WO2011031897A1 - Microorganisms and methods for the co-production of isopropanol with primary alcohols, diols and acids - Google Patents
Microorganisms and methods for the co-production of isopropanol with primary alcohols, diols and acids Download PDFInfo
- Publication number
- WO2011031897A1 WO2011031897A1 PCT/US2010/048318 US2010048318W WO2011031897A1 WO 2011031897 A1 WO2011031897 A1 WO 2011031897A1 US 2010048318 W US2010048318 W US 2010048318W WO 2011031897 A1 WO2011031897 A1 WO 2011031897A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- coa
- pathway
- isopropanol
- exogenous nucleic
- nucleic acids
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
Definitions
- the present invention relates generally to biosynthetic processes, and more specifically to organisms having n-propanol and isopropanol, 1,4-butanediol and isopropanol, 1,3-butanediol and isopropanol or methylacrylic and isopropanol biosynthetic capability.
- Isopropanol is a colorless, flammable liquid that mixes completely with most solvents, including water.
- the largest use for IPA is as a solvent, including its well known yet small use as "rubbing alcohol,” which is a mixture of IPA and water.
- rubbing alcohol is a mixture of IPA and water.
- IPA is found in many everyday products such as paints, lacquers, thinners, inks, adhesives, general-purpose cleaners, disinfectants, cosmetics, toiletries, de-icers, and pharmaceuticals.
- Low-grade IPA is also used in motor oils.
- the second largest use is as a chemical intermediate for the production of isopropylamines, isopropylethers, and isopropyl esters. Isopropanol can potentially be dehydrated to form propylene, a polymer precursor with an annual market of more than 2 million metric tons.
- IPA isopropanol
- Isopropanol is manufactured by two petrochemical routes. The predominant process entails the hydration of propylene either with or without sulfuric acid catalysis. Secondarily, IPA is produced via hydrogenation of acetone, which is a by-product formed in the production of phenol and propylene oxide. High-priced propylene is currently driving costs up and margins down throughout the chemical industry motivating the need for an expanded range of low cost feedstocks.
- n-Propanol can be potentially used as a gasoline substitute. It is currently used as a multi- purpose solvent in the pharmaceutical industry, for surface coatings and in ink formulations. It is used as a building block for resins and esters, propyl amines and halides. It is also used for packaging and food contact applications. Global production of n-propanol in 2005 was more than 140,000 metric tonnes. n-Propanol is manufactured by the catalytic hydrogenation of propionaldehyde.
- Propionaldehyde is itself produced via the oxo process, by hydroformylation of ethylene using carbon monoxide and hydrogen in the presence of a catalyst such as cobalt octacarbonyl or a rhodium complex. It is formed naturally in small amounts in many fermentation processes. For example, microbial production of very small quantities of n-propanol has been detected from certain species of Clostridium via threonine catabolism and from yeast in beer fermentation. No existing microorganism has been reported to produce 1-propanol from sugars in significant amounts.
- 1,4-Butanediol 14-BDO is a polymer intermediate and industrial solvent with a global market of about 3 billion lb/year.
- BDO is currently produced from petrochemical precursors, primarily acetylene, maleic anhydride, and propylene oxide.
- acetylene is reacted with 2 molecules of formaldehyde in the Reppe synthesis reaction (Kroschwitz and Grant,
- 1,4-butanediol Downstream, 14-BDO can be further transformed; for example, by oxidation to gamma-butyrolactone, which can be further converted to pyrrolidone and N-methyl-pyrrolidone, or hydrogenolysis to tetrahydrofuran.
- gamma-butyrolactone which can be further converted to pyrrolidone and N-methyl-pyrrolidone, or hydrogenolysis to tetrahydrofuran.
- 1,3-Butanediol 13-BDO is a four carbon diol commonly used as an organic solvent for food flavoring agents. It is also used as a co-monomer for polyurethane and polyester resins and is widely employed as a hypoglycaemic agent.
- Optically active 13-BDO is a useful starting material for the synthesis of biologically active compounds and liquid crystals.
- a substantial commercial use of 1,3-butanediol is subsequent dehydration to afford 1 ,3 -butadiene (Ichikawa, / Mol. Catalysis. 256 : 106- 112 (2006)) , a 25 billion lb/yr petrochemical used to manufacture synthetic rubbers (e.g., tires), latex, and resins.
- 13-BDO is traditionally produced from acetylene via its hydration. The resulting acetaldehyde is then converted to 3-hydroxybutyraldehdye which is subsequently reduced to form 1,3-BDO.
- Methylacrylic acid is a key precursor of methyl methacrylate (MMA), a chemical intermediate with a global demand in excess of 4.5 billion pounds per year, much of which is converted to polyacrylates.
- MMA methyl methacrylate
- the conventional process for synthesizing methyl methacrylate involves the conversion of hydrogen cyanide (HCN) and acetone to acetone cyanohydrin which then undergoes acid assisted hydrolysis and esterification with methanol to give MAA.
- MAA can easily be converted into MAA via esterification with methanol. No existing microorganism has been reported to produce MAA from sugars in significant amounts. Microbial organisms and methods for effectively co-producing commercial quantities of n- propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol are described herein and include related advantages.
- the invention provides non-naturally occurring microbial organisms having an n-propanol pathway and an isopropanol pathway.
- the embodiments disclosed herein relate to a non-naturally occurring microbial organism that includes a microbial organism having an n- propanol and an isopropanol pathway, where the n-propanol pathway includes at least one exogenous nucleic acid encoding an n-propanol pathway enzyme expressed in a sufficient amount to produce n-propanol and where the isopropanol pathway includes at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol.
- the n-propanol pathway includes a
- the invention provides a non-naturally occurring microbial organism that includes a microbial organism having an n-propanol and an isopropanol pathway, where the n-propanol pathway includes a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol and where the isopropanol pathway includes a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol.
- the first set encodes n-propanol pathway enzymes including a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate reductase and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate kinase, a propionyl phosphate reductase and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate reductase and a propanol dehydrogenase.
- the second set encodes isopropanol pathway enzymes including an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase or an acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an isopropanol dehydrogenase.
- isopropanol pathway enzymes including an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase or an acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an isopropanol dehydrogenase.
- the invention provides a non-naturally occurring microbial organism having a first set of exogenous nucleic acids encoding n-propanol pathway enzymes and a second set of exogenous nucleic acids encoding isopropanol pathway enzymes, where the first set encodes a PEP carboxykinase or a PEP carboxylase; a malate dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA mutase; a methylmalonyl-CoA decarboxylase; and a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a propionyl-CoA:phosphate propanoyltransferase and a propionyl phosphate reductase;
- the invention provides a non-naturally occurring microbial organism having a first set of exogenous nucleic acids encoding n-propanol pathway enzymes and a second set of exogenous nucleic acids encoding isopropanol pathway enzymes, where the first set encodes a PEP carboxykinase or a PEP carboxylase; a threonine deaminase; and a 2-oxobutanoate decarboxylase and a propanol dehydrogenase; or a 2-oxobutanoate dehydrogenase, a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a 2-oxobutanoate dehydrogenase, a propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate reductase and a propanol dehydrogenase; or
- dehydrogenase a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate reductase and a propanol dehydrogenase
- the second set encodes a pyruvate kinase; a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a pyruvate formate lyase activating enzyme and a formate
- dehydrogenase an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase or an acetoacetyl- CoA hydrolase or an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an isopropanol dehydrogenase.
- the invention provides a non-naturally occurring microbial organism having a first set of exogenous nucleic acids encoding n-propanol pathway enzymes and a second set of exogenous nucleic acids encoding isopropanol pathway enzymes, where the first set encodes a pyruvate kinase; a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a pyruvate formate lyase activating enzyme and a formate
- dehydrogenase an acetyl-CoA carboxylase; a malonyl-CoA reductase; a malonate semialdehyde reductase; propionyl-CoA synthase; and a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate reductase and propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate kinase, a propionyl phosphate reductase and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a pro
- the invention provides a non-naturally occurring microbial organism having a first set of exogenous nucleic acids encoding n-propanol pathway enzymes and a second set of exogenous nucleic acids encoding isopropanol pathway enzymes, where the first set encodes a lactate dehydrogenase; a lactate-CoA transferase; a lactyl-CoA dehydratase; acryloyl CoA reductase; and a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a propionyl- CoA:phosphate propanoyltransferase, a propionyl phosphate reductase and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl- CoA synthetase, a propionat
- the invention provides a non-naturally occurring microbial organism having an n-propanol pathway, the n-propanol pathway including at least one exogenous nucleic acid encoding an n-propanol pathway enzyme expressed in a sufficient amount to produce n- propanol.
- the n-propanol pathway includes a propionaldehyde dehydrogenase, a propanol dehydrogenase, a propionyl-CoA:phosphate propanoyltransferase, a propionyl-CoA hydrolase, a propionyl-CoA transferase, a propionyl-CoA synthetase, a propionate kinase, a propionate reductase or a propionyl phosphate reductase.
- the invention provides a non-naturally occurring microbial organism having an n-propanol pathway, the n-propanol pathway including a set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n- propanol, the set of exogenous nucleic acids encoding a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate reductase and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate kinase, a propionyl phosphate reductase and a propanol dehydrogenase; or
- embodiments disclosed herein relate to a method for producing n-propanol and isopropanol that includes culturing the aformentioned non-naturally occurring microbial organisms. In still other aspect, embodiments disclosed herein relate to a method for producing n-propanol that includes culturing the aforementioned non-naturally occurring micribial organisms.
- the invention provides non-naturally occurring microbial organisms having an isopropanol pathway and a 1,4-butanediol (14-BDO) pathway, a 1,3-butanediol (13-BDO) pathway or a methylacrylic acid (MAA) pathway.
- the embodiments disclosed herein relate to a non-naturally occurring microbial organism that includes a microbial organism having a 1,4-butanediol and an isopropanol pathway, where the 1,4-butanediol pathway includes at least one exogenous nucleic acid encoding a 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce 1,4-butanediol and where the isopropanol pathway includes at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol.
- the embodiments disclosed herein relate to a non-naturally occurring microbial organism that includes a microbial organism having a 1,3- butanediol and an isopropanol pathway, where the 1,3-butanediol pathway includes at least one exogenous nucleic acid encoding a 1,3-butanediol pathway enzyme expressed in a sufficient amount to produce 1,3-butanediol and where the isopropanol pathway includes at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol.
- the embodiments disclosed herein relate to a non-naturally occurring microbial organism that includes a microbial organism having a methylacrylic acid and an isopropanol pathway, where the methylacrylic acid pathway includes at least one exogenous nucleic acid encoding a methylacrylic acid pathway enzyme expressed in a sufficient amount to produce methylacrylic acid and where the isopropanol pathway includes at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol.
- the isopropanol pathway comprises an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an isopropanol dehydrogenase.
- the 14-BDO pathway comprises a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4- hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming), a 4- hydroxybutyraldehyde reductase, a 4-hydroxybutyrate reductase; a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-phosphate reductase, or a 4- hydroxybutyryl-CoA reductase (alcohol-forming).
- the 13-BDO pathway comprises a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4- hydroxybutyryl-CoA synthetase, a 4-hydroxybutyrate kinase, a phosphotrans-4- hydroxybutyrylase, a 4-hydroxybutyryl-CoA dehydratase, a crotonase, a 3-hydroxybutyryl-CoA reductase (aldehyde forming), a 3 -hydroxybutyraldehyde reductase, a 3-hydroxybutyryl-CoA reductase (alcohol-forming), a 3-hydroxybutyryl-CoA transferase, a 3-hydroxybutyryl-CoA synthetase, a 3-hydroxybutyryl-CoA hydrolase, or a 3-hydroxybutyrate reduc
- the MAA pathway comprises a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4- hydroxybutyryl-CoA synthetase, a 4-hydroxybutyrate kinase, a phosphotrans-4- hydroxybutyrylase, a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase, a methacrylyl-CoA hydrolase, a 3- hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA synthetase, a 3- hydroxyisobutyryl-CoA hydrolase, a 3-hydroxyisobutyrate dehydratase, a 3-
- the invention provides a non-naturally occurring microbial organism that includes a microbial organism having an 14-BDO and an isopropanol pathway, where the 14- BDO pathway includes a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO and where the isopropanol pathway includes a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol.
- the invention provides a non-naturally occurring microbial organism that includes a microbial organism having an 13-BDO and an isopropanol pathway, where the 13- BDO pathway includes a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO and where the isopropanol pathway includes a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol.
- the invention provides a non-naturally occurring microbial organism that includes a microbial organism having an methylacrylic acid and an isopropanol pathway, where the methylacrylic acid pathway includes a first set of exogenous nucleic acids encoding methylacrylic acid pathway enzymes expressed in a sufficient amount to produce methylacrylic acid and where the isopropanol pathway includes a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol.
- methylacrylic acid pathways passing through a 3-hydroxyisobutyrate intermediate can be applied for 3-hydroxyisobutyrate production as opposed to methylacrylic acid production if the downstream enzyme, that is, a dehydratase, is omitted (see Figures 7 and 8).
- the non-naturally occurring organism would produce 3-hydroxyisobutyrate instead of methylacrylic acid.
- the non-naturally occumng organism could alternatively produce a mixture of 3-hydroxyisobutyate and methylacrylic acid.
- the maximum molar yields of ATP and product will be unchanged regardless of whether methylacrylic acid or 3-hydroxyisobutyrate is produced.
- 3 -hydroxyisobutyric acid expressed by a microbial organism of the invention can be chemically converted to methylacrylic acid.
- 3- hydroxyisobutyric acid, or ⁇ -hydroxyisobutyric acid can be dehydrated to form methylacrylic acid as decribed, for example, in U.S. Patent No. 7,186,856.
- embodiments disclosed herein relate to a method for producing 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol that includes culturing the aformentioned non-naturally occurring microbial organisms.
- Figure 1 shows an exemplary pathway for co-production of n-propanol and isopropanol from glucose.
- Figure 2 shows an exemplary pathway for co-production of n-propanol and isopropanol from glucose.
- Glc - glucose PEP - phosphoenolpyruvate, PYR - pyruvate, FOR - formate, ACCOA - acetyl-CoA, AACOA - acetoacetyl-CoA, ACAC- acetoacetate, AC - acetone, PPOH-2 - isopropanol, OAA - oxaloacetate, THR - threonine, 2-OBUT - 2- oxobutanoate, PPCOA - propionyl-CoA, PPA - propionate, PPAL- propionaldehyde, PPPi - propionyl phosphate, PPOH-1 - n-propanol.
- Figure 3 shows an exemplary pathway for co-production of n-propanol and isopropanol from glucose.
- Glc - glucose PEP - phosphoenolpyruvate, PYR - pyruvate, FOR - formate, ACCOA - acetyl-CoA, AACOA - acetoacetyl-CoA, ACAC- acetoacetate, AC - acetone, PPOH-2 - isopropanol, MALCOA - malonyl-CoA, MALAL- malonate semialdehyde, 3HP- 3-hydroxypropionate, PPCOA - propionyl-CoA, PPA - propionate, PPAL- propionaldehyde, PPPi - propionyl phosphate, PPOH-1 - n-propanol.
- Figure 4 shows an exemplary pathway for co-production of n-propanol and isopropanol from glucose.
- Glc - glucose PEP - phosphoenolpyruvate, PYR - pyruvate, FOR - formate, ACCOA - acetyl-CoA, AACOA - acetoacetyl-CoA, ACAC- acetoacetate, AC - acetone, PPOH-2 - isopropanol, LAC- D-lactate, LACCOA- lactoyl-CoA, ACRYLCOA- acryloyl-CoA, PPCOA - propionyl-CoA, PPA - propionate, PPAL - propionaldehyde, PPPi - propionyl phosphate, PPOH-1 - n-propanol.
- Figure 5 shows an exemplary pathway for coproduction of 1,4-BDO and isopropanol from glucose.
- Figure 6 shows an exemplary pathway for coproduction of 1,3-BDO and isopropanol from glucose.
- Figure 7 shows an exemplary pathway for coproduction of methyacrylic acid and isopropanol from glucose.
- Figure 8 shows an exemplary pathway for coproduction of methyacrylic acid and isopropanol from glucose.
- Embodiments of the present invention provide non-naturally occurring microbial organisms having redox-balanced anaerobic pathways for co-production of n-propanol and isopropanol from 3 phosphoenolpyruvate (PEP) molecules as exemplified in Figures 1-4.
- Some advantages of this co-production strategy include: (1) the co-production affords the maximum theoretical yield of n-propanol and isopropanol at 1.33 moles total/mole of glucose; and (2) the pathway for co-production is completely redox balanced and has a net positive yield of ATP. This facilitates a completely anaerobic production of the C3 alcohols as opposed to culturing microbial organisms having the isopropanol pathway alone, which requires aeration for regeneration of NAD.
- Embodiments of the present invention also provide non-naturally occurring microbial organisms that can co-produce n-propanol and isopropanol from renewable resources as shown in Figures 1-4.
- the organisms include all enzymes utilized in the co-production of n-propanol and isopropanol from acetyl-CoA and propionyl-CoA.
- Formate can be converted to carbon dioxide by a formate dehydrogenase that provides an additional reducing equivalent that can be used for n-propanol and isopropanol syntheses.
- reducing equivalents can be obtained from other steps in the pathway, such as, the glycolysis pathway during conversion of glucose to phospheonolpyruvate, pyruvate dehydrogenase or pyruvate ferredoxin oxidoreductase during conversion of pyruvate to acetyl-CoA, or 2-oxobutanoate dehydrogenase during conversion of 2-oxobutanoate to propionyl-CoA.
- Embodiments of the present invention also provide non-naturally occurring microbial organisms that can produce n-propanol via propionyl-CoA. This conversion is carried out by two different enzymes: an aldehyde and alcohol dehydrogenase or in one step by a bifunctional
- propionyl-CoA can be converted into propionyl phosphate and then transformed into propionaldehyde by an acyl phosphate reductase.
- propionyl-CoA can be converted to propionate then to propionyl phosphate by a propionyl-CoA hydrolase, transferase, or synthetase and a propionate dinase, respectively.
- propionate can be converted to propionaldehyde by a propionate reductase.
- Pathways for production of propionyl-CoA are exemplified in Figures 1-4.
- the pathway for production of propionyl-CoA proceeds via oxaloacetate as exemplified in
- Oxaloacetate is converted to propionyl-CoA by means of the reductive TCA cycle, a methylmutase, a decarboxylase, and a decarboxylase.
- An epimerase may be required to convert the (R) stereoisomer of methylmalonyl-CoA to the (S) configuration.
- the pathway for production of propionyl-CoA proceeds via threonine as exemplified in Figure 2.
- Oxaloacetate is converted into threonine by the native threonine pathway engineered for high yields. It is then deaminated to form 2-oxobutanoate and subsequently converted into propionyl- CoA.
- 2-oxobutanoate is converted to propionaldehyde by a decarboxylase, which is then reduced to n-propanol by a propanol dehydrogenase.
- the pathway for production of propionyl-CoA proceeds via malonyl-CoA as exemplified in Figure 3.
- Acetyl-CoA is carboxylated to form malonyl-CoA. This is then reduced to malonate semialdehyde, and subsequently transformed into 3-hydroxypropionate (3HP).
- 3HP is converted into propionyl-CoA via propionyl-CoA synthase.
- the pathway for production of propionyl-CoA proceeds via lactate as exemplified in Figure 4. Pyruvate is reduced to form lactate which is then activated to form lactoyl-CoA. The lactoyl-CoA is dehydrated to form acryloyl-CoA and then reduced to generate propionyl-CoA.
- Embodiments of the present invention also provide non-naturally occurring microbial organisms that can produce isopropanol via acetyl-CoA.
- Isopropanol production is achieved via conversion of acetyl-CoA by an acetoacetyl-CoA thiolase, an acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase,, an acetoacetate decarboxylase, and an isopropanol dehydrogenase as exemplified in Figures 1-4.
- the pathway for production of acetyl-CoA from glucose proceeds via phosphoenolpyruvate (PEP).
- PEP phosphoenolpyruvate
- Glucose is converted into PEP by the native glycolysis pathway of the microbial organism.
- PEP is converted to pyruvate by pyruvate kinase and then to acetyl-CoA by pyruvate dehydrogenase or pyruvate ferredoxin oxidoreductase.
- pyruvate is converted to acetyl-CoA and formate by pyruvate formate lyase. The formate is then converted to carbon dioxide and hydrogen by a formate dehydrogenase.
- Embodiments of the present invention provide alternate methods for coproduction of isopropanol with the compounds 14-BDO, 13-BDO and MAA.
- the production of isopropanol proceeds via acetyl-CoA as described above. Alone this route is not redox-balanced and thus requires aeration to achieve high isopropanol yields.
- Embodiments described herein use this route and combine it with pathways for synthesizing the coproducts 1,4-butanediol (14-BDO), 1,3-butanediol (13-BDO) and methylacrylic acid (MAA).
- Coproduction routes are redox- balanced under anaerobic conditions as opposed to the requirement of oxygen if isopropanol is produced solely through acetone. Coproduction also provides related advantages, such as, the ease of separating isopropanol from other fermentation products due it its low boiling point (82°C) relative to 14-BDO (230°C), 13-BDO (203°C) and MAA (163°C) and the coproduction using any of the microbial organisms described herein provides that maximum theoretical yield of the carbon from glucose is afforded.
- Embodiments of the present invention provide non-naturally occurring microbial organisms that can produce 14-BDO via succinyl-CoA or in some aspects via succinate.
- succinyl-CoA is converted to succinic semialdehyde by a succinyl-CoA reductase.
- succinate can be converted to succinic semialdehyde by a succinate reductase.
- succinic semialdehyde is reduced to 4-hydroxybutyrate by 4-hydroxybutyrate
- dehydrogenase Activation of 4-HB to its acyl-CoA is catalyzed by a CoA transferase or synthetase.
- 4-HB can be converted into 4-hydroxybutyryl-phosphate and subsequently transformed into 4-HB-CoA by a phosphotrans-4-hydroxybutyrylase.
- 4-HB-CoA is then converted to 14-BDO by either a bifunctional CoA-dependent aldehyde/alcohol dehydrogenase, or by two separate enzymes with aldehyde and alcohol dehydrogenase activity.
- Yet another alternative that bypasses the 4-HB -CoA intermediate is direct reduction of 4-HB to 4-hydroxybutyrylaldehyde by a carboxylic acid reductase.
- 4-Hydroxybutyrylaldehyde is subsequently reduced to 14-BDO by an alcohol dehydrogenase.
- Yet another route that bypasses 4-HB-CoA entails reducing 4-hydroxybutyryl-phosphate to 4-hydroxybutyraldehyde by a phosphate reductase.
- Embodiments of the present invention provide non-naturally occurring microbial organisms that can produce 13-BDO via succinyl-CoA or in some aspects via succinate. Production of 13-BDO also proceeds through 4-hydroxybutyryl-CoA, formed as described above. In this route, 4- hydroxybutyryl-CoA is dehydrated and isomerized to form crotonyl-CoA. The dehydration and vinylisomerisation reactions are catalyzed by a bifunctional enzyme, 4-hydroxybutyryl-CoA dehydratase. Crotonyl-CoA is then hydrated to 3-hydroxybutyryl-CoA. Removal of the CoA moiety and concurrent reduction yields 3-hydroxybutyraldehyde.
- 3- hydroxybutyryl-CoA is converted to 3-hydroxybutyrate by a 3-hydroxybutyryl-CoA transferase, hydrolase, or synthetase and then reduced by a 3-hydroxybutyrate reductase to yield 3- hydroxybutyraldehyde. Finally reduction of the aldehyde by 3-hydroxybutyraldehyde reductase yields 13-BDO.
- Embodiments of the present invention provide non-naturally occurring microbial organisms that can produce MAA via two alternative routes.
- the first route proceeds through 4- hydroxybutyryl-CoA, formed as described above.
- 4-Hydroxybutyryl-CoA is converted to 3- hydroxyisobutyryl-CoA by a methyl mutase.
- the CoA moiety of 3-Hydroxyisobutyryl-CoA is then removed by a CoA transferase, hydrolase or synthetase. Finally, dehydration of the 3- hydroxy group yields MAA.
- 3-hydroxyisobutyryl-CoA is converted to methyacrylyl-CoA by a 3-hydroxyisobutyryl-CoA dehydratase and then the CoA moiety is removed by a CoA transferase, hydrolase or synthetase to yield MAA.
- succinyl-CoA is converted to methylmalonyl-CoA by methylmalonyl-CoA mutase.
- An epimerase may be required to convert the (R) stereoisomer of methylmalonyl-CoA to the (S) configuration.
- a CoA-dependent aldehyde dehydrogenase then converts
- methylmalonyl-CoA to methylmalonate semialdehyde.
- the CoA moiety of (R)- methylmalonyl-CoA or (S)-methylmalonyl-CoA is removed by a CoA transferase, hydrolase or synthetase to form methylmalonate, which is then converted to the semialdehyde by a reductase. Reduction of the aldehyde to 3-hydroxyisobutyrate, followed by dehydration, yields MAA.
- methylmalonyl-CoA is converted to 3-hydroxyisobutyrate by an alcohol-forming CoA reductase.
- Embodiments of the present invention provide non-naturally occurring microbial organisms having pathways for production of succinyl-CoA as exemplified in Figures 5-8.
- the pathway for production of succinyl-CoA proceeds via oxaloacetate.
- Oxaloacetate is converted to succinyl-CoA by means of the reductive TCA cycle, including a malate dehydrogenase, a fumerase, a fumarate reducatase and a succinyl-CoA transferase or alternatively a succinyl-CoA synthetase.
- Engineering these pathways into a microorganism involves cloning an appropriate set of genes encoding a set of enzymes into a production host described herein, optimizing fermentation conditions, and assaying product formation following fermentation.
- a production host for the production of n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol one or more exogenous DNA sequence(s) can be expressed in a microorganism.
- the microorganism can have endogenous gene(s) functionally disrupted, deleted or overexpressed.
- the metabolic modifications disclosed herein enable the production of n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol using renewable feedstock.
- the invention provides non-naturally occurring microbial organisms that include at least one exogenous nucleic acid that encode an n-propanol pathway enzyme expressed in a sufficient amount to produce n-propanol.
- the invention provides non-naturally occurring microbial organisms that include at least one exogenous nucleic acid that encode an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol.
- the invention provides methods for co-producing n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol. Such methods involve culturing the microbial organisms described herein.
- non-naturally occurring when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species.
- Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species.
- Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon.
- Exemplary metabolic polypeptides include enzymes or proteins within an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or MAA biosynthetic pathways.
- a metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides or, functional fragments thereof. Exemplary metabolic modifications are disclosed herein.
- isolated when used in reference to a microbial organism are intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments.
- an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments.
- Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.
- the terms "microbial,” “microbial organism” or “microorganism” is intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya.
- the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi.
- the term also includes cell cultures of any species that can be cultured for the production of a biochemical.
- n-propanol is intended to mean a primary alcohol with the molecular formula of C 3 3 ⁇ 40 and a molecular mass of 60.1 g/mol.
- N-propanol is also known in the art as 1- propanol, 1 -propyl alcohol, n-propyl alcohol, propan-l-ol, or simply propanol.
- N-propanol is an isomer of isopropanol.
- isopropanol is intended to mean a secondary alcohol, with the molecular formula of C 3 3 ⁇ 40 and a molecular mass of 60.1 g/mol, wherein the alcohol carbon is attached to two other carbons. This attachment is sometimes shown as (CH 3 ) 2 CHOH.
- isopropanol is also known in the art as propan-2-ol, 2-propanol or the abbreviation IPA.
- Isopropanol is an isomer of n-propanol.
- 1,4-butanediol is intended to mean an alcohol derivative of the alkane butane, carrying two hydroxyl groups which has the chemical formula C 4 H 10 O 2 and a molecular mass of 90.12 g/mol.
- the chemical compound 1,4-butanediol also is known in the art as 1,4- BDO and is a chemical intermediate or precursor for a family of compounds commonly referred to as the BDO family of compounds.
- 1,3-butanediol is intended to mean one of four stable isomers of butanediol having the chemical formula C4H10O2 and a molecular mass of 90.12 g/mol.
- the chemical compound 1,3-butanediol is known in the art as 13-BDO or ⁇ -butane glycol and is also a chemical intermediate or precursor for a family of compounds commonly referred to as the BDO family of compounds.
- Co A or "coenzyme A” is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system.
- Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.
- the term "substantially anaerobic" when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media.
- the term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.
- "Exogenous” as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism.
- the molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid.
- the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism.
- the term refers to an activity that is introduced into the host reference organism.
- the source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the host.
- the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism.
- heterologous refers to a molecule or activity derived from a source other than the referenced species whereas
- homologous refers to a molecule or activity derived from the host microbial organism.
- exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.
- the non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration.
- stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.
- suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway.
- E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species.
- Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.
- ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms.
- mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides.
- Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor.
- Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable.
- Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity.
- Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities.
- Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.
- Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism.
- An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species.
- a specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase.
- a second example is the separation of mycoplasma 5 '-3' exonuclease and Drosophila DNA polymerase III activity.
- the DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.
- paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions.
- Paralogs can originate or derive from, for example, the same species or from a different species.
- microsomal epoxide hydrolase epoxide hydrolase I
- soluble epoxide hydrolase epoxide hydrolase II
- Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor.
- Groups of paralogous protein families include HipA homologs, lucif erase genes, peptidases, and others.
- a nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species.
- a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein.
- Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.
- Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity.
- Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined.
- a computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art.
- Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.
- amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters:
- the invention provides a non-naturally occurring microbial organism, including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway having at least one exogenous nucleic acid encoding an n-propanol pathway enzyme expressed in a sufficient amount to produce n-propanol, the n-propanol pathway including a propionaldehyde dehydrogenase, a propanol dehydrogenase, a propionyl-
- CoA phosphate propanoyltransferase, a propionyl-CoA hydrolase, a propionyl-CoA transferase, a propionyl-CoA synthetase, a propionate kinase, a propionate reductase or a propionyl phosphate reductase
- the isopropanol pathway comprising at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol
- the isopropanol pathway including an acetyl-CoA acetyl thiolase, an acetoacetyl- CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an isopropanol dehydrogenase.
- the microbial organism has an acetyl-CoA pathway having at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, the acetyl-CoA pathway including a pyruvate kinase, a pyruvate dehydrogenase, a pyruvate ferredoxin oxidoreductase, a pyruvate formate lyase, a pyruvate formate lyase activating enzyme, or a formate dehydrogenase.
- the microbial organism has a propionyl-CoA pathway having at least one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme expressed in a sufficient amount to produce propionyl-CoA, the propionyl-CoA pathway including a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a methylmalonyl-CoA mutase, a methylmalonyl-CoA epimerase or a methylmalonyl-CoA decarboxylase.
- the propionyl-CoA pathway includes a pyruvate carboxylase or a methylmalonyl-CoA
- the microbial organism has a propionyl-CoA pathway having at least one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme expressed in a sufficient amount to produce propionyl-CoA, the propionyl-CoA pathway including a PEP carboxykinase, a PEP carboxylase, a threonine deaminase, or a 2-oxobutanoate dehydrogenase.
- the n-propanol pathway includes 2-oxobutanoate decarboxylase.
- the microbial organism has a propionyl-CoA pathway having at least one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme expressed in a sufficient amount to produce propionyl-CoA, the propionyl-CoA pathway including an acetyl- CoA carboxylase, a malonyl-CoA reductase, a malonate semialdehyde reductase or propionyl- CoA synthase.
- the microbial organism has a propionyl-CoA pathway having at least one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme expressed in a sufficient amount to produce propionyl-CoA, the propionyl-CoA pathway including a lactate dehydrogenase, a lactate-CoA transferase, a lactyl-CoA dehydratase or acryloyl CoA reductase.
- the invention provides a non-naturally occurring microbial organism, including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway having a first set of exogenous nucleic acids encoding n- propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the first set of exogenous nucleic acids encoding a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate reductase and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate kinase, a propionyl phosphate
- the microbial organism has an acetyl-CoA pathway having a third set of exogenous nucleic acids encoding acetyl-CoA pathway enzymes expressed in a sufficient amount to produce acetyl-CoA, the third set of exogenous nucleic acids encoding a pyruvate kinase; and a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a pyruvate formate lyase activating enzyme and a formate
- the microbial organism has a propionyl-CoA pathway having a third set of exogenous nucleic acids encoding propionyl-CoA pathway enzymes expressed in a sufficient amount to produce propionyl-CoA, the third set of exogenous nucleic acids encoding a PEP carboxykinase or a PEP carboxylase; a malate dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA mutase; and a methylmalonyl-CoA decarboxylase.
- the third set of exogenous nucleic acids further encodes a methylmalonyl-CoA epimerase, a pyruvate carboxylase or a methylmalonyl-CoA carboxytransferase.
- the microbial organism has a propionyl-CoA pathway having a third set of exogenous nucleic acids encoding propionyl-CoA pathway enzymes expressed in a sufficient amount to produce propionyl-CoA, said third set of exogenous nucleic acids encoding a PEP carboxykinase or a PEP carboxylase; a threonine deaminase; and a 2-oxobutanoate dehydrogenase.
- the third set of exogenous nucleic acids further encodes a methylmalonyl-CoA decarboxylase or a pyruvate carboxylase.
- the second set of exogenous nucleic acids further encodes a 2-oxobutanoate decarboxylase.
- the microbial organism has a propionyl-CoA pathway having a third set of exogenous nucleic acids encoding propionyl-CoA pathway enzymes expressed in a sufficient amount to produce propionyl-CoA, the third set of exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA reductase; a malonate semialdehyde reductase; and propionyl-CoA synthase.
- the microbial organism has a propionyl-CoA pathway having a third set of exogenous nucleic acids encoding a lactate dehydrogenase; a lactate-CoA transferase; a lactyl-CoA dehydratase; and acryloyl CoA reductase.
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway comprising a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the first set of exogenous nucleic acids encoding a PEP carboxykinase or a PEP carboxylase; a malate dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA mutase; a methylmalonyl-CoA decarboxylase; and a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a pro
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway comprising a first set of exogenous nucleic acids encoding n-popanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the first set of exogenous nucleic acids encoding a PEP carboxykinase or a PEP carboxylase; a threonine deaminase; and a 2-oxobutanoate decarboxylase and a propanol dehydrogenase; or a 2- oxobutanoate dehydrogenase, a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a 2-oxobutanoate dehydrogenase, a propionyl-CoA:phosphate propanoy
- dehydrogenase a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate kinase, a propionyl phosphate reductase and a propanol dehydrogenase; or a 2-oxobutanoate dehydrogenase, a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate reductase and a propanol dehydrogenase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding a pyruvate kinase; a pyruv
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway comprising a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the first set of exogenous nucleic acids encoding a pyruvate kinase; a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a pyruvate formate lyase activating enzyme and a formate dehydrogenase; an acetyl-CoA carboxylase; a malonyl-CoA reductase; a malonate semialdehyde reductase; propionyl-CoA
- propionyl-CoA phosphate propanoyltransferase, a propionyl phosphate reductase and propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate kinase, a propionyl phosphate reductase and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate reductase and a propanol dehydrogenase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway including a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the first set of exogenous nucleic acids encoding a lactate dehydrogenase; a lactate-CoA transferase; a lactyl- CoA dehydratase; acryloyl CoA reductase; and a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate reductase and a propanol dehydrogenase; or a propionyl-CoA hydrolase or
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway, the n-propanol pathway comprising at least one exogenous nucleic acid encoding an n-propanol pathway enzyme expressed in a sufficient amount to produce n-propanol, the n-propanol pathway including a propionaldehyde dehydrogenase, a propanol dehydrogenase, a propionyl-CoA:phosphate propanoyltransferase, a propionyl-CoA hydrolase, a propionyl-CoA transferase, a propionyl- CoA synthetase, a propionate kinase, a propionate reductase, or a propionyl phosphate reductase.
- a propionaldehyde dehydrogenase a propanol dehydrogenase
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway, the n-propanol pathway comprising a set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the set of exogenous nucleic acids encoding a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate reductase and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate kinase, a propionyl phosphate reductase and a propanol
- the non-naturally occurring microbial organism having an n-propanol pathway also has a propionyl-CoA pathway including exogenous nucleic acids encoding propionyl-CoA pathway enzymes expressed in a sufficient amount to produce propionyl-CoA as exemplified herein.
- the exogenous nucleic acids encode a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a methylmalonyl- CoA mutase or a methylmalonyl-CoA decarboxylase.
- the exogenous nucleic acids further encode a methylmalonyl-CoA epimerase.
- the non-naturally occurring microbial organism having an n-propanol pathway can have a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, wherein the first set of exogenous nucleic acids encode a PEP carboxykinase or a PEP carboxylase; a malate dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase, a methylmalonyl-CoA decarboxylase; a propionaldehyde dehydrogenase and a propanol dehydrogenase.
- a PEP carboxykinase or a PEP carboxylase a mal
- the invention provides a non-naturally occurring microbial organism, including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway having at least one exogenous nucleic acid encoding an 14-BDO pathway enzyme expressed in a sufficient amount to produce 14-BDO, the 14-BDO pathway including a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4- hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming), a 4-hydroxybutyraldehyde reductase, a 4-hydroxybutyrate reductase; a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4- hydroxybutyryl-phosphate reduct
- the invention provides a non-naturally occurring microbial organism, including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway having at least one exogenous nucleic acid encoding an 13-BDO pathway enzyme expressed in a sufficient amount to produce 13-BDO, the 13-BDO pathway including a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4- hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA dehydratase, a crotonase, a 3-hydroxybutyryl-CoA reductase (aldehyde forming), a 3-hydroxybutyraldehyde reductase,
- the invention provides a non-naturally occurring microbial organism, including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway having at least one exogenous nucleic acid encoding an MAA pathway enzyme expressed in a sufficient amount to produce MAA, the MAA pathway including a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyrate kinase, a phosphotrans-4- hydroxybutyrylase, a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase, a methacrylyl-CoA hydro
- the isopropanol pathway including at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol, the isopropanol pathway including an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an isopropanol dehydrogenase.
- the microbial organism has an acetyl-CoA pathway having at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, the acetyl-CoA pathway including a pyruvate kinase, a pyruvate dehydrogenase, a pyruvate ferredoxin oxidoreductase, a pyruvate formate lyase, a pyruvate formate lyase activating enzyme, or a formate dehydrogenase.
- the microbial organism has a succinyl-CoA pathway having at least one exogenous nucleic acid encoding a succinyl-CoA pathway enzyme expressed in a sufficient amount to produce succinyl-CoA, the succinyl-CoA pathway including a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase or a succinyl-CoA synthetase.
- the succinyl-CoA pathway includes a pyruvate carboxylase or a methylmalonyl-CoA
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4- hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl- CoA reductase (aldehyde-forming); and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4- hydroxybutyrate reductase; and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-CoA ace
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4- hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde- forming); and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4- hydroxybutyrate kinase; a 4-hydroxybutyryl-phosphate reductase; and a 4- hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl- CoA acetyl
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4- hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-CoA acetyl
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4- hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; and a 4- hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl- CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde-forming); and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucle
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate reductase; and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase or an
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde- forming); and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a 4-hydroxybutyryl-phosphate reductase; and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an acety
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol- forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl- CoA transferase or a 4-hydroxybutyryl-CoA synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-CoA acetyl
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4- hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl- CoA dehydratase; a crotonase; a 3-hydroxybutyryl-CoA reductase (aldehyde forming); and a 3-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding is
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4- hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl- CoA dehydratase; a crotonase; a 3-hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA synthetase or a 3-hydroxybutyryl-CoA hydrolase; a 3-hydroxybutyrate reductase; and a 3 hydroxybutyraldeh
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4- hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl- CoA dehydratase; a crotonase; and a 3-hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isoprop
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl- CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3-hydroxybutyryl-CoA reductase (aldehyde forming); and a
- 3-hydroxybutyraldehyde reductase and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl- CoA acetyl thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an isopropanol dehydrogenase.
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl- CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3-hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA synthetase or a 3- hydroxybutyryl-CoA hydrolase; a 3-hydroxybutyrate reductase; and a 3 hydroxybutyraldehyde reducta
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3-hydroxybutyryl-CoA reductase (aldehyde forming); and a 3-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4 hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3-hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA synthetase or a 3- hydroxybutyryl-CoA hydrolase; a 3 -hydroxybutyrate reductase; and a 3 hydroxybutyraldehyde reductase,
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a crotonase; and a 3-hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-CoA
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl- CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; and a 3-hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl- CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA mutase; a 3- hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA synthetase or a 3- hydroxyisobutyryl-CoA hydrolase; and a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogenous
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl- CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA mutase; a 3- hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA hydrolase, and the isopropanol pathway comprising a second set of exogenous nucleic acids
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA mutase; a 3- hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA synthetase or a 3- hydroxyisobutyryl-CoA hydrolase; and a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogenous nucleic acids en
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4 hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA mutase; a 3- hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA hydrolase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding iso
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA mutase; a 3- hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA synthetase or a 3- hydroxyisobutyryl-CoA hydrolase; and a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogenous nucleic acids
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA mutase; a 3- hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA hydrolase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding iso
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA mutase; a 3- hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA synthetase or a 3- hydroxyisobutyryl-CoA hydrolase; and a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropan
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4 hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA mutase; a 3- hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA hydrolase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a methylmalonyl-CoA mutase; a methylmalonyl-CoA reductase (aldehyde forming); a 3-hydroxyisobutyrate dehydrogenase; and a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an acetoacety
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase; a methylmalonyl- CoA transferase, a methylmalonyl-CoA synthetase, or a methylmalonyl-CoA hydrolase; a methylmalonate reductase; a 3-hydroxyisobutyrate dehydrogenase; and a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a methylmalonyl-CoA mutase; a methylmalonyl- Co A transferase, a methylmalonyl- CoA synthetase or a methylmalonyl-CoA hydrolase; a methylmalonate reductase; a 3- hydroxyisobutyrate dehydrogenase; and a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase; a methylmalonyl- CoA reductase (alcohol forming); and a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-Co
- the microbial organism has an acetyl-CoA pathway having a third set of exogenous nucleic acids encoding acetyl-CoA pathway enzymes expressed in a sufficient amount to produce acetyl-CoA, the third set of exogenous nucleic acids encoding a pyruvate kinase; and a pyruvate dehydrogenase or a pyruvate ferredoxin
- oxidoreductase or a pyruvate formate lyase, a pyruvate formate lyase activating enzyme and a formate dehydrogenase.
- the microbial organism has a succinyl-CoA pathway having a third set of exogenous nucleic acids encoding succinyl-CoA pathway enzymes expressed in a sufficient amount to produce succinyl-CoA, the third set of exogenous nucleic acids encoding a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase and a succinyl-CoA synthetase.
- the third set of exogenous nucleic acids further encodes a pyruvate carboxylase or a methylmalonyl- CoA carboxytransferase.
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a pyruvate carboxylase, a methylmalonyl-CoA carboxy transferase,
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a pyruvate carboxylase, a methylmalonyl-CoA carboxy transferase, a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transfera
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a pyruvate carboxylase, a methylmalonyl-CoA carboxy transferase, a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4- hydroxy
- the invention provides a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a pyruvate carboxylase, a methylmalonyl-CoA carboxytransferase, a methylmalonyl-CoA mutase, a methylmalonyl-CoA epimerase, a methylmalonyl-CoA transferase, a methylmalonyl-CoCoA epi
- the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
- the invention provides a non-naturally occurring microbial organism having an n-propanol and isopropanol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of
- the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of an n-propanol and isopropanol pathway, such as that shown in Figure 1.
- the invention provides a non-naturally occurring microbial organism having an n-propanol and isopropanol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of
- propionaldehyde propionyl phosphate to propionaldehyde, phosphoenolpyruvate to pyruvate, pyruvate to oxaloacetate, pyruvate to acetyl-CoA, pyruvate to acetyl-CoA and formate, formate to CO2, 2 acetyl-CoA substrates to 1 acetoacetyl-CoA product, acetoacetyl-CoA to acetoacetate, acetoacetate to acetone, acetone to isopropanol.
- the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of an n-propanol and isopropanol pathway, such as that shown in Figure 2.
- the invention provides a non-naturally occurring microbial organism having an n-propanol and isopropanol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of
- the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of an n-propanol and isopropanol pathway, such as that shown in Figure 3.
- the invention provides a non-naturally occurring microbial organism having an n-propanol and isopropanol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of pyruvate to D-lactate, D-lactate to lactoyl-CoA, lactoyl-CoA to acryloyl-CoA, acryloyl-CoA to propionyl- CoA, propionyl-CoA to propionaldehyde, propionaldehyde to n-propanol, propionyl-CoA to propionyl phosphate, propionyl-CoA to propionate, propionate to propionyl phosphate, propionate to propionaldehyde, propionyl phosphate to propionaldehyde, pyruvate to acetyl- CoA, pyr
- the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of an n-propanol and isopropanol pathway, such as that shown in Figure 4.
- the invention provides a non-naturally occurring microbial organism having an n-propanol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or proten that converst a strabstrate to a product selected from the group consisting of propionyl-CoA to propionaldehyde, propionaldehyde to n-propanol, propionyl-CoA to propionyl phosphate, propionyl-CoA to propionate, propionate to propionyl phosphate, propionate to a product selected from the group consisting of propionyl-CoA to propionaldehyde, propionaldehyde to n-propanol, propionyl-CoA to propionyl phosphate, propionyl-CoA to propionate, propionate to propionyl phosphate, propionate to
- the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of an n-propanol pathway, such as that shown in Figures 1-4.
- the invention provides a non-naturally occurring microbial organism having an 14-BDO and an isopropanol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of
- the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of an n-propanol and isopropanol pathway, such as that shown in Figure 5.
- the invention provides a non-naturally occurring microbial organism having an 13-BDO and an isopropanol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of
- the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of an n-propanol and isopropanol pathway, such as that shown in Figure 6.
- the invention provides a non-naturally occurring microbial organism having an MA A and an isopropanol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of
- the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of an n-propanol and isopropanol pathway, such as that shown in Figure 7 and 8.
- the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding an n- propanol, an isopropanol, a 14-BDO, a 13-BDO and/or MAA pathway enzyme expressed in a sufficient amount to produce an intermediate of an n-propanol, an isopropanol, a 14-BDO, a 13- BDO and/or MAA pathway.
- an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or MAA pathway is exemplified in Figures 1-8. Therefore, in addition to a microbial organism containing an n-propanol and an isopropanol, a 14-BDO and an isopropanol, a 13-BDO and an isopropanol or a MAA and an isopropanol pathway that produces n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol, the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding an n-propanol, an isopropanol, a 14- BDO, a 13-BDO and/or MAA pathway enzyme, where the microbial organism produces
- any of the pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of Figures 1-8, can be utilized to generate a non-naturally occurring microbial organism that produces any pathway intermediate or product, as desired.
- a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to produce a desired product.
- a non-naturally occurring microbial organism that produces an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or MAA intermediate can be utilized to produce the intermediate as a desired product.
- the invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product.
- reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.
- the non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic pathways.
- nucleic acids for some or all of a particular n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic pathway can be expressed.
- a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression.
- the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthesis.
- a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA.
- the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed n- propanol, isopropanol, 14-BDO, 13-BDO and/or MAApathway-encoding nucleic acid and up to all encoding nucleic acids for one or more n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic pathways.
- n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid.
- exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins.
- exogenous expression of all enzymes or proteins in a pathway for production of n-propanol and isopropanol can be included, such as a PEP carboxykinase or a PEP carboxylase; a malate dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase; a methylmalonyl-CoA decarboxylase; and a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a propion
- nucleic acids to introduce in an expressible form will, at least, parallel the n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAApathway deficiencies of the selected host microbial organism.
- a non-naturally occurring microbial organism of the invention can have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or twenty one, up to all nucleic acids encoding the enzymes or proteins constituting an n-propanol, an isopropanol, a 14- BDO, a 13-BDO and/or a MAA biosynthetic pathway disclosed herein.
- the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthesis or that confer other useful functions onto the host microbial organism.
- One such other functionality can include, for example, augmentation of the synthesis of one or more of the n- propanol, isopropanol, 14-BDO, 13-BDO and/or MAA pathway precursors such as
- a host microbial organism is selected such that it produces the precursor of an n- propanol, an isopropanol, a 14-BDO, a 13-BDO and/or a MAA pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism.
- phosphoenolpyruvate and pyruvate are produced naturally in a host organism such as E. coli.
- a host organism can be engineered to increase production of a precursor, as disclosed herein.
- a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or a MAA pathway.
- a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA.
- it can be useful to increase the synthesis or accumulation of an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or a MAA pathway product to, for example, drive n-propanol, isopropanol, 14-BDO, 13- BDO and/or MAA pathway reactions toward n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA production.
- Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described n-propanol and/or isopropanol pathway enzymes or proteins.
- Over expression of the enzyme or enzymes and/or protein or proteins of the n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes.
- Naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, producing n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA, through overexpression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or twenty one, that is, up to all nucleic acids encoding n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic pathway enzymes or proteins.
- a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic pathway.
- exogenous expression of the encoding nucleic acids is employed.
- Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user.
- endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element.
- an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time.
- an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.
- any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention.
- the nucleic acids can be introduced so as to confer, for example, an n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol biosynthetic pathway onto the microbial organism.
- encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic capability.
- a non-natural occurring microbial organism having an n-propanol and an isopropanol, a 14-BDO and an isopropanol, a 13-BDO and an isopropanol or a MAA and an isopropanol biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the combination of propionaldehyde dehydrogenase and isopropanol dehydrogenase, or alternatively propionyl-CoA synthase and acetyl-CoA acetyl thiolase, or alternatively lactate dehydrogenase and acetyl-CoA thiolase, or alternatively a succinyl-CoA reductase and 4-hydroxybutyryl-CoA reductase (alcohol-forming), or alternatively crotonase and acetoacetate decarboxylase, or alternatively 4-
- any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention.
- any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, PEP carboxykinase, acetyl-CoA acetyl thiolase and propanol dehydrogenase, or alternatively pyruvate kinase, acetoacetate decarboxylase and 2-oxobutanoate dehydrogenase, or alternatively propionyl-CoA:phosphate propanoyltransferase, propionyl phosphate reductase and isopropanol dehydrogenase, or alternatively lactate-CoA transferase and lactyl-CoA dehydratase and pyruvate formate
- any combination of four or more enzymes or proteins of a biosynthetic pathway as disclosed herein for example, pyruvate carboxylase, malate dehydrogenase, methylmalonyl-CoA epimerase and acetoacetyl-CoA hydrolase, or alternatively acetyl-CoA acetyl thiolase, isopropanol dehydrogenase,
- propionaldehyde dehydrogenase and propanol dehydrogenase or alternatively acetyl-CoA carboxylase, malonyl-CoA reductase, malonate semialdehyde and acetoacetate decarboxylase, or alternatively, acryloyl CoA reductase, acetoacetyl-CoA transferase, acetoacetate
- hydroxyisobutyrate dehydratase can be included in a non-naturally occurring microbial organism of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.
- exogenous nucleic acid when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein.
- two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism
- the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids.
- exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids.
- the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.
- non-naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes.
- one alternative to produce n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA other than use of the n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA producers is through addition of another microbial organism capable of converting an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or a MAA pathway intermediate to n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA.
- One such procedure includes, for example, the fermentation of a microbial organism that produces an n-propanol, an isopropanol, a 14-BDO, a
- n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol pathway intermediate can then be used as a substrate for a second microbial organism that converts the n-propanol, isopropanol,
- 14- BDO, 13-BDO and/or MAA pathway intermediate to n-propanol, isopropanol, 14-BDO, 13- BDO and/or MAA.
- the n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA pathway intermediate can be added directly to another culture of the second organism or the original culture of the n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA pathway intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps.
- the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol.
- biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized.
- the biosynthesis of n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product.
- n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA also can be biosynthetically produced from microbial organisms through co-culture or co- fermentation using two organisms in the same vessel, where the first microbial organism produces a propionyl-CoA, succinyl-CoA and/or an acetyl-CoA intermediate and the second microbial organism converts the intermediate(s) to n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA.
- Sources of encoding nucleic acids for an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or a MAA pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction.
- species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human.
- Exemplary species for such sources include, for example, Escherichia coli, Acetobacter pasteurians, Acidanus brierleyi, Acinetobacter baylyi Acinetobacter calcoaceticus, Acinetobacter sp. Strain M-l, Actinobacillus succino genes, Anaerobio spirillum
- Bradyrhizobium japonicum USDA110 Caenorhabditis elegans, Campylobacter jejuni, Chlamydomonas reinhardtii, Chloroflexus aurantiacus, Clostridium acetobutylicum,
- Clostridium propionicum Clostridium saccharobutylicum, Clostridium
- Helicobacter pylori Homo sapiens, Klebsiella pneumonia MGH78578, Kluyveromyces lactis, Lactobacillus casei, Lactobacillus plantarum WCFS1, Lactococcus lactis, Leuconostoc mesenteroides, Mannheimia succiniciproducens, marine gamma proteobacterium HTCC2080, Mesorhizobium loti, Metallosphaera sedula, Methylobacterium extorquens, Moorella thermoacetica, Mycobacterium smegmatis, Mycobacterium tuberculosis, Oryctolagus cuniculus, Plasmodium ovale, Porphyromonas gingivalis, Propionibacterium acnes, Propionibacterium fredenreichii sp.
- Penicillium chrysogenum Penicillium chrysogenum, Porphyromonas gingivalis ATCC 33277, Pseudomonas mendocina, Streptomyces griseus subsp.griseus NBRC 13350 as well as other exemplary species disclosed herein are available as source organisms for corresponding genes.
- the metabolic alterations allowing biosynthesis of n- propanol, isopropanol, 14-BDO, 13-BDO and/or MAA described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.
- n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic pathway exists in an unrelated species
- n-propanol, isopropanol, 14- BDO, 13-BDO and/or MAA biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ.
- teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize n-propanol, isopropanol, 14- BDO, 13-BDO and/or MAA.
- Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes.
- Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobio spirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.
- Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger and Pichia pastoris.
- E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering.
- Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae.
- Other particulalarly useful host organisms include microbial organisms which naturally produce sufficient quantities of propionyl-CoA and/or acetyl-CoA for co-production of n-propanol and isopropanol. Examples of such organisms include, but are not limited to, Clostrium propionicum, Escherichia coli and Propionibacterium freudenreichii subsp.
- Methods for constructing and testing the expression levels of a non-naturally occurring n- propanol-, isopropanol-, 14-BDO-, 13-BDO- and/or MAA-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).
- Exogenous nucleic acid sequences involved in a pathway for production of n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation.
- conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation for exogenous expression in E.
- nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired.
- targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired.
- removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., /. Biol. Chem. 280:4329-4338 (2005)).
- genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells.
- a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells.
- An expression vector or vectors can be constructed to include one or more n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism.
- Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media.
- Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.
- both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors.
- the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art.
- Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
- nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA
- PCR polymerase chain reaction
- immunoblotting for expression of gene products
- Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (e.g., >10 4 ). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al., Biomol. Eng 22: 11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax.
- Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example, selectivity/specificity - for conversion of non-natural substrates; temperature stability - for robust high temperature processing; pH stability - for bioprocessing under lower or higher pH conditions; substrate or product tolerance - so that high product titers can be achieved; binding (K m ) - broadens substrate binding to include non-natural substrates; inhibition (K ; ) - to remove inhibition by products, substrates, or key intermediates; activity (kcat) - increases enzymatic reaction rates to achieve desired flux; expression levels - increases protein yields and overall pathway flux; oxygen stability - for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity - for operation of an aerobic enzyme in the absence of oxygen.
- EpPCR Principal et al., J Theor.Biol 234:497-509 (2005) introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions by the addition of Mn 2+ ions, by biasing dNTP concentrations, or by other conditional variations.
- the five step cloning process to confine the mutagenesis to the target gene of interest involves: 1) error-prone PCR amplification of the gene of interest; 2) restriction enzyme digestion; 3) gel purification of the desired DNA fragment; 4) ligation into a vector; 5) transformation of the gene variants into a suitable host and screening of the library for improved performance.
- This method can generate multiple mutations in a single gene simultaneously, which can be useful.
- a high number of mutants can be generated by EpPCR, so a high-throughput screening assay or a selection method (especially using robotics) is useful to identify those with desirable characteristics.
- Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., Nucleic Acids Res 32:el45 (2004); and Fujii et al., Nat.Protoc. 1:2493-2497 (2006)) has many of the same elements as epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats. Adjusting the Mn 2+ concentration can vary the mutation rate somewhat.
- DNA or Family Shuffling typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes.
- Fragments prime each other and recombination occurs when one copy primes another copy (template switch).
- This method can be used with >lkbp DNA sequences.
- this method introduces point mutations in the extension steps at a rate similar to error-prone PCR.
- the method can be used to remove deleterious, random and neutral mutations that might confer antigenicity.
- Staggered Extension (StEP) (Zhao et al., Nat.Biotechnol 16:258-261 (1998)) entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec).
- Random Priming Recombination random sequence primers are used to generate many short DNA fragments complementary to different segments of the template.
- Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al., Nat.Biotechnol 19:354-359 (2001)) employs Dnase I fragmentation and size fractionation of ssDNA.
- Homologous fragments are hybridized in the absence of polymerase to a complementary ssDNA scaffold. Any overlapping unhybridized fragment ends are trimmed down by an exonuc lease. Gaps between fragments are filled in, and then ligated to give a pool of full-length diverse strands hybridized to the scaffold (that contains U to preclude amplification). The scaffold then is destroyed and is replaced by a new strand complementary to the diverse strand by PCR amplification. The method involves one strand (scaffold) that is from only one parent while the priming fragments derive from other genes; the parent scaffold is selected against. Thus, no reannealing with parental fragments occurs. Overlapping fragments are trimmed with an exonuclease.
- Recombined Extension on Truncated templates entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates.
- RTT Truncated templates
- No DNA endonucleases are used. Unidirectional ssDNA is made by DNA polymerase with random primers or serial deletion with exonuclease. Unidirectional ssDNA are only templates and not primers. Random priming and exonucleases don't introduce sequence bias as true of enzymatic cleavage of DNA shuffling/RACHITT.
- RETT can be easier to optimize than StEP because it uses normal PCR conditions instead of very short extensions. Recombination occurs as a component of the PCR steps-no direct shuffling. This method can also be more random than StEP due to the absence of pauses.
- DOGS Oligonucleotide Gene Shuffling
- this method can be used to control the tendency of other methods such as DNA shuffling to regenerate parental genes.
- This method can be combined with random mutagenesis (epPCR) of selected gene segments. This can be a good method to block the reformation of parental sequences. No endonucleases are needed. By adjusting input concentrations of segments made, one can bias towards a desired backbone. This method allows DNA shuffling from unrelated parents without restriction enzyme digests and allows a choice of random mutagenesis methods.
- ITCHY Incremental Truncation for the Creation of Hybrid Enzymes
- THIO-ITCHY Thio-Incremental Truncation for the Creation of Hybrid Enzymes
- ITCHY Thio-Incremental Truncation for the Creation of Hybrid Enzymes
- SCRATCHY combines two methods for recombining genes, ITCHY and DNA shuffling. (Lutz et al., Proc Natl Acad Sci U.S.A. 98: 11248-11253 (2001)) SCRATCHY combines the best features of ITCHY and DNA shuffling. First, ITCHY is used to create a comprehensive set of fusions between fragments of genes in a DNA homology-independent fashion. This artificial family is then subjected to a DNA-shuffling step to augment the number of crossovers.
- SCRATCHY is more effective than DNA shuffling when sequence identity is below 80%.
- RNDM Random Drift Mutagenesis
- RNDM is usable in high throughput assays when screening is capable of detecting activity above background. RNDM has been used as a front end to DOGS in generating diversity. The technique imposes a requirement for activity prior to shuffling or other subsequent steps; neutral drift libraries are indicated to result in higher/quicker improvements in activity from smaller libraries. Though published using epPCR, this could be applied to other large-scale mutagenesis methods.
- Sequence Saturation Mutagenesis is a random mutagenesis method that: 1) generates pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage; this pool is used as a template to 2) extend in the presence of "universal" bases such as inosine; 3) replication of a inosine-containing complement gives random base incorporation and, consequently, mutagenesis.
- overlapping oligonucleotides are designed to encode "all genetic diversity in targets" and allow a very high diversity for the shuffled progeny.
- this technique one can design the fragments to be shuffled. This aids in increasing the resulting diversity of the progeny.
- sequence/codon biases to make more distantly related sequences recombine at rates approaching those observed with more closely related sequences. Additionally, the technique does not require physically possessing the template genes.
- Nucleotide Exchange and Excision Technology NexT exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation.
- the gene is reassembled using internal PCR primer extension with proofreading polymerase.
- the sizes for shuffling are directly controllable using varying dUPT::dTTP ratios. This is an end point reaction using simple methods for uracil incorporation and cleavage.
- Other nucleotide analogs, such as 8-oxo-guanine, can be used with this method.
- GSSMTM Gene Site Saturation Mutagenesis
- the starting materials are a supercoiled dsDNA plasmid containing an insert and two primers which are degenerate at the desired site of mutations.
- Primers carrying the mutation of interest anneal to the same sequence on opposite strands of DNA.
- the mutation is typically in the middle of the primer and flanked on each side by -20 nucleotides of correct sequence.
- Dpnl is used to digest dam-methylated DNA to eliminate the wild- type template.
- This technique explores all possible amino acid substitutions at a given locus (i.e., one codon).
- the technique facilitates the generation of all possible replacements at a single-site with no nonsense codons and results in equal to near-equal representation of most possible alleles.
- This technique does not require prior knowledge of the structure, mechanism, or domains of the target enzyme. If followed by shuffling or Gene Reassembly, this technology creates a diverse library of recombinants containing all possible combinations of single-site up- mutations. The utility of this technology combination has been demonstrated for the successful evolution of over 50 different enzymes, and also for more than one property in a given enzyme.
- Combinatorial Cassette Mutagenesis involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations.
- Combinatorial Multiple Cassette Mutagenesis is essentially similar to CCM except it is employed as part of a larger program: 1) Use of epPCR at high mutation rate to 2) ID hot spots and hot regions and then 3) extension by CMCM to cover a defined region of protein sequence space. (Reetz, M. T., S. Wilensek, D. Zha, and K. E. Jaeger, 2001, Directed Evolution of an Enantioselective Enzyme through Combinatorial Multiple-Cassette Mutagenesis.
- conditional ts mutator plasmids allow increases of 20- to 4000- X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required.
- This technology is based on a plasmid-derived mutD5 gene, which encodes a mutant subunit of DNA polymerase III. This subunit binds to endogenous DNA polymerase III and compromises the proofreading ability of polymerase III in any strain that harbors the plasmid. A broad-spectrum of base substitutions and frameshift mutations occur.
- the mutator plasmid should be removed once the desired phenotype is achieved; this is accomplished through a temperature sensitive origin of replication, which allows for plasmid curing at 41°C. It should be noted that mutator strains have been explored for quite some time (e.g., see Low et al., J. Mol. Biol. 260:359-3680 (1996)). In this technique very high spontaneous mutation rates are observed. The conditional property minimizes non- desired background mutations. This technology could be combined with adaptive evolution to enhance mutagenesis rates and more rapidly achieve desired phenotypes.
- LTM Look-Through Mutagenesis
- Gene Reassembly is a DNA shuffling method that can be applied to multiple genes at one time or to creating a large library of chimeras (multiple mutations) of a single gene. (Tunable
- GeneReassemblyTM (TGRTM) Technology supplied by Verenium Corporation) Typically this technology is used in combination with ultra-high-throughput screening to query the represented sequence space for desired improvements.
- TGRTM GeneReassemblyTM
- This technique allows multiple gene recombination independent of homology. The exact number and position of cross-over events can be pre- determined using fragments designed via bioinformatic analysis. This technology leads to a very high level of diversity with virtually no parental gene reformation and a low level of inactive genes. Combined with GSSMTM, a large range of mutations can be tested for improved activity.
- the method allows "blending" and "fine tuning" of DNA shuffling, e.g. codon usage can be optimized.
- PDA Silico Protein Design Automation
- This technology uses in silico structure- based entropy predictions in order to search for structural tolerance toward protein amino acid variations. Statistical mechanics is applied to calculate coupling interactions at each position. Structural tolerance toward amino acid substitution is a measure of coupling.
- this technology is designed to yield desired modifications of protein properties while maintaining the integrity of structural characteristics.
- the method computationally assesses and allows filtering of a very large number of possible sequence variants (10 50 ).
- the choice of sequence variants to test is related to predictions based on the most favorable thermodynamics. Ostensibly only stability or properties that are linked to stability can be effectively addressed with this technology.
- the method has been successfully used in some therapeutic proteins, especially in engineering immunoglobulins. In silico predictions avoid testing extraordinarily large numbers of potential variants. Predictions based on existing three-dimensional structures are more likely to succeed than predictions based on hypothetical structures. This technology can readily predict and allow targeted screening of multiple simultaneous mutations, something not possible with purely experimental technologies due to exponential increases in numbers.
- ISM Iterative Saturation Mutagenesis
- Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques.
- the invention provides a method for producing n-propanol and isopropanol that includes culturing a non- naturally occurring microbial organism, including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway having at least one exogenous nucleic acid encoding an n-propanol pathway enzyme expressed in a sufficient amount to produce n-propanol, the n-propanol pathway including a
- propionaldehyde dehydrogenase a propanol dehydrogenase, a propionyl-CoA:phosphate propanoyltransferase, a propionyl-CoA hydrolase, a propionyl-CoA transferase, a propionyl-
- CoA synthetase a propionate kinase, a propionate reductase or a propionyl phosphate reductase
- the isopropanol pathway comprising at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol
- the isopropanol pathway including an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an isopropanol dehydrogenase.
- the method includes a microbial organism having an acetyl-CoA pathway having at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, the acetyl-CoA pathway including a pyruvate kinase, a pyruvate dehydrogenase, a pyruvate ferredoxin oxidoreductase, a pyruvate formate lyase, a pyruvate formate lyase activating enzyme, or a formate dehydrogenase.
- the method includes a microbial organism having a propionyl-CoA pathway having at least one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme expressed in a sufficient amount to produce propionyl-CoA, the propionyl-CoA pathway including a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a methylmalonyl- CoA mutase, a methylmalonyl- Co A epimerase or a methylmalonyl- Co A decarboxylase.
- the propionyl-CoA pathway includes a pyruvate carboxylase or a methylmalonyl- CoA carboxytransferase.
- the method includes a microbial organism having a propionyl- CoA pathway having at least one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme expressed in a sufficient amount to produce propionyl-CoA, the propionyl-CoA pathway including a PEP carboxykinase, a PEP carboxylase, a threonine deaminase, or a 2- oxobutanoate dehydrogenase.
- the n-propanol pathway includes
- the method includes a microbial organism having a propionyl - CoA pathway having at least one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme expressed in a sufficient amount to produce propionyl-CoA, the propionyl-CoA pathway including an acetyl-CoA carboxylase, a malonyl-CoA reductase, a malonate semialdehyde reductase or propionyl-CoA synthase.
- the method includes a microbial organism having a propionyl- CoA pathway having at least one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme expressed in a sufficient amount to produce propionyl-CoA, the propionyl-CoA pathway including a lactate dehydrogenase, a lactate-CoA transferase, a lactyl-CoA dehydratase or acryloyl CoA reductase.
- the invention provides a method for producing n-propanol and isopropanol that includes culturing a non-naturally occurring microbial organism, including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway having a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the first set of exogenous nucleic acids encoding a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a propionyl- CoA:phosphate propanoyltransferase, a propionyl phosphate reductase and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl- CoA synthe
- the method includes a microbial organism having an acetyl-CoA pathway having a third set of exogenous nucleic acids encoding acetyl-CoA pathway enzymes expressed in a sufficient amount to produce acetyl-CoA, the third set of exogenous nucleic acids encoding a pyruvate kinase; and a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a pyruvate formate lyase activating enzyme and a formate dehydrogenase.
- the method includes a microbial organism having a propionyl- CoA pathway having a third set of exogenous nucleic acids encoding propionyl-CoA pathway enzymes expressed in a sufficient amount to produce propionyl-CoA, the third set of exogenous nucleic acids encoding a PEP carboxykinase or a PEP carboxylase; a malate dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA mutase; and a methylmalonyl-CoA decarboxylase.
- the third set of exogenous nucleic acids further encodes a methylmalonyl-CoA epimerase or a pyruvate carboxylas.
- the method includes a microbial organism having a propionyl- CoA pathway having a third set of exogenous nucleic acids encoding propionyl-CoA pathway enzymes expressed in a sufficient amount to produce propionyl-CoA, said third set of exogenous nucleic acids encoding a PEP carboxykinase or a PEP carboxylase; a threonine deaminase; and a 2-oxobutanoate dehydrogenase.
- the third set of exogenous nucleic acids further encodes a methylmalonyl-CoA decarboxylase or a pyruvate carboxylase.
- the second set of exogenous nucleic acids further encodes a 2-oxobutanoate
- the method includes a microbial organism having a propionyl- CoA pathway having a third set of exogenous nucleic acids encoding propionyl-CoA pathway enzymes expressed in a sufficient amount to produce propionyl-CoA, the third set of exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA reductase; a malonate semialdehyde reductase; and propionyl-CoA synthase.
- the method includes a microbial organism having a propionyl- CoA pathway having a third set of exogenous nucleic acids encoding a lactate dehydrogenase; a lactate- CoA transferase; a lactyl-CoA dehydratase; and acryloyl CoA reductase.
- the invention provides a method for producing n-propanol and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway comprising a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the first set of exogenous nucleic acids encoding a PEP carboxykinase or a PEP carboxylase; a malate dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase; a methylmalonyl- CoA mutase; a methylmalonyl-CoA decarboxylase; and a propionaldehy
- the invention provides a method for producing n-propanol and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway comprising a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the first set of exogenous nucleic acids encoding a PEP carboxykinase or a PEP carboxylase; a threonine deaminase; and a 2- oxobutanoate decarboxylase and a propanol dehydrogenase; or a 2-oxobutanoate dehydrogenase, a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a 2-oxobutan
- dehydrogenase a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate reductase and a propanol dehydrogenase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding a pyruvate kinase; a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a pyruvate formate lyase activating enzyme and a formate dehydrogenase; an acetyl-CoA acetyl thiolase; an acetoace
- the second set of exogenous nucleic acids further encodes a pyruvate carboxylase or a methylmalonyl-CoA carboxytransferase.
- the invention provides a method for producing n-propanol and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway comprising a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the first set of exogenous nucleic acids encoding a pyruvate kinase; a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a pyruv
- the invention provides a method for producing n-propanol and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway and an isopropanol pathway, the n-propanol pathway including a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the first set of exogenous nucleic acids encoding a lactate dehydrogenase; a lactate-CoA transferase; a lactyl-CoA dehydratase;
- dehydrogenase an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase or an acetoacetyl- CoA hydrolase or an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an isopropanol dehydrogenase.
- the invention provides a method for producing n-propanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway, the n-propanol pathway comprising at least one exogenous nucleic acid encoding an n-propanol pathway enzyme expressed in a sufficient amount to produce n- propanol, the n-propanol pathway including a propionaldehyde dehydrogenase, a propanol dehydrogenase, a propionyl-CoA:phosphate propanoyltransierase, a propionyl-CoA hydrolase, a propionyl-CoA transferase, a propionyl-CoA synthetase, a propionate kinase, a propionate reductase, or a propionyl phosphate reductase.
- the invention provides a method for producing n-propanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an n-propanol pathway, the n-propanol pathway comprising a set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, the set of exogenous nucleic acids encoding a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a propionyl-CoA:phosphate propanoyltransierase, a propionyl phosphate reductase and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA transferase or a propionyl-CoA synthetase, a propionate kinase, a propionyl
- the method for producing an propanol includes culturing the non-naturally occurring microbial organism having an n-propanol pathway that also has a propionyl-CoA pathway including exogenous nucleic acids encoding propionyl-CoA pathway enzymes expressed in a sufficient amount to produce propionyl-CoA as exemplified herein.
- the exogenous nucleic acids encode a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a methylmalonyl-CoA mutase, or a methylmalonyl-CoA decarboxylase.
- the exogenous nucleic acids further encode a methylmalonyl- CoA epimerase.
- the method for producing an propanol includes culturing the non-naturally occurring microbial organism having an n-propanol pathway that has a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, wherein the first set of exogenous nucleic acids encode a PEP carboxykinase or a PEP carboxylase; a malate dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase; a methylmalonyl- CoA decarboxylase; a propionaldehyde dehydrogenase and a propanol dehydrogenase
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism, including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway having at least one exogenous nucleic acid encoding an 14-BDO pathway enzyme expressed in a sufficient amount to produce 14-BDO, the 14-BDO pathway including a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde- forming), a 4-hydroxybutyraldehyde reductase, a 4-hydroxybutyrate reductase; a 4- hydroxybutyrate kinase, a
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism, including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway having at least one exogenous nucleic acid encoding an 13-BDO pathway enzyme expressed in a sufficient amount to produce 13-BDO, the 13-BDO pathway including a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyrate kinase, a phosphotrans-4- hydroxybutyrylase, a 4-hydroxybutyryl-CoA dehydratase, a crotonase, a 3-hydroxybutyryl-CoA reductase (aldeh
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism, including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway having at least one exogenous nucleic acid encoding an MAA pathway enzyme expressed in a sufficient amount to produce MAA, the MAA pathway including a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl- CoA synthetase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4- hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA transferase, a methacrylyl-CoA transfer
- the isopropanol pathway including at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol, the isopropanol pathway including an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an isopropanol dehydrogenase.
- the microbial organism has an acetyl-CoA pathway having at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, the acetyl-CoA pathway including a pyruvate kinase, a pyruvate dehydrogenase, a pyruvate ferredoxin oxidoreductase, a pyruvate formate lyase, a pyruvate formate lyase activating enzyme, or a formate dehydrogenase.
- the microbial organism has a succinyl-CoA pathway having at least one exogenous nucleic acid encoding a succinyl-CoA pathway enzyme expressed in a sufficient amount to produce succinyl-CoA, the succinyl-CoA pathway including a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase or a succinyl-CoA synthetase.
- the succinyl-CoA pathway includes a pyruvate carboxylase or a methylmalonyl-CoA
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA reductase
- aldehyde-forming (aldehyde-forming); and a 4-hydroxybutyraldehyde reductase
- the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase or an acetoacetyl- CoA hydrolase or an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an isopropanol dehydrogenase.
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate reductase; and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl- CoA ace
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde-forming); and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a 4- hydroxybutyryl -phosphate reductase; and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 44 ydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol-forming); and a 4-hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding iso
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde-forming); and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate reductase; and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl- CoA acetyl th
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA reductase (aldehyde-forming); and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a 4- hydroxybutyryl-phosphate reductase; and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids en
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; and a 4-hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogen
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3-hydroxybutyryl-CoA reductase (aldehyde forming); and a
- the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl- CoA acetyl thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an isopropanol dehydrogenase.
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3-hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA synthetase or a 3- hydroxybutyryl-CoA hydrolase; a
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3- hydroxybutyryl-CoA reductase (aldehyde forming); and a 3 -hydroxybutyraldehyde reductase, and the isopropanol pathway
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3- hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA synthetase or a 3-hydroxybutyryl- CoA hydrolase; a 3-hydroxybutyrate reduc
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; and a 3- hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropan
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; and a 3-hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids en
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3- hydroxybutyryl-CoA reductase (aldehyde forming); and a 3-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3- hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA synthetase or a 3-hydroxybutyryl- CoA hydrolase; a 3-hydroxybutyrate
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3- hydroxybutyryl-CoA reductase (aldehyde forming); and a 3-hydroxybutyraldehyde reductase, and the isopropanol pathway comprising a second set of
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4 hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a 3- hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA synthetase or a 3-hydroxybutyryl- CoA hydrolase; a 3 -hydroxybutyrate reducta
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a crotonase; and a 3-hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding a succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; and a 3- hydroxybutyryl-CoA reductase (alcohol-forming), and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isoprop
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4- hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl- CoA synthetase; a 4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA transferase, a 3- hydroxyisobutyryl-CoA synthetase or a 3-hydroxyisobutyryl-CoA hydrolase; and a 3- hydroxyisobutyrate dehydratas
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4- hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl- CoA synthetase; a 4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA hydrolase, and the is
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4- hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4- hydroxybutyrylase; a 4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA synthetase or a 3-hydroxyisobutyryl-CoA hydrolase; and a 3- hydroxyisobutyrate dehydratase, and the iso
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinyl-CoA reductase; a 4- hydroxybutyrate dehydrogenase; a 4 hydroxybutyrate kinase; a phosphotrans-4- hydroxybutyrylase; a 4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA hydrolase, and the isopropano
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinate reductase; a 4- hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl- CoA synthetase; a 4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA transferase, a 3- hydroxyisobutyryl-CoA synthetase or a 3-hydroxyisobutyryl-CoA hydrolase; and a 3- hydroxyisobutyrate dehydratase, and the is
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinate reductase; a 4- hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl- CoA synthetase; a 4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA hydrolase, and the isopropanol
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinate reductase; a 4- hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate kinase; a phosphotrans-4- hydroxybutyrylase; a 4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA synthetase or a 3-hydroxyisobutyryl-CoA hydrolase; and a 3- hydroxyisobutyrate dehydratase, and the isopropanol pathway
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a succinate reductase; a 4- hydroxybutyrate dehydrogenase; a 4 hydroxybutyrate kinase; a phosphotrans-4- hydroxybutyrylase; a 4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA hydrolase, and the isopropanol pathway comprising
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a methylmalonyl-CoA mutase; a methylmalonyl-CoA reductase (aldehyde forming); a 3-hydroxyisobutyrate dehydrogenase; and a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase; a methylmalonyl-CoA transferase, a methylmalonyl-CoA synthetase, or a methylmalonyl-CoA hydrolase; a methylmalonate reductase; a 3- hydroxyisobutyrate dehydrogenase; and a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogen
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a methylmalonyl-CoA mutase; a methylmalonyl-CoA transferase, a methylmalonyl-CoA synthetase or a methylmalonyl-CoA hydrolase; a methylmalonate reductase; a 3-hydroxyisobutyrate dehydrogenase; and a 3- hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzyme
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids a methylmalonyl-CoA mutase; a methylmalonyl-CoA reductase (alcohol forming); and a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway comprising a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, the second set of exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an acetoacetyl
- the microbial organism has an acetyl-CoA pathway having a third set of exogenous nucleic acids encoding acetyl-CoA pathway enzymes expressed in a sufficient amount to produce acetyl-CoA, the third set of exogenous nucleic acids encoding a pyruvate kinase; and a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a pyruvate formate lyase activating enzyme and a formate dehydrogenase.
- the microbial organism has a succinyl-CoA pathway having a third set of exogenous nucleic acids encoding succinyl-CoA pathway enzymes expressed in a sufficient amount to produce succinyl-CoA, the third set of exogenous nucleic acids encoding a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase and a succinyl-CoA synthetase.
- the third set of exogenous nucleic acids further encodes a methylmalonyl-CoA epimerase, a pyruvate carboxylase or a methylmalonyl-CoA carboxytransferase.
- the invention provides a method for producing 14-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway including a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, the first set of exogenous nucleic acids encoding a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a pyruvate carboxylase, a methylmalonyl-CoA carboxytransferase, a succinyl-CoA reductase, a succinate reductase, a 4-
- oxidoreductase a pyruvate formate lyase, a pyruvate formate lyase activating enzyme, a formate dehydrogenase, an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetyl- CoA hydrolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase, and an isopropanol dehydrogenase.
- the invention provides a method for producing 13-BDO and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway including a first set of exogenous nucleic acids encoding 13-BDO pathway enzymes expressed in a sufficient amount to produce 13-BDO, the first set of exogenous nucleic acids encoding PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a pyruvate carboxylase, a methylmalonyl-CoA carboxytransferase, a succinyl-CoA reductase, a succinate reductase, a 4- hydroxy
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a pyruvate carboxylase, a methylmalonyl-CoA carboxytransferase, a succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate dehydrogenase
- the invention provides a method for producing MAA and isopropanol that includes culturing a non-naturally occurring microbial organism including a microbial organism having an MAA pathway and an isopropanol pathway, the MAA pathway including a first set of exogenous nucleic acids encoding MAA pathway enzymes expressed in a sufficient amount to produce MAA, the first set of exogenous nucleic acids encoding a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a pyruvate carboxylase, a methylmalonyl-CoA carboxytransferase, a methylmalonyl-CoA mutase, a methylmalonyl-CoA epimerase, a methylmalon
- the exogenous nucleic acid is a heterologous nucleic acid.
- the conditions include substantially anaerobic culture conditions.
- Suitable purification and/or assays to test for the production of n-propanol, isopropanol, 14- BDO, 13-BDO and/or MAA can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art.
- HPLC High Performance Liquid Chromatography
- GC-MS Gas Chromatography-Mass Spectroscopy
- LC-MS Liquid Chromatography-Mass Spectroscopy
- the release of product in the fermentation broth can also be tested with the culture supernatant.
- Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art.
- the individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art.
- Various alcohols can be quantified by gas chromatography by using a flame ionization detector as described in Atsumi et al. Metab Eng (2007) and Hanai et al.
- n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA can be separated from other components in the culture using a variety of methods well known in the art.
- separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.
- any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention.
- the n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA producers can be cultured for the biosynthetic production of n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA.
- the recombinant strains are cultured in a medium with a carbon source and other essential nutrients. It is highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in U.S. publication 2009/0047719, filed August 10, 2007.
- Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein.
- the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH.
- the growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.
- the growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism.
- Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch.
- Other sources of carbohydrate include, for example, renewable feedstocks and biomass.
- Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks.
- Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
- carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
- renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA.
- the n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol microbial organisms of the invention also can be modified for growth on syngas as its source of carbon.
- one or more proteins or enzymes are expressed in the n- propanol, isopropanol, 14-BDO, 13-BDO and/or MAA producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.
- Synthesis gas also known as syngas or producer gas
- syngas is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues.
- Syngas is a mixture primarily of H 2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely 3 ⁇ 4 and CO, syngas can also include C0 2 and other gases in smaller quantities.
- synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, C0 2 .
- the Wood-Ljungdahl pathway catalyzes the conversion of CO and 3 ⁇ 4 to acetyl-CoA and other products such as acetate.
- Organisms capable of utilizing CO and syngas also generally have the capability of utilizing C0 2 and CO 2 H 2 mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway.
- Independent conversion of CO 2 to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved.
- Many acetogens have been shown to grow in the presence of CO 2 and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:
- non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize C0 2 and H 2 mixtures as well for the production of acetyl- Co A and other desired products.
- the Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch.
- the methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA.
- the reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase,
- methyltetrahydrofolate orrinoid protein methyltransferase for example, AcsE
- corrinoid iron- sulfur protein for example, nickel-protein assembly protein
- nickel-protein assembly protein for example, AcsF
- ferredoxin for example, ferredoxin
- acetyl-CoA synthase carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC).
- Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase,
- NAD(P)H ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase.
- the reducing equivalents extracted from CO and/or H 2 by carbon monoxide dehydrogenase and hydrogenase are utilized to fix C0 2 via the reductive TCA cycle into acetyl- CoA or acetate.
- Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase/phosphotransacetylase, and acetyl-CoA synthetase.
- Acetyl-CoA can be converted to an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or a MAA precursors, glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis.
- a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate.
- a carbon source such as a carbohydrate.
- Such compounds include, for example, n-propanol, isopropanol, 14-BDO, 13- BDO and/or MAA and any of the intermediate metabolites in the n-propanol, isopropanol, 14- BDO, 13-BDO and/or MAA pathway.
- All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the n-propanol, isopropanol, 14- BDO, 13-BDO and/or MAA biosynthetic pathways.
- the invention provides a non- naturally occurring microbial organism that produces and/or secretes n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA pathway when grown on a carbohydrate or other carbon source.
- n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol producing microbial organisms of the invention can initiate synthesis from an intermediate, for example, succinyl-CoA, propionyl-CoA and/or acetyl-CoA.
- the non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding an n-propanol, an isopropanol, al4-BDO, a 13-BDO and/or a MAA pathway enzyme or protein in sufficient amounts to produce n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA.
- the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of n-propanol, isopropanol, 14-BDO, 13- BDO and/or MAA resulting in intracellular concentrations between about 0.1-200 mM or more.
- the intracellular concentration of n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA is between about 3-150 mM, particularly between about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more.
- Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non-naturally occurring microbial organisms of the invention.
- culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions.
- Exemplary anaerobic conditions have been described previously and are well known in the art.
- Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication 2009/0047719, filed August 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art.
- n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA producers can synthesize n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA at intracellular
- n- propanol, isopropanol, 14-BDO, 13-BDO and/or MAA producing microbial organisms can produce n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA intracellularly and/or secrete the product into the culture medium.
- the culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.
- one exemplary growth condition for achieving biosynthesis of n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol includes anaerobic culture or fermentation conditions.
- the non- naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions.
- anaerobic conditions refers to an environment devoid of oxygen.
- Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation.
- Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen.
- the percent of oxygen can be maintained by, for example, sparging the culture with an N2/CO2 mixture or other suitable non-oxygen gas or gases.
- the culture conditions described herein can be scaled up and grown continuously for manufacturing of n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol.
- Exemplary growth procedures include, for example, fed- batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art.
- Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA.
- the continuous and/or near-continuous production of n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA will include culturing a non-naturally occurring n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase.
- Continuous culture under such conditions can be include, for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose. Fermentation procedures are well known in the art.
- fermentation for the biosynthetic production of n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.
- n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA producers of the invention for continuous production of substantial quantities of n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol
- the n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA producers also can be, for example, simultaneously subjected to chemical synthesis procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical conversion to convert the product to other compounds, if desired.
- growth condition for achieving biosynthesis of n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol can include the addition of an osmoprotectant to the culturing conditions.
- the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant.
- an osmoprotectant means a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress.
- Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin,
- the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used.
- the amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about lOmM, no more than about 50mM, no more than about lOOmM or no more than about 500mM.
- metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S.
- Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA.
- OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable microorganisms which overproduce the target product.
- the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth.
- OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism.
- the OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data.
- OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions.
- OptKnock computational framework allows the construction of model formulations that allow an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems.
- SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system.
- This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.
- metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock.
- SimPheny® and OptKnock For illustration of the invention, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation.
- OptKnock computation framework for modeling and simulation.
- Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art.
- the methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions.
- Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes.
- the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set.
- One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene.
- These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur.
- an optimization method termed integer cuts. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions.
- the integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®. The methods exemplified herein allow the construction of cells and organisms that
- biosynthetically produce a desired product including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®.
- the set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.
- the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum-growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism's ability to self-optimize under selective pressures.
- the OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry.
- the identification of optimal gene/reaction knockouts requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the resulting network overproduces the biochemical of interest (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)).
- An in silico stoichiometric model of E. coll metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Patent No. 7,127,379.
- the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth-coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of deletions.
- integer cuts an optimization technique, termed integer cuts. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above.
- This example describes exemplary pathways for co-production of n-propanol and isopropanol. Novel pathways for co-producing n-propanol and isopropanol and related products are described herein.
- This invention provides four alternate methods for co-production of n-propanol and isopropanol.
- the production of isopropanol in E. coli has been described previously (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007)). Briefly, acetyl CoA is converted into acetoacetyl CoA, transformed into acetoacetate, decarboxylated to form acetone and then reduced to form isopropanol ( Figures 1-4).
- the microbial organisms and methods described herein combine this known route with four novel pathways for synthesizing n-propanol.
- This co- production will provide completely redox balanced routes for production of the C3 alcohols, i.e. n-propanol and isopropanol, allowing for anaerobic production as opposed to the requirement of oxygen if isopropanol is produced solely via acetone as described by Hanai et al., supra.
- One advantage to the co-production of n-propanol and isopropanol using any of the pathways described herein is that the maximum theoretical yield of the C3 alcohols is afforded:
- Isopropanol production is achieved via conversion of acetyl-CoA by an acetoacetyl-CoA thiolase, an acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl- CoA synthetase,, an acetoacetate decarboxylase, and an isopropanol dehydrogenase as exemplified in Figures 1-4. Isopropanol production has been described for recombinant E. coli following expression of two heterologous genes from C.
- acetobutylicum thl and adc encoding acetoacetyl- CoA thiolase and acetoacetate decarboxylase, respectively
- C. beijerinckii adh encoding a secondary alcohol dehydrogenase
- acetoacetyl-CoA acetate: Co A transferase activity
- the conversion of acetoacetyl-CoA to acetoacetate can alternately be catalyzed by an enzyme with acetoacetyl-CoA hydrolase or acetoacetyl-CoA synthetase activities.
- Acetoacetyl-CoA thiolase (also known as acetyl-CoA acetyltransferase) converts two molecules of acetyl-CoA into one molecule each of acetoacetyl-CoA and CoA.
- Exemplary acetoacetyl- CoA thiolase enzymes include the gene products of atoB from E. coli (Martin et al.,
- Acetoacetyl-CoA transferase catalyzes the conversion of acetoacetyl-CoA to acetoacetate while transferring the CoA moiety to a CoA acceptor molecule.
- Many transferases have broad specificity and thus may utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3- mercaptopropionate, propionate, vinylacetate, butyrate, among others.
- Acetoacetyl-CoA:acetate:CoA transferase converts acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA.
- Exemplary enzymes include the gene products of atoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007), ctfAB from C. acetobutylicum (Jojima et al., Appl Microbiol Biotechnol 77: 1219-1224 (2008), and ctfAB from Clostridium
- a succinyl-CoA:3-ketoacid CoA transferase can also catalyze the conversion of the 3-ketoacyl-CoA, acetoacetyl-CoA, to the 3-ketoacid, acetoacetate.
- succinate As opposed to acetoacetyl-CoA:acetate:CoA transferase, SCOT employs succinate as the CoA acceptor instead of acetate.
- Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J Biol Chem 272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein Expr Purif 53:396-403 (2007)), and Homo sapiens (Fukao et al., Genomics 68:144-151 (2000); Tanaka et al., Mol Hum Reprod 8: 16-23 (2002)). Yet another transferase capable of this conversion is butyryl-CoA:acetoacetate CoA-transferase.
- Exemplary enzymes can be found in Fusobacterium nucleatum (Barker et al, J Bacteriol l52(l):20l-1 (1982)), Clostridium SB4 (Barker et &L, J Biol Chem 253(4): 1219-25 (1978)), and Clostridium acetobutylicum (Wiesenborn et al., Appl Environ Microbiol 55(2):323-9 (1989)).
- F 1857 and FN1856 are located adjacent to many other genes involved in lysine fermentation and are thus very likely to encode an acetoacetate:butyrate CoA transferase (Kreimeyer, et al., J Biol Chem 282 (10) 7191-7197 (2007)). Additional candidates from
- Porphyrmonas gingivalis and Thermoanaerobacter tengcongensis can be identified in a similar fashion (Kreimeyer, et al., J Biol Chem 282 (10) 7191-7197 (2007)). These genes/proteins are identified below in Table 2.
- a CoA synthetase can also catalyze the removal of the CoA moiety from acetoacetyl-CoA.
- One candidate enzyme, ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) couples the conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP.
- ACD ADP-forming acetyl-CoA synthetase
- Haloarcula marismortui annotated as a succinyl-CoA synthetase accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch Microbiol 182:277-287 (2004)).
- the ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl- CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., supra).
- Another candidate CoA synthetase is succinyl-CoA synthetase.
- the sucCD genes of E. coli form a succinyl-CoA synthetase complex which naturally catalyzes the formation of succinyl-CoA from succinate with the concaminant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochem. 24:6245-6252 (1985)).
- These genes/proteins are identified below in Table 4.
- CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochemical Journal 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas- Maceiras et al., Biochem. J. 395: 147-155 (2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez- Bianco et al., J. Biol. Chem.
- Acetoacetyl-CoA can also be converted to acetoacetate by a CoA hydrolase.
- Acetoacetyl-CoA hydrolase enzyme candidates include acyl-CoA hydrolase, 3-hydroxyisobutyryl-CoA hydrolase, acetyl-CoA hydrolase, and dicarboxylic acid thioesterase.
- a short-chain acyl-CoA hydrolase in rat liver mitochondria was found to accept acetoacetyl-CoA as a substrate; however, the gene associated with this enzyme has not been identified to date (Svensson et al. Eur. J. Biochem., 239:526-531 (1996)).
- 3-Hydroxyisobutyryl-CoA hydrolase efficiently catalyzes the conversion of 3- hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valine degradation (Shimomura et al., / Biol Chem. 269: 14248-14253 (1994)).
- Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomura et al., supra ;Shimomura et al., Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra). The H.
- sapiens enzyme also accepts 3- hydroxybutyryl-CoA and 3-hydroxypropionyl-CoA as substrates (Shimomura et al., supra).
- Candidate genes by sequence homology include hibch of Saccharomyces cerevisiae and BC_2292 of Bacillus cereus. These genes/proteins are identified below in Table 6.
- acetyl-CoA hydrolases (EC 3.1.2.1) have broad substrate specificity and thus represent suitable candidate enzymes.
- the enzyme from Rattus norvegicus brain (Robinson et al., Res. Commun. 71 :959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA.
- the enzyme from the mitochondrion of the pea leaf also has a broad substrate specificity, with demonstrated activity on acetyl-CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, and crotonyl-CoA (Zeiher et al., Plant. Physiol. 94:20-27 (1990)).
- the acetyl-CoA hydrolase, ACH1 from S. cerevisiae represents another candidate hydrolase (Buu et al., /. Biol. Chem. 278: 17203-17209 (2003)) .
- Another candidate hydrolase is the human dicarboxylic acid thioesterase, acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J Biol. Chem. 280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which can also hydrolyze a broad range of Co A thioesters (Naggert et al., J Biol. Chem.
- E. coli thioester hydrolases include the gene products of tesA (Bonner et al., Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005); and (Zhuang et al., FEBS Lett. 516:161-163 (2002)), paal (Song et al., J Biol. Chem. 281:11028-11038 (2006)), md ybdB (Leduc et al., J Bacteriol. 189:7112-7126 (2007)). These genes/proteins are identified below in Table 8.
- Yet another candidate hydrolase is the glutaconate CoA-transferase from Acidaminococcus fermentans. This enzyme was transformed by site-directed mutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS. Lett. 405:209-212 (1997)). This suggests that the enzymes encoding succinyl-CoA:3-ketoacid- CoA transferases and acetoacetyl-CoA:acetyl-CoA transferases may also serve as candidates for this reaction step but would require certain mutations to change their function. These genes/proteins are identified below in Table 9.
- Acetoacetate decarboxylase converts acetoacetate into carbon dioxide and acetone.
- Exemplary acetoacetate decarboxylase enzymes are encoded by the gene products of ode from C.
- the final step in the isopropanol synthesis pathway involves the reduction of acetone to isopropanol.
- Exemplary alcohol dehydrogenase enzymes capable of this transformation include adh from C. beijerinckii (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007); Jojima et al., Appl Microbiol Biotechnol 77: 1219-1224 (2008)) and adh from Thermoanaerobacter brockii (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007); Peretz et al, Anaerobe 3:259-270 (1997)).
- Additional characterized enzymes include alcohol dehydrogenases from Ralstonia eutropha (formerly Alcaligenes eutrophus) (Steinbuchel and Schlegel et al., Eur.J.Biochem_. 141:555-564 (1984)) and Phytomonas species (Uttaro and Opperdoes et al.,
- n-propanol utilize reduction of propionyl-CoA into propionaldehyde by a CoA-dependent aldehyde dehydrogenase that is then reduced further to form n-propanol ( Figures 1-4).
- This conversion is carried out by two different enzymes: an aldehyde and alcohol dehydrogenase or in one step by a bifunctional aldehyde/alcohol dehydrogenase.
- propionyl CoA can be converted into propionyl phosphate and then transformed into propionaldehyde by an acyl phosphate reductase.
- propionyl-CoA to propanol is catalyzed by either a bifunctional enzyme that has both the CoA-dependent aldehyde dehydrogenase and the alcohol dehydrogenase activities or by two different enzymes with the aldehyde and alcohol dehydrogenase activities.
- exemplary two-step oxidoreductases that convert an acyl-CoA to alcohol include those that transform substrates such as acetyl-CoA to ethanol (e.g., adhE from E. coli) (Kessler, FEBS. Lett. 281:59-63 (1991)) and butyryl-CoA to butanol (e.g. adhE2 from C.
- Another exemplary enzyme can convert malonyl-CoA to 3-HP.
- An NADPH-dependent enzyme with this activity has been characterized in Chloroflexus aurantiacus where it participates in the 3-hydroxypropionate cycle (Hugler, . Bacteriol. 184:2404-2410 (2002); and Strauss, Eur. J. Biochem. 215:633-643 (1993)).
- This enzyme with a mass of 300 kDa, is highly substrate- specific and shows little sequence similarity to other known oxidoreductases (Hugler, J.
- acyl-CoA molecules can be reduced by enzymes such as the jojoba (Simmondsia chinensis) FAR which encodes an alcohol-forming fatty acyl-CoA reductase. Its overexpression in E. coli resulted in FAR activity and the accumulation of fatty alcohol (Metz, Plant Physiology 122:635-644 (2000). These genes/proteins are identified below in Table 14.
- acyl-CoA dehydrogenases are capable of reducing an acyl-CoA to its corresponding aldehyde.
- Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acrl encoding a fatty acyl-CoA reductase, (Reiser, Journal of Bacteriology 179:2969-2975 (1997)) the Acinetobacter sp. M-l fatty acyl-CoA reductase, (Ishige et al., Appl. Environ.
- SucD of P. gingivalis is another succinate semialdehyde dehydrogenase (Takahashi, J. Bacteriol 182:4704-4710 (2000)).
- the enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another candidate as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski, J. Bacteriol. 175:377-385 (1993)).
- malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde.
- Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg, Science 318: 1782-1786 (2007); and Thauer, Science 318:1732-1733 (2007)).
- the enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp. (Alber et al., J. Bacteriol.
- the enzyme is encoded by Msed_0709 in Metallosphaera sedula (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg, Science 318:1782-1786 (2007)).
- a gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., J. Bacteriol 188:8551-8559 (2006).
- This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO2007141208 (2007)). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde.
- Exemplary genes encoding enzymes that catalyze the conversion of an aldehyde to alcohol include air A encoding a medium- chain alcohol dehydrogenase for C2-C14, (Tani, Appl. Environ. Microbiol. 66:5231-5235 (2000)) ADH2 from Saccharomyces cerevisiae, (Atsumi, Nature 451 :86-89 (2008)) yqhD from E. coli which has preference for molecules longer than C3, (Sulzenbacher et al., Journal of Molecular Biology 342:489-502 (2004)) and bdh I and bdh II from C.
- alcohol dehydrogenase or equivalently aldehyde reductase include air A encoding a medium- chain alcohol dehydrogenase for C2-C14, (Tani, Appl. Environ. Microbiol. 66:5231-5235 (2000)) ADH2 from Saccharomyces cerevisiae, (Atsum
- ADHl from Zymomonas mobilis has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita, Appl. Microbiol. Biotechnol. 22:249-254 (1985)).
- Enzymes exhibiting 3-hydroxybutyraldehyde reductase activity also fall into this category. Such enzymes have been characterized in Ralstonia eutropha, (Bravo J. Forensic Sci. 49:379-387 (2004)) Clostridium kluyveri (Wolff, Protein Expr. Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitnch et al., / Biol. Chem. 278:41552-41556 (2003)). Yet another gene candidate is the alcohol dehydrogenase adhi from Geobacillus thermoglucosidasius (Jeon et al., /. Biotechnal 135:127-133 (2008)). These genes/proteins are identified below in Table 18.
- Another exemplary enzyme is 3-hydroxyisobutyrate dehydrogenase which catalyzes the reversible oxidation of 3-hydroxyisobutyrate to methylmalonate semialdehyde.
- This enzyme participates in valine, leucine and isoleucine degradation and has been identified in bacteria, eukaryotes, and mammals.
- the enzyme encoded by P84067 from Thermus thermophilus HB8 has been structurally characterized (Lokanath et al., J Mol Biol 352:905-917 (2005)).
- the reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated using isotopically-labeled substrate (Manning, Biochem J 231:481-484 (1985)).
- the conversion of propanoyl-CoA to propanoyl phosphate can be catalyzed by a phosphate transferase.
- a phosphate transferase Among the phosphate acetyltransferases (EC 2.3.1.8), several enzymes including those from Bacillus subtilis, (Rado, Biochem. Biophys. Acta 321: 114-125 (1973)) Clostridium kluyveri, (Stadtman, Methods Enzymol 1 :596-599 (1955)) and Thermotoga maritima (Bock, / Bacteriol. 181: 1861-1867 (1999)) have been shown to have activity on propionyl-CoA.
- genes coding for these phosphate acetyltransferases as well as Escherichia coli pta gene will be utilized to catalyze this step. These genes/proteins are identified below in Table 20.
- Propionyl-CoA can be converted to propionate by a CoA hydrolase, synthetase or transferase.
- the hydrolysis of propionyl-CoA to propionate occurs in organic acid degradation pathways that proceed through the intermediate 2-oxobutanoate. This reaction is catalyzed by acyl-CoA hydrolase enzymes (EC 3.1.2.18).
- Propionyl-CoA is the preferred substrate of the short chin acyl-CoA hydrolase found in rat liver mitochondria (Alexson et al., Biochim Biophys. Acta.,
- propionyl-CoA hydrolase candidates include 3-hydroxyisobutyryl-CoA hydrolase, acetyl-CoA hydrolase, and dicarboxylic acid thioesterase.
- 3-Hydroxyisobutyryl-CoA hydrolase efficiently catalyzes the conversion of 3- hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valine degradation (Shimomura et al., / Biol Chem. 269: 14248-14253 (1994)).
- Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomura et al., ,w ⁇ ra;Shimomura et al., Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra). The H. sapiens enzyme also accepts 3- hydroxybutyryl-CoA and 3-hydroxypropionyl-CoA as substrates (Shimomura et al., supra).
- Candidate genes by sequence homology include hibch of Saccharomyces cerevisiae and BC_2292 of Bacillus cereus. These genes/proteins are identified below in Table 22.
- acetyl-CoA hydrolases (EC 3.1.2.1) have broad substrate specificity and thus represent suitable candidate enzymes.
- the enzyme from Rattus norvegicus brain (Robinson et al., Res. Commun. 71 :959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA.
- the acetyl-CoA hydrolase, ACH1 represents another candidate hydrolase (Buu et al., J. Biol. Chem. 278: 17203-17209 (2003)) .
- These genes/proteins are identified below in Table 23.
- Another candidate hydrolase is the human dicarboxylic acid thioesterase, acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J Biol. Chem. 280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which can also hydrolyze a broad range of Co A thioesters (Naggert et al., J Biol. Chem.
- E. coli thioester hydrolases include the gene products of tesA (Bonner et al., Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005); and (Zhuang et al., FEBS Lett. 516:161-163 (2002)), paal (Song et al., J Biol. Chem. 281:11028-11038 (2006)), md ybdB (Leduc et al., J Bacteriol. 189:7112-7126 (2007)). These genes/proteins are identified below in Table 24.
- Yet another candidate hydrolase is the glutaconate CoA-transferase from Acidaminococcus fermentans. This enzyme was transformed by site-directed mutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS. Lett. 405:209-212 (1997)). This suggests that the enzymes encoding succinyl-CoA:3-ketoacid- CoA transferases and acetoacetyl-CoA:acetyl-CoA transferases may also serve as candidates for this reaction step but would require certain mutations to change their function. These genes/proteins are identified below in Table 25.
- a CoA synthetase can also catalyze the removal of the CoA moiety from propionyl-CoA.
- One candidate enzyme, ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) couples the conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP.
- ACD ADP-forming acetyl-CoA synthetase
- Haloarcula marismortui annotated as a succinyl-CoA synthetase accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch Microbiol 182:277-287 (2004)).
- the ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl- CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., supra).
- Another candidate CoA synthetase is succinyl-CoA synthetase.
- the sucCD genes of E. coli form a succinyl-CoA synthetase complex which naturally catalyzes the formation of succinyl-CoA from succinate with the concaminant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochem. 24:6245-6252 (1985)).
- These genes/proteins are identified below in Table 27.
- CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochemical Journal 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas- Maceiras et al., Biochem. J. 395: 147-155 (2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez- Bianco et al., J. Biol. Chem.
- Propionyl-CoA transferase catalyzes the conversion of propionyl-CoA to propionate while transferring the CoA moiety to a CoA acceptor molecule.
- Many transferases have broad specificity and thus may utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3- mercaptopropionate, propionate, vinylacetate, butyrate, among others.
- Clostridium propionicum Clostridium propionicum (Selmer et al., Eur J Biochem 269, 372-380 (2002)).
- This enzyme can use acetate, (R)-lactate, (S)-lactate, acrylate, and butyrate as the CoA acceptor (Selmer et al., Eur J Biochem 269, 372-380 (2002); Schweiger and Buckel, FEBS Letters, 171(1) 79-84 (1984)).
- Close homologs can be found in, for example, Clostridium novyi NT, Clostridium beijerinckii NCIMB 8052, and Clostridium botulinum C str. Eklund.
- Ygfli encodes a propionyl CoA: succinate CoA transferase in E. coli (Haller et al., Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for example, Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909. These genes/proteins are identified below in Table 29.
- An additional candidate enzyme is the two-unit enzyme encoded by peal and pcaJ in
- Pseudomonas which has been shown to have 3-oxoadipyl-CoA/succinate transferase activity (Kaschabek et al., supra). Similar enzymes based on homology exist in Acinetobacter sp. ADPl (Kowalchuk et al., Gene 146:23-30 (1994)) and Streptomyces coelicolor. Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J.Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al.,
- a CoA transferase that can utilize acetate as the CoA acceptor is acetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Vanderwinkel et al., Biochem.Biophys.Res Commun. 33:902-908 (1968); Korolev et al., Acta Crystallogr.D Biol Crystallogr. 58:2116-2121 (2002)).
- This enzyme has also been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies et al., Appl Environ Microbiol 58: 1435-1439 (1992)), valerate
- the above enzymes may also exhibit the desired activities on propionyl-CoA.
- Additional exemplary transferase candidates are catalyzed by the gene products of catl, cat2, and cat3 of Clostridium kluyveri which have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively (Seedorf et al., swpra;Sohling et al., Eur.J Biochem. 212: 121-127 (1993);Sohling et al., J Bacteriol. 178:871-880 (1996)). Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem.
- Acidaminococcus fermentans reacts with diacid glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)).
- the genes encoding this enzyme are gctA and gctB.
- This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., Eur.J.Biochem. 118:315- 321 (1981)).
- the enzyme has been cloned and expressed in E. coli (Mack et al., Eur.J.Biochem. 226:41-51 (1994)).
- Propionate is activated to propionyl-phosphate by an enzyme with propionate kinase activity.
- Butyrate kinase (EC 2.7.2.7) carries out the reversible conversion of butyryl-phosphate to butyrate during acidogenesis in C. acetobutylicum (Cary et al., Appl. Environ. Microbiol 56: 1576-1583 (1990)).
- This enzyme is encoded by either of the two buk gene products (Huang et al., J Mol. Microbiol Biotechnol 2:33-38 (2000)). This enzyme was shown to accept propionate, isobutanoate and valerate as alternate substrates (Hartmanis, J. Biol. Chem., 262(2):617-21 (1987)).
- Other butyrate kinase enzymes are found in C. butyricum and C.
- tetanomorphum (Twarog et al., J Bacteriol. 86: 112-117 (1963)). These enzymes also accept propionate, isobutanoate and valerate as secondary substrates .
- Related enzyme isobutyrate kinase from Thermotoga maritima has also been expressed in E. coli and crystallized (Diao et al., E. Biol. Crystallogr. 59:1100-1102 (2003); and Diao et al., J Bacteriol. 191:2521-2529 (2009)). Aspartokinase catalyzes the ATP-dependent phosphorylation of aspartate and participates in the synthesis of several amino acids.
- the aspartokinase III enzyme in E. coli has a broad substrate range and the catalytic residues involved in substrate specificity have been elucidated (Keng et al., Arch. Biochem. Biophys. 335:73-81 (1996)).
- Two additional kinases in E. coli are also good candidates: acetate kinase and gamma-glutamyl kinase.
- the E. coli acetate kinase, encoded by ackA (Skarstedt et al., J. Biol. Chem.
- Example I the pathway for production of acetyl-CoA from glucose proceeds via phosphoenolpyruvate (PEP) ( Figures 1-4).
- PEP phosphoenolpyruvate
- Glucose is converted into PEP by the native glycolysis pathway of the microbial organism.
- PEP is converted to pyruvate by pyruvate kinase and then to acetyl-CoA by pyruvate dehydrogenase or pyruvate ferredoxin oxidoreductase.
- pyruvate is converted to acetyl-CoA and formate by pyruvate formate lyase.
- Formate is then converted to carbon dioxide by a formate dehydrogenase that also produces NADH.
- the acetyl-CoA produced by these pathways are then utilized for production of isopropanol as described in Example I or utilized for production of both n-propanol and isopropano
- the pyruvate dehydrogenase complex catalyzing the conversion of pyruvate to acetyl-CoA, has been extensively studied.
- the S. cerevisiae complex consists of an E2 (LATl) core that binds El (PDAl, PDB1), E3 (LPD1), and Protein X (PDX1) components (Pronk, Yeast 12:1607-1633 (1996)).
- E. cerevisiae complex consists of an E2 (LATl) core that binds El (PDAl, PDB1), E3 (LPD1), and Protein X (PDX1) components (Pronk, Yeast 12:1607-1633 (1996)).
- E. coli enzyme specific residues in the El component are responsible for substrate specificity (Bisswanger, J. Biol Chem. 256:815-822 (1981); Bremer, Eur. J Biochem. 8:535-540 (1969) and Gong et al., J. Biol
- PFOR Pyruvate ferredoxin oxidoreductase catalyzes the oxidation of pyruvate to form acetyl- CoA.
- the PFOR from Desulfovibrio africanus has been cloned and expressed in E. coli resulting in an active recombinant enzyme that was stable for several days in the presence of oxygen (Pieulle, J Bacteriol 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is believed to be conferred by a 60 residue extension in the polypeptide chain of the D. africanus enzyme. The M.
- thermoacetica PFOR is also well characterized (Menon, Biochemistry 36:8484-8494 (1997)) and was even shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui, J Biol Chem. 275:28494-28499 (2000)). Further, E. coli possesses an uncharacterized open reading frame, ydbK, that encodes a protein that is 51% identical to the M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E. coli has been described (Blaschkowski, Eur. J Biochem. 123:563-569 (1982)). Several additional PFOR enzymes are described in the following review (Ragsdale, Chem. Rev.
- flavodoxin reductases o.g.,fqrB from Helicobacter pylori or Campylobacter jejuni
- flavodoxin reductases o.g.,fqrB from Helicobacter pylori or Campylobacter jejuni
- Rnf-type proteins Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008)
- Herrmann, J. Bacteriol 190:784-791 (2008) provide a means to generate NADH or NADPH from the reduced ferredoxin generated by PFOR.
- Pyruvate formate lyase is an enzyme that catalyzes the conversion of pyruvate and CoA into acetyl-CoA and formate.
- Pyruvate formate lyase is a common enzyme in prokaryotic organisms that is used to help modulate anaerobic redox balance. Exemplary enzymes can be found in Escherichia coli (Knappe, FEMS. Microbiol Rev. 6:383-398 (1990)), Lactococcus lactis (Melchiorsen, Appl Microbiol Biotechnol 58:338-344(2002)), and Streptococcus mutans.
- a mitochondrial pyruvate formate lyase has also been identified in the eukaryote, Chlamydomonas reinhardtii.
- a formate hydrogen lyase enzyme can be employed to convert formate to carbon dioxide and hydrogen.
- An exemplary formate hydrogen lyase enzyme can be found in Escherichia coli.
- the E. coli formate hydrogen lyase consists of hydrogenase 3 and formate dehydrogenase-H
- a formate hydrogen lyase enzyme also exists in the hyperthermophilic archaeon, Thermococcus litoralis (Takacs et al., BMC. Microbiol 8:88 (2008)). These genes/proteins are identified below in Table 39.
- Formate dehydrogenase activity is present in both E. coli and Saccharomyces cerevisiae among other organisms.
- S. cerevisiae contains two formate dehydrogenases, FDH1 and FDH2, that catalyze the oxidation of formate to C0 2 .
- FDH1 and FDH2 formate dehydrogenases
- FDH1 and FDH2 formate dehydrogenases
- Moth_2312 and Moth_2313 are actually one gene that is responsible for encoding the alpha subunit of formate dehydrogenase while the beta subunit is encoded by Moth_2314 (Pierce et al., Environ. Microbiol (2008); Andreesen, J. Bacteriol. 116:867-873 (1973); Li, J. Bacteriol 92:405-412 (1966) and Yamamoto, J. Biol. Chem.
- Sfum_2703 Another set of genes encoding formate dehydrogenase activity is encoded by Sfum_2703 through Sfum_2706 in Syntrophobacter fumaroxidans (Reda, Proc. Natl. Acad. Sci. U.S.A. 105:10654-10658 (2008); and de Bok et al., Eur. J. Biochem. 270:2476- 2485 (2003)). Similar to their M. thermoacetica counterparts, Sfum_2705 and Sfum_2706 are actually one gene. E. coli contains multiple formate dehydrogenases. These genes/proteins are identified below in Table 40.
- the pathway for production of propionyl-CoA proceeds via oxaloacetate ( Figure 1).
- PEP is converted into oxaloacetate either via PEP carboxykinase or PEP carboxylase.
- PEP is converted first to pyruvate by pyruvate kinase and then to oxaloacetate by methylmalonyl-CoA carboxytransferase or pyruvate carboxylase.
- Oxaloacetate is converted to propionyl-CoA by means of the reductive TCA cycle, a methylmutase, a decarboxylase, an epimerase and a decarboxylase.
- PEP Carboxykinase Although the net conversion of phosphoenolpyruvate to oxaloacetate is redox-neutral, the mechanism of this conversion is important to the overall energetics of the co- production pathway.
- the most desirable enzyme for the conversion of PEP to oxaloacetate is PEP carboxykinase which simultaneously forms an ATP while carboxylating PEP. In most organisms, however, PEP carboxykinase serves a gluconeogenic function and converts oxaloacetate to PEP at the expense of one ATP.
- S. cerevisiae is one such organism whose native PEP carboxykinase, PCK1, serves a gluconeogenic role (Valdes-Hevia, FEBS.
- E. coli is another such organism, as the role of PEP carboxykinase in producing oxaloacetate is believed to be minor when compared to PEP carboxylase, which does not form ATP, possibly due to the higher K m for bicarbonate of PEP carboxykinase (Kim, Appl Environ Microbiol 70:1238-1241 (2004)). Nevertheless, activity of the native E. coli PEP carboxykinase from PEP towards oxaloacetate has been recently demonstrated in ppc mutants of E. coli K-12 (Kwon, Journal of Microbiology and Biotechnology 16: 1448-1452 (2006)).
- PEP carboxykinase is quite efficient in producing oxaloacetate from PEP and generating ATP.
- PEP carboxykinase genes that have been cloned into E. coli include those from Mannheimia succiniciproducens (Lee, Biotechnol. Bioprocess Eng. 7:95-99 (2002)), Anaerobio spirillum succiniciproducens (Laivenieks, Appl Environ Microbiol 63:2273-2280 (1997)), and
- sequences and sequences for subsequent enzymes listed in this report can be used to identify homologue proteins in GenBank or other databases through sequence similarity searches (e.g. BLASTp).
- sequence similarity searches e.g. BLASTp.
- the resulting homologue proteins and their corresponding gene sequences provide additional DNA sequences for transformation into the host organism of choice.
- PEP carboxylase represents an alternative enzyme for the formation of oxaloacetate from PEP. Since the enzyme does not generate ATP upon decarboxylating oxaloacetate, its utilization decreases the maximum ATP yield of the production pathway and represents a less favorable alternative for converting oxaloacetate to PEP. Nevertheless, the maximum theoretical C3 alcohols yield of 1.33 mol/mol will remain unchanged if PEP carboxylase is utilized to convert PEP to oxaloacetate.
- S. cerevisiae does not naturally encode a PEP carboxylase, but exemplary organisms that possess genes that encode PEP carboxylase include E. coli (Kai, Arch. Biochem. Biophys. 414: 170-179 (2003)), Methylobacterium extorquens AMI (Arps, J. Bacteriol.
- Pyruvate kinase catalyzes the ATP-generating conversion of PEP to pyruvate and is encoded by the PYK1 (Burke, J. Biol. Chem. 258:2193-2201 (1983)) and PYK2 (Boles et al., J. Bacteriol. 179:2987- 2993 (1997)) genes in S. cerevisiae. In E. coli, this activity is catalyzed by the gene product of pykF and pykA.
- Methylmalonyl-CoA carboxytransferase catalyzes the conversion of pyruvate to oxaloacetate. Importantly, this reaction also simultaneously catalyzes the conversion of (S)- methylmalonyl-CoA to propionyl-CoA (see Figures 1 and 2).
- An exemplary methylmalonyl- CoA carboxytransferase which is comprised of 1.3S, 5S, and 12S subunits can be found in Propionibacterium freudenreichii (Thornton et al., J. Bacteriol 175:5301-5308 (1993)). These genes/proteins are identified below in Table 43.
- Pyruvate Kinase and Pyruvate Carboxylase A combination of enzymes can convert PEP to oxaloacetate with a stoichiometry identical to that of PEP carboxylase. These enzymes are encoded by pyruvate kinase, PYKl (Burke, J. Biol. Chem. 258:2193-2201 (1983)) or PYKl (Boles et al., J. Bacteriol, 179:2987-2993 (1997))and pyruvate carboxylase, PYCl (Walker, Biochem. Biophys. Res. Commun. 176: 1210-1217 (1991)) or PYCl (Walker, Biochem. Biophys. Res. Commun. 176: 1210-1217 (1991)). The latter genes/proteins are identified below in Table 44.
- Oxaloacetate can be converted to succinate by malate dehydrogenase, fumarase and fumarate reductase when the TCA cycle is operating in the reductive cycle.
- S. cerevisiae possesses three copies of malate dehydrogenase, MDH1 (McAlister-Henn, J. Bacteriol 169:5157-5166 (1987)) MDH1 (Minard, Mol. Cell. Biol. 11:370-380 (1991); and Gibson, /. Biol. Chem. 278:25628- 25636 (2003)), and MDH3 (Steffan, /. Biol. Chem.
- S. cerevisiae contains one copy of a fumarase-encoding gene, FUM1, whose product localizes to both the cytosol and mitochondrion (Sass, J. Biol. Chem. 278:45109-45116 (2003)).
- Fumarate reductase is encoded by two soluble enzymes, FRDS1 (Enomoto, DNA. Res. 3:263-267 (1996)) and FRDS2 (MuratsubaM, Arch. Biochem. Biophys. 352: 175-181 (1998)), which localize to the cytosol and promitochondrion.
- E. coli is known to have an active malate dehydrogenase. It has three fumarases encoded by fumA, B and C, each one of which is active under different conditions of oxygen availability. The fumarate reductase in E. coli is composed of four subunits. These genes/proteins are identified below in Table 45.
- Succinyl-CoA transferase catalyzes the conversion of succinyl-CoA to succinate while transferring the CoA moiety to a CoA acceptor molecule.
- Many transferases have broad specificity and thus may utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3- mercaptopropionate, propionate, vinylacetate, butyrate, among others.
- succinyl-CoA The conversion of succinate to succinyl-CoA is ideally carried by a transferase which does not require the direct consumption of an ATP or GTP. This type of reaction is common in a number of organisms. Perhaps the top candidate enzyme for this reaction step is succinyl-CoA:3- ketoacid-CoA transferase. This enzyme converts succinate to succinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid. Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., /. Biol. Chem.
- succinyl-CoA Acetyl- CoA transferase.
- the gene product of call of Clostridium kluyveri has been shown to exhibit succinyl-CoA: acetyl-CoA transferase activity (Sohling, J Bacteriol. 178:871-880 (1996)).
- the activity is present in Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283: 1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., /. Biol. Chem. 279:45337- 45346 (2004)).
- CoA acceptor is benzylsuccinate.
- Succinyl-CoA:(R)-Benzylsuccinate CoA- Transferase functions as part of an anaerobic degradation pathway for toluene in organisms such as Thauera aromatica (Leutwein and Heider, J. Bact. 183(14) 4288-4295 (2001)).
- Homologs can be found in Azoarcus sp. T, Aromatoleum aromaticum EbNl, and Geobacter
- ygfH encodes a propionyl CoA:succinate CoA transferase in E. coli (Haller et al., Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for example, Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909. These genes/proteins are identified below in Table 49.
- Succinyl-CoA Synthetase The product of the LSC1 and LSC2 genes of S. cerevisiae and the sucC and sucD genes of E. coli naturally form a succinyl-CoA synthetase complex that catalyzes the formation of succinyl- CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Przybyla-Zawilask et al., Eur. J. Biochem. 258(2):736-743 (1998) and Buck et al., /. Gen. Microbiol. 132(6): 1753-1762 (1986)). These genes/proteins are identified below in Table 50.
- Succinyl-CoA can be converted into (R)-methylmalonyl-CoA by methylmalonyl-CoA mutase (MCM).
- MCM methylmalonyl-CoA mutase
- E. coli the reversible adenosylcobalamin-dependant mutase participates in a three- step pathway leading to the conversion of succinate to propionate (Haller, Biochemistry 39:4622-9 (2000)).
- MCM is encoded by genes scpA in Escherichia coli (Haller, Biochemistry 39: 4622-4629 (2000); and Bobik, Anal. Bioanal Chem. 375:344-349 (2003)) and mutA in Homo sapiens (Padovani, Biochemistry 45:9300-9306 (2006)).
- MCM contains alpha and beta subunits and is encoded by two genes.
- Exemplary gene candidates encoding the two-subunit protein are Propionibacterium fredenreichii sp. shermani mutA and mutB (Korotkova, J Biol Chem. 279:13652-13658 (2004)) and Methylobacterium extorquens mcmA and mcmB (Korotkova, J Biol Chem. 279: 13652-13658 (2004)). These genes/proteins are identified below in Table 51.
- M. extorquens forms a complex with methylmalonyl-CoA mutase, stimulates in vitro mutase activity, and possibly protects it from irreversible inactivation (Korotkova, J Biol Chem. 279: 13652-13658 (2004)).
- the M. extorquens meaB gene product is highly similar to the product of the E. coli argK gene (BLASTp: 45% identity, e-value: 4e-67) which is adjacent to scpA on the chromosome.
- methylmalonyl-CoA mutase gene on the chromosome are identified below in Table 53.
- Methylmalonyl-CoA Epimerase Methylmalonyl-CoA Epimerase
- Methylmalonyl- CoA epimerase is the enzyme that interconverts (R)-methylmalonyl- CoA and (S)-methylmalonyl-CoA.
- MMCE is an essential enzyme in the breakdown of odd- numbered fatty acids and of the amino acids valine, isoleucine, and methionine.
- Methylmalonyl- CoA epimerase is present in organisms such as Bacillus subtilis (YqjC) (Haller, Biochemistry. 39:4622-4629 (2000)), Homo sapiens (YqjC) (Fuller, Biochem.
- MMCE activity is required if the employed methylmalonyl-CoA decarboxylase or methylmalonyl- Co A carboxytransferase requires the (S) stereoisomer of methylmalonyl-CoA.
- Methylmalonyl-CoA decarboxylase is a biotin-independent enzyme that catalyzes the conversion of methylmalonyl-CoA to propionyl-CoA in E. coli (Benning, Biochemistry.
- PEP is converted into oxaloacetate either via PEP carboxykinase or PEP carboxylase as described in Example III.
- PEP is converted first to pyruvate by pyruvate kinase and then to oxaloacetate by methylmalonyl-CoA carboxytransferase or pyruvate carboxylase as described in Example III.
- Oxaloacetate is converted into threonine by the native threonine pathway engineered for high yields.
- 2- oxobutanoate is converted to propionaldehyde by a decarboxylase, which is then reduced to n- propanol by a propanol dehydrogenase.
- Threonine Deaminase The conversion of threonine to 2-oxobutanoate (or 2-ketobutyrate) can be accomplished by a threonine deaminase. It is encoded by one or more genes selected from ilvA (Calhoun et al., J. Biol. Chem. 248(10):3511-6, (1973)) and tdcB (Umbarger et al. , /. Bacteriol. 73(1):105-12, (1957); Datta et al., Proc. Natl. Acad. Sci. U S A 84(2): 393-7(1987)).
- RhodospiriUum rubrum represents an additional exemplary organism containing threonine deaminase (Feldberg et al., Eur. J. Biochem. 21(3): 438-46 (1971); U.S. Patent 5,958,745). Details for exemplary enzymes for carrying out this transformation are shown below. These genes/proteins are identified below in Table 56. Table 56.
- 2-oxobutanoate (2-ketobutyrate) can be converted to propionyl-CoA via a pyruvate formate lyase and a pyruvate formate lyase activating enzyme.
- the pyruvate formate lyase is encoded by gene selected from pflB and tdcE, while the pyruvate formate lyase activating enzyme is encoded by a pflA gene. Details for these exemplary genes for carrying out this transformation are already listed.
- 2-oxobutanoate can be converted to propionyl-CoA by means of pyruvate dehydrogenase, pyruvate ferredoxin oxidoreductase (PFOR), or any other enzyme with 2- ketoacid dehydrogenase functionality.
- PFOR pyruvate ferredoxin oxidoreductase
- Such enzymes are also capable of converting pyruvate to acetyl-CoA.
- Exemplary pyruvate dehydrogenase enzymes are present in E. coli (Bisswanger, H., J. Biol. Chem. 256:815-822 (1981); Bremer, J., Eur.J. Biochem. 8:535-540 (1969); Gong et al, J. Biol. Chem.
- Exemplary PFOR enzymes include, for example, the enzyme from Desulfovibrio africanus which has been cloned and expressed in E. coli, resulting in an active recombinant enzyme that was stable for several days in the presence of oxygen (Pieulle et al., J. Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is reported to be conferred by a 60 residue extension in the polypeptide chain of the D. africanus enzyme. The M.
- thermoacetica PFOR is also well characterized (Menon et al. Biochemistry 36:8484-8494 (1997)) and was shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui et al. J. Biol. Chem. 275:28494-28499 (2000)). Further, E. coli possesses an uncharacterized open reading frame, ydbK, that encodes a protein that is 51% identical to the M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E. coli has been described (Blaschkowski et al., Eur. J. Biochem. 123:563-569 (1982)).
- PFOR enzymes can be identified by the following GenBank accession and/or GI numbers as shown below. Several additional PFOR enzymes have been described (Ragsdale, Chem. Rev. 103:2333-2346 (2003)). These genes/proteins are identified below in Table 58.
- a keto acid decarboxylase can catalyze the conversion of 2-oxobutanoate to propionaldehyde.
- 2-keto acid decarboxylases have been identified. Enzyme candidates for this step are pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (4.1.1.7), alpha- ketoglutarate decarboxylase (EC 4.1.1.71), branched-chain alpha-keto-acid decarboxylase
- indolepyruvate decarboxylase (EC 4.1.1.74), and indolepyruvate decarboxylase (EC 4.1.1.74).
- decarboxylases are NADH-independent, they utilize thiamine diphosphate as a cofactor, and the interaction of the substrate with the enzyme-bound cofactor is thought to be the rate-limiting step for enzyme activation (Hubner, Eur. J Biochem. 92: 175-181 (1978)).
- Pyruvate decarboxylase and benzoylformate decarboxylase have broad substrate ranges for diverse keto-acids and have been characterized in structural detail. Fewer alpha-ketoglutarate and branched-chain alpha-ketoacid decarboxylases have been characterized experimentally; however these enzymes also appear to decarboxylate a variety of keto-acid substrates.
- Pyruvate decarboxylase also termed keto-acid decarboxylase
- the enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate (22).
- the PDC from Zymomonas mobilis, encoded by pdc has been a subject of directed engineering studies that altered the affinity for different substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)).
- the PDC from Saccharomyces cerevisiae has also been extensively studied, engineered for altered activity, and functionally expressed in E. coli (Li, Biochemistry.
- benzoylformate decarboxylase has a broad substrate range and has been the target of enzyme engineering studies.
- the enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Polovnikova et al, Biochemistry 42: 1820-1830 (2003); and Hasson et al., Biochemistry 37:9918-9930 (1998)).
- Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally and non- naturally occurring substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)).
- This enzyme has been further modified by directed engineering (Lingen et al., Chembiochem. 4:721-726 (2003); and Lingen, Protein Eng 15:585- 593 (2002)).
- Additional gene candidates from Pseudomonas stutzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using a growth selection system developed in Pseudomonas putida (Henning et al, Appl Environ. Microbiol. 72:7510-7517 (2006)). These genes/proteins are identified below in Table 60.
- a third enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD).
- the substrate range of this class of enzymes has not been studied to date.
- the KDC from Mycobacterium tuberculosis (Tian, Proc Natl Acad Sci U S. A 102: 10670-10675 (2005)) has been cloned and functionally expressed in other internal projects at Genomatica. However, it is not an ideal candidate for strain engineering because it is large (-130 kD) and GC-rich. KDC enzyme activity has been detected in several species of Rhizobia including Bradyrhizobium japonicum and Mesorhizobium loti (Green, J Bacteriol.
- KDC-encoding gene(s) have not been isolated in these organisms, the genome sequences are available and several genes in each genome are annotated as putative KDCs.
- a KDC from Euglena gracilis has also been characterized but the gene associated with this activity has not been identified to date (Shigeoka, Arch. Biochem. Biophys. 288:22-28 (1991)).
- the first twenty amino acids starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (Shigeoka, Arch. Biochem. Biophys. 288:22-28 (1991)).
- the gene could be identified by testing candidate genes containing this N-terminal sequence for KDC activity.
- a fourth candidate enzyme for catalyzing this step is branched chain alpha-ketoacid
- BCKA decarboxylase
- Lactococcus lactis has been characterized on a variety of branched and linear substrates including 2-oxobutanoate, 2- oxohexanoate, 2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate (Smit et al., Appl Environ Microbiol 71 :303-311 (2005)).
- the enzyme has been structurally characterized (Berthold et al., Acta Crystallogr. D Biol Crystallogr. 63:1217-1224 (2007)).
- IPDA Indolepyruvate decarboxylase
- Recombinant branched chain alpha-keto acid decarboxylase enzymes derived from the El subunits of the mitochondrial branched-chain keto acid dehydrogenase complex from Homo sapiens and Bos taurus have been cloned and functionally expressed in E. coli (Wynn, J. Biol. Chem. 267: 12400-12403 (1992); Davie, J. Biol. Chem. 267: 16601-16606 (1992) and Wynn et al., J. Biol. Chem. 267: 1881-1887 (1992)).
- ACC acetyl-CoA carboxylase complex
- E. coli Feiberg et al., . Biol. Chem. 279: 26066-26073 (2004)
- yeast Zhang, Proc. Natl. Acad. Sci. U S. A 101 :5910-5915 (2004)
- Bacillus subtilis Feiberg et al., J. Biol. Chem.
- ACC is composed of four subunits encoded by accA, accB, accC and accD (Choi- Rhee, /. Biol. Chem. 278:30806-30812 (2003)). Expression of two subunits, accB and accC, is autoregulated by the gene product of accB (James, J. Biol. Chem. 279:2520-2527 (2004)). In yeast, the enzyme is encoded by two genes, hfal and accl.
- the gene bpll encoding a biotin: apoprotein ligase, is required for enzyme function.
- Metallosphaera sedula the acyl-CoA carboxylase holoenzyme is a multimer composed of subunits encoded by three genes: Msed_0148 (biotin/lipoyl attachment), Msed_0147 (biotin carboxylase), and Msed_1375 (carboxyl transferase).
- the enzyme has been purified and characterized and was found to be bifunctional, reacting with acetyl-CoA and propionyl-CoA (Hugler, Eur. J. Biochem. 270:736-744 (2003)).
- malonyl-CoA Reductase and Malonate Semialdehyde Reductase The reduction of malonyl-CoA to 3-HP can be accomplished by a bifunctional malonyl-CoA reductase with aldehyde dehydrogenase and alcohol dehydrogenase functionality.
- An NADPH- dependent enzyme with this activity has been characterized in Chloroflexus aurantiacus where it participates in the 3-hydroxypropionate cycle (Hugler, J. Bacteriol. 184:2404-2410 (2002); and Strauss, Eur. J. Biochem. 215:633-643 (1993)).
- This enzyme with a mass of 300 kDa, is highly substrate- specific and shows little sequence similarity to other known oxidoreductases (Hugler, /. Bacteriol. 184:2404-2410 (2002)). No enzymes in other organisms have been shown to catalyze this specific reaction; however there is bioinformatic evidence that other organisms may have similar pathways (Klatt, Environ. Microbiol. 9:2067-2078 (2007). Enzyme candidates in other organisms including Roseiflexus castenholzii, Erythrobacter sp. NAPl and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity. These genes/proteins are identified below in Table 65.
- malonyl-CoA is first reduced to malonate semialdehyde (MSA) by malonate-semialdehyde dehydrogenase or malonyl-CoA reductase. MSA is subsequently converted to 3-HP by 3-HP- dehydrogenase.
- Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3- hydroxypropionate cycle in thermoacidophilic archael bacteria (Berg, Science. 318:1782-1786 (2007); and Thauer, Science. 318: 1732-1733 (2007)).
- the enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et a/, J.
- malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius. These genes/proteins are identified below in Table 66.
- the subsequent conversion of malonic semialdehyde to 3-HP can be accomplished by enzyme with 3-HP dehydrogenase activity.
- Three enzymes are known to catalyze this conversion: NADH-dependent 3-hydroxypropionate dehydrogenase, NADPH-dependent malonate semialdehyde reductase, and NADH-dependent 3-hydroxyisobutyrate dehydrogenases.
- An NADH-dependent 3-hydroxypropionate dehydrogenase is thought to participate in beta- alanine biosynthesis pathways from propionate in bacteria and plants (Rathinasabapathi, Journal of Plant Pathology 159:671-674 (2002); and Stadtman, A. J. Am. Chem. Soc. 77:5765-5766 (1955)).
- dehydrogenase activity in Alcaligenes faecalis M3A has also been identified (Liao, U.S. Patent Publication 2005-0221466 (2005); and Liao, U.S. Patent Publcation 2005-0221466 (2005)). Additional gene candidates from other organisms including Rhodobacter spaeroides can be inferred by sequence similarity. These genes/proteins are identified below in Table 67.
- Enzymes exhibiting a 4-hydroxybutyrate activity may also be able to convert malonic semialdehyde to 3-HP, as the chemical transformation is very similar.
- Such enzymes have been characterized in Ralstonia eutropha (Bravo, / Forensic Sci. 49:379-387 (2004)), Clostridium kluyveri (Wolff, Protein Expr. Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitnch et al., J. Biol. Chem. 278:41552-41556 (2003)).
- Activity of these enzymes on malonic semialdehyde has not been demonstrated experimentally to date. However, since these enzymes have been studied in other internal projects at Genomatica they could easily be tested for 3-HP dehydrogenase activity.
- These genes/proteins are identified below in Table 68.
- Propionyl-CoA Synthase The conversion of 3-hydroxypropionate (3HP) to propionyl-CoA is accomplished by a propionyl-CoA synthase. This step is known to be catalyzed by a single fusion protein of 201 KDa in Chloroflexus aurantiacus (Alber, J Biol. Chem. 277:12137-12143 (2002)).
- the protein is comprised of a CoA ligase, an enoyl-CoA hydratase and an enoyl-CoA reductase.
- the enzyme has been purified 30-fold to near homogeneity and has a veiy large native molecular mass between 500 and 800 kDa.
- thermoacidophilic Metallosphaera sedula (and members of the Sulfolobaceae family), this function is catalyzed by three different enzymes, a 3- hydroxypropionyl-CoA synthetase that activates 3HP to its CoA ester, a 3-hydroxypropionyl- CoA dehydratase that converts 3 -HP- CoA to aery loyl- CoA followed by the reduction of the latter to form propionyl-CoA.
- a 3-HP-CoA synthetase had been reported (Alber, / Bacteriol. 190: 1383-1389 (2008)).
- the gene encoding the protein has been sequenced and gene encoding a homologous protein identified in the genome of Sulfolobus tokodaii; similar genes were found in S. solfataricus and S. acidocaldarius. The gene was heterologously expressed in Escherichia coli. These genes/proteins are identified below in Table 69.
- lactate dehydrogenase EC 1.1.1.27
- lactate dehydrogenases Many lactate dehydrogenases have been described in detail (Garvie, Microbiol Rev 44: 106- 139
- the fermentative lactate dehydrogenase of Escherichia coli will be the first candidate to be overexpressed for converting pyruvate to lactate (Bunch, Microbiology 143 ( Pt 1), 187- 195 (1997)).
- Other lactate dehydrogenase candidates will be utilized for this step including those with low Km for pymvate that favors the formation of lactate, such as lactate
- Plasmodium falciparum (Brown et al., Biochemistry 43:6219-6229 (2004)), and Thermotoga maritime (Auerbach et al., Structure. 6:769-781 (1998)). These genes/proteins are identified below in Table 72.
- lactate-CoA transferase activity associated with propionate CoA-transferase (EC 2.8.3.1).
- Clostridium propionicum ferments alanine via the nonrandomising pathway with acryloyl-CoA as characteristic intermediate.
- lactate is activated to lactoyl-CoA by the enzyme propionate:acetyl-CoA CoA- transferase (EC 2.8.3.1, or propionate CoA-transferase) using propionyl-CoA or acetyl-CoA as a coenzyme A donor (Schweiger, FEBS Lett. 171 :79-84 (1984)).
- the enzyme exhibited rather broad substrate specificities for monocarboxylic acids including acrylate, propionate and butyrate whereas dicarboxylic acids were not used. Gene coding for this enzyme was cloned (Selmer, Eur. J Biochem. 269:372-380 (2002)). Other propionate CoA-transferase can be candidates for this step include homologues of Clostridium propionicum propionate CoA- transferase. These genes/proteins are identified below in Table 73.
- lactoyl-CoA dehydratase EC 4.2.1.54
- Clostridium propionicum ferments alanine via the nonrandomising pathway with acryloyl-CoA as characteristic intermediate (Schweiger, FEBS Lett. 171:79-84 (1984)).
- lactoyl-CoA is dehydrated to acryloyl-CoA by the lactoyl-CoA dehydratase
- acryloyl-CoA reductase catalyzes the irreversible NADH-dependent formation of propionyl-CoA from acryloyl-CoA.
- the enzyme has been purified and the N-termini of the subunits of the enzyme have been determined (Hetzel et al., Eur. J Biochem. 270:902-910 (2003)).
- the N-terminus of the dimeric propionyl-CoA dehydsrogenase subunit is similar to those of butyryl-CoA dehydrogenases from several Clostridia and related anaerobes (up to 55% sequence identity).
- the N-termini of the ⁇ and ⁇ subunits share 40% and 35% sequence identities with those of the A and B subunits of the electron-transferring flavoprotein (ETF) from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes.
- ETF electron-transferring flavoprotein
- the propionyl-CoA synthase is a natural fusion protein of 201 kDa consisting of a CoA ligase, an enoyl-CoA hydratase, and an enoyl-CoA reductase.
- the enzyme catalyzes the conversion from 3-hydroxypropionate to 3-hydroxypropionyl-CoA to acryloyl- CoA then to propionyl-CoA. This enzyme can be utilized in whole or in part for its enoyl-CoA reductase activity.
- the gene/protein is identifed below in Table 76.
- This example describes exemplary pathways for co-production of 1,4-butanediol (1,4-BDO) and isopropanol. Novel pathways for co-producing 1,4-butanediol (1,4-BDO) and isopropanol and related products are described herein.
- central metabolism intermediates are first channeled into succinyl-CoA.
- succinyl-CoA phosphoenolpyruvate (PEP) is converted into oxaloacetate either via PEP carboxykinase or PEP carboxylase.
- PEP is converted first to pyruvate by pyruvate kinase and then to oxaloacetate by methylmalonyl-CoA carboxytransferase or pyruvate carboxylase.
- Oxaloacetate is then converted to succinyl-CoA by means of the reductive TCA cycle.
- Succinyl-CoA is then converted to succinic semialdehyde by a CoA- dependent aldehyde dehydrogenase.
- succinate can be converted to succinic semialdehyde by a succinate reductase.
- succinic semialdehyde is reduced to 4- hydroxybutyrate by 4-hydroxybutyrate dehydrogenase.
- 4-HB Activation of 4-HB to its acyl-CoA is catalyzed by a CoA transferase or synthetase.
- 4-HB can be converted into 4- hydroxybutyryl-phosphate and subsequently transformed into 4-HB-CoA by a phosphotrans-4- hydroxybutyrylase.
- 4-HB -CoA is then converted to 14-BDO by either a bifunctional CoA- dependent aldehyde/alcohol dehydrogenase, or by two separate enzymes with aldehyde and alcohol dehydrogenase activity.
- This example describes exemplary pathways for co-production of 1,3-butanediol (13-BDO) and isopropanol. Novel pathways for co-producing 1,3-butanediol (13-BDO) and isopropanol and related products are described herein.
- the coproduction route to 1,3-butanediol (13-BDO) and isopropanol, shown in Figure 6, also proceeds through 4-hydroxybutyryl-CoA, formed as described in Example VI. In this route, 4-hydroxybutyryl-CoA is dehydrated and isomerized to form crotonyl-CoA.
- Succinate can be converted to succinic semialdehyde by a carboxylic acid reductase, bypassing the formation of succinyl-CoA.
- 4-HB can be phosphorylated to 4-HB- phosphate by a kinase, then subsequently converted to 4-HB-CoA.
- 3-hydroxybutyryl- CoA can be de-acylated by a CoA hydrolase, transferase or synthetase, then subsequently reduced to 3-hydroxybutyraldehyde by a carboxylic acid reductase.
- Pathways for production of isopropanol proceed as described above in Examples I and II.
- This example describes exemplary pathways for co-production of methylacrylic acid (MAA) and isopropanol. Novel pathways for co-producing methylacrylic acid (MAA) and isopropanol and related products are described herein.
- Two coproduction routes to methylacrylic acid (MAA) are shown in Figures 7 and 8.
- the route shown in Figure 7 proceeds through 4-hydroxybutyryl-CoA, formed as described previously.
- 4-Hydroxybutyryl-CoA is converted to 3-hydroxyisobutyryl- CoA by a methyl mutase.
- the CoA moiety of 3-Hydroxyisobutyryl-CoA is then removed by a CoA transferase, hydrolase or synthetase.
- succinyl-CoA is formed through the reductive TCA cycle, then converted to methylmalonyl-CoA by methylmalonyl-CoA mutase.
- An epimerase may be required to convert the (R) stereoisomer of methylmalonyl-CoA to the (S) configuration.
- a CoA-dependent aldehyde dehydrogenase then converts
- methylmalonyl-CoA to methylmalonate semialdehyde. Reduction of the aldehyde to 3- hydroxyisobutyrate, followed by dehydration, yields MAA. Alternately, methylmalonyl-CoA is converted to 3-hydroxyisobutyrate by an alcohol-forming CoA reductase. In yet another alternate route, methylmalonyl-CoA is converted to methylmalonate by a CoA hydrolase, transferase or synthetase. Methylmalonate is subsequently converted to methylmalonate semialdehyde by a carboxylic acid reductase. Methylmalonate semialdehyde is converted to MAA as described previously.
- 1,3-Butanediol (1,3-BDO) or Methylacrylic acid (MAA) This example describes the enzyme classification system for the exemplary pathways described in Examples VII and IX for production of 1,4-butanediol (1,4-BDO), 1,3-butanediol (1,3-BDO) or methylacrylic acid (MAA).
- Exemplary enzymes for production of isopropanol from acetyl- CoA are described in Example I and exemplary enzymes for production acetyl-CoA from glucose are described in Example II.
- PEP carboxykinase which simultaneously forms an ATP while carboxylating PEP.
- PEP carboxykinase serves a gluconeogenic function and converts oxaloacetate to PEP at the expense of one ATP.
- S. cerevisiae is one such organism whose native PEP carboxykinase, PCK1, serves a gluconeogenic role (Valdes-Hevia et al., FEBS. Lett.
- E. coli is another such organism, as the role of PEP carboxykinase in producing oxaloacetate is believed to be minor when compared to PEP carboxylase, which does not form ATP, possibly due to the higher K m for bicarbonate of PEP carboxykinase (Kim, et al., Appl Environ Microbiol 70:1238-1241 (2004)). Nevertheless, activity of the native E. coli PEP carboxykinase from PEP towards oxaloacetate has been recently demonstrated in ppc mutants of E. coli K-12 (Kwon et al., Journal of Microbiology and Biotechnology 16: 1448-1452 (2006)).
- PEP carboxykinase is quite efficient in producing oxaloacetate from PEP and generating ATP.
- PEP carboxykinase genes that have been cloned into E. coli include those from Mannheimia succiniciproducens (Lee et al., Gene. Biotechnol. Bioprocess Eng. 7:95-99 (2002)),
- sequences and sequences for subsequent enzymes listed in this report can be used to identify homologue proteins in GenBank or other databases through sequence similarity searches (e.g. BLASTp).
- sequence similarity searches e.g. BLASTp.
- the resulting homologue proteins and their corresponding gene sequences provide additional DNA sequences for transformation into the host organism of our choice.
- PEP Carboxylase PEP carboxylase represents an alternative enzyme for the formation of oxaloacetate from PEP.
- S. cerevisiae does not naturally encode a PEP carboxylase, but exemplary organisms that possess genes that encode PEP carboxylase include E. coli (Kai et al., Arch. BioChem. Biophys. 414: 170-179 (2003)), Methylobacterium extorquens AMI (Arps et al., J. Bacteriol. 175:3776- 3783 (1993)), and Corynebacterium glutamicum (Eikmanns et al, Mol. Gen. Genet. 218:330- 339 (1989)). These genes/proteins are identified below in Table 78.
- Pyruvate kinase catalyzes the ATP-generating conversion of PEP to pyruvate and is encoded by the PYK1 (Burke et al., J. Biol. Chem. 258:2193-2201 (1983)) and PYK2 (Boles et al., J. Bacteriol.
- methylmalonyl-CoA carboxytransferase which is comprised of 1.3S, 5S, and 12S subunits can be found in Propionibacteriumfreudenreichii (Thornton et al., J. Bacteriol. 175:5301-5308 (1993)). These genes/proteins are identified below in Table 79.
- a combination of enzymes can convert PEP to oxaloacetate with a stoichiometry identical to that of PEP carboxylase.
- These enzymes are encoded by pyruvate kinase, PYK1 (Burke et al., J. Biol. Chem. 258:2193-2201 (1983)) or PYK2 (Boles et al., J. Bacteriol. 179:2987-2993 (1997)), and pyruvate carboxylase, PYC1 (Walker et al., BioChem. Biophys. Res. Commun. 176:1210- 1217 (1991)) or PYC2 (224).
- Some candidates for pyruvate carboxylase function are identified below in Table 80.
- Oxaloacetate can be converted to succinate by malate dehydrogenase, fumarase and fumarate reductase when the TCA cycle is operating in the reductive cycle.
- S. cerevisiae possesses three copies of malate dehydrogenase, MDH1 (McAlister-Henn et al., J. Bacteriol. 169:5157-5166 (1987)), MDH2 (Gibson J. Biol. Chem. 278:25628-25636 (2003); and Minard et al., Mol. Cell Biol. 11:370-380 (1991)), and MDH3 (Steffan et al., /. Biol. Chem.
- S. cerevisiae contains one copy of a fumarase-encoding gene, FUM1, whose product localizes to both the cytosol and mitochondrion (Sass et al., J. Biol. Chem. 278:45109-45116 (2003)).
- Fumarate reductase is encoded by two soluble enzymes, FRDS1 (Enomoto et al., DNA. Res. 3:263-267 (1996)) and FRDS2 (Muratsubaki et al., Arch. BioChem. Biophys.
- E. coli is known to have an active malate dehydrogenase. It has three fumarases encoded by fumA, B and C, each one of which is active under different conditions of oxygen availability. The fumarate reductase in E. coli is composed of four subunits. These genes/proteins are identified below in Table 81.
- Succinyl-CoA transferase catalyzes the conversion of succinyl-CoA to succinate while transferring the CoA moiety to a CoA acceptor molecule.
- Many transferases have broad specificity and thus may utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3- mercaptopropionate, propionate, vinylacetate, butyrate, among others.
- the conversion of succinate to succinyl-CoA is ideally carried by a transferase which does not require the direct consumption of an ATP or GTP.
- succinyl-CoA:3- ketoacid-CoA transferase This enzyme converts succinate to succinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid.
- Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein. Expr. Purif.
- succinyl-CoA Acetyl- CoA transferase.
- the gene product of call of Clostridium kluyveri has been shown to exhibit succinyl-CoA: acetyl-CoA transferase activity (Sohling et al., J Bacteriol. 178:871-880 (1996)).
- the activity is present in Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem. 279:45337- 45346 (2004)).
- These genes/proteins are identified below in Table 83.
- CoA acceptor is benzylsuccinate.
- Succinyl-CoA:(R)-Benzylsuccinate CoA- Transferase functions as part of an anaerobic degradation pathway for toluene in organisms such as Thauera aromatica (Leutwein and Heider, J. Bact. 183(14) 4288-4295 (2001)).
- Homologs can be found in Azoarcus sp. T, Aromatoleum aromaticum EbNl, and Geobacter
- ygfli encodes a propionyl CoA:succinate CoA transferase in E. coli (Haller et al., Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for example, Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909. These genes/proteins are identified below in Table 85.
- the product of the LSC1 and LSC2 genes of S. cerevisiae and the sucC and sucD genes of E. coli naturally form a succinyl-CoA synthetase complex that catalyzes the formation of succinyl- CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Bravo et al., /. Forensic Sci. 49:379-387 (2004)).
- These genes/proteins are identified below in Table 86.
- Pyruvate formate lyase is an enzyme that catalyzes the conversion of pyruvate and CoA into acetyl-CoA and formate.
- Pyruvate formate lyase is a common enzyme in prokaryotic organisms that is used to help modulate anaerobic redox balance. Exemplary enzymes can be found in Escherichia coli (Knappe et al., FEMS. Microbiol Rev.
- a formate hydrogen lyase enzyme can be employed to convert formate to carbon dioxide and hydrogen.
- An exemplary formate hydrogen lyase enzyme can be found in Escherichia coli.
- the E. coli formate hydrogen lyase consists of hydrogenase 3 and formate dehydrogenase-H (Maeda et al., Appl Microbiol Biotechnol 77:879-890 (2007)). It is activated by the gene product ⁇ (Maeda et al., Appl Microbiol Biotechnol 77:879-890 (2007)).
- a formate hydrogen lyase enzyme also exists in the hyperthermophilic archaeon, Thermococcus litoralis (Takacs et al., Microbiol 8:88 2008)). These genes/proteins are identified below in Table 89.
- Formate dehydrogenase activity is present in both E. coli and Saccharomyces cerevisiae among other organisms.
- S. cerevisiae contains two formate dehydrogenases, FDH1 and FDH2, that catalyze the oxidation of formate to C0 2 (Overkamp et al., Yeast 19:509-520 (2002)).
- FDH1 and FDH2 formate dehydrogenases
- Moth_2312 and Moth_2313 are actually one gene that is responsible for encoding the alpha subunit of formate dehydrogenase while the beta subunit is encoded by Moth_2314 (Andreesen et al., J. Bacteriol. 116:867-873 (1973); Li et al., J.
- Sfum_2703 Another set of genes encoding formate dehydrogenase activity is encoded by Sfum_2703 through Sfum_2706 in Syntrophobacter fumaroxidans (de Bok, et al., Eur. J. BioChem. 270:2476-2485 (2003); and Reda et al., Proc. Natl. Acad. Sci. U S. A. 105: 10654-10658 (2008)). Similar to their M. thermoacetica counterparts, Sfum_2705 and Sfum_2706 are actually one gene. E. coli contains multiple formate dehydrogenases. These genes/proteins are identified below in Table 90.
- the pyruvate dehydrogenase complex catalyzing the conversion of pyruvate to acetyl-CoA, has been extensively studied.
- the S. cerevisiae complex consists of an E2 (LATl) core that binds El (PDA1, PDB1), E3 (LPD1), and Protein X (PDX1) components (Pronk et al., Yeast 12: 1607- 1633 (1996)).
- E. coli enzyme specific residues in the El component are responsible for substrate specificity (Bisswanger / Z o/ Chem. 256:815-822. (1981); Bremer / BioChem.
- Some maMAAlian PDH enzymes complexes can react on alternate substrates such as 2-oxobutanoate (Paxton et al., BioChem. J. 234:295-303 (1986)). These genes/proteins are identified below in Table 91. Table 91.
- PFOR Pyruvate ferredoxin oxidoreductase catalyzes the oxidation of pyruvate to form acetyl- CoA.
- the PFOR from Desulfovibrio africanus has been cloned and expressed in E. coli resulting in an active recombinant enzyme that was stable for several days in the presence of oxygen (Pieulle et al., J Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is believed to be conferred by a 60 residue extension in the polypeptide chain of the D. africanus enzyme. The M.
- thermoacetica PFOR is also well characterized (Menon et al., Biochemistry 36:8484-8494 (1997)) and was even shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui et al., J Biol Chem. 275:28494-28499 (2000)). Further, E. coli possesses an uncharacterized open reading frame, ydbK, encoding a protein that is 51% identical to the M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E. coli has been described (Blaschkowski et al., / BioChem. 123:563-569 (1982)).
- Succinic semialdehyde dehydrogenase (CoA-dependent), also referred to as succinyl-CoA reductase, is a CoA- and NAD(P)H- dependent oxidoreductase that reduces succinyl-CoA to its corresponding aldehyde.
- Exemplary enzymes are encoded by the sucD gene in Clostridium kluyveri (Sohling et al., J Bacteriol 178:871-80 (1996); and Sohling et al., J Bacteriol. 178:871- 880 (1996)) and the sucD gene of P. gingivalis (Takahashi et al., J. Bacteriol.
- acrl YP_047869.1 50086359 Acinetobacter calcoaceticus acrl AAC45217 1684886 Acinetobacter baylyi
- 4-Hydroxybutyrate dehydrogenase catalyzes the NAD(P)H dependent reduction of succinic semialdehyde to 4-HB. Enzymes exhibiting this activity are found in Ralstonia eutropha (Bravo et al., J. Forensic Sci. 49:379-387 (2004)), Clostridium kluyveri (Wolff et al., Protein Expr. Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitnch et al.,. J. Biol. Chem. 278:41552- 41556 (2003)).
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
MX2012003025A MX2012003025A (en) | 2009-09-09 | 2010-09-09 | Microorganisms and methods for the co-production of isopropanol with primary alcohols, diols and acids. |
BR112012005296A BR112012005296A2 (en) | 2009-09-09 | 2010-09-09 | microorganisms and methods for the co-production of isopropanol with primary alcohols, dios and acids. |
JP2012528907A JP2013504326A (en) | 2009-09-09 | 2010-09-09 | Microorganisms and methods for the co-production of isopropanol and primary alcohols, diols and acids |
SG2012016408A SG179048A1 (en) | 2009-09-09 | 2010-09-09 | Microorganisms and methods for the co-production of isopropanol with primary alcohols, diols and acids |
EP10816105A EP2475775A4 (en) | 2009-09-09 | 2010-09-09 | Microorganisms and methods for the co-production of isopropanol with primary alcohols, diols and acids |
CN2010800504376A CN102625845A (en) | 2009-09-09 | 2010-09-09 | Microorganisms and methods for the co-production of isopropanol with primary alcohols, diols and acids |
CA2773694A CA2773694A1 (en) | 2009-09-09 | 2010-09-09 | Microorganisms and methods for the co-production of isopropanol with primary alcohols, diols and acids |
ZA2012/01468A ZA201201468B (en) | 2009-09-09 | 2012-02-28 | Microorganisms and methods for the co-production of isopropanol with primary alcohols, diols and acids |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US24095909P | 2009-09-09 | 2009-09-09 | |
US61/240,959 | 2009-09-09 | ||
US25465009P | 2009-10-23 | 2009-10-23 | |
US61/254,650 | 2009-10-23 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2011031897A1 true WO2011031897A1 (en) | 2011-03-17 |
Family
ID=43732800
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2010/048318 WO2011031897A1 (en) | 2009-09-09 | 2010-09-09 | Microorganisms and methods for the co-production of isopropanol with primary alcohols, diols and acids |
Country Status (11)
Country | Link |
---|---|
US (2) | US8715971B2 (en) |
EP (2) | EP2933338A3 (en) |
JP (1) | JP2013504326A (en) |
KR (1) | KR20120068021A (en) |
CN (2) | CN102625845A (en) |
BR (1) | BR112012005296A2 (en) |
CA (1) | CA2773694A1 (en) |
MX (1) | MX2012003025A (en) |
SG (1) | SG179048A1 (en) |
WO (1) | WO2011031897A1 (en) |
ZA (2) | ZA201201468B (en) |
Cited By (37)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012058603A1 (en) | 2010-10-29 | 2012-05-03 | Novozymes A/S | Recombinant n-propanol and isopropanol production |
WO2012080421A1 (en) * | 2010-12-17 | 2012-06-21 | Total Petrochemicals Research Feluy | Process for producing propylene from syngas via fermentative propanol production and dehydration |
WO2012177599A2 (en) * | 2011-06-22 | 2012-12-27 | Genomatica, Inc. | Microorganisms for producing n-propanol 1, 3-propanediol, 1,2-propanediol or glycerol and methods related thereto |
WO2013028519A1 (en) | 2011-08-19 | 2013-02-28 | Genomatica, Inc. | Microorganisms and methods for producing 2,4-pentadienoate, butadiene, propylene, 1,3-butanediol and related alcohols |
JP2013042701A (en) * | 2011-08-24 | 2013-03-04 | Hiroshima Univ | Microorganism manipulated to produce 2-propanol |
WO2013178699A1 (en) | 2012-05-31 | 2013-12-05 | Novozymes A/S | Isopropanol production by bacterial hosts |
WO2014038216A1 (en) | 2012-09-10 | 2014-03-13 | 三菱レイヨン株式会社 | Method for producing methacrylic acid and/or ester thereof |
WO2014038214A1 (en) | 2012-09-10 | 2014-03-13 | 三菱レイヨン株式会社 | Method for producing methacrylic acid ester |
WO2014068010A1 (en) | 2012-10-31 | 2014-05-08 | Novozymes A/S | Isopropanol production by bacterial hosts |
US8728804B2 (en) | 2010-10-29 | 2014-05-20 | Novozymes A/S | Polypeptides having succinyl-CoA: acetoacetate transferase activity and polynucleotides encoding same |
WO2014076232A2 (en) | 2012-11-19 | 2014-05-22 | Novozymes A/S | Isopropanol production by recombinant hosts using an hmg-coa intermediate |
EP2743352A1 (en) * | 2011-08-11 | 2014-06-18 | Mitsui Chemicals, Inc. | Method for producing isopropyl alcohol by continuous culture |
CN103930558A (en) * | 2011-04-01 | 2014-07-16 | 基因组股份公司 | Microorganisms for producing methacrylic acid and methacrylate esters and methods related thereto |
WO2014152434A2 (en) | 2013-03-15 | 2014-09-25 | Genomatica, Inc. | Microorganisms and methods for producing butadiene and related compounds by formate assimilation |
JP5628288B2 (en) * | 2010-03-09 | 2014-11-19 | 三井化学株式会社 | Highly productive isopropyl alcohol-producing bacteria |
WO2014160846A3 (en) * | 2013-03-28 | 2014-12-04 | The Procter & Gamble Company | Microorganisms and methods for producing propionic acid |
CN104284974A (en) * | 2012-03-06 | 2015-01-14 | 利戈斯股份有限公司 | Recombinant host cells for the production of malonate |
WO2015015784A1 (en) | 2013-08-01 | 2015-02-05 | 三菱レイヨン株式会社 | METHOD FOR PRODUCING METHACRYLYL-CoA |
WO2015084633A1 (en) | 2013-12-03 | 2015-06-11 | Genomatica, Inc. | Microorganisms and methods for improving product yields on methanol using acetyl-coa synthesis |
EP2734627A4 (en) * | 2011-07-20 | 2015-07-22 | Genomatica Inc | Methods for increasing product yields |
WO2015158716A1 (en) | 2014-04-16 | 2015-10-22 | Novamont S.P.A. | Process for the production of 1,4-butanediol |
WO2016044713A1 (en) | 2014-09-18 | 2016-03-24 | Genomatica, Inc. | Non-natural microbial organisms with improved energetic efficiency |
WO2016100910A1 (en) * | 2014-12-19 | 2016-06-23 | Novozymes A/S | Recombinant host cells for the production of 3-hydroxypropionic acid |
KR20160108546A (en) | 2014-03-07 | 2016-09-19 | 미쯔비시 레이온 가부시끼가이샤 | Method for producing methacrylic acid ester, and novel methacrylic acid ester synthase |
WO2016196233A1 (en) | 2015-05-30 | 2016-12-08 | Genomatica, Inc. | Vinylisomerase-dehydratases, alkenol dehydratases, linalool dehydratases and/ crotyl alcohol dehydratases and methods for making and using them |
EP3004362A4 (en) * | 2013-06-05 | 2017-01-11 | Lanzatech New Zealand Limited | Recombinant microorganisms exhibiting increased flux through a fermentation pathway |
WO2017075208A1 (en) | 2015-10-30 | 2017-05-04 | Genomatica, Inc. | Methanol dehydrogenase fusion proteins |
US9909150B2 (en) | 2012-11-05 | 2018-03-06 | Genomatica, Inc. | Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 1,2-propanediol, n-propanol, 1,3-propanediol, or glycerol related thereto |
KR101860442B1 (en) * | 2011-06-27 | 2018-05-24 | 삼성전자주식회사 | Genetic Modification for Production of 3-hydroxypropionic acid |
WO2019102030A1 (en) | 2017-11-27 | 2019-05-31 | Novamont S.P.A. | Process for the production of 1,4-butanediol from renewable sources and polyesters obtained therefrom |
WO2019152375A1 (en) | 2018-01-30 | 2019-08-08 | Genomatica, Inc. | Fermentation systems and methods with substantially uniform volumetric uptake rate of a reactive gaseous component |
WO2020006058A2 (en) | 2018-06-26 | 2020-01-02 | Genomatica, Inc. | Engineered microorganisms with g3p---> 3pg enzyme and/or fructose-1,6-bisphosphatase including those having synthetic or enhanced methylotrophy |
US10597684B2 (en) | 2013-12-27 | 2020-03-24 | Genomatica, Inc. | Methods and organisms with increased carbon flux efficiencies |
US10676766B2 (en) | 2015-10-23 | 2020-06-09 | The Regents Of The University Of California | Biological production of methyl methacrylate |
US10829789B2 (en) | 2016-12-21 | 2020-11-10 | Creatus Biosciences Inc. | Methods and organism with increased xylose uptake |
WO2021245228A2 (en) | 2020-06-04 | 2021-12-09 | Novamont S.P.A. | Process for purifying a mixture of diols |
IT202100030572A1 (en) | 2021-12-02 | 2023-06-02 | Novamont Spa | 1,3-BUTANDIOL PURIFIED FROM A MIXTURE OF DIOLS |
Families Citing this family (40)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2304039B1 (en) | 2008-06-17 | 2019-08-21 | Genomatica, Inc. | Microorganisms and methods for the biosynthesis of fumarate, malate, and acrylate |
AU2009291825B2 (en) | 2008-09-10 | 2016-05-05 | Genomatica, Inc. | Microorganisms for the production of 1,4-butanediol |
EP2373781A4 (en) | 2008-12-16 | 2012-10-10 | Genomatica Inc | Microorganisms and methods for conversion of syngas and other carbon sources to useful products |
VN29235A1 (en) | 2009-06-04 | 2012-03-26 | ||
US8420375B2 (en) * | 2009-06-10 | 2013-04-16 | Genomatica, Inc. | Microorganisms and methods for carbon-efficient biosynthesis of MEK and 2-butanol |
CN102762735B (en) | 2009-10-13 | 2016-08-03 | 基因组股份公司 | Produce 1,4-butanediol, 4-hydroxybutyraldehyde, 4-maloyl group-COA, putrescine and the microorganism of related compound and correlation technique thereof |
SG181607A1 (en) | 2009-12-10 | 2012-07-30 | Genomatica Inc | Methods and organisms for converting synthesis gas or other gaseous carbon sources and methanol to 1,3-butanediol |
US8445244B2 (en) | 2010-02-23 | 2013-05-21 | Genomatica, Inc. | Methods for increasing product yields |
US8372610B2 (en) | 2010-09-15 | 2013-02-12 | Ls9, Inc. | Production of odd chain fatty acid derivatives in recombinant microbial cells |
AU2012272856A1 (en) | 2011-06-22 | 2013-05-02 | Genomatica, Inc. | Microorganisms for producing 1,4-butanediol and methods related thereto |
CA2846234A1 (en) * | 2011-08-22 | 2013-02-28 | Suganit Systems, Inc. | Production of bio-butanol and related products |
CN103082292B (en) * | 2011-11-02 | 2015-03-04 | 深圳华大基因研究院 | Use of Roseburia for the treatment and prevention of obesity-related diseases |
JP6415326B2 (en) * | 2011-12-16 | 2018-10-31 | ブラスケム エス.エー. | Modified microorganism and method for producing butadiene using the same |
WO2013126855A1 (en) * | 2012-02-23 | 2013-08-29 | The Regents Of The University Of California | Atp driven direct photosynthetic production of fuels and chemicals |
CN104508136A (en) * | 2012-05-30 | 2015-04-08 | 新西兰郎泽科技公司 | Recombinant microorganisms and uses therefor |
CN104685058B (en) | 2012-06-04 | 2020-07-07 | 基因组股份公司 | Microorganisms and methods for making 4-hydroxybutyrate, 1, 4-butanediol, and related compounds |
WO2014004625A1 (en) * | 2012-06-26 | 2014-01-03 | Genomatica, Inc. | Microorganisms for producing ethylene glycol using synthesis gas |
EP2909325A4 (en) * | 2012-10-22 | 2016-05-25 | Genomatica Inc | Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing succinate related thereto |
CN104797713A (en) * | 2012-11-27 | 2015-07-22 | 昭和电工株式会社 | Method for producing 1,4-butanediol, and microorganism |
US9650651B2 (en) * | 2013-03-14 | 2017-05-16 | Rathin Datta | Method for production of n-propanol and other C3-containing products from syngas by symbiotic co-cultures of anaerobic microorganisms |
EP2970068B1 (en) | 2013-03-15 | 2021-07-28 | Genomatica, Inc. | Process and systems for obtaining 1,4-butanediol from fermentation broths |
KR102113254B1 (en) | 2013-08-23 | 2020-05-21 | 삼성전자주식회사 | A screening method of identification of gene for 1,4-BDO production |
WO2015034948A1 (en) | 2013-09-03 | 2015-03-12 | Myriant Corporation | A process for manufacturing acrylic acid, acrylonitrile and 1,4-butanediol from 1,3-propanediol |
WO2015035244A1 (en) * | 2013-09-05 | 2015-03-12 | Braskem S/A | Modified microorganism and methods of using same for producing butadiene and 1-propanol and/or 1,2-propanediol |
CN104298866B (en) * | 2014-09-30 | 2017-06-06 | 杭州电子科技大学 | Reacting furnace dynamic modelling method in a kind of Claus sulphur recovery process |
EP3273782B9 (en) | 2015-02-27 | 2022-07-13 | White Dog Labs, Inc. | Mixotrophic fermentation method for making acetone, isopropanol, and other bioproducts, and mixtures thereof |
WO2016168681A1 (en) * | 2015-04-15 | 2016-10-20 | William Marsh Rice University | Iterative platform for the synthesis of alpha functionalized products |
WO2016176347A1 (en) * | 2015-04-29 | 2016-11-03 | William Marsh Rice University | Synthesis of omega-1 functionalized products and derivatives thereof |
WO2016176339A1 (en) * | 2015-04-28 | 2016-11-03 | William Marsh Rice University | Synthesis of omega-phenyl products and derivatives thereof |
WO2016168708A1 (en) * | 2015-04-16 | 2016-10-20 | William Marsh Rice University | Synthesis of omega functionalized methylketones, 2-alcohols, 2-amines, and derivatives thereof |
US9881793B2 (en) | 2015-07-23 | 2018-01-30 | International Business Machines Corporation | Neutral hard mask and its application to graphoepitaxy-based directed self-assembly (DSA) patterning |
CN108291194A (en) * | 2015-09-02 | 2018-07-17 | 积水化学工业株式会社 | The production method of recombinant cell, the manufacturing method of recombinant cell and 1,4- butanediols |
EP3362567A4 (en) * | 2015-10-13 | 2019-03-27 | Lanzatech New Zealand Limited | Genetically engineered bacterium comprising energy-generating fermentation pathway |
KR101828551B1 (en) * | 2016-01-11 | 2018-02-13 | 한국과학기술원 | Recombinant Variant Microorganism Having a Producing Ability of Malonic Acid and Method for Preparing Malonic Acid Using the Same |
GB201605354D0 (en) | 2016-03-30 | 2016-05-11 | Zuvasyntha Ltd | Modified enzyme |
US11541105B2 (en) | 2018-06-01 | 2023-01-03 | The Research Foundation For The State University Of New York | Compositions and methods for disrupting biofilm formation and maintenance |
FR3086670B1 (en) * | 2018-09-28 | 2024-05-31 | Ifp Energies Now | PROCESS FOR PRODUCING ALCOHOLS WITH CLOSTRIDIUM ON SOLID SUPPORT |
AU2021223603A1 (en) | 2020-02-21 | 2022-09-01 | Braskem S.A. | Production of ethanol with one or more co-products in yeast |
CA3195088C (en) | 2021-02-08 | 2024-04-23 | Fungmin Liew | Recombinant microorganisms and their use in the production of 3-hydroxypropionate [3-hp] |
US20240067994A1 (en) | 2022-08-24 | 2024-02-29 | Braskem S.A. | Process for the recovery of low-boiling point components from an ethanol stream |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4167583A (en) * | 1973-07-03 | 1979-09-11 | Bayer Aktiengesellschaft | Microbial composition and formulations |
US20080293125A1 (en) * | 2007-04-18 | 2008-11-27 | Gevo, Inc. | Engineered microorganisms for producing isopropanol |
WO2009028582A1 (en) * | 2007-08-29 | 2009-03-05 | Research Institute Of Innovative Technology For The Earth | Transformants capable of producing isopropanol |
US20090081746A1 (en) * | 2007-02-09 | 2009-03-26 | The Regents Of The University Of California | Biofuel production by recombinant microorganisms |
Family Cites Families (131)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4076948A (en) | 1968-10-10 | 1978-02-28 | El Paso Products Company | Process for treatment of adipic acid mother liquor |
US3513209A (en) | 1968-08-19 | 1970-05-19 | Du Pont | Method of making 1,4-cyclohexadiene |
GB1230276A (en) | 1968-12-09 | 1971-04-28 | ||
US3965182A (en) | 1969-10-02 | 1976-06-22 | Ethyl Corporation | Preparation of aniline from phenol and ammonia |
JPS4831084B1 (en) | 1970-09-04 | 1973-09-26 | ||
GB1344557A (en) | 1972-06-23 | 1974-01-23 | Mitsubishi Petrochemical Co | Process for preparing 1,4-butanediol |
JPS5543759B2 (en) | 1972-06-28 | 1980-11-07 | ||
DE2455617C3 (en) | 1974-11-23 | 1982-03-18 | Basf Ag, 6700 Ludwigshafen | Process for the production of butanediol and / or tetrahydrofuran via the intermediate stage of γ-butyrolactone |
DE2501499A1 (en) | 1975-01-16 | 1976-07-22 | Hoechst Ag | PROCESS FOR THE PRODUCTION OF BUTANDIOL- (1.4) |
US4190495A (en) | 1976-09-27 | 1980-02-26 | Research Corporation | Modified microorganisms and method of preparing and using same |
US4301077A (en) | 1980-12-22 | 1981-11-17 | Standard Oil Company | Process for the manufacture of 1-4-butanediol and tetrahydrofuran |
JPS60114197A (en) | 1983-11-25 | 1985-06-20 | Agency Of Ind Science & Technol | Preparation of dicarboxylic acid by bacterium |
US4871667A (en) | 1984-11-26 | 1989-10-03 | Agency Of Industrial Science & Technology | Process for preparing muconic acid |
US4652685A (en) | 1985-11-15 | 1987-03-24 | General Electric Company | Hydrogenation of lactones to glycols |
DE3783081T2 (en) | 1986-06-11 | 1993-04-15 | Michigan Biotech Inst | METHOD FOR PRODUCING AMBER ACID BY ANAEROBIC FERMENTATION. |
US5168055A (en) | 1986-06-11 | 1992-12-01 | Rathin Datta | Fermentation and purification process for succinic acid |
US5143834A (en) | 1986-06-11 | 1992-09-01 | Glassner David A | Process for the production and purification of succinic acid |
US5182199A (en) | 1987-05-27 | 1993-01-26 | Hartley Brian S | Thermophilic ethanol production in a two-stage closed system |
DE68928956T2 (en) | 1988-04-27 | 1999-07-29 | Daicel Chem | Process for the preparation of optically active 1,3-butanediol |
JP3043063B2 (en) | 1989-04-27 | 2000-05-22 | バイオコントロール システムズ インコーポレイテッド | Precipitation test for microorganisms |
US5192673A (en) | 1990-04-30 | 1993-03-09 | Michigan Biotechnology Institute | Mutant strain of C. acetobutylicum and process for making butanol |
US5079143A (en) | 1990-05-02 | 1992-01-07 | The Upjohn Company | Method of indentifying compounds useful as antiparasitic drugs |
US5173429A (en) | 1990-11-09 | 1992-12-22 | The Board Of Trustees Of The University Of Arkansas | Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism |
IL100572A (en) | 1991-01-03 | 1997-01-10 | Lepetit Spa | Amides of antibiotic ge 2270 factors their preparation and pharmaceutical compositions containing them |
US5416020A (en) | 1992-09-29 | 1995-05-16 | Bio-Technical Resources | Lactobacillus delbrueckii ssp. bulgaricus strain and fermentation process for producing L-(+)-lactic acid |
US6136577A (en) | 1992-10-30 | 2000-10-24 | Bioengineering Resources, Inc. | Biological production of ethanol from waste gases with Clostridium ljungdahlii |
US5807722A (en) | 1992-10-30 | 1998-09-15 | Bioengineering Resources, Inc. | Biological production of acetic acid from waste gases with Clostridium ljungdahlii |
FR2702492B1 (en) | 1993-03-12 | 1995-05-24 | Rhone Poulenc Chimie | Production process by fermentation of itaconic acid. |
US5487987A (en) | 1993-09-16 | 1996-01-30 | Purdue Research Foundation | Synthesis of adipic acid from biomass-derived carbon sources |
US5521075A (en) | 1994-12-19 | 1996-05-28 | Michigan Biotechnology Institute | Method for making succinic acid, anaerobiospirillum succiniciproducens variants for use in process and methods for obtaining variants |
US5504004A (en) | 1994-12-20 | 1996-04-02 | Michigan Biotechnology Institute | Process for making succinic acid, microorganisms for use in the process and methods of obtaining the microorganisms |
US5700934A (en) | 1995-03-01 | 1997-12-23 | Dsm N.V. | Process for the preparation of epsilon-caprolactam and epsilon-caprolactam precursors |
US5478952A (en) | 1995-03-03 | 1995-12-26 | E. I. Du Pont De Nemours And Company | Ru,Re/carbon catalyst for hydrogenation in aqueous solution |
US5863782A (en) | 1995-04-19 | 1999-01-26 | Women's And Children's Hospital | Synthetic mammalian sulphamidase and genetic sequences encoding same |
US5686276A (en) | 1995-05-12 | 1997-11-11 | E. I. Du Pont De Nemours And Company | Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism |
FR2736927B1 (en) | 1995-07-18 | 1997-10-17 | Rhone Poulenc Fibres & Polymer | ENZYMES HAVING AMIDASE ACTIVITY, GENETIC TOOLS AND HOST MICROORGANISMS FOR OBTAINING SAME AND HYDROLYSIS PROCESS USING THE SAME |
US5573931A (en) | 1995-08-28 | 1996-11-12 | Michigan Biotechnology Institute | Method for making succinic acid, bacterial variants for use in the process, and methods for obtaining variants |
US5869301A (en) | 1995-11-02 | 1999-02-09 | Lockhead Martin Energy Research Corporation | Method for the production of dicarboxylic acids |
US5770435A (en) | 1995-11-02 | 1998-06-23 | University Of Chicago | Mutant E. coli strain with increased succinic acid production |
KR19990087330A (en) | 1996-02-27 | 1999-12-27 | 로저 윌킨슨 | Cloning and Expression of the Gene Encoding Secondary Alcohol Dehydrogenase of Thermoaerobacter Ethanolicus 39E and Biochemical Characterization of the Enzyme |
US5958745A (en) | 1996-03-13 | 1999-09-28 | Monsanto Company | Methods of optimizing substrate pools and biosynthesis of poly-β-hydroxybutyrate-co-poly-β-hydroxyvalerate in bacteria and plants |
KR100387301B1 (en) | 1996-07-01 | 2003-06-12 | 바이오 엔지니어링 리소스 인코포레이티드 | Biological production of products from waste gases |
KR100459818B1 (en) | 1996-09-02 | 2004-12-03 | 이. 아이. 두퐁 드 느무르 앤드 컴퍼니 | Process for the preparation of epsilon-caprolactam |
AU6169498A (en) | 1997-02-13 | 1998-09-08 | James Madison University | Methods of making polyhydroxyalkanoates comprising 4-hydroxybutyrate monomer units |
KR100516986B1 (en) | 1997-02-19 | 2005-09-26 | 코닌클리즈케 디에스엠 엔.브이. | Process for the preparation of caprolactam in the absence of catalysts by contacting 6-aminocaproic acid derivatives with superheated steam |
US6274790B1 (en) | 1997-04-14 | 2001-08-14 | The University Of British Columbia | Nucleic acids encoding a plant enzyme involved in very long chain fatty acid synthesis |
KR19990013007A (en) | 1997-07-31 | 1999-02-25 | 박원훈 | Transformed Escherichia Coli S373 (BTCC 8818 P) and Production Method of Succinic Acid Using the Same |
JPH11103863A (en) | 1997-10-08 | 1999-04-20 | Nippon Shokubai Co Ltd | Maleate isomerase gene |
US6280986B1 (en) | 1997-12-01 | 2001-08-28 | The United States Of America As Represented By The Secretary Of Agriculture | Stabilization of pet operon plasmids and ethanol production in bacterial strains lacking lactate dehydrogenase and pyruvate formate lyase activities |
US20030087381A1 (en) | 1998-04-13 | 2003-05-08 | University Of Georgia Research Foundation, Inc. | Metabolically engineered organisms for enhanced production of oxaloacetate-derived biochemicals |
WO1999053035A1 (en) | 1998-04-13 | 1999-10-21 | The University Of Georgia Research Foundation, Inc. | Pyruvate carboxylase overexpression for enhanced production of oxaloacetate-derived biochemicals in microbial cells |
US6159738A (en) | 1998-04-28 | 2000-12-12 | University Of Chicago | Method for construction of bacterial strains with increased succinic acid production |
US6432686B1 (en) * | 1998-05-12 | 2002-08-13 | E. I. Du Pont De Nemours And Company | Method for the production of 1,3-propanediol by recombinant organisms comprising genes for vitamin B12 transport |
US6444784B1 (en) | 1998-05-29 | 2002-09-03 | Exxonmobil Research & Engineering Company | Wax crystal modifiers (LAW657) |
DE19856136C2 (en) | 1998-12-04 | 2002-10-24 | Pasteur Institut | Method and device for the selection of accelerated proliferation of living cells in suspension |
WO2000046405A2 (en) | 1999-02-02 | 2000-08-10 | Bernhard Palsson | Methods for identifying drug targets based on genomic sequence data |
US6686310B1 (en) | 1999-02-09 | 2004-02-03 | E. I. Du Pont De Nemours And Company | High surface area sol-gel route prepared hydrogenation catalysts |
US6365376B1 (en) | 1999-02-19 | 2002-04-02 | E. I. Du Pont De Nemours And Company | Genes and enzymes for the production of adipic acid intermediates |
WO2000052183A1 (en) | 1999-03-05 | 2000-09-08 | Monsanto Technology Llc | Multigene expression vectors for the biosynthesis of products via multienzyme biological pathways |
KR20020048910A (en) | 1999-05-21 | 2002-06-24 | 카길 다우 엘엘씨 | Method and materials for the synthesis of organic products |
US6852517B1 (en) | 1999-08-30 | 2005-02-08 | Wisconsin Alumni Research Foundation | Production of 3-hydroxypropionic acid in recombinant organisms |
US6897055B2 (en) * | 1999-11-25 | 2005-05-24 | Degussa Ag | Nucleotide sequences coding for the genes sucC and sucD |
US6660857B2 (en) | 2000-02-03 | 2003-12-09 | Dsm N.V. | Process for the preparation of ε-caprolactam |
US6878861B2 (en) | 2000-07-21 | 2005-04-12 | Washington State University Research Foundation | Acyl coenzyme A thioesterases |
EA006106B1 (en) | 2000-07-25 | 2005-08-25 | Эммаус Фаундейшн, Инк. | Method for stable continuous production of ethanol |
EP1343874B1 (en) | 2000-11-20 | 2012-10-03 | Cargill, Incorporated | Cells comprising lactyl-coa dehydratase, 3-hydroxypropionyl-coa dehydratase or a multienzyme complex isolated from megasphaera elsdenii and chloroflexus aurantiacus for the production of 3-hydroxypropionic acid |
CA2430270A1 (en) | 2000-11-22 | 2002-05-30 | Cargill Dow Polymers, Llc | Methods and materials for the synthesis of organic products |
CN1358841A (en) | 2000-12-11 | 2002-07-17 | 云南省微生物研究所 | Yunnan streptin |
CA2433529A1 (en) | 2000-12-28 | 2002-07-11 | Toyota Jidosha Kabushiki Kaisha | Process for producing prenyl alcohol |
CA2434224C (en) | 2001-01-10 | 2013-04-02 | The Penn State Research Foundation | Method and system for modeling cellular metabolism |
US7127379B2 (en) | 2001-01-31 | 2006-10-24 | The Regents Of The University Of California | Method for the evolutionary design of biochemical reaction networks |
WO2002070730A2 (en) | 2001-03-01 | 2002-09-12 | The Regents Of The University Of California | Models and methods for determining systemic properties of regulated reaction networks |
US6743610B2 (en) | 2001-03-30 | 2004-06-01 | The University Of Chicago | Method to produce succinic acid from raw hydrolysates |
CN1279013C (en) | 2001-05-07 | 2006-10-11 | 嘉吉有限公司 | Process for preparing carboxylic acids and derivatives thereof |
CA2356540A1 (en) | 2001-08-30 | 2003-02-28 | Emory University | Expressed dna sequences involved in mitochondrial functions |
AU2002353970A1 (en) | 2001-11-02 | 2003-05-19 | Rice University | Recycling system for manipulation of intracellular nadh availability |
ATE432338T1 (en) | 2002-01-18 | 2009-06-15 | Novozymes As | ALANINE 2,3-AMINOMUTASE |
JPWO2003066863A1 (en) | 2002-02-06 | 2005-06-02 | 昭和電工株式会社 | Reductase gene for α-substituted-α, β-unsaturated carbonyl compounds |
US20030224363A1 (en) | 2002-03-19 | 2003-12-04 | Park Sung M. | Compositions and methods for modeling bacillus subtilis metabolism |
JP2005521929A (en) | 2002-03-29 | 2005-07-21 | ジェノマティカ・インコーポレイテッド | Human metabolic models and methods |
AU2003234790A1 (en) | 2002-05-10 | 2003-11-11 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing mevalonic acid |
US7856317B2 (en) | 2002-06-14 | 2010-12-21 | Genomatica, Inc. | Systems and methods for constructing genomic-based phenotypic models |
US7826975B2 (en) | 2002-07-10 | 2010-11-02 | The Penn State Research Foundation | Method for redesign of microbial production systems |
CA2491753A1 (en) | 2002-07-10 | 2004-03-04 | The Penn State Research Foundation | Method for determining gene knockout strategies |
WO2004007688A2 (en) | 2002-07-15 | 2004-01-22 | Kosan Biosciences, Inc. | Metabolic pathways for starter units in polyketide biosynthesis |
MXPA05003382A (en) | 2002-10-04 | 2005-06-22 | Du Pont | Process for the biological production of 1,3-propanediol with high yield. |
US7734420B2 (en) | 2002-10-15 | 2010-06-08 | The Regents Of The University Of California | Methods and systems to identify operational reaction pathways |
AU2003287625A1 (en) | 2002-11-06 | 2004-06-03 | University Of Florida | Materials and methods for the efficient production of acetate and other products |
JP4275666B2 (en) | 2003-02-24 | 2009-06-10 | 財団法人地球環境産業技術研究機構 | Highly efficient hydrogen production method using microorganisms |
RU2268300C2 (en) | 2003-04-07 | 2006-01-20 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" | METHOD FOR PREPARING L-AMINO ACIDS BY USING MICROORGANISMS POSSESSING ENHANCED EXPRESSION OF pckA GENE |
CN1190400C (en) * | 2003-06-02 | 2005-02-23 | 大连理工大学 | Method for extracting and separating 1,3-propylene glycol from microbial fermented liquor |
US7927859B2 (en) | 2003-08-22 | 2011-04-19 | Rice University | High molar succinate yield bacteria by increasing the intracellular NADH availability |
JP4619291B2 (en) | 2003-09-17 | 2011-01-26 | 三菱化学株式会社 | Method for producing non-amino organic acid |
US7244610B2 (en) | 2003-11-14 | 2007-07-17 | Rice University | Aerobic succinate production in bacteria |
FR2864967B1 (en) | 2004-01-12 | 2006-05-19 | Metabolic Explorer Sa | ADVANCED MICROORGANISM FOR THE PRODUCTION OF 1,2-PROPANEDIOL |
DE602005018898D1 (en) | 2004-01-19 | 2010-03-04 | Dsm Ip Assets Bv | BIOCHEMICAL SYNTHESIS OF 6-AMINOCAPRONIC ACID |
US7608700B2 (en) | 2004-03-08 | 2009-10-27 | North Carolina State University | Lactobacillus acidophilus nucleic acid sequences encoding stress-related proteins and uses therefor |
DE102004031177A1 (en) | 2004-06-29 | 2006-01-19 | Henkel Kgaa | New odoriferous gene products from Bacillus licheniformis and improved biotechnological production processes based on them |
US7262046B2 (en) | 2004-08-09 | 2007-08-28 | Rice University | Aerobic succinate production in bacteria |
EP1781797B1 (en) | 2004-08-27 | 2016-10-19 | Rice University | Mutant e. coli strain with increased succinic acid production |
EP2434015B1 (en) | 2004-09-09 | 2013-11-20 | Research Institute Of Innovative Technology For The Earth | DNA fragment having promoter function |
KR20070065870A (en) | 2004-09-17 | 2007-06-25 | 라이스 유니버시티 | High succinate producing bacteria |
WO2006069174A2 (en) | 2004-12-22 | 2006-06-29 | Rice University | Simultaneous anaerobic production of isoamyl acetate and succinic acid |
JP2006204255A (en) | 2005-01-31 | 2006-08-10 | Canon Inc | ACETYL-CoA ACYLTRANSFERASE GENE-BROKEN POLYHYDROXYALKANOATE-PRODUCING MICROORGANISM, AND METHOD FOR PRODUCING POLYHYDROXYALKANOATE THEREWITH |
WO2006109312A2 (en) | 2005-04-15 | 2006-10-19 | Vascular Biogenics Ltd. | Compositions containing beta 2-glycoprotein i-derived peptides for the prevention and/or treatment of vascular disease |
KR100679638B1 (en) | 2005-08-19 | 2007-02-06 | 한국과학기술원 | Microorganisms transformed with gene encoding formate ddehydrogenase d or e and method for preparing succinic acid using the same |
KR100676160B1 (en) | 2005-08-19 | 2007-02-01 | 한국과학기술원 | Microorganisms transformed with gene encoding malic enzyme and method for preparing succinic acid using the same |
JP2009507493A (en) | 2005-09-09 | 2009-02-26 | ジェノマティカ・インコーポレイテッド | Methods and organisms for growth-linked succinate production |
US9297028B2 (en) | 2005-09-29 | 2016-03-29 | Butamax Advanced Biofuels Llc | Fermentive production of four carbon alcohols |
EP1948814B1 (en) | 2005-10-26 | 2018-11-21 | Butamax (TM) Advanced Biofuels LLC | Fermentive production of four carbon alcohols |
KR20070096348A (en) * | 2006-03-23 | 2007-10-02 | 주식회사 엘지화학 | Mutants having a producing ability of 1,4-butanediol and method for preparing 1,4-bdo using the same |
US8206970B2 (en) | 2006-05-02 | 2012-06-26 | Butamax(Tm) Advanced Biofuels Llc | Production of 2-butanol and 2-butanone employing aminobutanol phosphate phospholyase |
DE102006025821A1 (en) | 2006-06-02 | 2007-12-06 | Degussa Gmbh | An enzyme for the production of Mehylmalonatsemialdehyd or Malonatsemialdehyd |
US8017364B2 (en) | 2006-12-12 | 2011-09-13 | Butamax(Tm) Advanced Biofuels Llc | Solvent tolerant microorganisms |
AU2008229076B2 (en) * | 2007-03-16 | 2014-05-15 | Genomatica, Inc. | Compositions and methods for the biosynthesis of 1,4-butanediol and its precursors |
US20080274522A1 (en) | 2007-05-02 | 2008-11-06 | Bramucci Michael G | Method for the production of 2-butanone |
US9080187B2 (en) | 2007-05-17 | 2015-07-14 | The Board Of Trustees Of The University Of Illinois | Methods and compositions for producing solvents |
CN101679187B (en) * | 2007-06-01 | 2013-06-05 | 赢创罗姆有限责任公司 | A process for preparing methacrylic acid or methacrylic esters |
EP2017344A1 (en) | 2007-07-20 | 2009-01-21 | Nederlandse Organisatie voor toegepast- natuurwetenschappelijk onderzoek TNO | Production of itaconic acid |
US7947483B2 (en) * | 2007-08-10 | 2011-05-24 | Genomatica, Inc. | Methods and organisms for the growth-coupled production of 1,4-butanediol |
KR101042242B1 (en) * | 2007-09-07 | 2011-06-17 | 한국과학기술원 | Mutants having a producing ability of 1,4-butanediol and method for preparing 1,4-butanediol using the same |
KR20100087695A (en) | 2007-10-12 | 2010-08-05 | 더 리전트 오브 더 유니버시티 오브 캘리포니아 | Microorganism engineered to produce isopropanol |
CN101230363B (en) * | 2007-11-11 | 2012-04-18 | 江南大学 | Method for preparing (R)-styrene glycol by employing asymmetric conversion of recombinant strain |
US7803589B2 (en) * | 2008-01-22 | 2010-09-28 | Genomatica, Inc. | Methods and organisms for utilizing synthesis gas or other gaseous carbon sources and methanol |
ES2656790T3 (en) | 2008-03-27 | 2018-02-28 | Genomatica, Inc. | Microorganisms for the production of adipic acid and other compounds |
EP2304039B1 (en) | 2008-06-17 | 2019-08-21 | Genomatica, Inc. | Microorganisms and methods for the biosynthesis of fumarate, malate, and acrylate |
AU2009291825B2 (en) | 2008-09-10 | 2016-05-05 | Genomatica, Inc. | Microorganisms for the production of 1,4-butanediol |
US8344188B2 (en) | 2008-10-16 | 2013-01-01 | Maverick Biofuels, Inc. | Methods and apparatus for synthesis of alcohols from syngas |
EP2373781A4 (en) * | 2008-12-16 | 2012-10-10 | Genomatica Inc | Microorganisms and methods for conversion of syngas and other carbon sources to useful products |
US8530210B2 (en) * | 2009-11-25 | 2013-09-10 | Genomatica, Inc. | Microorganisms and methods for the coproduction 1,4-butanediol and gamma-butyrolactone |
US8445244B2 (en) * | 2010-02-23 | 2013-05-21 | Genomatica, Inc. | Methods for increasing product yields |
-
2010
- 2010-09-09 MX MX2012003025A patent/MX2012003025A/en active IP Right Grant
- 2010-09-09 SG SG2012016408A patent/SG179048A1/en unknown
- 2010-09-09 JP JP2012528907A patent/JP2013504326A/en active Pending
- 2010-09-09 EP EP15155588.5A patent/EP2933338A3/en not_active Withdrawn
- 2010-09-09 US US12/878,980 patent/US8715971B2/en active Active
- 2010-09-09 CN CN2010800504376A patent/CN102625845A/en active Pending
- 2010-09-09 KR KR1020127008948A patent/KR20120068021A/en not_active Application Discontinuation
- 2010-09-09 BR BR112012005296A patent/BR112012005296A2/en not_active IP Right Cessation
- 2010-09-09 WO PCT/US2010/048318 patent/WO2011031897A1/en active Application Filing
- 2010-09-09 CN CN201710897714.0A patent/CN107586753A/en active Pending
- 2010-09-09 CA CA2773694A patent/CA2773694A1/en not_active Abandoned
- 2010-09-09 EP EP10816105A patent/EP2475775A4/en not_active Withdrawn
-
2012
- 2012-02-28 ZA ZA2012/01468A patent/ZA201201468B/en unknown
-
2013
- 2013-02-27 ZA ZA2013/01486A patent/ZA201301486B/en unknown
-
2014
- 2014-01-29 US US14/167,693 patent/US20140377820A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4167583A (en) * | 1973-07-03 | 1979-09-11 | Bayer Aktiengesellschaft | Microbial composition and formulations |
US20090081746A1 (en) * | 2007-02-09 | 2009-03-26 | The Regents Of The University Of California | Biofuel production by recombinant microorganisms |
US20080293125A1 (en) * | 2007-04-18 | 2008-11-27 | Gevo, Inc. | Engineered microorganisms for producing isopropanol |
WO2009028582A1 (en) * | 2007-08-29 | 2009-03-05 | Research Institute Of Innovative Technology For The Earth | Transformants capable of producing isopropanol |
Non-Patent Citations (1)
Title |
---|
See also references of EP2475775A4 * |
Cited By (63)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5628288B2 (en) * | 2010-03-09 | 2014-11-19 | 三井化学株式会社 | Highly productive isopropyl alcohol-producing bacteria |
US8932845B2 (en) | 2010-03-09 | 2015-01-13 | Mitsui Chemicals, Inc. | Highly productive isopropyl alcohol-producing bacterium |
US8728804B2 (en) | 2010-10-29 | 2014-05-20 | Novozymes A/S | Polypeptides having succinyl-CoA: acetoacetate transferase activity and polynucleotides encoding same |
WO2012058603A1 (en) | 2010-10-29 | 2012-05-03 | Novozymes A/S | Recombinant n-propanol and isopropanol production |
WO2012080421A1 (en) * | 2010-12-17 | 2012-06-21 | Total Petrochemicals Research Feluy | Process for producing propylene from syngas via fermentative propanol production and dehydration |
CN103930558A (en) * | 2011-04-01 | 2014-07-16 | 基因组股份公司 | Microorganisms for producing methacrylic acid and methacrylate esters and methods related thereto |
WO2012177599A3 (en) * | 2011-06-22 | 2013-02-21 | Genomatica, Inc. | Microorganisms for producing n-propanol 1, 3-propanediol, 1,2-propanediol or glycerol and methods related thereto |
WO2012177599A2 (en) * | 2011-06-22 | 2012-12-27 | Genomatica, Inc. | Microorganisms for producing n-propanol 1, 3-propanediol, 1,2-propanediol or glycerol and methods related thereto |
KR101860442B1 (en) * | 2011-06-27 | 2018-05-24 | 삼성전자주식회사 | Genetic Modification for Production of 3-hydroxypropionic acid |
EP2734627A4 (en) * | 2011-07-20 | 2015-07-22 | Genomatica Inc | Methods for increasing product yields |
JPWO2013022070A1 (en) * | 2011-08-11 | 2015-03-05 | 三井化学株式会社 | Isopropyl alcohol production method by continuous culture |
US9150885B2 (en) | 2011-08-11 | 2015-10-06 | Mitsui Chemicals, Inc. | Method for producing isopropyl alcohol by continuous culture |
EP2743352A4 (en) * | 2011-08-11 | 2015-03-11 | Mitsui Chemicals Inc | Method for producing isopropyl alcohol by continuous culture |
TWI563093B (en) * | 2011-08-11 | 2016-12-21 | Mitsui Chemicals Inc | Method of producing isopropyl alcohol by continuous culture |
EP2743352A1 (en) * | 2011-08-11 | 2014-06-18 | Mitsui Chemicals, Inc. | Method for producing isopropyl alcohol by continuous culture |
KR20140057346A (en) * | 2011-08-19 | 2014-05-12 | 게노마티카 인코포레이티드 | Microorganisms and methods for producing 2,4-pentadienoate, butadiene, propylene, 1,3-butanediol and related alcohols |
KR102043381B1 (en) | 2011-08-19 | 2019-11-11 | 게노마티카 인코포레이티드 | Microorganisms and methods for producing 2,4-pentadienoate, butadiene, propylene, 1,3-butanediol and related alcohols |
EP2744906A4 (en) * | 2011-08-19 | 2015-07-22 | Genomatica Inc | Microorganisms and methods for producing 2,4-pentadienoate, butadiene, propylene, 1,3-butanediol and related alcohols |
WO2013028519A1 (en) | 2011-08-19 | 2013-02-28 | Genomatica, Inc. | Microorganisms and methods for producing 2,4-pentadienoate, butadiene, propylene, 1,3-butanediol and related alcohols |
JP2013042701A (en) * | 2011-08-24 | 2013-03-04 | Hiroshima Univ | Microorganism manipulated to produce 2-propanol |
CN104284974B (en) * | 2012-03-06 | 2018-06-26 | 利戈斯股份有限公司 | For generating the recombinant host cell of malonic acid |
CN104284974A (en) * | 2012-03-06 | 2015-01-14 | 利戈斯股份有限公司 | Recombinant host cells for the production of malonate |
WO2013178699A1 (en) | 2012-05-31 | 2013-12-05 | Novozymes A/S | Isopropanol production by bacterial hosts |
WO2014038216A1 (en) | 2012-09-10 | 2014-03-13 | 三菱レイヨン株式会社 | Method for producing methacrylic acid and/or ester thereof |
US10851392B2 (en) | 2012-09-10 | 2020-12-01 | Mitsubishi Chemical Corporation | Method for producing methacrylic acid ester |
EP3395951A2 (en) | 2012-09-10 | 2018-10-31 | Mitsubishi Chemical Corporation | Method for producing methacrylic acid ester |
KR20140092386A (en) | 2012-09-10 | 2014-07-23 | 미쯔비시 레이온 가부시끼가이샤 | Method for producing methacrylic acid and/or ester thereof |
KR20170018099A (en) | 2012-09-10 | 2017-02-15 | 미쯔비시 레이온 가부시끼가이샤 | Method for producing methacrylic acid and/or ester thereof |
WO2014038214A1 (en) | 2012-09-10 | 2014-03-13 | 三菱レイヨン株式会社 | Method for producing methacrylic acid ester |
US10294500B2 (en) | 2012-09-10 | 2019-05-21 | Mitsubishi Chemical Corporation | Method for producing methacrylic acid and/or ester thereof |
WO2014068010A1 (en) | 2012-10-31 | 2014-05-08 | Novozymes A/S | Isopropanol production by bacterial hosts |
US11629363B2 (en) | 2012-11-05 | 2023-04-18 | Genomatica, Inc. | Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 1,2-propanediol, n-propanol, 1,3-propanediol, or glycerol related thereto |
US9909150B2 (en) | 2012-11-05 | 2018-03-06 | Genomatica, Inc. | Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 1,2-propanediol, n-propanol, 1,3-propanediol, or glycerol related thereto |
WO2014076232A2 (en) | 2012-11-19 | 2014-05-22 | Novozymes A/S | Isopropanol production by recombinant hosts using an hmg-coa intermediate |
WO2014152434A2 (en) | 2013-03-15 | 2014-09-25 | Genomatica, Inc. | Microorganisms and methods for producing butadiene and related compounds by formate assimilation |
WO2014160846A3 (en) * | 2013-03-28 | 2014-12-04 | The Procter & Gamble Company | Microorganisms and methods for producing propionic acid |
EP3004362A4 (en) * | 2013-06-05 | 2017-01-11 | Lanzatech New Zealand Limited | Recombinant microorganisms exhibiting increased flux through a fermentation pathway |
US10323264B2 (en) | 2013-08-01 | 2019-06-18 | Mitsubishi Chemical Corporation | Method for producing methacrylyl-CoA |
KR20160025011A (en) | 2013-08-01 | 2016-03-07 | 미쯔비시 레이온 가부시끼가이샤 | METHOD FOR PRODUCING METHACRYLYL-CoA |
US11667939B2 (en) | 2013-08-01 | 2023-06-06 | Mitsubishi Chemical Corporation | Method for producing methacrylyl-CoA |
WO2015015784A1 (en) | 2013-08-01 | 2015-02-05 | 三菱レイヨン株式会社 | METHOD FOR PRODUCING METHACRYLYL-CoA |
US10808262B2 (en) | 2013-12-03 | 2020-10-20 | Genomatica, Inc. | Microorganisms and methods for improving product yields on methanol using acetyl-CoA synthesis |
EP3967747A1 (en) | 2013-12-03 | 2022-03-16 | Genomatica, Inc. | Microorganisms and methods for improving product yields on methanol using acetyl-coa synthesis |
EP4296364A2 (en) | 2013-12-03 | 2023-12-27 | Genomatica, Inc. | Microorganisms and methods for improving product yields on methanol using acetyl-coa synthesis |
WO2015084633A1 (en) | 2013-12-03 | 2015-06-11 | Genomatica, Inc. | Microorganisms and methods for improving product yields on methanol using acetyl-coa synthesis |
US10597684B2 (en) | 2013-12-27 | 2020-03-24 | Genomatica, Inc. | Methods and organisms with increased carbon flux efficiencies |
EP3744830A1 (en) | 2013-12-27 | 2020-12-02 | Genomatica, Inc. | Methods and organisms with increased carbon flux efficiencies |
KR20160108546A (en) | 2014-03-07 | 2016-09-19 | 미쯔비시 레이온 가부시끼가이샤 | Method for producing methacrylic acid ester, and novel methacrylic acid ester synthase |
US10570426B2 (en) | 2014-03-07 | 2020-02-25 | Mitsubishi Chemical Corporation | Method for producing methacrylic acid ester and novel methacrylic acid ester synthetase |
WO2015158716A1 (en) | 2014-04-16 | 2015-10-22 | Novamont S.P.A. | Process for the production of 1,4-butanediol |
WO2016044713A1 (en) | 2014-09-18 | 2016-03-24 | Genomatica, Inc. | Non-natural microbial organisms with improved energetic efficiency |
EP3741865A1 (en) | 2014-09-18 | 2020-11-25 | Genomatica, Inc. | Non-natural microbial organisms with improved energetic efficiency |
WO2016100910A1 (en) * | 2014-12-19 | 2016-06-23 | Novozymes A/S | Recombinant host cells for the production of 3-hydroxypropionic acid |
WO2016196233A1 (en) | 2015-05-30 | 2016-12-08 | Genomatica, Inc. | Vinylisomerase-dehydratases, alkenol dehydratases, linalool dehydratases and/ crotyl alcohol dehydratases and methods for making and using them |
US10676766B2 (en) | 2015-10-23 | 2020-06-09 | The Regents Of The University Of California | Biological production of methyl methacrylate |
WO2017075208A1 (en) | 2015-10-30 | 2017-05-04 | Genomatica, Inc. | Methanol dehydrogenase fusion proteins |
US10829789B2 (en) | 2016-12-21 | 2020-11-10 | Creatus Biosciences Inc. | Methods and organism with increased xylose uptake |
WO2019102030A1 (en) | 2017-11-27 | 2019-05-31 | Novamont S.P.A. | Process for the production of 1,4-butanediol from renewable sources and polyesters obtained therefrom |
WO2019152375A1 (en) | 2018-01-30 | 2019-08-08 | Genomatica, Inc. | Fermentation systems and methods with substantially uniform volumetric uptake rate of a reactive gaseous component |
WO2020006058A2 (en) | 2018-06-26 | 2020-01-02 | Genomatica, Inc. | Engineered microorganisms with g3p---> 3pg enzyme and/or fructose-1,6-bisphosphatase including those having synthetic or enhanced methylotrophy |
WO2021245228A2 (en) | 2020-06-04 | 2021-12-09 | Novamont S.P.A. | Process for purifying a mixture of diols |
IT202100030572A1 (en) | 2021-12-02 | 2023-06-02 | Novamont Spa | 1,3-BUTANDIOL PURIFIED FROM A MIXTURE OF DIOLS |
WO2023099650A1 (en) | 2021-12-02 | 2023-06-08 | Novamont S.P.A. | 1,3-butanediol purified from a mixture of diols |
Also Published As
Publication number | Publication date |
---|---|
MX2012003025A (en) | 2012-06-27 |
ZA201201468B (en) | 2013-05-29 |
CA2773694A1 (en) | 2011-03-17 |
CN102625845A (en) | 2012-08-01 |
ZA201301486B (en) | 2014-10-29 |
US8715971B2 (en) | 2014-05-06 |
EP2475775A4 (en) | 2013-03-20 |
EP2475775A1 (en) | 2012-07-18 |
CN107586753A (en) | 2018-01-16 |
EP2933338A3 (en) | 2016-01-06 |
SG179048A1 (en) | 2012-04-27 |
EP2933338A2 (en) | 2015-10-21 |
KR20120068021A (en) | 2012-06-26 |
US20140377820A1 (en) | 2014-12-25 |
JP2013504326A (en) | 2013-02-07 |
US20110201068A1 (en) | 2011-08-18 |
BR112012005296A2 (en) | 2019-01-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8715971B2 (en) | Microorganisms and methods for the co-production of isopropanol and 1,4-butanediol | |
US11708589B2 (en) | Microorganisms for producing 1,3-butanediol and methods related thereto | |
US8993285B2 (en) | Organisms for the production of isopropanol, n-butanol, and isobutanol | |
US9139853B2 (en) | Organisms for the production of cyclohexanone | |
US9284581B2 (en) | Methods and organisms for converting synthesis gas or other gaseous carbon sources and methanol to 1,3-butanediol | |
EP2462221B1 (en) | Semi-synthetic terephthalic acid via microorganisms that produce muconic acid | |
US20100021978A1 (en) | Methods and organisms for production of 3-hydroxypropionic acid | |
US20240141397A1 (en) | Microorganisms and methods for the production of biosynthesized target products having reduced levels of byproducts | |
US20140363864A1 (en) | Microorganisms and methods for the production of 1,4-cyclohexanedimethanol | |
CA2796118A1 (en) | Microorganisms and methods for the production of ethylene glycol | |
WO2012177601A2 (en) | Microorganisms for producing isobutanol and methods related thereto | |
AU2013202621A1 (en) | Organisms for the production of isopropanol, n-butanol, and isobutanol | |
AU2013203166A1 (en) | Microorganisms for producing 1,3-butanediol and methods related thereto |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 201080050437.6 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10816105 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12012500463 Country of ref document: PH |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2111/CHENP/2012 Country of ref document: IN |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2773694 Country of ref document: CA Ref document number: 2012528907 Country of ref document: JP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1201000995 Country of ref document: TH Ref document number: MX/A/2012/003025 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2010816105 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 20127008948 Country of ref document: KR Kind code of ref document: A |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112012005296 Country of ref document: BR |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01E Ref document number: 112012005296 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112012005296 Country of ref document: BR Kind code of ref document: A2 Effective date: 20120309 |