WO2011071682A1 - Methods and organisms for converting synthesis gas or other gaseous carbon sources and methanol to 1,3-butanediol - Google Patents

Methods and organisms for converting synthesis gas or other gaseous carbon sources and methanol to 1,3-butanediol Download PDF

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WO2011071682A1
WO2011071682A1 PCT/US2010/057525 US2010057525W WO2011071682A1 WO 2011071682 A1 WO2011071682 A1 WO 2011071682A1 US 2010057525 W US2010057525 W US 2010057525W WO 2011071682 A1 WO2011071682 A1 WO 2011071682A1
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coa
microbial organism
protein
reductase
encoding
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PCT/US2010/057525
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English (en)
French (fr)
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Anthony P. Burgard
Mark J. Burk
Priti Pharkya
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Genomatica, Inc.
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Priority to EP10836412.6A priority Critical patent/EP2510102A4/en
Priority to KR1020127017813A priority patent/KR20120120493A/ko
Priority to SG2012042537A priority patent/SG181607A1/en
Priority to MX2012006602A priority patent/MX2012006602A/es
Priority to RU2012128843/10A priority patent/RU2012128843A/ru
Priority to BR112012014062A priority patent/BR112012014062A2/pt
Priority to JP2012543135A priority patent/JP2013513384A/ja
Priority to CA2783096A priority patent/CA2783096A1/en
Priority to CN2010800635936A priority patent/CN102753698A/zh
Publication of WO2011071682A1 publication Critical patent/WO2011071682A1/en
Priority to ZA2012/04217A priority patent/ZA201204217B/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • 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
    • C12P7/16—Butanols
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates generally to biosynthetic processes and more specifically to organisms capable of using carbohydrates, methanol, synthesis gas and other gaseous carbon sources in the production of commodity chemicals.
  • 1,3-butanediol (1,3-BDO) is a four carbon diol 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.
  • acetylene has been replaced by the less expensive ethylene as a source of acetaldehyde.
  • 1,3-BDO is 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 1,3-BDO is a useful starting material for the synthesis of biologically active compounds and liquid crystals.
  • 1,3-butanediol Another use of 1,3-butanediol is that its dehydration affords 1,3 -butadiene (Ichikawa et al., /. Molecular Catalysis A-Chemical, 231: 181-189 (2005); Ichikawa et al., /. Molecular Catalysis A-Chemical, 256: 106-112 (2006)), a chemical used to manufacture synthetic rubbers (e.g.tires), latex, and resins.
  • Synthesis gas is a mixture of primarily H 2 and CO that can be obtained via gasification of any organic feedstock, such as coal, coal oil, natural gas, biomass, or waste organic matter.
  • Any organic feedstock such as coal, coal oil, natural gas, biomass, or waste organic matter.
  • Numerous gasification processes have been developed, and most designs are based on partial oxidation, where limiting oxygen avoids full combustion, of organic materials at high temperatures (500-1500°C) to provide syngas as a 0.5: 1-3: 1 H 2 /CO mixture.
  • Steam is sometimes added to increase the hydrogen content, typically with increased C0 2 production through the water gas shift reaction.
  • Methanol is most commonly produced industrially from the syngas components, CO, and H 2 , via catalysis.
  • coal is the main substrate used for industrial production of syngas, which is traditionally used for heating and power and as a feedstock for Fischer-Tropsch synthesis of methanol and liquid hydrocarbons.
  • syngas is traditionally used for heating and power and as a feedstock for Fischer-Tropsch synthesis of methanol and liquid hydrocarbons.
  • Many large chemical and energy companies employ coal gasification processes on large scale and there is experience in the industry using this technology.
  • syngas is inexpensive.
  • pathways as in organisms such as Clostridium spp., that utilize syngas effectively.
  • Clostridium and related bacteria are strict anaerobes that are intolerant to high concentrations of certain products such as butanol, thus limiting titers and commercialization potential.
  • the Clostridia also produce multiple products, which presents separations issues in obtaining a desired product.
  • Finally development of facile genetic tools to manipulate Clostridial genes is in its infancy; therefore, they are not readily amenable to genetic engineering to improve yield or production characteristics of a desired product.
  • Feedstock flexibility relies on the introduction of methods that enable access and use of a wide range of materials as primary feedstocks for chemical manufacturing.
  • the reliance on petroleum based feedstocks for either acetylene or ethylene warrants the development of a renewable feedstock based route to 1,3-butanediol and to butadiene.
  • embodiments disclosed herein relate to a non-naturally occurring microbial organism having a 1,3-butanediol (1,3-BDO) pathway that includes at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme or protein expressed in a sufficient amount to produce 1,3-BDO.
  • the 1,3-BDO pathway includes Methanol methyltransferase (MtaB),
  • Corrinoid protein (MtaC), Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), Methyltetrahydrofolate: comnoid protein methyltransferase (AcsE), Corrinoid iron- sulfur protein (AcsD), Nickel-protein assembly protein (AcsF & CooC), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB & AcsC), Carbon monoxide dehydrogenase (AcsA), Hydrogenase (Hyd), Acetoacetyl-CoA thiolase (AtoB), Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 3-oxobutyraldehyde reductase (ketone reducing), 3-hydroxybutyraldehyde reductase, Acetoacetyl-CoA
  • Acetoacetyl-CoA synthetase or Acetoacetate reductase.
  • embodiments disclosed herein relate to a non-naturally occurring microbial organism having a 1,3-butanediol (1,3-BDO) pathway that includes at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme or protein expressed in a sufficient amount to produce 1,3-BDO.
  • the 1,3-BDO pathway includes Formate dehydrogenase,
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), Corrinoid iron- sulfur protein (AcsD), Nickel-protein assembly protein (AcsF & CooC), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB & AcsC), Carbon monoxide dehydrogenase (AcsA), Hydrogenase (Hyd), Acetoacetyl-CoA thiolase (AtoB), Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 3-oxobutyraldehyde reductase (ketone reducing), 3-hydroxybutyraldehyde reductase, Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), 3-oxobutyraldehyde reductase (aldehyde reducing), 4-hydroxy,
  • Acetoacetyl-CoA synthetase or Acetoacetate reductase.
  • embodiments disclosed herein relate to a method for producing 1,3-BDO that includes culturing the aforementioned non-naturally occurring microbial organisms under conditions and for a sufficient period of time to produce 1,3-BDO.
  • Figure 1 shows a diagram depicting the Wood-Ljungdahl pathway and formation routes for acetate and ethanol.
  • the transformations that are characteristic of organisms capable of growth on synthesis gas are 1) CO dehydrogenase, 2) hydrogenase, 3) energy-conserving hydrogenase (ECH), and 4) bi-functional CO dehydrogenase/acetyl-CoA synthase.
  • Figure 2 shows the complete Wood-Ljungdahl pathway that enables the conversion of gases including CO, C0 2 , and/or H 2 to acetyl-CoA which is subsequently converted to cell mass and products such as ethanol or acetate.
  • 10FTHF 10-formyltetrahydrofolate
  • 5MTHF 5-methyltetrahydrofolate
  • ACTP acetyl phosphate
  • CFeSp corrinoid iron sulfur protein
  • FOR formate
  • MeOH methanol
  • METHF methyltetrahydrofolate
  • MLTHF MLTHF:
  • THF tetrahydrofolate
  • Figure 3 depicts pathways from acetoacetyl-CoA to 1,3-butanediol.
  • the enzymatic steps are: A) acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), B) 3-oxobutyraldehyde reductase (ketone reducing), C) 3-hydroxybutyraldehyde reductase, D) acetoacetyl-CoA reductase (CoA-dependent, alcohol forming) , E) 3-oxobutyraldehyde reductase (aldehyde reducing), F) 4-hydroxy,2-butanone reductase, G) acetoacetyl-CoA reductase (ketone reducing), H) 3-hydroxybutyryl-CoA reductase (aldehyde forming), I) 3-hydroxybutyryl-CoA reductase (alcohol forming), J) 3-hydroxy
  • Figure 4 shows a biosynthetic metabolic pathway for the conversion of carbohydrates, such as glucose, gases including CO, C0 2 , and/or H 2 , and methanol to acetyl-CoA and further to 1,3- butanediol.
  • carbohydrates such as glucose, gases including CO, C0 2 , and/or H 2
  • methanol acetyl-CoA
  • ACTP acetyl phosphate
  • CFeSp corrinoid iron sulfur protein
  • FOR formate
  • MeOH methanol
  • METHF methyltetrahydrofolate
  • MLTHF MLTHF
  • THF tetrahydrofolate
  • Figure 5 shows a biosynthetic metabolic pathway for the conversion of carbohydrates, such as glucose, gases including CO, C0 2 , and/or H 2 and methanol to acetyl-CoA, and further to 1,3- butanediol.
  • carbohydrates such as glucose, gases including CO, C0 2 , and/or H 2 and methanol
  • acetyl-CoA 1,3- butanediol.
  • the specific enzymatic transformations that are engineered into a production host are numbered and shown in the figure.
  • THF tetrahydrofolate
  • Figure 6 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91 (lane2), Mta98/99 (lanes 3 and 4) cell extracts with size standards (lane 5) and controls of M. thermoacetica CODH (Moth_1202/1203) or Mtr (Moth_l 197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and 1000 ng).
  • Figure 7 shows cuvettes used in a methyl viologen assay. A blank is on the right and a cuvette with reduced methyl viologen is on the left. Note, the stoppers and vacuum grease on top of each are used to keep the reactions anaerobic.
  • Figure 8 shows a spectrogram of ACS90 cell extracts assayed for transfer of CH 3 from added CH 3 -THF to purified M. thermoacetica corrinoid protein.
  • Figure 9 shows from left to right, anaerobic growth of recombinant E. coli MG1655 in N 2 and CO for 36 hr at 37 °C, empty vector, ACS90, and ACS91.
  • Figure 10 shows an exemplary flux distribution that can produce a 1.09 mol/mol yield of 1,3- butanediol on glucose by emplying the Wood-Ljungdahl pathway enzymes in combination with a 1,3-butanediol biosynthetic pathway.
  • Figure 11 shows an exemplary flux distribution that can produce a 1.2 mol/mol yield of 1,3- butanediol on glucose by employing enzymes that allow the co-utilization of methanol as a substrate.
  • the present invention relates to developing and using microorganisms capable of utilizing carbohydrates, methanol, syngas and/or other gaseous carbon sources to produce 1,3-butanediol.
  • the invention further relates to expanding the product range of syngas -utilizing microorganisms and generating recombinant organisms capable of utilizing syngas to produce 1,3-butanediol and optimizing their yields, titers, and productivities.
  • Development of a recombinant organism for example, Escherichia coli or other organisms suitable for commercial scale up, that can efficiently utilize syngas as a substrate for growth and for chemical production provides cost- advantaged processes for renewable chemical and fuel manufacturing. The organisms can be optimized and tested rapidly and at reasonable costs.
  • the potential of syngas as a feedstock resides in its ability to be efficiently and cost-effectively converted into chemicals and fuels of interest.
  • Two main technologies for syngas conversion are Fischer-Tropsch processes and fermentative processes.
  • the Fischer- Tropsch (F-T) technology has been developed since World War II and involves inorganic and metal-based catalysts that allow efficient production of methanol or mixed hydrocarbons as fuels.
  • the drawbacks of F-T processes are: 1) a lack of product selectivity, which results in difficulties separating desired products; 2) catalyst sensitivity to poisoning; 3) high energy costs due to high temperatures and pressures required; and 4) the limited range of products available at commercially competitive costs.
  • syngas has been shown to serve as a carbon and energy source for many anaerobic microorganisms that can convert this material into products such as ethanol, acetate and hydrogen.
  • the main benefits of fermentative conversion of syngas are the selectivity of organisms for production of single products, greater tolerance to syngas impurities, lower operating temperatures and pressures, and potential for a large portfolio of products from syngas.
  • the main drawbacks of fermentative processes are that organisms known to convert syngas tend to generate only a limited range of chemicals, such as ethanol and acetate, and are not efficient producers of other chemicals, the organisms lack established tools for genetic manipulation, and the organisms are sensitive to end products at high concentrations.
  • the present invention relates to the generation of microorganisms that are effective at producing 1,3-butanediol from syngas or other gaseous carbon sources.
  • the organisms and methods of the present invention allow production of 1,3-butanediol at costs that are significantly advantaged over both traditional petroleum-based products and products derived directly from glucose, sucrose or lignocellulosic sugars.
  • the invention provides a non-naturally occurring microorganism capable of utilizing syngas or other gaseous carbon sources to produce 1,3-butanediol in which the parent microorganism lacks the natural ability to utilize syngas, as shown in Figure 4 and 5.
  • one or more proteins or enzymes are expressed in the microorganism, thereby conferring a pathway to utilize syngas or other gaseous carbon source to produce a desired product.
  • the invention provides a non-naturally occurring microorganism that has been genetically modified, for example, by expressing one or more exogenous proteins or enzymes that confer an increased efficiency of production of 1,3-butanediol, where the parent microorganism has the ability to utilize syngas or other gaseous carbon source to produce a desired product.
  • the invention relates to generating a microorganism with a new metabolic pathway capable of utilizing syngas as well as generating a microorganism with improved efficiency of utilizing syngas or other gaseous carbon source to produce 1,3-butanediol.
  • the present invention additionally provides a non-naturally occurring microorganism expressing genes encoding enzymes that catalyze reactions associated with the carbonyl-branch of the Wood-Ljungdahl pathway in conjunction with a MtaABC-type methyltransferase system.
  • a non-naturally occurring microorganism expressing genes encoding enzymes that catalyze reactions associated with the carbonyl-branch of the Wood-Ljungdahl pathway in conjunction with a MtaABC-type methyltransferase system.
  • Such an organism is capable of converting methanol, a relatively inexpensive organic feedstock that can be derived from synthesis gas, and gases comprising CO, C0 2 , and/or H 2 into acetyl-CoA, cell mass, and products.
  • the present invention further provides pathways that can achieve an increased yield of 1,3-butandiol on carbohydrate feedstocks over what would be naturally expected, that is about 1 mol 1,3-butanediol/mol of glucose, by providing an efficient mechanism for fixing the carbon present in methanol or carbon dioxide, fed exogenously or produced endogenously, into acetyl-CoA.
  • Escherichia coli is an industrial workhorse organism with an unrivaled suite of genetic tools.
  • Engineering the capability to convert C0 2 , Co, and/or H 2 into acetyl-CoA, the central metabolite from which all cell mass components and many valuable products can be derived, into a foreign host such as E. coli can be accomplished following the expression of exogenous genes that encode various proteins of the Wood-Ljungdahl pathway.
  • This pathway is highly active in acetogenic organisms such as Moorella thermoacetica (formerly, Clostridium thermoaceticum), which has been the model organism for elucidating the Wood-Ljungdahl pathway since its isolation in 1942 (Fontaine et al., J Bacteriol.
  • the Wood-Ljungdahl pathway comprises two branches: the Eastern, or methyl, branch that allows the conversion of C0 2 to methyltetrahydrofolate (Me-THF) and the Western, or carbonyl, branch that allows the conversion of methyl-THF, CO, and Coenzyme-A into acetyl-CoA (see Figures 1 and 2).
  • the invention provides a non-naturally occurring microorganism expressing genes that catalyze both branches of the Wood-Ljungdahl pathway, in addition to genes for the production of 1,3-butanediol.
  • Such an organism is capable of converting gases comprising CO, C02, and/or H 2 into acetyl-CoA, 1,3-butanediol, cell mass, and products.
  • Such an organism is also capable of producing 1,3-butanediol from carbohydrates at the stoichiometric optimum yield.
  • the Wood-Ljungdahl enzymes provide the means to produce 12 moles of 1,3-butanediol for every 11 moles of glucose as opposed to 1 mol 1,3-butanediol/l mol glucose which would be attainable in the absence of the Wood-Ljungdahl pathway enzymes.
  • the invention additionally provides a non-naturally occurring microorganism expressing genes encoding enzymes that catalyze the carbonyl-branch of the Wood-Ljungdahl pathway in conjunction with a MtaABC-type methy transferase system, in addition to genes for the production of 1,3-butanediol.
  • Such an organism is capable of converting methanol, a relatively inexpensive organic feedstock that can be derived from synthesis gas, and gases comprising CO, C0 2 , and/or H 2 into acetyl-CoA, 1,3-butanediol, cell mass, and products.
  • the organism can utilize methanol exclusively or in combination with carbohydrate feedstocks such as glucose to produce 1,3-butanediol at high yield.
  • 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 H 2 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 .
  • gaseous carbon sources such as syngas comprising CO and/or C0 2 can be utilized by non-naturally occurring microorganisms of the invention to produce 1,3-butanediol.
  • syngas Although generally exemplified herein as syngas, it is understood that any source of gaseous carbon comprising CO and/or C0 2 can be utilized by the non-naturally occurring
  • the invention relates to non-naturally occurring microorganisms that are capable of utilizing CO and/or C0 2 as a carbon source.
  • the Wood-Ljungdahl pathway catalyzes the conversion of CO and H 2 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 C0 2 /H 2 mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway.
  • H 2 -dependent conversion of C0 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.
  • non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize CO2 and 3 ⁇ 4 mixtures as well for the production of acetyl- Co A and other desired products, such as 1,3-butanediol.
  • 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 corrinoid protein methyltransferase (for example, AcsE), corrinoid iron- sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, C00C).
  • the non-naturally occurring microorganisms of the invention can use syngas or other gaseous carbon sources providing CO and/or CO2 to produce 1,3-butanediol.
  • additional sources include, but are not limited to, production of CO2 as a byproduct in ammonia and hydrogen plants, where methane is converted to CO2; combustion of wood and fossil fuels; production of CO2 as a byproduct of fermentation of sugar in the brewing of beer, whisky and other alcoholic beverages, or other fermentative processes; thermal decomposition of limestone, CaCC>3, in the manufacture of lime, CaO; production of CO2 as byproduct of sodium phosphate manufacture; and directly from natural carbon dioxide springs, where it is produced by the action of acidified water on limestone or dolomite.
  • Acetogens such as Moorella thermoacetica, C. ljungdahlii and C. carboxidivorans, can grow on a number of carbon sources ranging from hexose sugars to carbon monoxide.
  • Hexoses such as glucose
  • EMP Embden-Meyerhof-Parnas
  • PFOR pyruvate:ferredoxin oxidoreductase
  • Acetyl- CoA can be used to build biomass precursors or can be converted to acetate which produces energy via acetate kinase and phosphotransacetylase.
  • the overall conversion of glucose to acetate, energy, and reducing equivalents is
  • Acetogens extract even more energy out of the glucose to acetate conversion while also maintaining redox balance by further converting the released C0 2 to acetate via the Wood- Ljungdahl pathway:
  • n in the above equation signifies that this conversion is an energy generating endeavor, as many acetogens can grow in the presence of C0 2 via the Wood-Ljungdahl pathway even in the absence of glucose as long as hydrogen is present to supply the necessary reducing equivalents.
  • the Wood-Ljungdahl pathway illustrated in Figure 1 , is coupled to the creation of Na + or H + ion gradients that can generate ATP via an Na + - or H + - dependant ATP synthase, respectively Muller V. Energy conservation in acetogenic bacteria. Appl Environ Microbiol 69:6345-6353 (2003). Based on these known transformations, acetogens also have the capacity to utilize CO as the sole carbon and energy source. Specifically, CO can be oxidized to produce reducing equivalents and C0 2 , or directly assimilated into acetyl-CoA which is subsequently converted to either biomass or acetate. 4 CO + 2 H 2 0 ⁇ CH 3 COOH + 2 C0 2
  • Hydrogenic bacteria such as R. rubrum can also generate energy from the conversion of CO and water to hydrogen (see Figure 1) (Simpma et al., Critical Reviews in Biotechnology, 26.1 :41-65 (2006)).
  • a central mechanism is the coordinated action of an energy converting hydrogenase (ECH) and CO dehydrogenase.
  • ECH energy converting hydrogenase
  • CO dehydrogenase supplies electrons from CO which are then used to reduce protons to H 2 by ECH, whose activity is coupled to energy-generating proton translocation.
  • the net result is the generation of energy via the water-gas shift reaction.
  • the processes disclosed herein for the biosynthetic production of 1,3-BDO via syngas involve sustainable manufacturing practices that utilize renewable feedstocks, reduce energy intensity and lower greenhouse gas emissions.
  • biobased-l,3-BDO represents a renewable route to butadiene in small end-use facilities where no transport of this flammable and reactive chemical is required.
  • 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 a 1,3-butanediol biosynthetic pathway.
  • 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 is 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. Therefore, 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.
  • microbial As used herein, 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. Therefore, 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
  • 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.
  • 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. Therefore, 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. When used in reference to a biosynthetic activity, 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. Similarly, 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. The term “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.
  • exogenous nucleic acid when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acid refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such 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.
  • the non-naturally occurring microbal 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 can be greater than 50 generations, including indefinitely.
  • Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.
  • An 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.
  • orthologs 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.
  • 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, luciferase 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 can 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.
  • Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm can be as set forth below.
  • amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on.
  • Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept-16-1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.
  • Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.
  • the present invention provides a non-naturally occurring microbial organism having a 1,3-butanediol (1,3-BDO) pathway that includes at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme or protein expressed in a sufficient amount to produce 1,3-BDO.
  • 1,3-BDO 1,3-butanediol
  • the 1,3-BDO pathway includes Methanol methyltransferase (MtaB), Corrinoid protein (MtaC), Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD), Nickel-protein assembly protein (AcsF & CooC), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB & AcsC), Carbon monoxide dehydrogenase (AcsA), Hydrogenase (Hyd), Acetoacetyl-CoA thiolase (AtoB), Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 3-oxobutyraldehyde reductase (ketone
  • Methyltetrahydrofolate corrinoid protein methyltransferase (MtaA), 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase (CoA- dependent, aldehyde forming), 13) 3-oxobutyraldehyde reductase (ketone reducing), and 14) 3- hydroxybutyraldehyde reductase; C: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC),
  • Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), and 14) 4-hydroxy,2-butanone reductase; F: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (MtaA), 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA transferase, hydrolase, or synthetase, 13) Acetoacetate reductase, 14) 3-oxobutyraldehyde reductase (ketone reducing), and 15) 3-hydroxybutyraldehyde reductase; G: 1) Methanol methyltransferase (MtaB), 2) Corrinoid
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (MtaA), 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase (ketone reducing), 13) 3-hydroxybutyryl-CoA transferase, hydrolase, or synthetase, 14) 3- hydroxybutyrate reductase, and 15) 3-hydroxybutyraldehyde reductase; I: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein
  • Acetoacetyl-CoA transferase, hydrolase, or synthetase 13) 3 -hydroxybutyrate dehydrogenase, 14) 3 -hydroxybutyrate reductase, and 15) 3-hydroxybutyraldehyde reductase.
  • the non-naturally occurring microbial organism can include two exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in some embodiments, three exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, four exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, five exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, six exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme in other embodiments, seven exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, eight exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, nine exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, 10 exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments,
  • any of the at least one exogenous nucleic acid can be a heterologous nucleic acid and the non-naturally occurring microbial organism can be constructed for culturing in a substantially anaerobic culture medium.
  • Such microbial organisms can use a carbon feedstock selected from 1) methanol and CO, 2) methanol, C0 2 , and H 2 , 3) methanol, CO, C0 2 , and H 2 , 4) methanol and synthesis gas comprising CO and H 2 , 5) methanol and synthesis gas comprising CO, C0 2 , and H 2 , 6) one or more carbohydrates, 7) methanol and one or more carbohydrates, and 8) methanol.
  • Exemplary carbohydrates include, but are not limited to, glucose, sucrose, xylose, arabinose, and glycerol.
  • the present invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a 1,3-butanediol (1,3-BDO) pathway comprising at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme or protein expressed in a sufficient amount to produce 1,3-BDO said 1,3-BDO pathway comprising Formate dehydrogenase, Formyltetrahydrofolate synthetase, Methenyltetrahydrofolate cyclohydrolase, Methylenetetrahydrofolate dehydrogenase, Methylenetetrahydrofolate reductase, Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), Corrinoid iron- sulfur protein (AcsD), Nickel-protein assembly protein (AcsF & CooC), Ferredoxin (Orf7), Acetyl-CoA synthase
  • the 1,3-BDO pathway enzymes are a set of enzymes selected from: A: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3) Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 7) Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein (AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB & AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd), 13) Acetoacetyl-CoA
  • Methyltetrahydrofolate corrinoid protein methy transferase (AcsE), 7) Corrinoid iron- sulfur protein (AcsD), 8) Nickel-protein assembly protein (AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB & AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd), 13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl- Co A reductase (CoA-dependent, aldehyde forming), 15) 3-oxobutyraldehyde reductase (ketone reducing), and 16) 3-hydroxybutyraldehyde reductase; C: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase,
  • the non-naturally occurring microbial organism can include two exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in some embodiments, three exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, four exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, five exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, six exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme in other embodiments, seven exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, eight exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, nine exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments, 10 exogenous nucleic acids each encoding a 1,3-BDO pathway enzyme, in other embodiments,
  • any of the at least one exogenous nucleic acid can be a heterologous nucleic acid and the non-naturally occurring microbial organism can be constructed for culturing in a substantially anaerobic culture medium.
  • Such microbial organisms can use a carbon feedstock selected from 1) CO, 2) C0 2 and H 2 , 3) CO, C0 2 , and H 2 , 4) synthesis gas comprising CO and H 2 , 5) synthesis gas comprising CO, C0 2 , and H 2 , and 6) one or more carbohydrates.
  • Exemplary carbohydrates include, but are not limted to, glucose, sucrose, xylose, arabinose, and glycerol.
  • the invention provides a non-naturally occurring microbial organisms having a 1,3-butanediol pathways, 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 carbon dioxide to FOR, FOR to 10FTHF, lOFTHFto METHF, METHF to MLTHF, MLTHF to 5MTHF, methanol to CH 3 -MtaC, CH 3 -MtaC to 5MTHF, 5MTHF to CFeSp-CH 3 , CFeSp-CH 3 to acetyl-CoA, acetyl- CoA to acetoacetyl-CoA, acetoacetyl-CoA to 3-hydroxybutryl-CoA, 3-hydroxybutryl-CoA to 3- hydroxybutyraldehyde, 3 -hydroxybutry aldehyde to 1,3-BDO, 3-hydroxy
  • 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 a 1,3-butanediol pathway, such as those shown in Figures 4 and 5.
  • the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a 1,3-butanediol pathway enzyme or protein expressed in a sufficient amount to produce an intermediate of a 1,3- butanediol pathway.
  • 1,3-butanediol pathway are exemplified in Figures 4 and 5.
  • the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a 1,3- butanediol pathway enzyme, where the microbial organism produces a 1,3-butanediol pathway intermediate, for example, acetyl-CoA, acetoacetyl-CoA, acetoacetate, 3-hydroxybutryl-CoA, 3- hydroxybutyrate, 3-oxobutyraldehyde, 4-hydroxy-2-butanone, and 3 -hydroxybutry aldehyde.
  • any of the pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of Figures 4 and 5, 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 a 1,3-butanediol pathway intermediate can be utilized to produce the intermediate as a desired product.
  • non-naturally occurring organisms of the invention can be used to produce, for example, acetoacetate, 3- hydroxybutyrate, 3-oxobutyraldehyde, 3-hydroxybutyraldehyde, or 4-hydroxy-2-butanone.
  • any of these intermediate products can be produced in a separate organism utilizing the pathways shown in Figures 4 and 5.
  • non-naturally occurring organisms described herein have three capabilities which are depicted in Figure 4: 1) a functional methyltransferase system allowing the production of 5-methyl-tetrahydrofolate (Me-THF) from methanol and THF, 2) the ability to combine CO, Coenzyme A, and the methyl group of Me-THF to form acetyl-CoA, and 3) the ability to synthesize 1,3-butanediol from acetyl-CoA.
  • Me-THF 5-methyl-tetrahydrofolate
  • the latter can be facilitated by the generation of pyruvate from a carbohydrate via glycolysis.
  • Glycolysis is an anaerobic metabolic pathway that is found in the cytoplasm of cells in all living organisms. The process converts one molecule of glucose into two molecules of pyruvate, while providing two net molecules of ATP:
  • Such non-naturally occurring organisms can 'fix' carbon from exogenous CO and/or C0 2 and methanol to synthesize acetyl-CoA, cell mass, and products.
  • implementing the pathway to form acetyl-CoA from methanol and syngas is energetically favorable compared to utilizing the full Wood-Ljungdahl pathway.
  • the direct conversion of synthesis gas to acetate is an energetically neutral process (see Figures 1 and 2). Specifically, one ATP molecule is consumed during the formation of formyl-THF by formyl-THF synthase and one ATP molecule is produced during the production of acetate via acetate kinase.
  • the pathways disclosed herein circumvent the ATP consumption by ensuring that the methyl group on the methyl branch product, methyl-THF, is obtained from methanol rather than C0 2 . This ensures that acetate formation has a positive ATP yield that can help support cell growth and maintenance.
  • a host organism engineered with these capabilities that also possesses the capability for anapleurosis (e.g., E. coli) can grow on the methanol and syngas-generated acetyl- CoA in the presence of a suitable external electron acceptor such as nitrate. This electron acceptor is used to accept electrons from the reduced quinone formed via succinate
  • a further benefit of adding an external electron acceptor is that additional energy for cell growth, maintenance, and product formation can be generated from respiration of acetyl-CoA.
  • another non-naturally occurring microbial organism has a pyruvate ferredoxin oxidoreductase (PFOR) enzyme or other enzymes that facilitate the conversion of pyruvate into acetyl-CoA or vice versa into the strain to facilitate the synthesis of biomass precursors in the absence of an external electron acceptor.
  • PFOR ferredoxin oxidoreductase
  • a further characteristic of such non-naturally occurring organisms is the capability for extracting reducing equivalents from molecular hydrogen.
  • the organisms described in this invention can produce acetyl-CoA, cell mass, and targeted chemicals, more specifically 1,3-BDO, from: 1) methanol and CO, 2) methanol, C0 2 , and H 2 , 3) methanol, CO, C0 2 , and H 2 , 4) methanol and synthesis gas comprising CO and H 2 , 5) methanol and synthesis gas comprising CO, C0 2 , and H 2 , 6) one or more carbohydrates, 7) methanol and one or more carbohydrates, and 8) methanol.
  • Exemplary carbohydrates include, but are not limited to, glucose, sucrose, xylose, arabinose, and glycerol.
  • Successfully engineering these pathways into an organism involves identifying an appropriate set of enzymes, cloning their corresponding genes into a production host, optimizing the stability and expression of these genes, optimizing fermentation conditions, and assaying for product formation following fermentation.
  • a number of enzymes that catalyze each step of the pathways used the conversion of synthesis gas and methanol to acetyl-CoA, and further to 1,3- butanediol are described below.
  • To engineer a production host for the utilization of syngas and methanol one or more exogenous DNA sequence(s) encoding the enzymes of these pathways can be expressed in the microorganism.
  • MtaB is a zinc protein that catalyzes the transfer of a methyl group from methanol to MtaC, a corrinoid protein.
  • Exemplary genes encoding MtaB and MtaC can be found in methanogenic archaea such as Methanosarcina barkeri (Maeder et al., /. Bacteriol. 188.22:7922-7931 (2006)) and Methanosarcina acetivorans (Galagan et al., Genome Res. 12/4:532-542 (2002)), as well as the acetogen, Moorella thermoacetica (Das et al., Proteins 67.1 : 167-176 (2007)).
  • MtaB and MtaC genes are adjacent to one another on the chromosome as their activities are tightly interdependent.
  • the protein sequences of various MtaB and MtaC encoding genes in M. barkeri, M. acetivorans, and M. thermoaceticum can be identified by their following GenBank accession numbers: Protein GenBank ID GI number Organism
  • the MtaBl and MtaCl genes, YP_304299 and YP_304298, from M. barkeri were cloned into E. coli and sequenced (Sauer et al., Eur. J. Biochem. 243.3:670-677 (1997)). The crystal structure of this methanol-cobalamin methyltransferase complex is also available Hagemeier et al., Proc. Natl. Acad. Sci. U.S.A. 103.50: 18917-18922 (2006)).
  • the MtaB genes, YP_307082 and YP_304612, in M. barkeri were identified by sequence homology to YP_304299.
  • MtaB encoding genes show little or no similarity to methyltransferases that act on alternative substrates such as trimethylamine, dimethylamine, monomethylamine, or dimethylsulfide.
  • the MtaC genes, YP_307081 and YP_304611, were identified based on their proximity to the MtaB genes and also their homology to YP_304298.
  • the three sets of MtaB and MtaC genes from M. acetivorans have been genetically, physiologically, and biochemically characterized Pritchett and Metcalf, Mol. Microbiol. 56.5: 1183-1194 (2005)).
  • M. thermoacetica MtaB gene was identified based on homology to the methanogenic MtaB genes and also by its adjacent chromosomal proximity to the methanol-induced corrinoid protein, MtaC, which has been crystallized (Zhou et al., Acta Crystallogr.Sect.F Struct. Biol Cryst. Commun. 61 Pt.
  • MtaA is zinc protein that catalyzes the transfer of the methyl group from MtaC to either
  • MtaA Coenzyme M in methanogens or methyltetrahydrofolate in acetogens.
  • MtaA can also utilize methylcobalamin as the methyl donor.
  • Exemplary genes encoding MtaA can be found in methanogenic archaea such as Methanosarcina barkeri (Maeder et al., /. Bacteriol. 188.22 7922-7931 (2006)) and Methanosarcina acetivorans (Galagan et al., Genome Res. 12.4:532-542 (2002)), as well as the acetogen, Moorella thermoacetica (Das et al., Proteins 67.1: 167-176 (2007)).
  • MtaA proteins that catalyze the transfer of the methyl group from C3 ⁇ 4- MtaC are difficult to identify bioinformatically as they share similarity to other corrinoid protein methyl transferases and are not oriented adjacent to the MtaB and MtaC genes on the chromosomes. Nevertheless, a number of MtaA encoding genes have been characterized. The protein sequences of these genes in M. barkeri and M. acetivorans can be identified by the following GenBank accession numbers:
  • MtaA gene YP_304602, from M. barkeri was cloned, sequenced, and functionally overexpressed in E. coli (Harms and Thauer, Eur. J. Biochem. 135.3:653-659 (1996).
  • MtaAl is used for growth on methanol, whereas MtaA2 is dispensable even though methane production from methanol is reduced in MtaA2 mutants (Bose et al. /. Bacteriol. 190.11:4017-4026 (2008).
  • MtaA homologs in M. barkeri and M.
  • M. thermoacetica acetivorans that are as yet uncharacterized, but can also catalyze corrinoid protein methyltransferase activity.
  • Putative MtaA encoding genes in M. thermoacetica were identified by their sequence similarity to the characterized methanogenic MtaA genes. Specifically, three M. thermoacetica genes show high homology (>30 sequence identity) to YP_304602 from M. barkeri. Unlike methanogenic MtaA proteins that naturally catalyze the transfer of the methyl group from CH 3 - MtaC to Coenzyme M, an M. thermoacetica MtaA can transfer the methyl group to
  • ACS/CODH is the central enzyme of the carbonyl branch of the Wood-Ljungdahl pathway. It catalyzes the reversible reduction of carbon dioxide to carbon monoxide and also the synthesis of acetyl-CoA from carbon monoxide, Coenzyme A, and the methyl group from a methylated corrinoid-iron-sulfur protein.
  • the corrinoid-iron- sulfur-protein is methylated by
  • methyltetrahydrofolate via a methyltransferase expression of ACS/CODH in a foreign host involves introducing one or more of the following proteins: Methyltetrahydrofolate:corrinoid, protein methyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD), Nickel-protein assembly protein (AcsF), Ferredoxin (OrfT), Acetyl-CoA synthase (AcsB and AcsC), Carbon monoxide dehydrogenase (AcsA), Nickel-protein assembly protein (CooC).
  • the genes used for carbon-monoxide dehydrogenase/acetyl-CoA synthase activity typically reside in a limited region of the native genome that may be an extended operon (Ragsdale, S.W., Crit. Rev. Biochem. Mol. Biol. 39.3: 165-195 (2004); Morton et al., /. Biol. Chem. 266.35:23824- 23828 (1991); Roberts et al., Proc. Natl. Acad. Sci. U.S.A. 86.1:32-36 (1989)).
  • Each of the genes in this operon from the acetogen, M. thermoacetica has already been cloned and expressed actively in E.
  • the hydrogenogenic bacterium Carboxydothermus hydro genoformans
  • the acetyl-CoA synthase enzyme complex lacks carbon monoxide dehydrogenase due to a frameshift mutation (Wu et al. PLos Genet. 1.5:e65 (2005)), whereas in strain DSM 6008, a functional unframeshifted full-length version of this protein has been purified (Svetlitchnyi et al., Proc. Natl. Acad. Sci. U.S.A. 101.2:446-451 (2004)).
  • the protein sequences of the C. hydro genojormans genes from strain Z-2901 can be identified by the following GenBank accession numbers:
  • the methanogenic archaeon, Methanosarcina acetivorans can also grow on carbon monoxide, exhibits acetyl-CoA synthase/carbon monoxide dehydrogenase activity, and produces both acetate and formate (Lessner et al., Proc. Natl. Acad. Sci. U.S.A. 103.47: 17921-17926 (2006)).
  • This organism contains two sets of genes that encode ACS/CODH activity (Rother and Metcalf, Proc. Natl. Acad. Sci. U.S.A. 101.48: 16929-16934 (2004)).
  • the protein sequences of both sets of M. acetivorans genes can be identified by the following GenBank accession numbers:
  • the AcsC, AcsD, AcsB, AcsE, and AcsA proteins are commonly referred to as the gamma, delta, beta, epsilon, and alpha subunits of the methanogenic CODH/ACS. Homologs to the epsilon encoding genes are not present in acetogens such as M. thermoacetica or
  • CODH encoding genes are located outside of the ACS/CODH operons. These enzymes provide a means for extracting electrons (or reducing equivalents) from the conversion of carbon monoxide to carbon dioxide. The reducing equivalents are then passed to acceptors such as oxidized ferredoxin, NADP+, water, or hydrogen peroxide to form reduced ferredoxin, NADPH, H 2 , or water, respectively. In some cases, hydrogenase encoding genes are located adjacent to a CODH.
  • Rhodospirillum rubrum the encoded CODH/hydrogenase proteins form a membrane-bound enzyme complex that has been indicated to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO to C0 2 and H 2 (Fox et al., /. Bacteriol. 178.21:6200-6208 (1996)).
  • the CODH-I of C. hydrogenoformans and its adjacent genes have been proposed to catalyze a similar functional role based on their similarity to the R. rubrum CODH/hydrogenase gene cluster (Wu et al., PLoS Genet. 1.5:e65 (2005)).
  • CODH-II The crystal structure of the CODH-II is also available (Dobbek et al., Science 293.5533: 1281-1285 (2001)).
  • GenBank accession numbers The protein sequences of exemplary CODH and hydrogenase genes can be identified by the following GenBank accession numbers:
  • PFOR pyruvate ferredoxin oxidoreductase
  • Oxygen stability is relatively uncommon in PFORs but can 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 and Ragsdale Biochemistry 36.28:8484-8494 (1997)) and was shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui and Ragsdale, /. Biol. Chem. 275.37:28494-28499 (2000)).
  • E. coli possesses an uncharacterized open reading frame, ydbK, that encodes a protein that is 51% identical to the M. thermoacetica PFOR.
  • pyruvate dehydrogenase can transform pyruvate into acetyl- CoA with the concomitant reduction of a molecule of NAD into NADH. It is a multi-enzyme complex that catalyzes a series of partial reactions which results in acylating oxidative decarboxylation of pyruvate.
  • the enzyme comprises of three subunits: the pyruvate
  • E3 dehydrogenase
  • This enzyme is naturally present in several organisms, including E. coli and S. cerevisiae.
  • specific residues in the El component are responsible for substrate specificity (Bisswanger, H., /. Biol. Chem. 256:815-82 (1981); Bremer, J., Eur. J. Biochem. 8:535-540 (1969); Gong et al., /. Biol. Chem. 275: 13645-13653 (2000)).
  • Enzyme engineering efforts have improved the E. coli PDH enzyme activity under anaerobic conditions (Kim et al., /. Bacteriol.
  • Azotobacter vinelandii are available (Mattevi et al., Science 255: 1544-1550 (1992)). Yet another enzyme that can catalyze this conversion is pyruvate formate lyase. This enzyme 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 encoded by pflB (Knappe and Sawers, FEMS. Microbiol Rev.
  • E. coli possesses an additional pyruvate formate lyase, encoded by tdcE, that catalyzes the conversion of pyruvate or 2-oxobutanoate to acetyl-CoA or propionyl-CoA, respectively (Hesslinger et al., Mol.
  • pflB and tdcE from E. coli require the presence of pyruvate formate lyase activating enzyme, encoded by pflA.
  • a short protein encoded by yfiD in E. coli can associate with and restore activity to oxygen-cleaved pyruvate formate lyase (Vey et al., ProcNatl. Acad. Sci. U.S.A. 105: 16137-16141 (2008). Note that pflA and pflB from E. coli were expressed in S.
  • acetyl-CoA is obtained in the cytosol by first decarboxylating pyruvate to form acetaldehyde; the latter is oxidized to acetate by acetaldehyde dehydrogenase and subsequently activated to form acetyl-CoA by acetyl-CoA synthetase.
  • Acetyl-CoA synthetase is a native enzyme in several other organisms including E. coli (Kumari et al., /. Bacteriol.
  • acetate can be activated to form acetyl-CoA by acetate kinase and phosphotransacetylase.
  • Acetate kinase first converts acetate into acetyl-phosphate with the accompanying use of an ATP molecule.
  • Acetyl-phosphate and CoA are next converted into acetyl-CoA with the release of one phosphate by phosphotransacetylase.
  • Both acetate kinase and phosphotransacetlyase are well-studied enzymes in several Clostridia and Methanosarcina thermophila
  • Yet another way of converting pyruvate to acetyl-CoA is via pyruvate oxidase. Pyruvate oxidase converts pyruvate into acetate, using ubiquione as the electron acceptor. In E. coli, this activity is encoded by poxB. PoxB has similarity to pyruvate decarboxylase of S. cerevisiae and
  • the enzyme has a thiamin pyrophosphate cofactor (Koland and Gennis, Biochemistry 21:4438-4442 (1982)); O'Brien et al., Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, /. Biol. Chem. 255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD) cofactor.
  • Acetate can then be converted into acetyl-CoA by either acetyl-CoA synthetase or by acetate kinase and phosphotransacetylase, as described earlier. Some of these enzymes can also catalyze the reverse reaction from acetyl-CoA to pyruvate.
  • reducing equivalents are obtained by the conversion of CO and water to C0 2 via carbon monoxide dehydrogenase or directly from the activity of a hydrogen-utilizing hydrogenase which transfers electrons from H 2 to an acceptor such as ferredoxin, flavodoxin, FAD + , NAD + , or NADP + .
  • Native to E. coli and other enteric bacteria are multiple genes encoding up to four hydrogenases (Sawers, G., Antonie van Leeuwenhoek 66.1:57-88 1994); Sawers et al., /. Bacteriol. 168.1 :398- 404 (1986); Sawers and Boxer, Eur. J. Biochem. 156.2:265-275 (1986); Sawers et al., /.
  • E. coli or another host organism can provide sufficient hydrogenase activity to split incoming molecular hydrogen and reduce the corresponding acceptor.
  • E. coli or another host organism can provide sufficient hydrogenase activity to split incoming molecular hydrogen and reduce the corresponding acceptor.
  • the endogenous hydrogen-lyase enzymes of E. coli are hydrogenase 3, a membrane-bound enzyme complex using ferredoxin as an acceptor, and hydrogenase 4 that also uses a ferredoxin acceptor.
  • Hydrogenase 3 and 4 are encoded by the hyc and hyf gene clusters, respectively. Hydrogenase activity in E.
  • M. thermoacetica hydrogenases are suitable candidates should the production host lack sufficient endogenous hydrogenase activity.
  • M. thermoacetica can grow with C0 2 as the exclusive carbon source indicating that reducing equivalents are extracted from H 2 to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L., /. Bacteriol.
  • M. thermoacetica has homologs to several hyp, hyc, and hyf genes from E. coli. These protein sequences encoded for by these genes can be identified by the following GenBank accession numbers. In addition, several gene clusters encoding hydrogenase functionality are present in M. thermoacetica and their corresponding protein sequences are also provided below:
  • Hydrogenase 3 proteins are shown below:
  • Hydrogenase 4 proteins are shown below:
  • 1,3-butanediol production can be achieved in recombinant E. coli by various alternate pathways described in Figure 4 and Figure 5. All pathways first convert two molecules of acetyl-CoA into one molecule of acetoacetyl-CoA employing a thiolase.
  • Acetoacetyl-CoA thiolase 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., Nat. Biotechnol. 21.7:796-802 (2003)), thlA and MB from C. acetobutylicum (Hanai et al., Appl. Environ. Microbiol. 73.24:7814-7818
  • One pathway from acetoacetyl-CoA entails its reduction to 3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase (ketone reducing). This can subsequently be converted to 3- hydroxybutyraldehyde via a CoA-dependent aldehyde reductase called 3-hydroxybutyryl-CoA reductase.
  • 3-hydroxybutyraldehyde can eventually be reduced to the product 1,3-BDO by 3- hydroxybutyraldehyde reductase.
  • 3-hydroxybutyryl-CoA can be reduced directly to 1,3-BDO by an alcohol-forming CoA-dependent 3-hydroxybutyryl-CoA reductase.
  • Acetoacetyl-CoA reductase (ketone reducing) catalyzing the reduction of acetoacetyl-CoA to 3- hydroxybutyryl-CoA participates in the acetyl-CoA fermentation pathway to butyrate in several species of Clostridia and has been studied in detail (Jones and Woods, Microbiol. Rev. 50.4:484- 524 (1986)).
  • the enzyme from Clostridium acetobutylicum, encoded by hbd has been cloned and functionally expressed in E. coli (Youngleson et al., /. Bacteriol. 171.12:6800-6807 (1989)).
  • subunits of two fatty acid oxidation complexes in E. coli function as 3-hydroxyacyl-CoA dehydrogenases (Binstock and Schulz, Methods Enzymol. 71 Pt. C: 403-411 (1981)).
  • Other genes demonstrated to reduce acetoacetyl-CoA to 3- hydroxybutyryl-CoA are phbB from Zoogloea ramigera (Ploux et al., Eur. J. Biochem.
  • Hbd2 N-terminal domain in Clostridium kluyveri (Hillmer and Gottschalk, Biochim. Biophys. Acta 3334: 12-23 (1974)) and HSD17B10 in Bos taurus (Wakil et al., /. Biol. Chem. 207.2:631- 638 (1954)).
  • acyl-CoA dehydrogenases are capable of reducing an acyl-CoA to its corresponding aldehyde and can be used for catalyzing the 3-hydroxybutyryl-CoA reductase (aldehyde forming) activity.
  • Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acrl encoding a fatty acyl-CoA reductase (Reiser and Somerville, /. Bacteriol. 179.9:2969-2975 (1997)), the Acinetobacter sp. M-l fatty acyl-CoA reductase Ishige et al. Appl. Environ. Microbiol.
  • Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol. Biochem. 71.1:58-68 (2007)). Information related to these genes and proteins are show below:
  • 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 et al., Science 318.5857: 1782-1786 (2007); Thauer, R.K., Science 318.5857: 1732-1733).
  • the enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Suljolobus spp (Alber et al., /. Bacteriol.
  • the enzyme is encoded by Msed_0709 in Metallosphaera sedula ((Alber et al., /. Bacteriol. 188.24: 8551-8559 (2006); (Berg et al., Science
  • This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde
  • aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity.
  • malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate - semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent
  • Enzymes exhibiting 3-hydroxybutyraldehyde reductase activity have been characterized in Ralstonia eutropha (Bravo et al., /. Forensic Sci. 49.2:379-387 (2004)), Clostridium kluyveri (Wolff and Kenealy, Protein Expr. Purij. 6.2:206-212 (1995)) and Arabidopsis thaliana (Breit Regen et al., /. Biol. Chem. 278.42: 41552-41556 (2003)). Yet another gene is the alcohol dehydrogenase adhl from Geobacillus thermoglucosidasius (Jeon et al., /. Biotechnol. 135.2:127-133 (2008)). A summary of gene and protein information is shown below:
  • 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., /. Mol. Biol. 352.4:905-917 (2005)).
  • the reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated using isotopically-labeled substrate (Manning and Pollitt, Biochem. 7.231.2:481-484 (1985)).
  • Additional genes encoding this enzyme include 3hidh in Homo sapiens (Hawes et al. Methods Enzymol. 324:218-228 (2000)) and Oryctolagus cuniculus ( Hawes et al. Methods Enzymol. 324:218-228 (2000); Chowdhury et al., Biosci. Biotechnol. Biochem. 60.12:2043-2047 (1996)), mmsb in Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Aberhart and Hsu, J. Chem. Soc. (Perkin 1) 6: 1404-1406; Chowdhury et al., Biosci. Biotechnol. Biochem.
  • dehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol. 66.12:5231-5335 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et al., Nature 451.7174:86-89 (2008)), yqhD from E. coli which has preference for molecules longer than C3 (Sulzenbacher et al., /. Mol. Biol. 342.2:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum which converts butyraldehyde into butanol (Walter et al., /. Bacteriol.
  • the gene product of yqhD catalyzes the reduction of acetaldehyde, malondialdehyde, propionaldehyde, butyraldehyde, and acrolein using NADPH as the cofactor (Perez et al., /. Biol. Chem.
  • ADH1 from Zymomonas mobilis has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al., Appl. Microbiol. Biotchenol. 22:249-254 (1985)).
  • the protein sequences for each of these exemplary gene products, if available, can be found using the following GenBank accession numbers:
  • the alcohol-forming 3-hydroxybutyryl-CoA reductase can be catalyzed by_exemplary two-step oxidoreductases that convert an acyl-CoA to alcohol.
  • oxidoreductases that convert an acyl-CoA to alcohol.
  • These include enzymes that transform substrates such as acetyl-CoA to ethanol (e.g., adhE from E. coli (Kessler et al., FEBS Lett. 281.1-2:59-63 (1991)) and butyryl-CoA to butanol (e.g. adhE2 from C. acetobutylicum (Fontaine et al., /. Bacteriol. 184.3:821-830 (2002)).
  • substrates such as acetyl-CoA to ethanol (e.g., adhE from E. coli (Kessler et al., FEBS Lett. 281.1-2:59-63 (1991)) and buty
  • 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 et al., Plant Physiol. 122.3: 635-644 (2000)). Information related to FAR is shown below:
  • a second alternate pathway from acetoacetyl-CoA to 1,3-butanediol proceeds via the reduction of acetoacetyl-CoA into 3-oxobutyraldehyde via the CoA-dependent aldehyde forming acetoacetyl-CoA reductase.
  • 3-oxobutyraldehyde is next reduced to 3-hydroxybutyraldehyde by 3-oxobutyraldehyde reductase (ketone reducing), and eventually, this intermediate is reduced to 1,3-butanediol by a 3-hydroxybutyraldehyde reductase.
  • the enzymes and genes encoding these enzymes for each of these steps are listed below.
  • Exemplary candidates for acetoacetyl-CoA reductase that catalyzes the transformation of acetoacetyl-CoA into 3-oxobutyraldehyde are the same as those described for 3-hydroxybutyryl-CoA reductase (aldehyde forming) described herein above.
  • coli are encoded by malate dehydrogenase (mdh) and lactate dehydrogenase (IdhA).
  • mdh malate dehydrogenase
  • IdhA lactate dehydrogenase
  • lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on substrates of various chain lengths such as lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel and Schlegel, Eur. J. Biochem. 130.2:329-334 (1983)).
  • Conversion of the oxo functionality to the hydroxyl group can also be catalyzed by 2-ketol,3-butanediol reductase, an enzyme reported to be found in rat and in human placenta (Suda et al., Arch. Biochem. Biophys. 176.2:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun. 342.2:586-591 (1977)). All of these enzymes can be use as a 3-oxobutyraldehyde reductase.
  • An additional enzyme for this step is the mitochondrial 3- hydroxybutyrate dehydrogenase (bdh) from the human heart which has been cloned and characterized (Marks et al., /. Biol. Chem. 267.22: 15459-15463 (1992)).
  • This enzyme is a dehydrogenase that operates on a 3-hydroxyacid.
  • Methyl ethyl ketone (MEK) reductase or alternatively, 2-butanol dehydrogenase, catalyzes the reduction of MEK to form 2-butanol.
  • MEK Methyl ethyl ketone
  • Exemplary enzymes can be found in Rhodococcus ruber (Kosjek et al., Biotechnol. Bioeng. 86.1:55-62 (2004)) and Pyrococcus furiosus (van der Oost et al., Eur. J. Biochem. 268.10:3062-3068 (2001)). Information related to these proteins and genes is shown below:
  • Another pathway from acetoacetyl-CoA proceeds via its reduction to 4-hydroxy,2-butanone by the CoA-dependent, alcohol forming acetoacetyl-CoA reductase.
  • This intermediate is then reduced to 1,3-butanediol by 4-hydroxybutanone reductase.
  • 4-hydroxybutanone can also be formed from 3-oxobutyraldehyde by an aldehyde reducing 3-oxobutyraldehyde reductase.
  • Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming) can utilize the same enzymes as those for the alcohol-forming 3-hydroxybutyryl-CoA reductase.
  • 4-hydroxybutanone reductase activity can be obtained from the same genes as those described for 3-oxobutyraldehyde reductase. Additionally, a number of organisms can catalyze the reduction of 4-hydroxy,2-butanone to 1,3-butanediol, including those belonging to the genus Bacillus, Brevibacterium, Candida, and Klebsiella among others, as described by Matsuyama et al., US Patent No. 5,413,922.
  • Exemplary genes encoding enzymes that catalyze the conversion of an aldehyde to alcohol include alrA encoding a medium- chain alcohol dehydrogenase for C2-C14 (Tani et al., App. Environ. Microbiol. 66.12:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et al., Nature 451.7174:86-89 (2008)), yqhD from E. coli which has preference for molecules longer than C3 (Sulzenbacher et al., /. Mol. Biol.
  • ADH1 from Zymomonas mobilis has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al., App. Microbiol. Biotechnol. 22:249-254 (1985)).
  • acetoacetyl-CoA NP_349892.1 15896543
  • the conversion of acetoacetyl-CoA to acetoacetate can be carried out by a acetoacetyl-CoA transferase which conserves the energy stored in the CoA-ester bond.
  • transferase enzymes capable of catalyzing this transformation are provided below.
  • enzymes either naturally exhibit the desired acetoacetyl-CoA transferase activity or they can be engineered via directed evolution to accept acetetoacetyl-CoA as a substrate with increased efficiency.
  • Such enzymes either naturally or following directed evolution, are also suitable for catalyzing the conversion of 3-hydroxybutyryl-CoA to 3-hydroxybutyrate via a transferase mechanism.
  • Acetoacetyl-CoA transferase naturally converts acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA.
  • This enzyme can also accept 3-hydroxybutyryl-CoA as a substrate or could be engineered to do so.
  • Exemplary enzymes include the gene products of atoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007)), ctfAB from C.
  • Succinyl-CoA:3-ketoacid-CoA transferase naturally 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.
  • acetoacetyl-CoA and 3-hydroxybutyryl-CoA transferases are encoded by the gene products of catl , cat2, and cat3 of Clostridium kluyveri. These enzymes have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activity, respectively (Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008); Sohling and Gottschalk, J Bacteriol 178:871-880 (1996)). Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., /. Biol.
  • FN1857 and FN1856 are located adjacent to many other genes involved in lysine fermentation and are thus very can encode an acetoacetate:butyrate CoA transferase (Kreimeyer, et al., /. Biol. Chem. 282 (10) 7191-7197 (2007)). Additional genes/gene products from Porphyrmonas gingivalis and Thermoanaerobacter tengcongensis can be identified in a similar fashion (Kreimeyer, et al., /. Biol. Chem. 282 (10) 7191-7197 (2007)). Information related to these proteins and genes is shown below:
  • Acetoacetyl-CoA can be hydrolyzed to acetoacetate by acetoacetyl-CoA hydrolase.
  • 3-hydroxybutyryl-CoA can be hydrolyzed to 3-hydroxybutyate by 3-hydroxybutyryl-CoA hydrolase.
  • Many CoA hydrolases (EC 3.1.2.1) have broad substrate specificity and are suitable enzymes for these transformations either naturally or following enzyme engineering. Though the sequences were not reported, several acetoacetyl-CoA hydrolases were identified in the cytosol and mitochondrion of the rat liver (Aragon and Lowenstein, /. Biol. Chem. 258(8):4725- 4733 (1983)).
  • Rattus norvegicus brain can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA.
  • the acotl2 enzyme from the rat liver was shown to hydrolyze C2 to C6 acyl-
  • CoA molecules (Suematsu et al., Eur. J. Biochem. 268:2700-2709 (2001)). Though its sequence has not been reported, the enzyme from the mitochondrion of the pea leaf showed activity on acetyl-CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, and crotonyl-CoA (Zeiher and Randall, Plant.Physiol. 94:20-27 (1990)).
  • a glutaconate CoA-transferase from Acidaminococcus fermentans was transformed by site-directed mutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA, acetyl-CoA and 3- butenoyl-CoA (Mack and Buckel, FEBS Lett. 405:209-212 (1997)).
  • This indicates that the enzymes encoding succinyl-CoA:3-ketoacid-CoA transferases and acetoacetyl-CoA:acetyl-CoA transferases can also be used as hydrolases with certain mutations to change their function.
  • the acetyl-CoA hydrolase, ACH1 from S.
  • hydrolase enzyme 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., /. 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., /. Biol. Chem.
  • E. coli thioester hydrolases include the gene products of tesA (Bonner and Bloch, /. Biol. Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol. Rev. 29:263-279 (2005); Zhuang et al., FEBS Lett.
  • Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolase which has been described to efficiently catalyze 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 (1994); Shimomura et al., Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra (1994).
  • Candidate genes by sequence homology include hibch of Saccharomyces cerevisiae and BC 2292 of Bacillus cereus. BC_2292 was shown to demonstrate 3-hydroxybutyryl-CoA hydrolase activity and function as part of a pathway for 3- hydroxybutyrate synthesis when engineered into Escherichia coli (Lee et al., Appl. Microbiol. Biotechnol. 79:633-641 (2008)). Information related to these proteins and genes is shown below:
  • An alternative method for removing the CoA moiety from acetoacetyl-CoA or 3- hydroxybutyryl-CoA is to apply a pair of enzymes such as a phosphate-transferring
  • acyltransferase and a kinase to impart acetoacetyl-CoA or 3-hydroxybutyryl-CoA synthetase activity.
  • a phosphotransacylase and a kinase enzyme is referred to as a "synthetase.” This activity allows the net hydrolysis of the CoA-ester of either molecule with the simultaneous generation of ATP.
  • &i/fc/phosphotransbutyrylase (ptb) system from Clostridium acetobutylicum has been successfully applied to remove the CoA group from 3-hydroxybutyryl-CoA when functioning as part of a pathway for 3 -hydroxybutyrate synthesis (Tseng et al., Appl. Environ. Microbiol. 75(10):3137-3145 (2009)).
  • the ptb gene from C. acetobutylicum encodes an enzyme that can convert an acyl-CoA into an acyl-phosphate (Walter et al. Gene 134(1): p. 107- 11 (1993)); Huang et al. J Mol Microbiol Biotechnol 2(1): p.
  • Additional exemplary phosphate-transferring acyltransferases include phosphotransacetylase, encoded by pta.
  • the pta gene from E. coli encodes an enzyme that can convert acetyl-CoA into acetyl-phosphate, and vice versa (Suzuki, T. Biochim.Biophys.Acta 191:559-569 (1969)).
  • This enzyme can also utilize propionyl-CoA instead of acetyl-CoA forming propionate in the process (Hesslinger et al. Mol.Microbiol 27:477-492 (1998)).
  • Information related to these proteins and genes is shown below:
  • Exemplary kinases include the E. coli acetate kinase, encoded by ackA (Skarstedt and Silverstein J.Biol. Chem. 251 :6775-6783 (1976)), the C. acetobutylicum butyrate kinases, encoded by bukl and buk2 ((Walter et al. Gene 134(1):107-111 (1993); Huang et al. J Mol Microbiol Biotechnol 2(l):33-38 (2000)), and the E. coli gamma-glutamyl kinase, encoded by proB (Smith et al. J.Bacteriol.
  • the hydrolysis of acetoacetyl-CoA or 3-hydroxybutyryl-CoA can alternatively be carried out by a single enzyme or enzyme complex that exhibits acetoacetyl-CoA or 3-hydroxybutyryl-CoA synthetase activity.
  • This activity enables the net hydrolysis of the CoA-ester of either molecule, and in some cases, results in the simultaneous generation of ATP.
  • Additional exemplary CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochemical J. 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., /. Biol. Chem.
  • Metallosphaera sedula (Berg et al., Science 318: 1782-1786 (2007)). This function has been assigned to the Msed_1422 gene. Information related to these proteins and genes is shown below:
  • ADP- forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP.
  • ACD 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 (2004)).
  • the enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Musfeldt et al., supra; Brasen et al., supra (2004)). Information related to these proteins and genes is shown below: Protein GENBANK ID GI NUMBER ORGANISM
  • the conversion of 3-hydroxybutyrate to 3 -hydroxy butyraldehyde can be carried out by a 3- hydroxybutyrate reductase.
  • the conversion of acetoacetate to acetoacetaldehyde can be carried out by an acetoacetate reductase.
  • a suitable enzyme for these transformations is the aryl-aldehyde dehydrogenase, or equivalently a carboxylic acid reductase, from Nocardia iowensis.
  • Carboxylic acid reductase catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes (Venkitasubramanian et al., /. Biol. Chem.
  • npt gene product improved activity of the enzyme via post-transcriptional modification.
  • the npt gene encodes a specific phosphopantetheine transferase (PPTase) that converts the inactive apo-enzyme to the active holo-enzyme.
  • PPTase phosphopantetheine transferase
  • the natural substrate of this enzyme is vanillic acid, and the enzyme exhibits broad acceptance of aromatic and aliphatic substrates (Venkitasubramanian et al., in Biocatalysis in the Pharmaceutical and Biotechnology Industires, ed. R.N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton, FL. (2006)). Information related to these proteins and genes is shown below:
  • Additional car and npt genes can be identified based on sequence homology.
  • alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group is first activated through the ATP-dependent formation of an adenylate that is then reduced by AAR, EC 1.2.1.31.
  • NAD(P)H to yield the aldehyde and AMP.
  • this enzyme utilizes magnesium and requires activation by a PPTase.
  • Enzyme candidates for AAR and its corresponding PPTase are found in Saccharomyces cerevisiae (Morris et al., Gene 98: 141-145 (1991)), Candida albicans (Guo et al., Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe (Ford et al., Curr. Genet. 28: 131-137 (1995)).
  • the AAR from S. pombe exhibited significant activity when expressed in E.
  • Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate, but did not react with adipate, L-glutamate or diaminopimelate (Hijarrubia et al., /. Biol. Chem. 278:8250-8256 (2003)).
  • the gene encoding the P. chrysogenum PPTase has not been identified to date.
  • any of these CAR or CAR-like enzymes can exhibit 3-hydroxybutyrate or acetoacetate reductase activity or can be engineered to do so.
  • 3-hydroxybutyrate dehydrogenase catalyzes the reduction of acetoacetate to form 3-hydroxybutyrate.
  • Exemplary enzymes can be found in Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcus furiosus (van der et al., Eur.J.Biochem. 268:3062-3068 (2001)). Additional secondary alcohol dehydrogenase enzymes capable of this transformation include adh from C.
  • the Wood-Ljungdahl pathway comprises of two branches: the Eastern (or methyl) branch that enables the conversion of C0 2 to methyltetrahydrofolate (Me-THF) and the Western (or carbonyl) branch that enables the conversion of methyl-THF, CO, and Coenzyme-A into acetyl- CoA (Figure 5).
  • the Eastern (or methyl) branch that enables the conversion of C0 2 to methyltetrahydrofolate (Me-THF)
  • the Western (or carbonyl) branch that enables the conversion of methyl-THF, CO, and Coenzyme-A into acetyl- CoA
  • Figure 5 we describe a non- naturally occurring microorganism expressing genes encoding enzymes that catalyze the methyl and carbonyl branches of the Wood-Ljungdahl pathway. Such an organism is capable of converting CO, C0 2 , and/or H 2 into acetyl-CoA, cell mass, and products
  • a non-naturally occuring organism utilizing pathways shown in Figure 5 exhibit three capabilities: 1) a functional methyl branch of the Wood-Ljungdahl pathway which enables the conversion of THF and C0 2 to 5-methyl-tetrahydrofolate, 2) the ability to combine CO, Coenzyme A, and the methyl group of Me-THF to form acetyl-CoA, and 3) the ability to synthesize 1,3-butanediol from acetyl-CoA.
  • Such non-naturally occurring organisms are able to 'fix' carbon from exogenous CO and/or exogenous or endogenously produced C0 2 to synthesize acetyl-CoA, cell mass, and products.
  • a host organism engineered with these capabilities that also naturally possesses the capability for anaplerosis (e.g., E. coli) can grow on the syngas-generated acetyl-CoA in the presence of a suitable external electron acceptor such as nitrate.
  • This electron acceptor is required to accept electrons from the reduced quinone formed via succinate dehydrogenase.
  • a further advantage of adding an external electron acceptor is that additional energy for cell growth, maintenance, and product formation can be generated from respiration of acetyl-CoA.
  • An alternative strategy involves engineering a pyruvate ferredoxin oxidoreductase (PFOR) enzyme or other enzymes that convert pyruvate into acetyl-CoA into the strain to enable synthesis of biomass precursors in the absence of an external electron acceptor.
  • PFOR ferredoxin oxidoreductase
  • a further characteristic of the engineered organism is the capability for extracting reducing equivalents from molecular hydrogen.
  • a non-naturally occuring organism can produce acetyl-CoA, cell mass, and targeted chemicals, more specifically 1,3-butanediol, from: 1) CO, 2) C0 2 and H 2 , 3) CO, C0 2 , and H 2 , 4) synthesis gas comprising CO and H 2 , 5) synthesis gas comprising CO, C0 2 , and H 2 , and 6) one or more carbohydrates.
  • Exemplary carbohydrates include, but are not limited to, glucose, sucrose, xylose, arabinose, and glycerol.
  • the enzymes used for the conversion of CO, C0 2 , and/or H 2 to acetyl-CoA are shown in Figure 5.
  • a production host for the utilization of CO, C0 2 , and/or H 2 one or more exogenous DNA sequence(s) encoding these enzymes can be expressed in the microorganism.
  • Figure 5 described below are enzymes that can be incorporated to utilize CO, C0 2 , and/or H 2 .
  • Formate dehydrogenase is a two subunit selenocysteine-containing protein that catalyzes the incorporation of C0 2 into formate in Moorella thermoacetica (Andreesen and Ljungdahl, /.
  • 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. 10:2550-2573 (2008)).
  • Sfum_2703 Another set of genes encoding formate dehydrogenase activity with a propensity for C0 2 reduction is encoded by Sfum_2703 through Sfum_2706 in Syntrophobacter fumaroxidans (Reda et al., Proc. Natl. Acad. Sci. U.S.A. 105.31 :10654-10658 (2008); de Bok et al., Eur. J. Biochem. 270. 11: 2476-2485 (2003)). Similar to their M. thermoacetica counterparts,
  • Sfum_2705 and Sfum_2706 are actually one gene.
  • a similar set of genes presumed to carry out the same function are encoded by CHY_0731 , CHY_0732, and CHY_0733 in C.
  • Formyltetrahydrofolate synthetase ligates formate to tetrahydrofolate at the expense of one ATP.
  • This reaction is catalyzed by the gene product of Moth_0109 in M. thermoacetica (O'brien et al., Experientia Suppl.26:249 >-262 (1976); Lovell et al., Arch. Microbiol. 149.4:280-285 (1988); Lovell et al., Biochemistry 29.24:5687-5694 (1990)), FHS in Clostridium acidurici (Whitehead and Rabinowitz, /. Bacteriol. 167.1 :205-209 (1986); Whitehead and Rabinowitz, /. Bacteriol. 170.7:3255-3261 (1988)), and CHY_2385 in C. hydro genoformans (Wu et al., PLoS
  • cyclohydrolase and methylenetetrahydrofolate dehydrogenase are carried out by the bi- functional gene products of Moth_1516, o/D, and CHY_1878, respectively (Pierce et al.
  • cyclohydrolase and methylenetetrahydrofolate dehydrogenase are carried out by the bi- functional gene products of Moth_1516, o/D, and CHY_1878, respectively (Pierce et al.
  • This enzyme is encoded by metF in E. coli (Sheppard et al., /. Bacteriol. 181.3:718- 725 (1999)) and CHY_1233 in C. hydrogenoformans (Wu et al., PLoS Genet. ⁇ .5:&65 (2005)).
  • the M. thermoacetica genes, and its C. hydrogenoformans counterpart, are located near the CODH/ACS gene cluster, separated by putative hydrogenase and heterodisulfide reductase genes.
  • E. coli naturally possesses the capability for some of the required transformations ⁇ i.e., methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate dehydrogenase, methylenetetrahydrofolate reductase), the methyl branch enzymes from acetogens can have significantly higher (50 - 100X) specific activities than those from non-acetogens (Morton et al., Genetics and molecular biology of anaerobic bacteria, Ed. M. Sebald, New York: Springer Verlag (1992) pages 389-406).
  • Formate dehydrogenase may be specialized for anaerobic conditions (Ljungdahl and Andreesen, FEBS Lett. 54.2:279-282 (1975)) (1975). Therefore, various non-native versions of each of these can be expressed in the strain of E. coli capable of methanol and C0 2 , CO, and/or H 2 utilization. Specifically, these genes can be cloned and combined into an expression vector designed to express them as a set. Initially, a high or medium copy number vector can be chosen (using ColEl or P15A replicons).
  • An exemplary promoter is a strongly constitutive promoter such as lambda pL or an IPTG-inducible version of this, pL-lacO (Lutz and Bujard, Nucleic Acids Res. 25.6: 1203-1210 (1997)).
  • pL-lacO an IPTG-inducible version of this, pL-lacO
  • one 5' terminal promoter is placed upstream of the set of genes and each gene receives a consensus rbs element. The order of genes is based on the natural order whenever possible.
  • the genes are integrated into the E. coli chromosome. Enzyme assays are performed as described in (Ljungdahl and Andreesen, Methods Enzymol. 53:360-372 (1978); Yamamoto et al.. /. Biol. Chem. 258.3: 1826-1832 (1983); Lovell et al., Arch. Microbiol.
  • E. coli After strains of E. coli expressing both the carbonyl and methyl branches of the Wood- Ljungdahl pathway are constructed, they are assayed for the ability to utilize CO, C0 2 , and/or H 2 , for incorporation into acetyl-CoA, cell mass, 1,3-butanediol.
  • Initial conditions employ strictly anaerobically grown cells provided with exogenous glucose. Metabolizing glucose or other carbohydrates to acetyl-CoA provides one potential source of C0 2 that can be fixed via the Wood-Ljungdahl pathway. Alternatively, or in addition to glucose, nitrate can be added to the fermentation broth to serve as an electron acceptor and initiator of growth. Anaerobic growth of E.
  • Oxygen can also be provided as long as its intracellular levels are maintained below any inhibition threshold of the engineered enzymes.
  • Syngas' of a composition suitable for these experiments can also be employed. 13 C-labeled CO and/or C0 2 are provided to the cells and analytical mass spectrometry is employed to measure incorporation of the labeled carbon into acetate, 1,3- butanediol, and cell mass (e.g., proteinogenic amino acids).
  • Process considerations for a syngas fermentation include high biomass concentration and good gas-liquid mass transfer (Bredwell et al., Biotechnol. Prog. 15.5:834-844 (1999)).
  • the solubility of CO in water is somewhat less than that of oxygen.
  • Continuously gas-sparged fermentations can be performed in controlled fermenters with constant off-gas analysis by mass spectrometry and periodic liquid sampling and analysis by GC and HPLC.
  • the liquid phase can function in batch mode.
  • Fermentation products such as alcohols, organic acids, and residual glucose along with residual methanol are quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids.
  • the growth rate is determined by measuring optical density using a spectrophotometer (600 nm). All piping in these systems is glass or metal to maintain anaerobic conditions.
  • the gas sparging can be performed with glass frits to decrease bubble size and improve mass transfer. Various sparging rates are tested, ranging from about 0.1 to 1 vvm (vapor volumes per minute). To obtain accurate measurements of gas uptake rates, periodic challenges are performed in which the gas flow is temporarily stopped, and the gas phase composition is monitored as a function of time.
  • One method to increase the microbial concentration is to recycle cells via a tangential flow membrane from a sidestream.
  • Repeated batch culture can also be used, as previously described for production of acetate by Moorella (Sakai et al., /. Biosci. Bioeng. 99.3:252-258 (2005)).
  • Various other methods can also be used (Bredwell et al., Biotechnol. Prog. 15.5:834- 844 (1999); Datar et al., Biotechnol. Bioeng.86.5:58 ' -594 (2004)). Additional optimization can be tested such as overpressure at 1.5 atm to improve mass transfer (Najafpour and Younesi, Enzyme and Microbial Technology 38:223-228 (2006)).
  • a typical impurity profile is 4.5% CH 4 , 0.1% C 2 H 2 , 0.35% C 2 H 6 , 1.4% C 2 H 4 , and 150 ppm nitric oxide (Datar et al., Biotechnol. Bioeng.86.5:58 ' -594 (2004)).
  • Tars represented by compounds such as benzene, toluene, ethylbenzene, p-xylene, o-xylene, and naphthalene, are added at ppm levels to test for any effect on production.
  • 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 1,3-butanediol biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular 1,3- butanediol 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 1,3-butanediol 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 1,3-butanediol.
  • Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus , algae, cyanobacteria, 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, Cory neb acterium 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, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like.
  • Exemplary cyanobacteria include Acaryochloris marina MBIC11017, Anabaena sp. PCC 7120, Anabaena variabilis ATCC 29413, Agmenellum quadruplicatum, Chlorobium tepidum TLS, Cyanothece sp.
  • Thermosynechococcus elongatus BP-1 Trichodesmium erythraeum IMS 101.
  • Exemplary algae include Botryococcus braunii, Chlamydomonas reinhardii, Chlorella sp., Crypthecodinium cohnii, Cylindrotheca sp., Dunaliella primolecta, Isochrysis sp., Monallanthus salina,
  • E. coli is a particularly useful host organisms since it is a well characterized microbial organism suitable for genetic engineering.
  • Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce a desired product.
  • the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed 1,3-BDO pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more 1,3-butanediol biosynthetic pathways.
  • 1,3- butanediol 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 1,3-butanediol can be included, such as 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein (MtaC), 3) Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4) Methyltetrahydrofolate: corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacety
  • exogenous expression of all enzymes or proteins in a pathway for production of 1,3-butanediol can be included, such as 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase, 3) Methenyltetrahydrofolate cyclohydrolase, 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 7) Corrinoid iron-sulfur protein (AcsD), 8) Nickel-protein assembly protein (AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoA synthase (AcsB & AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd), 13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), 15) 3-oxobutyraldehyde reductase (aldehyde reducing), 16) 4-hydroxy,2-butanone reductase, 17) Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 18) 3-
  • 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 that is, up to all nucleic acids encoding the enzymes or proteins constituting a 1,3-butanediol biosynthetic pathway disclosed herein and shown in Figure 4 and 5.
  • the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize 1,3-butanediol 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 1,3-butanediol pathway precursors such as acetyl-CoA, acetoacetyl-CoA, acetoacetate, 3-hydroxybutyryl-CoA, 3-hydroxybutyrate, 4-hydroxy-2- butanone, 3-oxobutryaldehyde, or 3-hydroxybutryaldehyde.
  • a host microbial organism is selected such that it produces the precursor of a 1,3- butanediol 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.
  • acetyl-CoA is 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 a 1,3-butanediol pathway.
  • a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize 1,3-butanediol.
  • it can be useful to increase the synthesis or accumulation of a 1,3- butanediol pathway product to, for example, drive 1,3-butanediol pathway reactions toward 1,3- butanediol production.
  • Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described 1,3- butanediol pathway enzymes or proteins.
  • the enzyme or enzymes and/or protein or proteins of the 1,3-butanediol pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, producing 1,3- butanediol, through overexpression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen that is, up to all nucleic acids encoding the enzymes or proteins constituting a 1,3-butanediol biosynthetic pathway disclosed herein and shown in Figure 4 and 5.
  • 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 1,3-butanediol 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, a 1,3-butanediol 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 1,3-butanediol biosynthetic capability.
  • a non-naturally occurring microbial organism having a 1,3-butanediol biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins.
  • 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.
  • any combination of four, or more enzymes or proteins of a biosynthetic pathway as disclosed herein 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.
  • any combination of five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, enzymes and/or proteins of a biosynthetic pathway as disclosed herein 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.
  • Exemplary combinations of 17 exogenous enzymes or proteins of a biosynthetic pathway, as disclosed herein, included in a non-naturally occurring microbial organism of the invention include:
  • Methyltetrahydrofolate cyclohydrolase Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoid protein
  • Methyltetrahydrofolate cyclohydrolase Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoid protein
  • Methyltetrahydrofolate cyclohydrolase Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoid protein
  • Methyltetrahydrofolate cyclohydrolase Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoid protein
  • Methyltetrahydrofolate cyclohydrolase Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoid protein
  • Methyltetrahydrofolate cyclohydrolase Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoid protein
  • Methyltetrahydrofolate cyclohydrolase Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoid protein
  • Methyltetrahydrofolate cyclohydrolase Methenyltetrahydrofolate cyclohydrolase, 4) Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolate reductase, 6) Methyltetrahydrofolate:corrinoid protein
  • AcsE methyltransferase
  • AcsD Corrinoid iron-sulfur protein
  • AcsF Nickel-protein assembly protein
  • Ferredoxin Orf7, 10) Acetyl-CoA synthase (AcsB & AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12) Hydrogenase (Hyd), 13) Acetoacetyl-CoA thiolase (AtoB), 14) Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), and 15) 4- hydroxy, 2-butanone reductase.
  • Methyltetrahydrofolate corrinoid protein methyltransferase (MtaA), 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA transferase, hydrolase, or synthetase, 13) Acetoacetate reductase, 14) 3-oxobutyraldehyde reductase (ketone reducing), 15) 3-hydroxybutyraldehyde reductase;
  • Methyltetrahydrofolate corrinoid protein methyltransferase (MtaA), 4) Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA transferase, hydrolase, or synthetase, 13) Acetoacetate reductase, 14) 3-oxobutyraldehyde reductase (aldehyde reducing), 15) 4-hydroxy,2-
  • Methyltetrahydrofolate corrinoid protein methyltransferase (MtaA), 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase (ketone reducing), 13) 3-hydroxybutyryl-CoA transferase, hydrolase, or synthetase, 14) 3- hydroxybutyrate reductase, 15) 3 -hydroxybutyraldehyde reductase; M: 1) Methanol methyltransferase (MtaB), 2) Corrinoid
  • Methyltetrahydrofolate corrinoid protein methyltransferase (MtaA), 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA transferase, hydrolase, or synthetase, 13) 3-hydroxybutyrate dehydrogenase, 14) 3-hydroxybutyrate reductase, 15) 3 -hydroxybutyraldehyde reductase.
  • AcsE corrinoid protein methyltransferase
  • AcsD Corrinoid iron-
  • Exemplary combinations of 14 exogenous enzymes or proteins of a biosynthetic pathway, as disclosed herein, included in a non-naturally occurring microbial organism of the invention include any combination of 14 of the enzymes disclosed above in A-M or:
  • MtaB Methanol methyltransferase
  • MtaC Corrinoid protein
  • Methyltetrahydrofolate corrinoid protein methyltransferase (MtaA), 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10)
  • MtaB Methanol methyltransferase
  • MtaC Corrinoid protein
  • Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase (CoA- dependent, aldehyde forming), 13) 3-oxobutyraldehyde reductase (ketone reducing), and 14) 3- hydroxybutyraldehyde reductase.
  • AcsE corrinoid protein methyltransferase
  • AcsD Corrinoid iron-
  • MtaB Methanol methyltransferase
  • MtaC Corrinoid protein
  • Methyltetrahydrofolate corrinoid protein methyltransferase (MtaA), 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10)
  • Exemplary combinations of 13 exogenous enzymes or proteins of a biosynthetic pathway, as disclosed herein, included in a non-naturally occurring microbial organism of the invention include any combination of 13 of the enzymes disclosed above in A-P or:
  • Methyltetrahydrofolate corrinoid protein methyltransferase (MtaA), 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase (ketone reducing), and 13) 3-hydroxybutyryl-CoA reductase (alcohol forming),
  • Methyltetrahydrofolate corrinoid protein methyltransferase (MtaA), 4)
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8) Acetyl-CoA synthase (AcsB & AcsC), 9) Carbon monoxide dehydrogenase (AcsA), 10) Hydrogenase, 11) Acetoacetyl-CoA thiolase (AtoB), 12) Acetoacetyl-CoA reductase (CoA- dependent, alcohol forming), and 13) 4-hydroxy,2-butanone reductase.
  • AcsE corrinoid protein methyltransferase
  • AcsD Corrinoid iron-sulfur protein
  • AcsF & CooC Nickel-protein assembly protein
  • Ferredoxin Orf7
  • Exemplary combinations of 12 exogenous enzymes or proteins of a biosynthetic pathway, as disclosed herein, included in a non-naturally occurring microbial organism of the invention include any combination of 12 of the enzymes disclosed above in A-R.
  • combinations of 11 exogenous enzymes or proteins of a biosynthetic pathway, as disclosed herein, included in a non-naturally occurring microbial organism of the invention include any combination of 10 of the enzymes disclosed above in A-R.
  • Exemplary combinations of 9 exogenous enzymes or proteins of a biosynthetic pathway, as disclosed herein, included in a non-naturally occurring microbial organism of the invention include any combination of 8 of the enzymes disclosed above in A-R.
  • Exemplary combinations of 7 exogenous enzymes or proteins of a biosynthetic pathway, as disclosed herein, included in a non-naturally occurring microbial organism of the invention include any combination of 6 of the enzymes disclosed above in A-R, and so on down to any combination of 2 of the enzymes disclosed above in A-R.
  • the 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 1,3- butanediol other than use of the 1,3 -butanediol producers is through addition of another microbial organism capable of converting a 1,3 -butanediol pathway intermediate to 1,3- butanediol.
  • One such procedure includes, for example, the fermentation of a microbial organism that produces a 1,3 -butanediol pathway intermediate.
  • the 1,3 -butanediol pathway intermediate can then be used as a substrate for a second microbial organism that converts the 1,3 -butanediol pathway intermediate to 1,3-butanediol.
  • the 1,3 -butanediol pathway intermediate can be added directly to another culture of the second organism or the original culture of the 1,3-butanediol 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, 1,3-butanediol.
  • 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.
  • the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized.
  • the biosynthesis of 1,3-butanediol 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.
  • 1,3-butanediol 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 1,3-butanediol intermediate and the second microbial organism converts the intermediate to 1,3-butanediol.
  • Sources of encoding nucleic acids for a 1,3-butanediol 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, algae, insect, animal, and mammal, including human.
  • Exemplary species for such sources include, for example, Escherichia coli, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes.
  • 1,3-butanediol biosynthetic pathway exists in an unrelated species
  • 1,3-butanediol 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.
  • Methods for constructing and testing the expression levels of a non-naturally occurring 1,3- butanediol-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,
  • Exogenous nucleic acid sequences involved in a pathway for production of 1,3-butanediol 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,
  • 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 1,3-butanediol 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.
  • 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
  • the present invention provides a method for producing 1,3-BDO that includes culturing a non-naturally occurring microbial organism having a 1,3-BDO pathway having at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme or protein expressed in a sufficient amount to produce 1,3-BDO, under conditions and for a sufficient period of time to produce 1,3-BDO.
  • the 1,3-BDO pathway includes
  • Methanol methyltransferase (MtaB), Corrinoid protein (MtaC), Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), Methyltetrahydrofolate: corrinoid protein methyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD), Nickel-protein assembly protein (AcsF & CooC), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB & AcsC), Carbon monoxide dehydrogenase (AcsA), Hydrogenase (Hyd), Acetoacetyl-CoA thiolase (AtoB), Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 3-oxobutyraldehyde reductase (ketone reducing), 3- hydroxybutyral
  • Acetoacetyl-CoA reductase (ketone reducing), 3-hydroxybutyryl-CoA reductase (aldehyde forming), 3-hydroxybutyryl-CoA reductase (alcohol forming), 3-hydroxybutyryl-CoA transferase, 3-hydroxybutyryl-CoA hydrolase, 3-hydroxybutyryl-CoA synthetase, 3- hydroxybutyrate dehydrogenase, 3-hydroxybutyrate reductase, acetoacetyl-CoA transferase, acetoacetyl-CoA hydrolase, acetoacetyl-CoA synthetase, or acetoacetate reductase.
  • the present invention provides a method for producing 1,3-BDO that includes culturing a non-naturally occurring microbial organism having a 1,3-BDO pathway having at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme or protein expressed in a sufficient amount to produce 1,3-BDO, under conditions and for a sufficient period of time to produce 1,3-BDO.
  • the 1,3-BDO pathway includes Formate dehydrogenase, Formyltetrahydrofolate synthetase, Methenyltetrahydrofolate cyclohydrolase,
  • Methyltetrahydrofolate corrinoid protein methyltransferase (AcsE), Corrinoid iron- sulfur protein (AcsD), Nickel-protein assembly protein (AcsF & CooC), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB & AcsC), Carbon monoxide dehydrogenase (AcsA), Hydrogenase (Hyd), Acetoacetyl-CoA thiolase (AtoB), Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 3-oxobutyraldehyde reductase (ketone reducing), 3 -hydroxybutyraldehyde reductase, Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming) , 3-oxobutyraldehyde reductase (aldehyde reducing
  • culturing the non-naturally occurring microbial organism includes culturing under conditions and for a sufficient period of time to produce 1,3-BDO. In some embodiments, culturing is performed in a substantially anaerobic culture medium. In some embodiments, at least one exogenous nucleic acid of the microbial organism is a heterologous nucleic acid. As described above, the cultured non-naturally occurring microbial organisms can have any number of exogenous nucleic acids in a 1,3-BDO pathway including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, that is up to all the nucleic acids encoding a 1,3-BDO pathway.
  • Non-naturally occurring microbial organisms of the invention can utilize a carbon feedstock selected from 1) methanol and CO, 2) methanol, C0 2 , and H 2 , 3) methanol, CO, C0 2 , and H 2 , 4) methanol and synthesis gas comprising CO and H 2 , 5) methanol and synthesis gas comprising CO, C0 2 , and H 2 , 6) one or more carbohydrates, 7) methanol and one or more carbohydrates, and 8) methanol, in some embodiments or a carbon feedstock selected from 1) CO, 2) C0 2 and H 2 , 3) CO, C0 2 , and H 2 , 4) synthesis gas comprising CO and H 2 , 5) synthesis gas comprising CO, C0 2 , and H 2 , and 6) one or more carbohydrates, in other embodiments.
  • a carbon feedstock selected from 1) methanol and CO, 2) methanol, C0 2 , and H 2 , 3) methanol, CO, C0 2 ,
  • Suitable purification and/or assays to test for the production of 1,3-butanediol 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
  • the 1,3-butanediol 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 1,3- butanediol producers can be cultured for the biosynthetic production of 1,3-butanediol.
  • the recombinant strains are cultured in a medium with 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.
  • 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 United States Patent application serial No. 11/891,602, 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.
  • 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 syngas, methanol, or combinations of CO, C0 2 , hydrogen, and the like.
  • a carbon source such as syngas, methanol, or combinations of CO, C0 2 , hydrogen, and the like.
  • Such compounds include, for example, 1,3-butanediol and any of the intermediate metabolites in the 1,3-butanediol 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 1,3-butanediol biosynthetic pathways.
  • 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 a 1,3-butanediol pathway enzyme or protein in sufficient amounts to produce 1,3- butanediol. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce 1,3-butanediol. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve
  • 1,3-butanediol resulting in intracellular concentrations between about 0.1-200 mM or more.
  • the intracellular concentration of 1,3-butanediol 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.
  • the 1,3-butanediol producers can synthesize 1,3-butanediol at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, 1,3- butanediol producing microbial organisms can produce 1,3-butanediol intracellularly and/or secrete the product into the culture medium.
  • growth condition for achieving biosynthesis of 1,3-butanediol 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 refers to 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 10 mM, no more than about 50 mM, no more than about 100 mM or no more than about 500 mM.
  • 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 1,3- butanediol 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 refer 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 1,3-butanediol.
  • 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 1,3-butanediol.
  • the continuous and/or near-continuous production of 1,3-butanediol will include culturing a non-naturally occurring 1,3-butanediol 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 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. Briefly, fermentation for the biosynthetic production of 1,3-butanediol 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.
  • the 1,3-butanediol producers of the invention 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.
  • 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. 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 U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 1,3-butanediol.
  • One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework (Burgard et al., Biotechnol. Bioeng.
  • 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.
  • the metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. publication 2002/0168654, filed January 10, 2002, in International Patent No. PCT/US02/00660, filed January 10, 2002, and U.S. publication 2009/0047719, filed August 10, 2007.
  • Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®. This computational method and system is described in, for example, U.S.
  • 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®.
  • 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. coli 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.
  • a nucleic acid encoding a desired activity of a 1,3-butanediol pathway can be introduced into a host organism.
  • it can be desirable to modify an activity of a 1,3-butanediol pathway enzyme or protein to increase production of 1,3-butanediol.
  • known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule.
  • optimization methods can be applied to increase the activity of an enzyme or protein and/or decrease an inhibitory activity, for example, decrease the activity of a negative regulator.
  • 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 (for example, >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
  • 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 ), including broadening substrate binding to include non-natural substrates; inhibition (3 ⁇ 4), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increases enzymatic reaction rates to achieve desired flux; expression levels, to increase 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.
  • a number of exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of a 1,3- butanediol pathway enzyme or protein. Such methods include, but are not limited to EpPCR, which introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions (Pritchard et al., J Theor.Biol.
  • epRCA Error-prone Rolling Circle Amplification
  • 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
  • Nucleases such as Dnase I or EndoV
  • Staggered Extension StEP
  • RPR Random Priming Recombination
  • Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (Volkov et al, Nucleic Acids Res. 27:el8 (1999); and Volkov et al., Methods Enzymol. 328:456-463 (2000)); Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I fragmentation and size fractionation of single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol.
  • RACHITT Random Chimeragenesis on Transient Templates
  • Random Drift Mutagenesis in which mutations made via epPCR are followed by screening/selection for those retaining usable activity (Bergquist et al., Biomol. Eng.
  • Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage, which is used as a template to extend in the presence of "universal" bases such as inosine, and replication of an inosine- containing complement gives random base incorporation and, consequently, mutagenesis (Wong et al., Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et al., Anal. Biochem.
  • CCM Combinatorial Multiple Cassette Mutagenesis
  • LTM Look- Through Mutagenesis
  • Gene Reassembly which is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene
  • TGRTM GeneReassemblyTM
  • PDA Silico Protein Design Automation
  • the first step in the cloning and expression process is to express in E. coli the minimal set of genes (e.g., MtaA, MtaB, and MtaC) necessary to produce Me-THF from methanol. These methyltransferase activities require Coenzyme B] 2 (cobalamin) as a cofactor.
  • Coenzyme B] 2 cobalamin
  • a cascade of methyltransferase proteins mediate incorporation of methanol derived methyl groups into the acetyl-CoA synthase pathway.
  • Recent work indicates that MtaABC are encoded by Moth_1208-09 and Moth_2346.
  • MtaABC proteins from M. thermoacetica confers upon E. coli the ability to transfer methyl groups from methanol to tetrahydrofolate (THF) the recombinant strain is fed methanol at various concentrations.
  • Activity of the methyltransferase system is assayed anaerobically as described for vanillate as a methyl source in M. thermoacetica (Naidu and Ragsdale, /. Bacteriol. 183.11 :3276-3281 (2001)) or for Methanosarcina barkeri methanol methyltransferase (Sauer et al., Eur. J. Biochem.
  • thermoacetica cells are cultured in parallel and assayed anaerobically to confirm endogenous methyltransferase activity. Demonstration of dependence on exogenously added coenzyme B] 2 confirms methanokcorrinoid methyltransferase activity in E. coli.
  • methyltransferase expression is achieved, further work is performed towards optimizing the expression. Titrating the promoter in the expression vector enables the testing of a range of expression levels. This is then used as a guide towards the expression needed in single-copy, or allows the determination of whether or not a single-copy of these genes allows sufficient expression. If so, the methyltransferase genes are integrated into the chromosome as a single, synthetic operon. This entails targeted integration using RecET-based 'recombineering' (Angrand et al., Nucleic Acids Res. 27.17:el6 (1999); Muyrers et al., Nucleic Acids Res.
  • Bacillus sacB gene (Link et al., /. Bacteriol. 179.20:6228-6237 (1997)); in this way, markerless and scar less insertions at any location in the E. coli chromosome can be generated.
  • the final goal is a strain of E. coli K-12 expressing methanol :corrinoid methyltransferase activity under an inducible promoter and in single copy (chromosomally integrated).
  • entire ACS/CODH operons are assembled into low or medium copy number vectors such as pZA33-S (P15A-based) or pZS13-S (pSClOl-based).
  • the structure and sequence of the cloned genes are confirmed. Expression is monitored via protein gel electrophoresis of whole-cell lysates grown under strictly anaerobic conditions with the requisite metals (Ni, Zn, Fe) and coenzyme B] 2 provided. As necessary, the gene cluster is modified for E. coli expression by identification and removal of any apparent terminators and introduction of consensus ribosomal binding sites chosen from sites known to be effective in E. coli (Barrick et al., Nucleic Acids Res. 22.7: 1287-1295 (1994); Ringquist et al. Mol. Microbiol. 6.9: 1219-1229).
  • each gene cluster is cloned and expressed in a manner parallel to its native structure and expression. This helps ensure the desired stoichiometry between the various gene products— most of which interact with each other.
  • Once satisfactory expression of the CODH/ACS gene cluster under anaerobic conditions is achieved, the ability of cells expressing these genes to fix CO and/or C0 2 into cellular carbon is assayed.
  • Initial conditions employ strictly anaerobically grown cells provided with exogenous glucose as a carbon and energy source via substrate-level phosphorylation or anaerobic respiration with nitrate as an electron acceptor. Additionally, exogenously provided CH 3 -THF can be added to the medium.
  • the ACS/CODH genes are cloned and expressed in cells also expressing the methanol- methyltransferase system. This can be achieved by introduction of compatible plasmids expressing ACS/CODH into MTR-expressing cells. For added long-term stability, the
  • ACS/CODH and MTR genes can also be integrated into the chromosome. After strains of E. coli capable of utilizing methanol to produce Me-THF and of expressing active CODH/ACS gene are made, they are assayed for the ability to utilize both methanol and syngas for incorporation into acetyl-CoA, acetate, and cell mass. Initial conditions employ strictly anaerobically grown cells provided with exogenous glucose as a carbon and energy source. Alternatively, or in addition to glucose, nitrate can be added to the fermentation broth to serve as an electron acceptor and initiator of growth. Anaerobic growth of E.
  • Oxygen can also be provided as long as its intracellular levels are maintained below any inhibition threshold of the engineered enzymes. 13 C-labeled methanol, 13 C-labeled bicarbonate or 13 C-labeled CO are provided to the cells and analytical mass spectrometry is employed to measure incorporation of the labeled carbon into acetate and cell mass (e.g., proteinogenic amino acids).
  • An alternative or supplement to engineering the methanol methyltransferase system involves engineering the methyl branch of the Wood-Ljungdahl pathway to supply ACS/CODH with the methyl group. While E. coli possesses genes encoding enzymes capable of carrying out some of the necessary activities (fdh, metF,folD), it has been indicated that the methyl branch enzymes from acetogens may have significantly higher (50 - 100X) specific activities than those from non-acetogens (Morton et al., Genetics and molecular biology of anaerobic bacteria Springer Verlag, New York). The M.
  • thermoacetica versions include formate dehydrogenase (fdhA, Moth_2312 - Moth_2313 alpha, Moth_2314 beta), formyl-tetrahydrofolate (THF) synthetase (Moth_0109), methenyl-THF cyclohydrolase/methylene-THF dehydrogenase (folD, Moth_1516), methylene-THF reductase (metF, Moth_1191), and methyltransferase (acsE, Moth_1197).
  • methyltransferase (acsE, Moth_1197) that will be cloned as part of the CODH/ACS cluster, these genes are cloned and combined into an expression vector designed to express these as a set. Cloning and expression of the methyl branch genes will be undertaken as described above. Initially, a high or medium copy number vector will be chosen (using ColEl or P15A replicons). These genes can also be integrated into the E. coli chromosome.
  • the pyruvate ferredoxin oxidoreductase genes from M. thermoacetica, D. africanus, and E. coli can be cloned and expressed in strains exhibiting ACS/CODH activities. Conditions, promoters, etc., are described above. Given the large size of the PFOR genes and oxygen sensitivity of the corresponding enzymes, tests can be performed using low or single-copy plasmid vectors or single-copy chromosomal integrations. Activity assays described in ref. (Furdui and Ragsdale, /. Biol. Chem. 275.37:28494-28499 (2000)) can be applied to demonstrate activity. In addition, demonstration of growth on the gaseous carbon sources and methanol in the absence of an external electron acceptor will provide further evidence for PFOR activity in vivo.
  • the endogenous hydrogen-utilizing hydrogenase activity of the host organism can be tested by growing the cells as described above in the presence and absence of hydrogen. If a dramatic shift towards the formation of more reduced products during fermentation is observed (e.g., increased ethanol as opposed to acetate), this indicates that endogenous hydrogenase activity is sufficiently active. In this case, no heterologous hydrogenases are cloned and expressed. If the native enzymes do not have sufficient activity or reduce the needed acceptor, the genes encoding an individual hydrogenase complex can be cloned and expressed in strains exhibiting
  • the nonnative genes needed for 1,3-butanediol synthesis are cloned on expression plasmids as described previously.
  • the host strain also expresses methanol methyltransferase activity, CODH/ACS activity, and possibly PFOR and hydrogenase activities.
  • these (CODH/ACS, etc.) genes can be integrated into the genome and expressed from promoters that can be used constitutively or with inducers (i.e., PAl-lacOl is inducible in cells containing lad or is otherwise constitutive).
  • the base strain can be further modified by integration of a single copy of these genes at a neutral locus.
  • an artificial operon encoding the required genes.
  • This operon can be introduced using integrative plasmids and is coupled to counter-selection methods such as that allowed by the Bacillus sacB gene (Link et al., /. Bacteriol. 179.20:6228-6237 (1997)). In this way, markerless and scar less insertions at any location in the E. coli chromosome can be generated.
  • Optimization involves altering gene order as well as ribosomal binding sites and promoters.
  • the native atoB (b2224) gene of E. coli which can serve as an alternative to the C. acetobutylicum acetyl-coenzyme A [CoA] acetyltransferase required for 1,3-butanediol production
  • RecET-based methods are applied to integrate a stronger upstream promoter.
  • this gene is the last in an operon and the next gene downstream iyfaP) is both non-essential and in the opposite orientation. Therefore, polarity should not be an issue.
  • a cassette containing a selectable marker such as spectinomycin resistance or chloramphenicol resistance flanked by FRT or loxP sites is used to select for introduction of a strong constitutive promoter (e.g., pL).
  • a strong constitutive promoter e.g., pL.
  • This example describes the creation of E. coli plasmids that express the M. thermoacetica ACS/CODH operon genes including those used for CODH, ACS, methyltransferase, and the corrinoid iron-sulfur protein. This example further describes the expression these in E. coli resulting in observable CO oxidation activity, methyltransferase activity, and corrinoid iron- sulfur protein activity. Finally, this example demonstrates that E. coli tolerates high CO concentrations, and may even consume CO when the CO-utilizing gene products from M.
  • thermoacetica are expressed.
  • Expression vectors were chosen from the set described by Lutz and Bujard (Lutz and Bujard, Nucleic Acids Res. 25.6: 1203-1210 (1997)); these come with compatible replicons that cover a range of copy numbers. Additionally, each contains prAl-lacol ; this T7 early gene promoter is inducible by IPTG and can lead to very high levels of transcription in the presence of IPTG and represses in other conditions.
  • the ACS/CODH-encoding operon was cloned from Moth_1204 (cooC) to Moth_1197; a second version containing only Moth_1203 to Moth_1197 was also constructed. Both of these fragments (10 - 11 kbp) were confirmed by DNA sequence analysis.
  • Amounts of CODH in ACS90 and ACS91 were estimated at 50 ng by comparison to the control lanes.
  • a carbon monoxide oxidation assay (Seravalli et al., Biochemistry 43.13:3944-3955 (2004)) was used to test whether or not functional expression of the CODH-encoding genes from M.
  • thermoacetica was achieved. Cultures of E. coli MG1655 containing either an empty vector, or the vectors expressing "Acs90" or “Acs91” were grown in Terrific Broth under anaerobic conditions (with supplements of cyanocobalamin, ferrous iron, and reducing agents) until reaching medium to high density at which point, IPTG was added to a final concentration of 0.2 mM to induce the promoter. After 3.5 hrs of growth at 37 °C, the cells were harvested and spun down prior to lysis with lysozyme and mild detergents. There is a benchmark figure of M. thermoacetica CODH specific activity, 500 U at 55C or ⁇ 60U at 25 °C.
  • This assay employed reduction of methyl viologen in the presence of CO. This is measured at 578 nm in stoppered, anaerobic, glass cuvettes. Reactions positive for CO oxidation by CODH turned a deep violet color (see Figure 7). About 0.5% of the cellular protein was CODH as estimated by Western blotting; therefore, the data in Table 1 are approximately 50X less than the 500 U/mg activity of pure M. thermoacetica CODH. Nevertheless, this experiment did clearly demonstrate CO oxidation activity in recombinant E. coli with a much smaller amount in the negative controls. The small amount of CO oxidation (C3 ⁇ 4 viologen reduction) seen in the negative controls indicates that E. coli may have a limited ability to reduce C3 ⁇ 4 viologen.
  • This assay is an in vitro reaction that synthesizes acetyl-CoA from methyl-tetrahydrofolate, CO, and CoA using ACS/CODH, methy transferase, and CFeSP (Raybuck et al., Biochemistry 27.20:7698-7702 (1988)).
  • this assay can be used for a wide range of experiments, from testing one or more purified enzymes or cell extracts for activity, to determining the kinetics of the reaction under various conditions or with limiting amounts of substrate or enzyme.
  • Samples of the reaction taken at various time points are quenched with 1M HC1, which liberates acetate from the acetyl-CoA end product. After purification with Dowex columns, the acetate can be analyzed by chromatography, mass spectrometry, or by measuring radioactivity. The exact method can be determined by the specific substrates used in the reaction.
  • the reaction was assembled in the anaerobic chamber in assay vials filled with CO.
  • the total reaction volume was small compared to the vial volume, reagents were added prior to filling with CO, a gas-tight Hamilton syringe was used and the reagents were kept anaerobic.
  • the reaction ( ⁇ 60ul total) consisted of the cell extract (except #1), CoA, Ti(III)citrate, MES (except #6), purified ACS/CODH, 14C-methyl-tetrahydrofolate, methyl-viologen, and ferredoxin. Additionally, purified MeTr was added to #1, #4-6 and purified CFeSP was added to #1.
  • the reaction was carried out in the anaerobic chamber in a sand bath at 55°.
  • An initial sample was taken immediately, followed by samples at 30 minutes, 1 hour, and 2 hours. These time points are not exact, as the 6 conditions were run concurrently (since this experiment was primarily a qualitative one).
  • the 15 ⁇ samples were added to 15 ⁇ of 1M HCl in scintillation vials. After counting the reaction mixtures, it was determined that the corrinoid Fe-S protein in ACS90 extracts was active with total activity approaching approximately 1/5 of the positive control.
  • ACS/CODH operon Within the ACS/CODH operon is encoded an essential methyltransferase activity that catalyzes the transfer of C3 ⁇ 4 from methyl-tetrahydrofolate to the ACS complex as part of the synthesis of acetyl-CoA (i.e. this is the step that the methyl and carbonyl paths join together).
  • Mtr-encoding gene Within the operon in M. thermoacetica, the Mtr-encoding gene is Moth_l 197 and comes after the main CODH and ACS subunits. Therefore, Mtr activity would constitute indirect evidence that the more proximal genes can be expressed.
  • Mtr activity was assayed by spectroscopy. Specifically, methylated CFeSP, with Co(III), has a small absorption peak at ⁇ 450nm, while non-methylated CFeSP, with Co(I), has a large peak at ⁇ 390nm. This spectrum is due to both the cobalt and iron-sulfur cluster chromophores.
  • the CFeSP can spontaneously oxidize to Co(II), which creates a broad absorption peak at ⁇ 470nm (Seravalli et al., Biochemistry 38.18:5728-5735 (1999)). See Figure 8 for the results from E. coli cells containing ACS90.
  • E. coli cells containing ACS90 To test whether or not E. coli can grow anaerobically in the presence of saturating amounts of CO we made up 120 ml serum bottles with 50 ml of Terrific Broth medium (plus NiCl 2 , Fe(II)NH 4 S0 4 , and cyanocobalamin) in anaerobic conditions. One half of these bottles were equilibrated with nitrogen gas for 30 min.
  • ACS/CODH operon might be metabolizing some of the CO.
  • the second conclusion is significantly less certain than the first.
  • Wood- Ljungdahl pathway enzymes assimilate carbon in the form of formate, CO and/or C0 2 into acetyl-CoA, which can subsequently be converted to useful chemical products such as 1,3- butanediol.
  • the Wood-Ljungdahl pathway can also serve as a secondary carbon assimilation pathway during growth on other substrates such as glucose. Specifically, the conversion of one mole of glucose to two moles of acetyl-CoA generates both reducing equivalents and C0 2 .
  • the WL pathway enzymes can harness the reducing equivalents to convert the C0 2 and/or formate to additional acetyl-CoA that can be further used for 1,3-butanediol formation.
  • 1,3-Butanediol can be synthesized from acetyl-CoA by several alternate routes described previously in this application and shown in Figures 4 and 5.
  • the maximum achievable 1,3- butanediol yield for any of these pathways from glucose, for example, is 1 mol/mol (0.5 g/g) in the absence of the Wood-Ljungdahl pathway enzymes. Additional assimilation of C0 2 via Wood-Ljungdahl pathway further improves the yield to the stoichiometric theoretical maximum of 1.09 mol/mol (0.545 g/g).
  • a predicted flux distribution for achieving the maximum theoretical yield is shown in Figure 10.
  • methanol can be co-fed with a carboyhydrate such as glucose to increase the yield of 1,3-butanediol.
  • a carboyhydrate such as glucose
  • utilizing glucose and methanol in a 1.0:0.4 ratio affords an increase from 1 mol 1,3-butanediol/mol glucose (0.5 g/g) to 1.2 mol 1,3-butanediol/mol glucose.
  • a predicted flux distribution for achieving this increased yield is shown in Figure 11.
  • Bi2 biosynthetic pathways have been characterized in several organisms including Salmonella typhimurium LT2 (Roth et al., J. Bacteriol. 175:3303-3316), Lactobacillus reuteri CRL1098 (Harms and Thauer, Eur. J. Biochem. 235:653-659 (1996)) and Bacillus megaterium (Brey et al., /. Bacteriol. 167:623-630 (1986)).
  • Bacterial B] 2 biosynthesis pathways involve 20-30 genes clustered together in one or more operons. Two cobalamin biosynthesis pathways: late-insertion (aerobic only) and early-insertion (anaerobic) have been described (Scott, A.I., /. Org. Chem. 68:2529-2539 (2003)).
  • the final products of the biosynthesis of vitamin B 12 are 5'- deoxyadenosylcobalamin (coenzyme B] 2 ) and methylcobalamin (MeCbl).
  • Vitamin B] 2 is defined as cyanocobalamin (CNCbl) which is the form commonly prepared in industry. In this example, B] 2 refers to all three analogous molecules.
  • the anaerobic cobalamin biosynthesis pathway has been well-characterized in Salmonella typhimurium LT2 (Roth et al., /. Bacteriol. 175:3303-3316)). Pathway genes are clustered in a large operon termed the cob operon.
  • a plasmid containing the following 20 genes from the cob operon (pAR8827) was transformed into E. coli and conferred the ability to synthesize cobalamin de novo (Raux et al., /. Bacteriol. 178:753-767 (1996)).
  • the authors removed the known regulatory elements of cbiA and altered the RBS.
  • E coli for example, is unable to synthesize the corrin ring structure but encodes proteins that catalyze several reactions in the pathway (Raux et al. /. Bacteriol. 178:753-767 (1996)).
  • the cysG gene encodes a functional CysG, a multifunctional enzyme that converts uroporphyrinogen III to precorrin-2 (Hugler et al., /. Bacteriol. 184:2404-2410 (2002); Ishige al., Appl. Environ. Microbiol. 68: 1192-1195 (2002)).
  • the proteins encoded by cobTSU transform cobinamide to cobalamin and introduce the 5 ' -deoxyadenosyl group (Raux et al., supra (1996)).
  • S. cerevisiae is not able to synthesize B] 2 de novo, nor is it able to uptake the vitamin at detectable levels.
  • the S. cerevisiae genome encodes two proteins, Metlp and Met8p, that catalyze several B 12 pathway reactions.
  • Metlp is analogous to the uroporphyrinogen III transmethylase CysG of S. typhimurium, which catalyzes the first step of B12 biosynthesis from uroporphyrinogen III (Raux et al., Biochem. J. 338(pt. 3):701-708 (1999)).
  • the Met8p protein is a bifunctional protein with uroporphyrinogen III transmethylase activity and cobaltochelatase activity analogous to the CysG of B. megaterium (Raux et al., supra (1999)).
  • B 12 uptake requires a specific transport system (Sennett et al., Annu. Rev. Biochem. 50:1053-1086 (1981)).
  • the B 12 transport system of E. coli has been extensively studied. High-affinity transport across the outer membrane is calcium-dependent and mediated by a 66 kDa outer membrane porin, BtuB (Heller et al., /. Bacteriol. 161:896-903 (1985)).
  • BtuB interacts with the TonB energy transducing system (TonB-ExbB-ExbD), facilitating energy-dependent translocation and binding to periplasmic binding protein BtuF (WO/2007/141208; Atsumi et al., Nature 451 :86-89 (2008)).
  • Transport across the inner membrane is facilitated by an ABC type uptake system composed of BtuF, BtuD (ATP binding component) and BtuC (permease) (Binstock et al., Meth. Enzymol. 71(pt. C):403-411 (1981)). Crystal structures of the BtuCDF complex are available (Atsumi et al., supra (2008); Binstock et al., supra (1981)).
  • BtuE An additional protein, BtuE, is coexpressed in the btuCED operon, but this protein is not required for B 12 transport and its function is unknown (Rioux et al., Mol. Gen. Genet. 217:301-308 (1989)).
  • the btuCED operon is constitutively expressed.
  • GenBank identifiers and GI numbers of the genes associated with B]2 transport are listed below.
  • the B i2 uptake capability of an organism can be further improved by overexpressing genes encoding the requisite transport proteins, and reducing or eliminating negative regulatory control.
  • Overexpressing the btuBCDF genes leads to increased binding of B12 to membranes and increased rate of uptake into cells.
  • Another strategy is to remove regulatory control.
  • the btuB mRNA translation is directly repressed by B 12 at the 5' UTR (Nahvi et al., Chem. Biol. 9:1043 (2002)). This interaction may induce mRNA folding to block ribosome access to the translational start. Mutation or elimination of the B] 2 binding site removes inhibition and improves the efficiency of B] 2 uptake (US Patent No. 6,432,686, Bulthuis et al.).
  • S. typhimurium possesses both high and low affinity transporters for B] 2 .
  • the high affinity transporter is encoded by btuB (Rioux et al., /. Bacteriol. 171 :2986-2993 (1989)).
  • btuB Rioux et al., /. Bacteriol. 171 :2986-2993 (1989)
  • E. coli transport across the periplasmic membrane is predicted to occur via an ABC transport system, although this has not been characterized to date.
  • the B] 2 binding protein is encoded by btuD and btuE, and btuC is predicted to encode the permease.
  • Any or all of these genes can be introduced into an organism deficient in one or more components of cobalamin uptake to enable or increase the efficienty cobalamin uptake.

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CA2783096A1 (en) 2011-06-16
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