL: SF2854(yqeF)
    SFX: S3052(yqeF)
    SFV: SFV—2922(yqeF)
    SSN: SSON—2283(atoB) SSON—3004(yqeF)
    SBO: SBO—2736(yqeF)
    ECA: ECA1282(atoB)
    ENT: Ent638—3299
    SPE: Spro—0592
    HIT: NTHOI0932(atoB)
    XCC: XCC1297(atoB)
    XCB: XC—2943
    XCV: XCV1401(thlA)
    XAC: XAC1348(atoB)
    XOO: XOO1881(atoB)
    XOM: XOO—1778(XOO1778)
    VCH: VCA0690
    VCO: VC0395—0630
    VVU: VV2—0494 VV2—0741
    VVY: VVA1043 VVA1210
    VPA: VPA0620 VPA1123 VPA1204
    PPR: PBPRB 1112 PBPRB 1840
    PAE: PA2001(atoB) PA2553 PA3454 PA3589 PA3925
    PAU: PA14—38630(atoB)
    PPU: PP—2051(atoB) PP—2215(fadAx) PP—3754 PP—4636
    PPF: Pput—2009 Pput—2403 Pput—3523 Pput—4498
    PST: PSPTO—0957(phbA-1) PSPTO—3164(phbA-2)
    PSB: Psyr—0824 Psyr—3031
    PSP: PSPPH—0850(phbA1) PSPPH—2209(phbA2)
    PFL: PFL—1478(atoB-2) PFL—2321 PFL—3066 PFL—4330(atoB-2) PFL—5283
    PFO: Pfl—1269 Pfl—1739 Pfl—2074 Pfl—2868
    PEN: PSEEN3197 PSEEN3547(fadAx) PSEEN4635(phbA)
    PMY: Pmen—1138 Pmen—2036 Pmen—3597 Pmen—3662 Pmen—3820
    PAR: Psyc—0252 Psyc—1169
    PCR: Pcryo—0278 Pcryo—1236 Pcryo—1260
    PRW: PsycPRwf—2011
    ACI: ACIAD0694 ACIAD1612 ACIAD2516(atoB)
    SON: SO—1677(atoB)
    SDN: Sden—1943
    SFR: Sfri—1338 Sfri—2063
    SAZ: Sama—1375
    SBL: Sbal—1495
    SBM: Shew185—1489
    SBN: Sba1195—1525
    SLO: Shew—1667 Shew—2858
    SPC: Sputcn32—1397
    SSE: Ssed—1473 Ssed—3533
    SPL: Spea—2783
    SHE: Shewmr4—2597
    SHM: Shewmr7—2664
    SHN: Shewana3—2771
    SHW: Sputw3181—2704
    ILO: IL0872
    CPS: CPS—1605 CPS—2626
    PHA: PSHAa0908 PSHAa1454(atoB) PSHAa1586(atoB)
    PAT: Patl—2923
    SDE: Sde—3149
    PIN: Ping—0659 Ping—2401
    MAQ: Maqu—2117 Maqu—2489 Maqu—2696 Maqu—3162
    CBU: CBU—0974
    LPN: lpg1825(atoB)
    LPF: lp11789
    LPP: lpp1788
    NOC: Noc—1891
    AEH: Mlg—0688 Mlg—2706
    HHA: Hhal—1685
    HCH: HCH—05299
    CSA: Csa1—0301 Csa1—3068
    ABO: ABO—0648(fadAx)
    MMW: Mmwyl1—0073 Mmwyl1—3021 Mmwyl1—3053 Mmwyl1—3097 Mmwyl1—4182
    AHA: AHA—2143(atoB)
    CVI: CV—2088(atoB) CV—2790(phaA)
    RSO: RSc0276(atoB) RSc1632(phbA) RSc1637(bktB) RSc1761(RS02948)
    REU: Reut_A0138 Reut_A1348 Reut_A1353 Reut_B4561 Reut_B4738 Reut_B5587 Reut_C5943 Reut_C6062
    REH: H16_A0170H16_A0867H16_A0868H16_A0872H16_A1297H16_A1438(phaA) H16_A1445(bktB) H16_A1528H16_A1713H16_A1720H16_A1887H16_A2148H16_B0380H16_B0381H16_B0406H16_B0662H16_B0668H16_B0759H16_B1369H16_B1771
    RME: Rmet—0106 Rmet—1357 Rmet—1362 Rmet—5156
    BMA: BMA1316 BMA1321(phbA) BMA1436
    BMV: BMASAVP1_A1805(bktB) BMASAVP1_A1810(phbA)
    BML: BMA10299_A0086(phbA) BMA10299_A0091
    BMN: BMA10247—1076(bktB) BMA10247—1081(phbA)
    BXE: Bxe_A2273 Bxe_A2335 Bxe_A2342 Bxe_A4255 Bxe_B0377 Bxe_B0739 Bxe_C0332 Bxe_C574 Bxe_C0915
    BVI: Bcep—1808—0519 Bcep1808—1717 Bcep1808—2877 Bcep1808—3594 Bcep1808—4015 Bcep1808—5507 Bcep1808—5644
    BUR: Bcep18194_A3629 Bcep18194_A5080 Bcep18194_A5091 Bcep18194_A6102 Bcep18194_B0263 Bcep18194_B1439 Bcep18194_C6652 Bcep18194_C6802 Bcep18194_C6874 Bcep18194_C7118 Bcep18194_C7151 Bcep18194_C7332
    BCN: Bcen—1553 Bcen—1599 Bcen—2158 Bcen—2563 Bcen—2998 Bcen—6289
    BCH: Bcen2424—0542 Bcen2424—1790 Bcen2424—2772 Bcen2424—5368 Bcen2424—6232 Bcen2424—6276 BAM: Bamb—0447 Bamb—1728 Bamb—2824 Bamb—4717 Bamb—5771 Bamb—5969
    BPS: BPSL1426 BPSL1535(phbA) BPSL1540
    BPM: BURPS1710b—2325(bktB) BURPS1710b—2330(phbA) BURPS1710b—2453(atoB-2)
    BPL: BURPS1106A—2197(bktB) BURPS1106A—2202(phbA)
    BPD: BURPS668—2160(bktB) BURPS668—2165(phbA)
    BTE: BTH—12144 BTH—12256 BTH—12261
    PNU: Pnuc—0927
    BPE: BP0447 BP0668 BP2059
    BPA: BPP0608 BPP1744 BPP3805 BPP4216 BPP4361
    BBR: BB0614 BB3364 BB4250 BB4804 BB4947
    RFR: Rfer—0272 Rfer—1000 Rfer—1871 Rfer—2273 Rfer—2561 Rfer—2594 Rfer—3839
    POL: Bpro—1577 Bpro—2140 Bpro—3113 Bpro—4187
    PNA: Pnap—0060 Pnap—0458 Pnap—0867 Pnap—1159 Pnap—2136 Pnap—2804
    AAV: Aave—0031 Aave—2478 Aave—3944 Aave—4368
    AJS: Ajs—0014 Ajs—0124 Ajs—1931 Ajs—2073 Ajs—2317 Ajs—3548 Ajs—3738 Ajs—3776
    VEI: Veis—1331 Veis—3818 Veis—4193
    DAC: Daci—0025 Daci—0192 Daci—3601 Daci—5988
    MPT: Mpe_A1536 Mpe_A1776 Mpe_A1869 Mpe_A3367
    HAR: HEAR0577(phbA)
    MMS: mma—0555
    NEU: NE2262(bktB)
    NET: Neut—0610
    EBA: ebA5202 p2A409(tioL)
    AZO: azo0464(fadA1) azo0469(fadA2) azo2172(thlA)
    DAR: Daro—0098 Daro—3022
    HPA: HPAG1—0675
    HAC: Hac—0958(atoB)
    GME: Gmet—1719 Gmet—2074 Gmet—2213 Gmet—2268 Gmet—3302
    GUR: Gura—3043
    BBA: Bd0404(atoB) Bd2095
    DOL: Dole—0671 Dole—1778 Dole—2160 Dole—2187
    ADE: Adeh—0062 Adeh—2365
    AFW: Anae109—0064 Anae109—1504
    MXA: MXAN—3791
    SAT: SYN—02642
    SFU: Sfum—2280 Sfum—3582
    RPR: RP737
    RCO: RC1134 RC1135
    RFE: RF—0163(paaJ)
    RBE: RBE—0139(paaJ)
    RAK: A1C—05820
    RBO: A1I—07215
    RCM: A1E—04760
    PUB: SAR11—0428(thlA)
    MLO: mlr3847
    MES: Meso—3374
    PLA: Plav—1573 Plav—2783
    SME: SMa1450 SMc03879(phbA)
    ATU: Atu2769(atoB) Atu3475
    ATC: AGR_C—5022(phbA) AGR_L—2713
    RET: RHE_CH04018(phbAch) RHE_PC00068(ypc00040) RHE_PF00014(phbAf)
    RLE: RL4621(phaA) pRL100301 pRL120369
    BME: BME10274 BMEII0817
    BMF: BAB1—1783(phbA-1) BAB2—0790(phbA-2)
    BMS: BR1772(phbA-1) BRA0448(phbA-2)
    BMB: BruAb 1—1756(phbA-1) BruAb2—0774(phbA-2)
    BOV: BOV—1707(phbA-1)
    OAN: Oant—1130 Oant—3107 Oant—3718 Oant—4020
    BJA: bll0 226(atoB) bll3949 bll7400 bll17819 blr3724(phbA)
    BRA: BRADO0562(phbA) BRADO0983(pimB) BRADO3110 BRADO3134(atoB)
    BBT: BBta—3558 BBta—3575(atoB) BBta—5147(pimB) BBta—7072(pimB) BBta—7614(phbA)
    RPA: RPA0513(pcaF) RPA0531 RPA3715(pimB)
    RPB: RPB—0509 RPB—0525 RPB—1748
    RPC: RPC—0504 RPC—0636 RPC—0641 RPC—0832 RPC—1050 RPC—2005
    RPC—2194 RPC—2228
    RPD: RPD—0306 RPD—0320 RPD—3105 RPD—3306
    RPE: RPE—0168 RPE—0248 RPE—3827
    NWI: Nwi—3060
    XAU: Xaut—3108 Xaut—4665
    CCR: CC—0510 CC—0894 CC—3462
    SIL: SPO0142(bktB) SPO0326(phbA) SPO0773 SPO3408
    SIT: TM1040—0067 TM1040—2790 TM1040—3026 TM1040—3735
    RSP: RSP—0745 RSP—1354 RSP—3184
    RSH: Rsph17029—0022 Rsph17029—2401 Rsph7029—3179 Rsph17029—3921
    RSQ: Rsph17025—0012 Rsph17025—2466 Rsph17025—2833
    JAN: Jann—0262 Jann—0493 Jann—4050
    RDE: RD1—0025 RD1—0201(bktB) RD—3394(phbA)
    PDE: Pden—2026 Pden—2663 Pden—2870 Pden—2907 Pden—4811 Pden—5022
    DSH: Dshi—0074 Dshi—3066 Dshi—3331
    MMR: Mmar10—0697
    HNE: HNE—2706 HNE—3065 HNE—3133
    NAR: Saro—0809 Saro—1069 Saro—1222 Saro—2306 Saro—2349
    SAL: Sala—0781 Sala—1244 Sala—2896 Sala—3158
    SWI: Swit—0632 Swit—0752 Swit—2893 Swit—3602 Swit—4887 Swit—5019
    ELI: ELI—01475 ELI—06705 ELI—12035
    GBE: GbCGDNIH1—0447
    ACR: Acry—1847 Acry—2256
    RRU: Rru_A0274 Rru_A1380 Rru_A1469 Rru_A1946 Rru_A3387
    MAG: amb0842
    MGM: Mmc1—1165
    ABA: Acid345—3239
    BSU: BG11319(mmgA) BG13063(yhfS)
    BHA: BH1997 BH2029 BH3801(mmgA)
    BAN: BA3687 BA4240 BA5589
    BAR: GBAA3687 GBAA4240 GBAA5589
    BAA: BA—0445 BA—4172 BA—4700
    BAT: BAS3418 BAS3932 BAS5193
    BCE: BC3627 BC4023 BC5344
    BCA: BCE—3646 BCE—4076 BCE—5475
    BCZ: BCZK3329(mmgA) BCZK3780(thl) BCZK5044(atoB)
    BCY: Bcer98—2722 Bcer98—3865
    BTK: BT9727—3379(mmgA) BT9727—3765(thl) BT9727—5028(atoB)
    BTL: BALH—3262(mmgA) BALH—3642(fadA) BALH—4843(atoB)
    BLI: BL03925(mmgA)
    BLD: BLi03968(mmgA)
    BCL: ABC0345 ABC2989 ABC3617 ABC3891(mmgA)
    BAY: RBAM—022450
    BPU: BPUM—2374(yhfS) BPUM—2941 BPUM—3373
    OIH: OB06760B06890B26320B3013
    GKA: GK1658 GK3397
    SAU: SA0342 SA0534(vraB)
    SAV: SAV0354 SAV0576(vraB)
    SAM: MW0330 MW0531(vraB)
    SAR: SAR0351(thl) SAR0581
    SAS: SAS0330 SAS0534
    SAC: SACOL0426 SACOL0622(atoB)
    SAB: SAB0304(thl) SAB0526
    SAA: SAUSA300—0355 SAUSA300—0560(vraB)
    SAO: SAOUHSC—00336 SAOUHSC—00558
    SAJ: SaurJH9—0402
    SAH: SaurJH1—0412
    SEP: SE0346 SE2384
    SER: SERP0032 SERP0220
    SHA: SH0510(mvaC) SH2417
    SSP: SSP0325 SSP2145
    LMO: lmo1414
    LMF: LMOf2365—1433
    LIN: lin1453
    LWE: lwe1431
    LLA: L11745(thiL) L25946(fadA)
    LLC: LACR—1665 LACR—1956
    LLM: llmg—0930(thiL)
    SPY: SPy—0140 SPy—1637(atoB)
    SPZ: M5005_Spy—0119 M5005_Spy—0432 M5005_Spy—1344(atoB)
    SPM: spyM18—0136 spyM18—1645(atoB)
    SPG: SpyM3—0108 SpyM3—1378(atoB)
    SPS: SPs0110 SPs0484
    SPH: MGAS10270_Spy0121 MGAS10270_Spy0433 MGAS10270_Spy1461(atoB)
    SPI: MGAS10750_Spy0124 MGAS10750_Spy0452 MGAS10750_Spy1453(atoB)
    SPJ: MGAS2096_Spy0123 MGAS2096_Spy0451 MGAS2096_Spy1365(atoB)
    SPK: MGAS9429_Spy0121 MGAS9429_Spy0431 MGAS9429_Spy1339(atoB)
    SPF: SpyM50447(atoB2)
    SPA: M6_Spy0166 M6_Spy0466 M6_Spy1390
    SPB: M28_Spy0117 M28_Spy0420 M28_Spy1385(atoB)
    SAK: SAK—0568
    LJO: LJ1609
    LAC: LBA0626(thiL)
    LSA: LSA1486
    LDB: Ldb0879
    LBU: LBUL—0804
    LBR: LVIS—2218
    LCA: LSEI—1787
    LGA: LGAS—1374
    LRE: Lreu—0052
    EFA: EF1364
    OOE: OEOE—0529
    STH: STH2913 STH725 STH804
    CAC: CAC2873 CA_P0078(thiL)
    CPE: CPE2195(atoB)
    CPF: CPF—2460
    CPR: CPR—2170
    CTC: CTC00312
    CNO: NT01CX—0538 NT01CX—0603
    CDF: CD1059(thlA1) CD2676(thlA2)
    CBO: CB03200(thl)
    CBE: Cbei—0411 Cbei—3630
    CKL: CKL—3696(thlA1) CKL—3697(thlA2) CKL—3698(thlA3)
    AMT: Amet—4630
    AOE: Clos—0084 Clos—0258
    CHY: CHY—1288 CHY—1355(atoB) CHY—1604 CHY—1738
    DSY: DSY0632 DSY0639 DSY1567 DSY1710 DSY2402 DSY3302
    DRM: Dred—0400 Dred—1491 Dred—1784 Dred—1892
    SWO: Swol—0308 Swol—0675 Swol—0789 Swol—1486 Swol—1934 Swol—2051
    TTE: TTE0549(paaJ)
    MTA: Moth—1260
    MTU: Rv1135A Rv1323(fadA4) Rv3546(fadA5)
    MTC: MT1365(phbA)
    MBO: Mb1167 Mb1358(fadA4) Mb3576(fadA5) Mb3586c(fadA6)
    MBB: BCG—1197 BCG—1385(fadA4) BCG—3610(fadA5) BCG—3620c(fadA6)
    MLE: ML1158(fadA4)
    MPA: MAP2407c(fadA3) MAP2436c(fadA4)
    MAV: MAV—1544 MAV—1573 MAV—1863 MAV—5081
    MSM: MSMEG—2224 MSMEG—4920
    MUL: MUL—0357
    MVA: Mvan—1976 Mvan—1988 Mvan—4305 Mvan—4677 Mvan—4891
    MGI: Mflv—1347 Mflv—1484 Mflv—2040 Mflv—2340 Mflv—4356 Mflv—4368 mMC: Mmcs—1758 Mmcs—1769 Mmcs—3796 Mmcs—3864
    MKM: Mkms—0251 Mkms—1540 Mkms—1805 Mkms—1816 Mkms—2836 Mkms—3159 Mkms—3286 Mkms—3869 Mkms—3938 Mkms—4227 Mkms—4411 Mkms—4580 Mkms—4724 Mkms—4764 Mkms—4776
    MJL: Mjls—0231 Mjls—1739 Mjls—1750 Mjls—2819 Mjls—3119 Mjls—3235 Mjls—3800 Mjls—3850 Mjls—4110 Mjls—4383 Mjls—4705 Mjls—4876 Mjls—5018 Mjls—5063 Mjls—5075
    CGL: NCgl2309(cgl2392)
    CGB: cg2625(pcaF)
    CEF: CE0731 CE2295
    CJK: jk1543(fadA3)
    NFA: nfa10750(fadA4)
    RHA: RHA1_ro01455 RHA1_ro01623 RHA1_ro01876 RHA1_ro02517(catF)
    RHA1_ro03022 RHA1_ro03024 RHA1_ro03391 RHA1_ro03892 RHA1_ro04599 RHA1_ro05257 RHA1_ro08871
    SCO: SCO5399(SC8F4.03)
    SMA: SAV1384(fadA5) SAV2856(fadA1)
    ART: Arth—1160 Arth—2986 Arth—3268 Arth—4073
    NCA: Noca—1371 Noca—1797 Noca—1828 Noca—2764 Noca—4142
    TFU: Tfu—1520 Tfu—2394
    FRA: Francci3—3687
    FRE: Franean1—1044 Franean1—2711 Franean1—2726 Franean1—3929 Franean1—4037 Franean1—4577
    FAL: FRAAL2514 FRAAL2618 FRAAL5910(atoB)
    ACE: Acel—0626 Acel—0672
    SEN: SACE—1192(mmgA) SACE—2736(fadA6) SACE—4011(catF) SACE—6236(fadA4)
    STP: Strop—3610
    SAQ: Sare—1316 Sare—3991
    RXY: Rxyl—1582 Rxyl—1842 Rxyl—2389 Rxyl—2530
    FNU: FN0495
    BGA: BG01101(fadA)
    BAF: BAPKO—0110(fadA)
    LIL: LA0457(thiL1) LA0828(thiL2) LA4139(fadA)
    LIC: LIC10396(phbA)
    LBJ: LBJ=2862(paaJ-4)
    LBL: LBL—0209(paaJ-4)
    SYN: slr1993(phaA)
    SRU: SRU—1211(atoB) SRU—1547
    CHU: CHU—1910(atoB)
    GFO: GFO—1507(atoB)
    FJO: Fjoh—4612
    FPS: FP0770 FP1586 FP1725
    RRS: RoseRS—3911 RoseRS—4348
    RCA: Rcas—0702 Rcas—3206
    HAU: Haur—0522
    DRA: DR—1072 DR—1428 DR—1960 DR—2480 DR_A0053
    DGE: Dgeo—0755 Dgeo—1305 Dgeo—1441 Dgeo—1883
    TTH: TTC0191 TTC0330
    TTJ: TTHA0559
    TME: Tme1—1134
    FNO: Fnod—0314
    PMO: Pmob—0515
    HMA: rmAC0896(acaB3) rrnAC2815(aca2) rrnAC3497(yqeF) rrnB0240(aca1) rrnB0242(acaB2) rrnB0309(acaB1)
    TAC: Ta0582
    TVO: TVN0649
    PTO: PT01505
    APE: APE—2108
    SSO: SSO2377(acaB-4)
    STO: ST0514
    SAI: Saci—0963 Saci—1361(acaB1)
    MSE: Msed—0656
    PAI: PAE1220
    PIS: Pisl—0029 Pisl—1301
    PCL: Pcal—0781
    PAS: Pars—0309 Pars—1071
    CMA: Cmaq—1941

Exemplary HMG-CoA Synthase Nucleic Acids and Polypeptides

    HSA: 3157(HMGCS1) 3158(HMGCS2)
    PTR: 457169(HMGCS2) 461892(HMGCS1)
    MCC: 702553(HMGCS1) 713541(HMGCS2)
    MMU: 15360(Hmgcs2) 208715(Hmgcs1)
    RNO: 24450(Hmgcs2) 29637(Hmgcs1)
    CFA: 479344(HMGCS1) 607923(HMGCS2)
    BTA: 407767(HMGCS1)
    SSC: 397673(CH242-38B5.1)
    GGA: 396379(HMGCS1)
    XLA: 380091(hmgcs1) 447204(MGC80816)
    DRE: 394060(hmgcs1)
    SPU: 578259(LOC578259)
    DME: Dme1_CG4311(Hmgs)
    CEL: F25B4.6
    ATH: AT4G11820(BAP1)
    OSA: 4331418 4347614
    CME: CMM189C
    SCE: YML126C(ERG13)
    AGO: AGOS_ADL356C
    PIC: PICST—83020
    CAL: CaO19—7312(CaO19.7312)
    CGR: CAGL0H04081g
    SPO: SPAC4F8.14c(hcs)
    MGR: MGG—01026
    ANI: AN4923.2
    AFM: AFUA—3G10660 AFUA—8G07210
    AOR: A0090003000611 A0090010000487
    CNE: CNC5080 CNG02670
    UMA: UM05362.1
    ECU: ECU10—0510
    DDI: DDBDRAFT—0217522 DDB—0219924(hgsA)
    TET: TTHERM—00691190
    TBR: Tb927.8.6110
    YPE: YPO1457
    YPK: y2712(pksG)
    YPM: YP—1349(pksG)
    YPA: YPA—0750
    YPN: YPN—2521
    YPP: YPDSF—1517
    YPS: YPTB1475
    CBD: COXBU7E912—1931
    TCX: Tcr—1719
    DNO: DNO—0799
    BMA: BMAA1212
    BPS: BPSS1002
    BPM: BURPS1710b_A2613
    BPL: BURPS1106A_A1384
    BPD: BURPS668_A1470
    BTE: BTH_II1670
    MXA: MXAN—3948(tac) MXAN—4267(mvaS)
    BSU: BG10926(pksG)
    OIH: OB2248
    SAU: SA2334(mvaS)
    SAV: SAV2546(mvaS)
    SAM: MW2467(mvaS)
    SAR: SAR2626(mvaS)
    SAS: SAS2432
    SAC: SACOL2561
    SAB: SAB2420(mvaS)
    SAA: SAUSA300—2484
    SAO: SAOUHSC—02860
    SAJ: SaurJH9—2569
    SAH: SaurJH1—2622
    SEP: SE2110
    SER: SERP2122
    SHA: SH0508(mvaS)
    SSP: SSP0324
    LMO: lmo1415
    LMF: LMOf2365—1434(mvaS)
    LIN: lin1454
    LWE: lwe1432(mvaS)
    LLA: L13187(hmcM)
    LLC: LACR—1666
    LLM: llmg—0929(hmcM)
    SPY: SPy—0881(mvaS.2)
    SPZ: M5005_Spy—0687(mvaS.1)
    SPM: spyM18—0942(mvaS2)
    SPG: SpyM3—0600(mvaS.2)
    SPS: SPs1253
    SPH: MGAS10270_Spy0745(mvaS1)
    SPI: MGAS10750_Spy0779(mvaS1)
    SPJ: MGAS2096_Spy0759(mvaS1)
    SPK: MGAS9429_Spy0743(mvaS1)
    SPF: SpyM51121(mvaS)
    SPA: M6_Spy0704
    SPB: M28_Spy0667(mvaS.1)
    SPN: SP—1727
    SPR: spr1571(mvaS)
    SPD: SPD—1537(mvaS)
    SAG: SAG1316
    SAN: gbs1386
    SAK: SAK—1347
    SMU: SMU.943c
    STC: str0577(mvaS)
    STL: stu0577(mvaS)
    STE: STER—0621
    SSA: SSA—0338(mvaS)
    SSU: SSU05—1641
    SSV: SSU98—1652
    SGO: SGO—0244
    LPL: lp—2067(mvaS)
    LJO: LJ1607
    LAC: LBA0628(hmcS)
    LSA: LSA1484(mvaS)
    LSL: LSL—0526
    LDB: Ldb0881(mvaS)
    LBU: LBUL—0806
    LBR: LVIS—1363
    LCA: LSEI—1785
    LGA: LGAS—1372
    LRE: Lreu—0676
    PPE: PEPE—0868
    EFA: EF1363
    OOE: OEOE—0968
    LME: LEUM—1184
    NFA: nfa22120
    SEN: SACE—4570(pksG)
    BBU: BB0683
    BGA: BG0706
    BAF: BAPKO—0727
    FJO: Fjoh—0678
    HAL: VNG1615G(mvaB)
    HMA: rmAC1740(mvaS)
    HWA: HQ2868A(mvaB)
    NPH: NP2608A(mvaB—1) NP4836A(mvaB—2)

Exemplary Hydroxymethylglutaryl-CoA Reductase Nucleic Acids and Polypeptides

    HSA: 3156(HMGCR)
    PTR: 471516(HMGCR)
    MCC: 705479(HMGCR)
    MMU: 15357(Hmgcr)
    RNO: 25675(Hmgcr)
    CFA: 479182(HMGCR)
    BTA: 407159(HMGCR)
    GGA: 395145(RCJMB04-14 m24)
    SPU: 373355(LOC373355)
    DME: Dme1_CG10367(Hmgcr)
    CEL: F08F8.2
    OSA: 4347443
    SCE: YLR450W(HMG2) YML075C(HMG1)
    AGO: AGOS_AER152W
    CGR: CAGLOL11506g
    SPO: SPCC162.09c(hmg1)
    ANI: AN3817.2
    AFM: AFUA—1G11230 AFUA—2G03700
    AOR: A0090103000311 A0090120000217
    CNE: CNF04830
    UMA: UM03014.1
    ECU: ECU10—1720
    DDI: DDB—0191125(hmgA) DDB—0215357(hmgB)
    TBR: Tb927.6.4540
    TCR: 506831.40 509167.20
    LMA: LmjF30.3190
    VCH: VCA0723
    VCO: VC0395—0662
    VVU: VV2—0117
    VVY: VVA0625
    VPA: VPA0968
    VFI: VFA0841
    PAT: Patl—0427
    CBU: CBU—0030 CBU—0610
    CBD: COXBU7E912—0151 COXBU7E912—0622(hmgA)
    TCX: Tcr—1717
    DNO: DNO—0797
    CVI: CV—1806
    SUS: Acid—5728 Acid—6132
    SAU: SA2333(mvaA)
    SAV: SAV2545(mvaA)
    SAM: MW2466(mvaA)
    SAB: SAB2419c(mvaA)
    SEP: SE2109
    LWE: 1we0819(mvaA)
    LLA: L10433(mvaA)
    LLC: LACR—1664
    LLM: llmg—0931(mvaA)
    SPY: SPy—0880(mvaS.1)
    SPM: spyM18—0941(mvaS1)
    SPG: SpyM3—0599(mvaS.1)
    SPS: SPs1254
    SPH: MGAS10270_Spy0744
    SPI: MGAS10750_Spy0778
    SPJ: MGAS2096_Spy0758
    SPK: MGAS9429_Spy0742
    SPA: M6_Spy0703
    SPN: SP—1726
    SAG: SAG1317
    SAN: gbs1387
    STC: str0576(mvaA)
    STL: stu0576(mvaA)
    STE: STER—0620
    SSA: SSA—0337(mvaA)
    LPL: lp—0447(mvaA)
    LJO: LJ1608
    LSL: LSL—0224
    LBR: LVIS—0450
    LGA: LGAS—1373
    EFA: EF1364
    NFA: nfa22110
    BGA: BG0708(mvaA)
    SRU: SRU—2422
    FPS: FP2341
    MMP: MMP0087(hmgA)
    MMQ: MmarC5—1589
    MAC: MA3073(hmgA)
    MBA: Mbar_A1972
    MMA: MM—0335
    MBU: Mbur—1098
    MHU: Mhun—3004
    MEM: Memar—2365
    MBN: Mboo—0137
    MTH: MTH562
    MST: Msp—0584(hmgA)
    MSI: Msm—0227
    MKA: MK0355(HMG1)
    AFU: AF1736(mvaA)
    HAL: VNG1875G(mvaA)
    HMA: rrnAC3412(mvaA)
    HWA: HQ3215A(hmgR)
    NPH: NP0368A(mvaA—2) NP2422A(mvaA—1)
    TAC: TaO406m
    TVO: TVN1168
    PTO: PT01143
    PAB: PAB2106(mvaA)
    PFU: PF1848
    TKO: TK0914
    RCI: RCIX1027(hmgA) RCIX376(hmgA)
    APE: APE—1869
    IHO: Igni—0476
    HBU: Hbut—1531
    SSO: SS00531
    STO: ST1352
    SAI: Saci—1359
    PAI: PAE2182
    PIS: Pisl—0814
    PCL: Pcal—1085
    PAS: Pars—0796

Exemplary Mevalonate Kinase Nucleic Acids and Polypeptides

    HSA: 4598(MVK)
    MCC: 707645(MVK)
    MMU: 17855(Mvk)
    RNO: 81727(Mvk)
    CFA: 486309(MVK)
    BTA: 505792(MVK)
    GGA: 768555(MVK)
    DRE: 492477(zgc:103473)
    SPU: 585785(LOC585785)
    DME: Dme1_CG33671
    OSA: 4348331
    SCE: YMR208W(ERG12)
    AGO: AGOS_AER335W
    PIC: PICST—40742(ERG12)
    CGR: CAGL0F03861g
    SPO: SPAC13G6.11c
    MGR: MGG—06946
    ANI: AN3869.2
    AFM: AFUA—4G07780
    AOR: A0090023000793
    CNE: CNK01740
    ECU: ECU09—1780
    DDI: DDBDRAFT—0168621
    TET: TTHERM—00637680
    TBR: Tb927.4.4070
    TCR: 436521.9 509237.10
    LMA: LmjF31.0560
    CBU: CBU—0608 CBU—0609
    CBD: COXBU7E912—0620(mvk)
    LPN: lpg2039
    LPF: lpl2017
    LPP: lpp2022
    BBA: Bd1027(lmbP) Bd1630(mvk)
    MXA: MXAN—5019(mvk)
    OIH: OB0225
    SAU: SA0547(mvaK1)
    SAV: SAV0590(mvaK1)
    SAM: MW0545(mvaK1)
    SAR: SAR0596(mvaK1)
    SAS: SAS0549
    SAC: SACOL0636(mvk)
    SAB: SAB0540(mvaK1)
    SAA: SAUSA300—0572(mvk)
    SAO: SAOUHSC—00577
    SEP: SE0361
    SER: SERP0238(mvk)
    SHA: SH2402(mvaK1)
    SSP: SSP2122
    LMO: lmo0010
    LMF: LMOf2365—0011
    LIN: lin0010
    LWE: lwe0011(mvk)
    LLA: L7866(yeaG)
    LLC: LACR—0454
    LLM: llmg—0425(mvk)
    SPY: SPy—0876(mvaK1)
    SPZ: M5005_Spy—0682(mvaK1)
    SPM: spyM18—0937(mvaK1)
    SPG: SpyM3—0595(mvaK1)
    SPS: SPs1258
    SPH: MGAS10270_Spy0740(mvaK1)
    SPI: MGAS10750_Spy0774(mvaK1)
    SPJ: MGAS2096_Spy0753(mvaK1)
    SPK: MGAS9429_Spy0737(mvaK1)
    SPF: SpyM51126(mvaK1)
    SPA: M6_Spy0699
    SPB: M28_Spy0662(mvaK1)
    SPN: SP—0381
    SPR: spr0338(mvk)
    SPD: SPD—0346(mvk)
    SAG: SAG1326
    SAN: gbs1396
    SAK: SAK—1357(mvk)
    SMU: SMU.181
    STC: str0559(mvaK1)
    STL: stu0559(mvaK1)
    STE: STER—0598
    SSA: SSA—0333(mvaK1)
    SSU: SSU05—0289
    SSV: SSU98—0285
    SGO: SGO—0239(mvk)
    LPL: lp—1735(mvaK1)
    LJO: LJ1205
    LAC: LBA1167(mvaK)
    LSA: LSA0908(mvaK1)
    LSL: LSL—0685(eRG)
    LDB: Ldb0999(mvk)
    LBU: LBUL—0906
    LBR: LVIS—0858
    LCA: LSEI—1491
    LGA: LGAS—1033
    LRE: Lreu—0915
    PPE: PEPE—0927
    EFA: EF0904(mvk)
    OOE: OEOE—1100
    LME: LEUM—1385
    NFA: nfa22070
    BGA: BG0711
    BAF: BAPKO—0732
    FPS: FP0313
    MMP: MMP1335
    MAE: Maeo—0775
    MAC: MA0602(mvk)
    MBA: Mbar_A1421
    MMA: MM—1762
    MBU: Mbur—2395
    MHU: Mhun—2890
    MEM: Memar—1812
    MBN: Mboo—2213
    MST: Msp—0858(mvk)
    MSI: Msm—1439
    MKA: MK0993(ERG12)
    HAL: VNG1145G(mvk)
    HMA: rrnAC0077(mvk)
    HWA: HQ2925A(mvk)
    NPH: NP2850A(mvk)
    PTO: PT01352
    PHO: PH1625
    PAB: PAB0372(mvk)
    PFU: PF1637(mvk)
    TKO: TK1474
    RCI: LRC399(mvk)
    APE: APE—2439
    HBU: Hbut—0877
    SSO: SS00383
    STO: ST2185
    SAI: Saci—2365(mvk)
    MSE: Msed—1602
    PAI: PAE3108
    PIS: Pisl—0467
    PCL: Pcal—1835

Exemplary Phosphomevalonate Kinase Nucleic Acids and Polypeptides

    HSA: 10654(PMVK)
    PTR: 457350(PMVK)
    MCC: 717014(PMVK)
    MMU: 68603(Pmvk)
    CFA: 612251(PMVK)
    BTA: 513533(PMVK)
    DME: Dme1_CG10268
    ATH: AT1G31910
    OSA: 4332275
    SCE: YMR220W(ERG8)
    AGO: AGOS_AER354W
    PIC: PICST—52257(ERG8)
    CGR: CAGL0F03993g
    SPO: SPAC343.01c
    MGR: MGG—05812
    ANI: AN2311.2
    AFM: AFUA—5G10680
    AOR: A0090010000471
    CNE: CNM00100
    UMA: UM00760.1
    DDI: DDBDRAFT—0184512
    TBR: Tb09.160.3690
    TCR: 507913.20 508277.140
    LMA: LmjF15.1460
    MXA: MXAN—5017
    OIH: OB0227
    SAU: SA0549(mvaK2)
    SAV: SAV0592(mvaK2)
    SAM: MW0547(mvaK2)
    SAR: SAR0598(mvaK2)
    SAS: SAS0551
    SAC: SACOL0638
    SAB: SAB0542(mvaK2)
    SAA: SAUSA300—0574
    SAO: SAOUHSC—00579
    SAJ: SaurJH9—0615
    SEP: SE0363
    SER: SERP0240
    SHA: SH2400(mvaK2)
    SSP: SSP2120
    LMO: lmo0012
    LMF: LMOf2365—0013
    LIN: lin0012
    LWE: lwe0013
    LLA: L10014(yebA)
    LLC: LACR—0456
    LLM: llmg—0427
    SPY: SPy—0878(mvaK2)
    SPZ: M5005_Spy—0684(mvaK2)
    SPM: spyM18—0939
    SPG: SpyM3—0597(mvaK2)
    SPS: SPs1256
    SPH: MGAS10270_Spy0742(mvaK2)
    SPI: MGAS10750_Spy0776(mvaK2)
    SPJ: MGAS2096_Spy0755(mvaK2)
    SPK: MGAS9429_Spy0739(mvaK2)
    SPF: SpyM51124(mvaK2)
    SPA: M6_Spy0701
    SPB: M28_Spy0664(mvaK2)
    SPN: SP—0383
    SPR: spr0340(mvaK2)
    SPD: SPD—0348(mvaK2)
    SAG: SAG1324
    SAN: gbs1394
    SAK: SAK—1355
    SMU: SMU.938
    STC: str0561(mvaK2)
    STL: stu0561(mvaK2)
    STE: STER—0600
    SSA: SSA—0335(mvaK2)
    SSU: SSU05—0291
    SSV: SSU98—0287
    SGO: SGO—0241
    LPL: lp—1733(mvaK2)
    LJO: LJ1207
    LAC: LBA1169
    LSA: LSA0906(mvaK2)
    LSL: LSL—0683
    LDB: Ldb0997(mvaK)
    LBU: LBUL—0904
    LBR: LVIS—0860
    LCA: LSEI—1092
    LGA: LGAS—1035
    LRE: Lreu—0913
    PPE: PEPE—0925
    EFA: EF0902
    NFA: nfa22090
    BGA: BG0710
    BAF: BAPKO—0731
    NPH: NP2852A
    SSO: SSO2988
    STO: ST0978
    SAI: Saci—1244

Exemplary Diphosphomevalonate Decarboxylase Nucleic Acids and Polypeptides

    HSA: 4597(MVD)
    PTR: 468069(MVD)
    MCC: 696865(MVD)
    MMU: 192156(Mvd)
    RNO: 81726(Mvd)
    CFA: 489663(MVD)
    GGA: 425359(MVD)
    DME: Dme1_CG8239
    SCE: YNR043W(MVD1)
    AGO: AGOS_AGL232C
    PIC: PICST—90752
    CGR: CAGL0C3630g
    SPO: SPAC24C9.03
    MGR: MGG—09750
    ANI: AN4414.2
    AFM: AFUA—4G07130
    AOR: A0090023000862
    CNE: CNL04950
    UMA: UM05179.1
    DDI: DDBDRAFT—0218058
    TET: TTHERM—00849200
    TBR: Tb10.05.0010 Tb10.61.2745
    TCR: 507993.330 511281.40
    LMA: LmjF18.0020
    CBU: CBU—0607(mvaD)
    CBD: COXBU7E912—0619(mvaD)
    LPN: lpg2040
    LPF: lp12018
    LPP: lpp2023
    TCX: Tcr—1734
    DNO: DNO—0504(mvaD)
    BBA: Bd1629
    MXA: MXAN—5018(mvaD)
    OIH: OB0226
    SAU: SA0548(mvaD)
    SAV: SAV0591(mvaD)
    SAM: MW0546(mvaD)
    SAR: SAR0597(mvaD)
    SAS: SAS0550
    SAC: SACOL0637(mvaD)
    SAB: SAB0541(mvaD)
    SAA: SAUSA300—0573(mvaD)
    SAO: SAOUHSC—00578
    SAJ: SaurJH9—0614
    SAH: SaurJH1—0629
    SEP: SE0362
    SER: SERP0239(mvaD)
    SHA: SH2401(mvaD)
    SSP: SSP2121
    LMO: lmo0011
    LMF: LMOf2365—0012(mvaD)
    LIN: lin0011
    LWE: lwe0012(mvaD)
    LLA: L9089(yeaH)
    LLC: LACR—0455
    LLM: llmg—0426(mvaD)
    SPY: SPy—0877(mvaD)
    SPZ: M5005_Spy—0683(mvaD)
    SPM: spyM18—0938(mvd)
    SPG: SpyM3—0596(mvaD)
    SPS: SPs1257
    SPH: MGAS10270_Spy0741(mvaD)
    SPI: MGAS10750_Spy0775(mvaD)
    SPJ: MGAS2096_Spy0754(mvaD)
    SPK: MGAS9429_Spy0738(mvaD)
    SPF: SpyM51125(mvaD)
    SPA: M6_Spy0700
    SPB: M28_Spy0663(mvaD)
    SPN: SP—0382
    SPR: spr0339(mvd1)
    SPD: SPD—0347(mvaD)
    SAG: SAG1325(mvaD)
    SAN: gbs1395
    SAK: SAK—1356(mvaD)
    SMU: SMU.937
    STC: str0560(mvaD)
    STL: stu0560(mvaD)
    STE: STER—0599
    SSA: SSA—0334(mvaD)
    SSU: SSU05—0290
    SSV: SSU98—0286
    SGO: SGO—0240(mvaD)
    LPL: lp—1734(mvaD)
    LJO: LJ1206
    LAC: LBA1168(mvaD)
    LSA: LSA0907(mvaD)
    LSL: LSL—0684
    LDB: Ldb0998(mvaD)
    LBU: LBUL—0905
    LBR: LVIS—0859
    LCA: LSEI—1492
    LGA: LGAS—1034
    LRE: Lreu—0914
    PPE: PEPE—0926
    EFA: EF0903(mvaD)
    LME: LEUM—1386
    NFA: nfa22080
    BBU: BB0686
    BGA: BG0709
    BAF: BAPKO—0730
    GFO: GFO—3632
    FPS: FP0310(mvaD)
    HAU: Haur—1612
    HAL: VNG0593G(dmd)
    HMA: rmAC1489(dmd)
    HWA: HQ1525A(mvaD)
    NPH: NP1580A(mvaD)
    PTO: PT00478 PT01356
    SSO: SSO2989
    STO: ST0977
    SAI: Saci—1245(mvd)
    MSE: Msed—1576

Exemplary Isopentenyl Phosphate Kinases (IPK) Nucleic Acids and Polypeptides

    Methanobacterium thermoautotrophicum gi|2621082
    Methanococcus jannaschii DSM 2661 gi|1590842
    Methanocaldococcus jannaschii gi|1590842
    Methanothermobacter thermautotrophicus gi|2621082
    Picrophilus torridus DSM9790 (IG-57) gi|48477569
    Pyrococcus abyssi gi|14520758
    Pyrococcus horikoshii OT3 gi|3258052
    Archaeoglobus fulgidus DSM4304 gi|2648231

Exemplary Isopentenyl-Diphosphate Delta-Isomerase (IDI) Nucleic Acids and Polypeptides

    HSA: 3422(IDI1) 91734(IDI2)
    PTR: 450262(IDI2) 450263(IDI1)
    MCC: 710052(LOC710052) 721730(LOC721730)
    MMU: 319554(Idi1)
    RNO: 89784(Idi1)
    GGA: 420-459(IDI1)
    XLA: 494671(LOC494671)
    XTR: 496783(idi2)
    SPU: 586184(LOC586184)
    CEL: K06H7.9(idi-1)
    ATH: AT3G02780(IPP2)
    OSA: 4338791 4343523
    CME: CMB062C
    SCE: YPL117C(IDI1)
    AGO: AGOS_ADL268C
    PIC: PICST—68990(IDI1)
    CGR: CAGL0J06952g
    SPO: SPBC106.15(idi1)
    ANI: AN0579.2
    AFM: AFUA—6G11160
    AOR: A0090023000500
    CNE: CNA02550
    UMA: UM04838.1
    ECU: ECU02—0230
    DDI: DDB—0191342(ipi)
    TET: TTHERM—00237280 TTHERM—00438860
    TBR: Tb09.211.0700
    TCR: 408799.19 510431.10
    LMA: LmjF35.5330
    EHI: 46.t00025
    ECO: b2889(idi)
    ECJ: JW2857(idi)
    ECE: Z4227
    ECS: ECs3761
    ECC: c3467
    ECI: UT189_C3274
    ECP: ECP—2882
    ECV: APECO1—3638
    ECW: EcE24377A—3215(idi)
    ECX: EcHS_A3048
    STY: STY3195
    STT: t2957
    SPT: SPA2907(idi)
    SEC: SC2979(idi)
    STM: STM3039(idi)
    SFL: SF2875(idi)
    SFX: S3074
    SFV: SFV—2937
    SSN: SSON—3042 SSON—3489(yhfK)
    SBO: SBO—3103
    SDY: SDY—3193
    ECA: ECA2789
    PLU: plu3987
    ENT: Ent638—3307
    SPE: Spro—2201
    VPA: VPA0278
    VFI: VF0403
    PPR: PBPRA0469(mvaD)
    PEN: PSEEN4850
    CBU: CBU—0607(mvaD)
    CBD: COXBU7E912—0619(mvaD)
    LPN: lpg2051
    LPF: lpl2029
    LPP: lpp2034
    TCX: Tcr—1718
    HHA: Hhal—1623
    DNO: DNO—0798
    EBA: ebA5678 p2A143
    DVU: DVU1679(idi)
    DDE: Dde—1991
    LIP: LI1134
    BBA: Bd1626
    AFW: Anae109—4082
    MXA: MXAN—5021(fni)
    RPR: RP452
    RTY: RT0439(idi)
    RCO: RC0744
    RFE: RF—0785(fni)
    RBE: RBE—0731(fni)
    RAK: A1C—04190
    RBO: A1I—04755
    RCM: A1E—02555
    RRI: A1G—04195
    MLO: mlr6371
    RET: RHE_PD00245(ypd00046)
    XAU: Xaut—4134
    SIL: SPO0131
    SIT: TM1040—3442
    RSP: RSP—0276
    RSH: Rsph17029—1919
    RSQ: Rsph17025—1019
    JAN: Jann—0168
    RDE: RD1—0147(idi)
    DSH: Dshi—3527
    BSU: BG11440(ypgA)
    BAN: BA1520
    BAR: GBAA1520
    BAA: BA—2041
    BAT: BAS1409
    BCE: BC1499
    BCA: BCE—1626
    BCZ: BCZK1380(fni)
    BCY: Bcer98—1222
    BTK: BT9727—1381(fni)
    BTL: BALH—1354
    BLI: BL02217(fni)
    BLD: BLi02426
    BAY: RBAM—021020(fni)
    BPU: BPUM—2020(fni)
    OIH: OB0537
    SAU: SA2136(fni)
    SAV: SAV2346(fni)
    SAM: MW2267(fni)
    SAR: SAR2431(fni)
    SAS: SAS2237
    SAC: SACOL2341(fni)
    SAB: SAB2225c(fni)
    SAA: SAUSA300-2292(fni)
    SAO: SAOUHSC—02623
    SEP: SE1925
    SER: SERP1937(fni-2)
    SHA: SH0712(fni)
    SSP: SSP0556
    LMO: lmo1383
    LMF: LMOf2365—1402(fni)
    LIN: lin1420
    LWE: lwe1399(fni)
    LLA: L11083(yebB)
    LLC: LACR—0457
    LLM: llmg—0428(fni)
    SPY: SPy—0879
    SPZ: M5005_Spy—0685
    SPM: spyM18—0940
    SPG: SpyM3—0598
    SPS: SPs1255
    SPH: MGAS10270_Spy0743
    SPI: MGAS10750_Spy0777
    SPJ: MGAS2096_Spy0756
    SPK: MGAS9429_Spy0740
    SPF: SpyM51123(fni)
    SPA: M6_Spy0702
    SPB: M28_Spy0665
    SPN: SP—0384
    SPR: spr0341(fni)
    SPD: SPD—0349(fni)
    SAG: SAG1323
    SAN: gbs1393
    SAK: SAK—1354(fni)
    SMU: SMU.939
    STC: str0562(idi)
    STL: stu0562(idi)
    STE: STER—0601
    SSA: SSA—0336
    SGO: SGO—0242
    LPL: lp—1732(idi1)
    LJO: LJ1208
    LAC: LBA1171
    LSA: LSA0905(idi)
    LSL: LSL—0682
    LDB: Ldb0996(fni)
    LBU: LBUL—0903
    LBR: LVIS—0861
    LCA: LSEI—1493
    LGA: LGAS—1036
    LRE: Lreu—0912
    EFA: EF0901
    OOE: OEOE—1103
    STH: STH1674
    CBE: Cbei—3081
    DRM: Dred—0474
    SWO: Swol—1341
    MTA: Moth—1328
    MTU: Rv1745c(idi)
    MTC: MT1787(idi)
    MBO: Mb1774c(idi)
    MBB: BCG—1784c(idi)
    MPA: MAP3079c
    MAV: MAV—3894(fni)
    MSM: MSMEG—1057(fni) MSMEG—2337(fni)
    MUL: MUL—0380(idi2)
    MVA: Mvan—1582 Mvan—2176
    MGI: Mflv—1842 Mflv—4187
    mMC: Mmcs—1954
    MKM: Mkms—2000
    MJL: Mjls—1934
    CGL: NCg12223(cg12305)
    CGB: cg2531(idi)
    CEF: CE2207
    CDI: DIP1730(idi)
    NFA: nfa19790 nfa22100
    RHA: RHA1_ro00239
    SCO: SCO6750(SC5F2A.33c)
    SMA: SAV1663(idi)
    LXX: Lxx23810(idi)
    CMI: CMM—2889(idiA)
    AAU: AAur—0321(idi)
    PAC: PPA2115
    FRA: Francci3—4188
    FRE: Franean1—5570
    FAL: FRAAL6504(idi)
    KRA: Krad—3991
    SEN: SACE 2627(idiB—2) SACE—5210(idi)
    STP: Strop—4438
    SAQ: Sare—4564 Sare—4928
    RXY: Rxyl—0400
    BBU: BB0684
    BGA: BG0707
    SYN: sll1556
    SYC: syc2161_c
    SYF: Synpcc7942—1933
    CYA: CYA—2395(fni)
    CYB: CYB—2691(fni)
    TEL: tll1403
    ANA: all4591
    AVA: Ava—2461 Ava_B0346
    TER: Tery—1589
    SRU: SRU—1900(idi)
    CHU: CHU—0674(idi)
    GFO: GFO—2363(idi)
    FJO: Fjoh—0269
    FPS: FP1792(idi)
    CTE: CT0257
    CCH: Cag—1445
    CPH: Cpha266—0385
    PVI: Cvib—1545
    PLT: Plut—1764
    RRS: RoseRS—2437
    RCA: Rcas—2215
    HAU: Haur—4687
    DRA: DR—1087
    DGE: Dgeo—1381
    TTH: TT_P0067
    TTJ: TTHB110
    MJA: MJ0862
    MMP: MMP0043
    MMQ: MmarC5—1637
    MMX: MmarC6—0906
    MMZ: MmarC7—1040
    MAE: Maeo—1184
    MVN: Mevan—1058
    MAC: MA0604(idi)
    MBA: Mbar_A1419
    MMA: MM—1764
    MBU: Mbur—2397
    MTP: Mthe—0474
    MHU: Mhun—2888
    MLA: Mlab—1665
    MEM: Memar 1814
    MBN: Mboo—2211
    MTH: MTH48
    MST: Msp—0856(fni)
    MSI: Msm—1441
    MKA: MK0776(lldD)
    AFU: AF2287
    HAL: VNG1818G(idi) VNG6081G(crt—1) VNG6445G(crt—2) VNG7060 VNG7149
    HMA: rrnAC3484(idi)
    HWA: HQ2772A(idiA) HQ2847A(idiB)
    NPH: NP0360A(idiB—1) NP4826A(idiA) NP5124A(idiB—2)
    TAC: Ta0102
    TVO: TVN0179
    PTO: PT00496
    PHO: PH1202
    PAB: PAB1662
    PFU: PF0856
    TKO: TK1470
    RCI: LRC397(fni)
    APE: APE—1765.1
    SMR: Smar—0822
    IHO: Igni13 0804
    HBU: Hbut—0539
    SSO: SS00063
    STO: ST2059
    SAI: Saci—0091
    MSE: Msed—2136
    PAI: PAE0801
    PIS: Pisl—1093
    PCL: Pcal—0017
    PAS: Pars—0051
    TPE: Tpen—0272

Exemplary Isoprene Synthase Nucleic Acids and Polypeptides Genbank Accession Nos. AY341431 AY316691 AY279379 AJ457070 AY182241

## Claims

1. Recombinant bacterial cells capable of increased production of isoprene, the cells comprising nucleic acids encoding:
(a) an isoprene synthase polypeptide, wherein the isoprene synthase polypeptide is encoded by a heterologous nucleic acid,
(b) an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide, and at least one of a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide and/or
one or more mevalonate (MVA) pathway polypeptides,

wherein the cells produce greater than 400 nmole/gwcm/hr of isoprene.

2. The cells of claim 1, wherein the cells produce at least 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, or 12,500 nmole/gwcm/hr of isoprene.

3. The cells of claim 1, wherein the cells produce between 400 nmole/gwcm/hr to 2.0×105 nmole/gwcm/hr of isoprene.

4. The cells of claim 1, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

5. The cells of claim 4, wherein the plant isoprene synthase polypeptide is a poplar isoprene synthase polypeptide.

6. The cells of claim 4, wherein the plant isoprene synthase polypeptide is a kudzu isoprene synthase polypeptide.

7. The cells of claim 1, wherein the nucleic acid encoding an IDI polypeptide of (b) is a heterologous nucleic acid encoding an IDI polypeptide.

8. The cells of claim 7, wherein the IDI polypeptide is a yeast IDI polypeptide.

9. The cells of claim 1, wherein the nucleic acid encoding an IDI polypeptide of (b) is a copy of an endogenous nucleic acid encoding an IDI polypeptide.

10. The cells of claim 1, wherein the nucleic acid encoding a DXS polypeptide of (c) is a heterologous nucleic acid encoding a DXS polypeptide.

11. The cells of claim 1, wherein the nucleic acid encoding a DXS polypeptide of (c) is a copy of an endogenous nucleic acid encoding a DXS polypeptide.

12. The cells of claim 1, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a heterologous nucleic acid.

13. The cells of claim 1, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a copy of an endogenous nucleic acid.

14. The cells of claim 1, wherein the cells comprise two or more polypeptides of the MVA pathway.

15. The cells of claim 1, wherein the cells comprise three or more polypeptides of the MVA pathway.

16. The cells of claim 1, wherein the cells comprise four or more polypeptides of the MVA pathway.

17. The cells of claim 1, wherein the cells comprise polypeptides of the entire MVA pathway.

18. The cells of claim 1, wherein the cells comprise polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

19. The cells of claim 1, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is over-expressed.

20. The cells of claim 19, wherein the over-expressed nucleic acid is cloned into a multicopy plasmid.

21. The cells of claim 19, wherein the over-expressed nucleic acid is placed under an inducible promoter or a constitutive promoter.

22. The cells of claim 1, wherein the cells are gram-positive bacterial cells or gram-negative bacterial cells.

23. The cells of claim 1, wherein the cells are selected from the group consisting of E. coli, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., and P. alcaligenes cells.

24. The cells of claim 1, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is in a vector.

25. The cells of claim 1, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is integrated into a chromosome of the cells.

26. The cells of claim 1, wherein the cells comprise nucleic acids encoding: a DXS polypeptide and one or more MVA pathway polypeptides.

27. The cells of claim 26, wherein the cells comprise nucleic acids encoding: a DXS polypeptide and polypeptides of the entire MVA pathway.

28. The cells of claim 1, wherein the cells comprise nucleic acids encoding: a DXS polypeptide and polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

29. Recombinant bacterial cells capable of increased production of isoprene, the cells comprising nucleic acids encoding:
(a) an isoprene synthase polypeptide, wherein the isoprene synthase polypeptide is encoded by a heterologous nucleic acid,
(b) an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide, and at least one of a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide and/or
one or more mevalonate (MVA) pathway polypeptides,

wherein the cells have an average volumetric productivity of isoprene greater than 0.1 mg/Lbroth/hr.

30. The cells of claim 29, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

31. The cells of claim 30, wherein the plant isoprene synthase polypeptide is a poplar isoprene synthase polypeptide.

32. The cells of claim 30, wherein the plant isoprene synthase polypeptide is a kudzu isoprene synthase polypeptide.

33. The cells of claim 29, wherein the nucleic acid encoding an IDI polypeptide of (b) is a heterologous nucleic acid encoding an IDI polypeptide.

34. The cells of claim 33, wherein the IDI polypeptide is a yeast IDI polypeptide.

35. The cells of claim 29, wherein the nucleic acid encoding an IDI polypeptide of (b) is a copy of an endogenous nucleic acid encoding an IDI polypeptide.

36. The cells of claim 29, wherein the nucleic acid encoding the DXS polypeptide is a heterologous nucleic acid encoding the DXS polypeptide.

37. The cells of claim 29, wherein the nucleic acid encoding the DXS polypeptide is a copy of an endogenous nucleic acid encoding the DXS polypeptide.

38. The cells of claim 29, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a heterologous nucleic acid.

39. The cells of claim 29, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a copy of an endogenous nucleic acid.

40. The cells of claim 29, wherein the cells comprise two or more polypeptides of the MVA pathway.

41. The cells of claim 29, wherein the cells comprise three or more polypeptides of the MVA pathway.

42. The cells of claim 29, wherein nucleic acids encode the cells comprise four or more polypeptides of the MVA pathway.

43. The cells of claim 29, wherein the cells comprise polypeptides of the entire MVA pathway.

44. The cells of claim 29, wherein the cells comprise polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

45. The cells of claim 29, wherein at least one of the nucleic acids encoding a polypeptide of any of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is over-expressed.

46. The cells of claim 45, wherein the over-expressed nucleic acid is cloned into a multicopy plasmid.

47. The cells of claim 45, wherein the overexpressed nucleic acid is placed under an inducible promoter or a constitutive promoter.

48. The cells of claim 29, wherein the cells are gram-positive bacterial cells or gram-negative bacterial cells.

49. The cells of claim 29, wherein the cells are selected from the group consisting of E. coli, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., and P. alcaligenes cells.

50. The cells of claim 29, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is in a vector.

51. The cells of claim 29, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is integrated into a chromosome of the cells.

52. The cells of claim 29, wherein the cells comprise nucleic acids encoding: a DXS polypeptide and one or more MVA pathway polypeptides.

53. The cells of claim 52, wherein the cells comprise nucleic acids encoding: a DXS polypeptide and (ii) polypeptides of the entire MVA pathway.

54. The cells of claim 29, wherein the cells comprise nucleic acids encoding: a DXS polypeptide and (ii) polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

55. Recombinant bacterial cells capable of increased production of isoprene, the cells comprising nucleic acids encoding:
(a) an isoprene synthase polypeptide, wherein the isoprene synthase polypeptide is encoded by a heterologous nucleic acid,
(b) an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide, and at least one of a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide and/or
one or more mevalonate (MVA) pathway polypeptides,

wherein the cells produce at least 0.0037 grams of isoprene per gram of dry cell mass (gdcm).

56. The cells of claim 55, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

57. The cells of claim 56, wherein the plant isoprene synthase polypeptide is a poplar isoprene synthase polypeptide.

58. The cells of claim 56, wherein the plant isoprene synthase polypeptide is a kudzu isoprene synthase polypeptide.

59. The cells of claim 55, wherein the nucleic acid encoding an IDI polypeptide of (b) is a heterologous nucleic acid encoding an IDI polypeptide.

60. The cells of claim 59, wherein the IDI polypeptide is a yeast IDI polypeptide.

61. The cells of claim 55, wherein the nucleic acid encoding an IDI polypeptide of (b) is a copy of an endogenous nucleic acid encoding an IDI polypeptide.

62. The cells of claim 55, wherein the nucleic acid encoding the DXS polypeptide is a heterologous nucleic acid encoding the DXS polypeptide.

63. The cells of claim 55, wherein the nucleic acid encoding the DXS polypeptide is a copy of an endogenous nucleic acid encoding the DXS polypeptide.

64. The cells of claim 55, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a heterologous nucleic acid.

65. The cells of claim 55, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a copy of an endogenous nucleic acid.

66. The cells of claim 55, wherein the cells comprise two or more polypeptides of the MVA pathway.

67. The cells of claim 55, wherein the cells comprise three or more polypeptides of the MVA pathway.

68. The cells of claim 55, wherein the cells comprise four or more polypeptides of the MVA pathway.

69. The cells of claim 55, wherein the cells comprise polypeptides of the entire MVA pathway.

70. The cells of claim 55, wherein the cells comprise polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

71. The cells of claim 55, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is over-expressed.

72. The cells of claim 71, wherein the over-expressed nucleic acid is cloned into a multicopy plasmid.

73. The cells of claim 71, wherein the overexpressed nucleic acid is placed under an inducible promoter or a constitutive promoter.

74. The cells of claim 55, wherein the cells are gram-positive bacterial cells or gram-negative bacterial cells.

75. The cells of claim 55, wherein the cells are selected from the group consisting of E. coli, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., and P. alcaligenes cells.

76. The cells of claim 55, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is in a vector.

77. The cells of claim 55, wherein at least one of the nucleic acids encoding a polypeptide of (a), (b), DXS polypeptide or MVA pathway polypeptide(s) is integrated into a chromosome of the cells.

78. The cells of claim 55, wherein the cells comprise nucleic acids encoding: a DXS polypeptide and one or more MVA pathway polypeptides.

79. The cells of claim 78, wherein the cells comprise nucleic acids encoding: a DXS polypeptide and polypeptides of the entire MVA pathway.

80. The cells of claim 55, wherein the cells comprise nucleic acids encoding: a DXS polypeptide and polypeptides of the lower MVA pathway, wherein the lower MVA pathway comprises mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonate decarboxylase (MVD).

81. The cells of claim 1 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one or more vectors.

82. The cells of claim 1 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one vector.

83. The cells of claim 29 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one or more vectors.

84. The cells of claim 29 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one vector.

85. The cells of claim 55 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one or more vectors.

86. The cells of claim 55 wherein the nucleic acids encoding the polypeptides of (a), (b), and DXS polypeptide and/or MVA pathway polypeptide(s) is in one vector.