# Patent

## Title
Microorganisms and methods for conversion of syngas and other carbon sources to useful products

## Abstract

A non-naturally occurring microbial organism having an isopropanol, 4-hydroxybutryate, or 1,4-butanediol pathway includes at least one exogenous nucleic acid encoding an isopropanol, 4-hydroxybutryate, or 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce isopropanol, 4-hydroxybutryate, or 1,4-butanediol. The aforementioned organisms are cultured to produce isopropanol, 4-hydroxybutryate, or 1,4-butanediol.

## Background
This application is a division of U.S. Ser. No. 13/371,278 filed Feb. 10, 2012, now U.S. Pat. No. 8,470,566, which is a continuation of U.S. Ser. No. 12/639,977 filed Dec. 16, 2009, now U.S. Pat. No. 8,129,155, which is a continuation-in-part of PCT Application Number PCT/US09/50757, filed Jul. 15, 2009 and further claims the benefit of priority of U.S. Provisional Application Ser. No. 61/138,108, filed Dec. 16, 2008, each of which the entire contents are incorporated herein by this reference.


BACKGROUND OF THE INVENTION
The present invention relates generally to biosynthetic processes and organisms capable of converting carbohydrates, methanol, synthesis gas and other gaseous carbon sources into higher-value chemicals.
Increasing the flexibility of cheap and readily available feedstocks and minimizing the environmental impact of chemical production are beneficial for a sustainable chemical industry. Feedstock flexibility relies on the introduction of methods that can access and use a wide range of materials as primary feedstocks for chemical manufacturing.
Isopropanol (IPA) is a colorless, flammable liquid that mixes completely with most solvents, including water. The largest use for IPA is as a solvent, including its well known yet small use as “rubbing alcohol,” which is a mixture of IPA and water. As a solvent, IPA is found in many everyday products such as paints, lacquers, thinners, inks, adhesives, general-purpose cleaners, disinfectants, cosmetics, toiletries, de-icers, and pharmaceuticals. Low-grade IPA is also used in motor oils. The second largest use is as a chemical intermediate for the production of isopropylamines (e.g. in agricultural products), isopropylethers, and isopropyl esters.
Isopropanol is manufactured by two petrochemical routes. The predominant process entails the hydration of propylene either with or without sulfuric acid catalysis. Secondarily, IPA is produced via hydrogenation of acetone, which is a by-product formed in the production of phenol and propylene oxide. High-priced propylene is currently driving costs up and margins down throughout the chemical industry motivating the need for an expanded range of low cost feedstocks.
4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB) is a 4-carbon carboxylic acid that has industrial potential as a building block for various commodity and specialty chemicals. In particular, 4-HB has the potential to serve as a new entry point into the 1,4-butanediol family of chemicals, which includes solvents, resins, polymer precursors, and specialty chemicals.
BDO is a valuable chemical for the production of high performance polymers, solvents, and fine chemicals. It is the basis for producing other high value chemicals such as tetrahydrofuran (THF) and gamma-butyrolactone (GBL). Uses of BDO include (1) polymers, (2) THF derivatives, and (3) GBL derivatives. In the case of polymers, BDO is a co-monomer for polybutylene terephthalate (PBT) production. PBT is a medium performance engineering thermoplastic made by companies such as DuPont and General Electric finding use in automotive, electrical, water systems, and small appliance applications. When converted to THF, and subsequently to polytetramethylene ether glycol (PTMEG), the Spandex and Lycra fiber and apparel industries are added to the markets served. PTMEG is also combined with BDO in the production of specialty polyester ethers (COPE). COPEs are high modulus elastomers with excellent mechanical properties and oil/environmental resistance, allowing them to operate at high and low temperature extremes. PTMEG and BDO also make thermoplastic polyurethanes processed on standard thermoplastic extrusion, calendaring, and molding equipment, and are characterized by their outstanding toughness and abrasion resistance. The GBL produced from BDO provides the feedstock for making pyrrolidones, as well as serving agrochemical market applications itself. The pyrrolidones are used as high performance solvents for extraction processes of increasing use in the electronics industry as well as use in pharmaceutical production.
BDO is produced by two main petrochemical routes with a few additional routes also in commercial operation. One route involves reacting acetylene with formaldehyde, followed by hydrogenation. More recently BDO processes involving butane or butadiene oxidation to maleic anhydride, followed by hydrogenation have been introduced. BDO is used almost exclusively as an intermediate to synthesize other chemicals and polymers.
Synthesis gas (syngas) is a mixture of primarily H2 and CO that can be obtained via gasification of 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, for example, 0.5:1-3:1 H2/CO mixture. Steam is sometimes added to increase the hydrogen content, typically with increased CO, production through the water gas shift reaction. Methanol is most commonly produced industrially from the syngas components, CO and H2, via catalysis
Today, coal is the main substrate used for industrial production of syngas, which is usually 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.
Overall, technology now exists for cost-effective production of syngas from a plethora of materials, including coal, biomass, wastes, polymers, and the like, at virtually any location in the world. Biomass gasification technologies are being practiced commercially, particularly for heat and energy generation.
Despite the availability of organisms that utilize syngas, such organisms are generally poorly characterized and are not well-suited for commercial development. For example, 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 isolating a desired product. Finally, development of facile genetic tools to manipulate clostridial genes is in its infancy, therefore, they are not currently amenable to rapid genetic engineering to improve yield or production characteristics of a desired product.
Thus, there exists a need to develop microorganisms and methods of their use to utilize carbohydrates, methanol, syngas and/or other gaseous carbon sources for the production of desired chemicals and fuels. More specifically, there exists a need to develop microorganisms for carbohydrate, methanol, and syngas utilization that also have existing and efficient genetic tools to enable their rapid engineering to produce valuable products at useful rates and quantities. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
In some aspects, the present invention provides a non-naturally occurring microbial organism having an isopropanol pathway that includes at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol. The isopropanol pathway enzyme includes a succinyl-CoA:3-ketoacid-CoA transferase.
In other aspects, the present invention provides a non-naturally occurring microbial organism having a 4-hydroxybutryate pathway that includes at least one exogenous nucleic acid encoding an 4-hydroxybutryate pathway enzyme expressed in a sufficient amount to produce 4-hydroxybutryate. The 4-hydroxybutryate pathway enzyme includes an acetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, a phosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyrate kinase.
In still other aspects, the present invention provides a non-naturally occurring microbial organism having a 1,4-butanediol pathway that includes at least one exogenous nucleic acid encoding a 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce 1,4-butanediol. The 1,4-butanediol pathway enzyme includes an acetoacetyl-CoA thiolase, a 3-Hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a 1,4-butanediol dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyrate kinase, and a 4-hydroxybutyrate reductase. Such an organism also includes an acetyl-CoA pathway having at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA. The acetyl-CoA pathway enzyme includes a corrinoid protein, a methyltetrahydrofolate:corrinoid protein methyltransferase, a corrinoid iron-sulfur protein, a nickel-protein assembly protein, a ferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and a hydrogenase.
In yet other aspects, the present invention provides a non-naturally occurring microbial organism having a 1,4-butanediol pathway that includes at least one exogenous nucleic acid encoding a 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce 1,4-butanediol. The 1,4-butanediol pathway enzyme includes an acetoacetyl-CoA thiolase, a 3-Hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a 1,4-butanediol dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyrate kinase, and a 4-hydroxybutyrate reductase. Such an organism also includes an acetyl-CoA pathway having at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA. The acetyl-CoA pathway enzyme includes an acetyl-CoA synthase, a formate dehydrogenase, a formyltetrahydrofolate synthetase, a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolate dehydrogenase, and a methylenetetrahydrofolate reductase.
In still further aspects, the present invention provides a non-naturally occurring microbial organism having an isopropanol pathway that includes at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol. The isopropanol pathway enzyme includes an acetoacetyl-CoA thiolase, an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, a phosphotransacetoacetylase, an acetoacetate kinase, an acetoacetate decarboxylase, and an isopropanol dehydrogenase. Such an organism also includes at least one exogenous nucleic acid encoding an acetyl-CoA enzyme expressed in a sufficient amount to produce acetyl-CoA. The acetyl-CoA pathway enzyme includes a methanol methyl transferase, a corrinoid protein, a methyltetrahydro-folate:corrinoid protein methyltransferase, a corrinoid iron-sulfur protein, a nickel-protein assembly protein, a ferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and a hydrogenase.
In still further aspects, the present invention provides a method for producing isopropanol that includes culturing a non-naturally occurring microbial organism having an isopropanol pathway. The pathway includes at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol under conditions and for a sufficient period of time to produce isopropanol. The isopropanol pathway includes a succinyl-CoA:3-ketoacid-CoA transferase.
In yet still further aspects, the present invention provides a method for producing 4-hydroxybutyrate that includes culturing a non-naturally occurring microbial organism having an 4-hydroxybutyrate pathway. The pathway includes at least one exogenous nucleic acid encoding an 4-hydroxybutyrate pathway enzyme expressed in a sufficient amount to produce 4-hydroxybutyrate under conditions and for a sufficient period of time to produce 4-hydroxybutyrate. The 4-hydroxybutyrate pathway includes an acetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, a phosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyrate kinase.
In still other aspects, the present invention provides a method for producing 1,4-butanediol that includes culturing a non-naturally occurring microbial organism having an 1,4-butanediol pathway. The pathway includes at least one exogenous nucleic acid encoding an 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce 1,4-butanediol under conditions and for a sufficient period of time to produce 1,4-butanediol. The 1,4-butanediol pathway includes an acetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a 1,4-butanediol dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyrate kinase, and a 4-hydroxybutyrate reductase. Such an organism also includes an acetyl-CoA pathway comprising at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA. The acetyl-CoA pathway enzyme includes a corrinoid protein, a methyltetrahydro-folate:corrinoid protein methyltransferase, a corrinoid iron-sulfur protein, a nickel-protein assembly protein, a ferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and a hydrogenase.
Finally, in some aspects, the present invention provides a method for producing 1,4-butanediol that includes culturing a non-naturally occurring microbial organism having an 1,4-butanediol pathway. The pathway includes at least one exogenous nucleic acid encoding an 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce 1,4-butanediol under conditions and for a sufficient period of time to produce 1,4-butanediol. The 1,4-butanediol pathway includes an acetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a 1,4-butanediol dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyrate kinase, and a 4-hydroxybutyrate reductase. Such an organism also includes an acetyl-CoA pathway having at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA. The acetyl-CoA pathway enzyme includes an acetyl-CoA synthase, a formate dehydrogenase, a formyltetrahydrofolate synthetase, a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolate dehydrogenase, and a methylenetetrahydrofolate reductase.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a diagram depicting the Wood-Ljungdahl pathway and formation routes for acetate and ethanol. The transformations that are typically unique to 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. Oxidation of hydrogen to 2 [H] or of CO with H2O to CO2 and 2 [H] provides reducing equivalents for the reduction of CO2 to formate, of methenyl-tetrahydrofolate (methenyl-THF) to methylene-tetrahydrofolate (methylene-THF), of methylene-THF to methyltetrahydrofolate (methyl-THF), and of CO2 to CO.
FIG. 2A shows the complete Wood-Ljungdahl pathway for the conversion of carbohydrates and/or gases including CO, CO2, and/or H2 to acetyl-CoA which is subsequently converted to cell mass and products such as ethanol or acetate. FIG. 2B shows a synthetic metabolic pathway for the conversion of carbohydrates and/or gases including CO, CO2, and/or H2, and methanol to acetyl-CoA and further to isopropanol. FIG. 2C shows a synthetic metabolic pathway for the conversion of carbohydrates and/or gases including CO, CO2, and/or H2, and methanol to acetyl-CoA and further to 4-hydroxybutyrate. FIG. 2D shows a synthetic metabolic pathway for the conversion of carbohydrates and/or gases including CO, CO2, and/or H2, and methanol to acetyl-CoA and further to 1,4-butanediol. In FIGS. 2A-D, the specific enzymatic transformations that can be engineered into a production host are numbered. Abbreviations: 10FTHF: 10-formyltetrahydrofolate, 5MTHF: 5-methyltetrahydrofolate, ACTP: acetyl phosphate, CFeSp: corrinoid iron sulfur protein, FOR: formate, MeOH: methanol, METHF: methyltetrahydrofolate, MLTHF: metheneyltetrahydrofolate, THF: tetrahydrofolate.
FIG. 3A shows a synthetic metabolic pathway for the conversion of carbohydrates and/or gases including CO, CO2, and/or H2 to acetyl-CoA, and further to isopropanol. FIG. 3B shows a synthetic metabolic pathway for the conversion of carbohydrates and/or gases including CO, CO2, and/or H2 to acetyl-CoA, and further to 4-hydroxybutyrate. FIG. 3C shows a synthetic metabolic pathway for the conversion of carbohydrates and/or gases including CO, CO2, and/or H2 to acetyl-CoA, and further to 1,4-butanediol. In FIGS. 3A-C the specific enzymatic transformations that can be engineered into a production host are numbered. Abbreviations: 10FTHF: 10-formyltetrahydrofolate, 5MTHF: 5-methyltetrahydrofolate, ACTP: acetyl phosphate, CFeSp: corrinoid iron sulfur protein, FOR: formate, MeOH: methanol, METHF: methyltetrahydrofolate, MLTHF: metheneyltetrahydrofolate, THF: tetrahydrofolate.
FIG. 4 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—1197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and 1000 ng).
FIG. 5 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. Stoppers and vacuum grease on top of each are used to keep the reactions anaerobic.
FIG. 6 shows a spectrogram of ACS90 cell extracts assayed for transfer of CH3 from added CH3-THF to purified M. thermoacetica corrinoid protein.
FIG. 7 shows anaerobic growth of recombinant E. coli MG1655 in N2 and CO for 36 hr at 37° C. From left to right: Empty vector, ACS90, and ACS91 are shown.
FIG. 8A shows a synthetic metabolic pathway and exemplary flux distribution for the conversion of carbohydrates and CO2 to acetyl-CoA, and further to isopropanol. FIG. 8B shows a synthetic metabolic pathway and exemplary flux distribution for the conversion of carbohydrates and CO2 to acetyl-CoA, and further to 4-hydroxybutyrate. FIG. 8C shows a synthetic metabolic pathway and exemplary flux distribution for the conversion of carbohydrates and CO2 to acetyl-CoA, and further to 1,4-butanediol. In FIGS. 8A-C, several enzymatic transformations that can be engineered into a production host are numbered.
FIG. 9 shows a synthetic metabolic pathway and exemplary flux distribution for the conversion of carbohydrates, methanol and CO2 to acetyl-CoA, and further to isopropanol. Several enzymatic transformations that can be engineered into a production host are numbered.
FIG. 10 plots the maximum yield of isopropanol or 4-hydroxybutyrate as a function of the glucose: methanol ratio.



DETAILED DESCRIPTION OF THE INVENTION
This invention is directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze the carbonyl-branch of the Wood-Ljungdahl pathway in conjunction with a MtaABC-type methyltransferase system. Such organisms are capable converting carbohydrates, methanol, a relatively inexpensive organic feedstock that can be derived from synthesis gas, and gasses including CO, CO2, and/or H2 into acetyl-CoA, cell mass, and products such as isopropanol (IPA), 4-hydroxybutyrate (4-HB), and 1,4-butanediol (BDO). Additional product molecules that can be produced by the teachings of this invention include but are not limited to ethanol, butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, adipic acid, 6-aminocaproic acid, hexamethylenediamine, 3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, acrylic acid, and long chain hydrocarbons, alcohols, acids, and esters. The invention is also directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze the carbonyl and methyl-branches of the Wood-Ljungdahl pathway. Such organisms are capable converting gasses including CO, CO2, and/or H2 into acetyl-CoA, cell mass, and products such as IPA, 4-HB, and BDO. Additional product molecules include but are not limited to ethanol, butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, adipic acid, 6-aminocaproic acid, hexamethylenediamine, 3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, acrylic acid, and long chain hydrocarbons, alcohols, acids, and esters.
In one embodiment, the invention provides non-naturally occurring microbial organisms capable of producing isopropanol, 4-hydroxybutyrate, or 1,4-butanediol from methanol and gaseous feedstocks such as mixtures of syngas. In other embodiments, the invention provides non-naturally occurring microbial organisms capable of producing isopropanol, 4-hydroxybutyrate, or 1,4-butanediol from mixtures of syngas, without the need for methanol. In further embodiments, the invention provides pathways for increased yield of isopropanol, 4-hydroxybutyrate or 1,4-butanediol on carbohydrate feedstocks over what would be naturally expected (e.g., one mol isopropanol, 4-hydroxybutyrate or 1,4-butanediol per one mole glucose consumed) by providing an efficient mechanism for fixing the carbon present in methanol or carbon dioxide, fed exogeneously or produced endogenously, into acetyl-CoA.
In another embodiment, the invention provides methods for producing isopropanol, 4-hydroxybutyrate, or 1,4-butanediol through culturing of these non-naturally occurring microbial organisms. Biotechnological processes utilizing these organisms will provide operational flexibility for isopropanol, 4-hydroxybutyrate, and 1,4-butanediol manufacturers to assure the lowest operating costs based on diversified feedstocks and optionally to offset volatility in market-driven commodity pricing of current feedstocks such as oil and natural gas. Furthermore, these processes will deliver sustainable manufacturing practices that utilize renewable feedstocks, reduce energy intensity and lower greenhouse gas emissions.
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, are metabolized first via Embden-Meyerhof-Parnas (EMP) glycolysis to pyruvate, which is then converted to acetyl-CoA via pyruvate:ferredoxin oxidoreductase (PFOR). 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 given by equation 1:

C6H12O6+4ADP+4Pi→2CH3COOH+2CO2+4ATP+8[H]  equation 1

Acetogens extract even more energy out of the glucose to acetate conversion while also maintaining redox balance by further converting the released CO2 to acetate via the Wood-Ljungdahl pathway.

2CO2+8[H]+n ADP+n Pi→CH3COOH+n ATP  equation 2

The coefficient n in the above equation signify that this conversion is an energy generating endeavor, as many acetogens can grow in the presence of CO2 via the Wood-Ljungdahl pathway even in the absence of glucose as long as hydrogen is present to supply the necessary reducing equivalents.

2CO2+4H2+n ADP+n Pi→CH3COOH+2H2O+n ATP  equation 3

The Wood-Ljungdahl pathway, illustrated in FIG. 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. 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 CO2, or directly assimilated into acetyl-CoA which is subsequently converted to either biomass or acetate.

4 CO+2H2O→CH3COOH+2 CO2  equation 4

Even higher acetate yields, however, can be attained when enough hydrogen is present to satisfy the requirement for reducing equivalents.

2 CO+2H2→CH3COOH  equation 5

Following from FIG. 1, the production of acetate via acetyl-CoA generates one ATP molecule, whereas the production of ethanol from acetyl-CoA does not and requires two reducing equivalents. Thus one might speculate that ethanol production from syngas will not generate sufficient energy for cell growth in the absence of acetate production. However, under certain conditions, Clostridium ljungdahlii produces mostly ethanol from synthesis gas (Klasson et al., Fuel 72:1673-1678 (1993)). indicating that some combination of the pathways

2CO2+6H2→CH3CH2OH+3H2O  equation 6

6CO+3H2O→CH3CH2OH+4CO2  equation 7

2CO+4H2CH3CH2OH+H2O  equation 8

does indeed generate enough energy to support cell growth. Hydrogenic bacteria such as R. rubrum can also generate energy from the conversion of CO and water to hydrogen (see FIG. 1) (Simpma et al., Critical Reviews in Biotechnology 26:41-65 (2006)). One important mechanism is the coordinated action of an energy converting hydrogenase (ECH) and CO dehydrogenase. The CO dehydrogenase supplies electrons from CO which are then used to reduce protons to H2 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.

Embodiments of the present invention describe the combination of (1) pathways for the conversion of synthesis gases including CO2, CO, and H2 with and without methanol to acetyl-CoA and (2) pathways for the conversion of acetyl-CoA to isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. As such, this invention provides production organisms and conversion routes with inherent yield advantages over organisms engineered to produce isopropanol, 4-hydroxybutyrate, or 1,4-butanediol from carbohydrate feedstocks using only the product pathways proceeding from acetyl-CoA (i.e., (2) of the combination described above). For example, the maximum theoretical yields of isopropanol, 4-hydroxybutyrate, and 1,4-butanediol from glucose are 1 mole per mole using only the metabolic pathways proceeding from acetyl-CoA as described herein. Specifically, 2 moles of acetyl-CoA are derived per mole of glucose via glycolysis and 2 moles of acetyl-CoA are required per mole of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. The net conversions are described by the following stoichiometric equations:

Isopropanol: C6H42O6+1.5O2→C3H8O+3CO2+2H2O

4-Hydroxybutyate: C6H12O6+1.5O2→C4H8O3+2CO2+2H2O

1,4-Butanediol: C6H12O6→C4H10O2+CH2O2+CO2 

On the other hand, gasification of glucose to its simpler components, CO and H2, followed by their conversion to isopropanol, 4-hydroxybutyrate, and 1,4-butanediol using the pathways described herein results in the following maximum theoretical yields:

Isopropanol: 6CO+6H2→1.333C3H8O+2CO2+0.667H2O

4-Hydroxybutyate: 6CO+6H2→1.333C4H8O3+0.667CO2+0.667H2O

1,4-Butanediol: 6CO+6H2→1.091C4H10O2+1.636CO2+0.545H2O

Note that the gasification of glucose can at best provide 6 moles of CO and 6 moles of H2. Similarly, the combination of certain syngas-utilization pathway components with the acetyl-CoA to isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathways results in high yields of these products from carbohydrates by providing an efficient mechanism for fixing the carbon present in carbon dioxide, fed exogeneously or produced endogenously, into acetyl-CoA (see below and also Example II).

Isopropanol: C6H12O6→1.333C3H8O+2CO2+0.667H2O

4-Hydroxybutyate: C6H12O6→1.333C4H8O3+0.667 CO2+0.667H2O

1,4-Butanediol: C6H12O6→1.091C4H10O2+1.636 CO2+0.545H2O

The maximum theoretical yields of isopropanol, 4-hydroxybutyrate, and 1,4-butanediol from synthesis gases or carbohydrates can be further enhanced by the addition of methanol as described below:

Isopropanol: CH4O+6CO+6H2→1.667C3H8O+2CO2+1.333H2O

4-Hydroxybutyate: CH4O+6 CO+6H2→1.667C4H8O3+0.333 CO2+1.333H2O

1,4-Butanediol: CH4O+6 CO+6H2→1.364C4H10O2+1.545 CO2+1.182H2O

Isopropanol: 2CH4O+6CO+6H2→2C3H8O+2CO2+2H2O

4-Hydroxybutyate: 2 CH4O+6 CO+6H2→2 C4H8O3+2 H2O

1,4-Butanediol: 2 CH4O+6 CO+6H2→1.636C4H10O2+1.455 CO2+1.818H2O

Isopropanol: CH4O+C6H12O6→1.667C3H8O+2CO2+1.333H2O

4-Hydroxybutyate: CH4O+C6H12O6→1.667C4H8O3+0.333 CO2+1.333H2O

1,4-Butanediol: CH4O+C6H12O6→1.364C4H10O2+1.545 CO2+1.182H2O

Isopropanol: 2CH4O+C6H12O6→2C3H8O+2CO2+2H2O

4-Hydroxybutyate: 2 CH4O+C6H12O6→2 C4H8O3+2 H2O

1,4-Butanediol: 2 CH4O+C6H12O6→1.636C4H10O2+1.455 CO2+1.818H2O

Thus, the organisms and conversion routes described herein provide an efficient means of converting carbohydrates to products such as isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. Additional product molecules that can be produced by the teachings of this invention include but are not limited to ethanol, butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, adipic acid, 6-aminocaproic acid, hexamethylenediamine, 3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, acrylic acid, and long chain hydrocarbons, alcohols, acids, and esters.
In some embodiments, the non-naturally occurring organisms of the present invention incorporating the Wood-Ljungdahl pathway can function in the presence of an electron acceptor other than CO2. Wood-Ljungdahl pathway enzymes are oxygen sensitive which can preclude the use of oxygen to improve the energetics of the organism. Under certain conditions, electron acceptor nitrate can block carbon assimilation by the Wood-Ljungdahl pathway, by transcriptional regulation of genes encoding Wood-Ljungdahl. Nonetheless, in some embodiments, a non-naturally occurring organism of the invention utilizing the Wood-Ljungdahl pathway can use nitrate as an electron acceptor to improve overall energetics, as described further below.
In addition to challenges in energy balance, a further challenge of incorporating the Wood-Ljungdahl pathway involves redox balance. Acetogens naturally make acetate as the sole product, not isopropanol. Some syngas utilizers such as Clostridium carboxydivorans have been shown to make multiple products (i.e., ethanol, butanol, acetate, and butyrate). Converting 2 acetyl-CoA molecules to one isopropanol provides 1 reducing equivalent. By contrast, the aforementioned products can produce 2 or more reducing equivalents.
Non-naturally occurring organisms of the present invention employing the combination of pathways disclosed herein can provide a yield of isopropanol, for example, on glucose up to 0.44 g/g glucose. This yield is improved over organism that do not incorporate the Wood-Ljungdahl pathway.
As used herein, the term “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 genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within an isopropanol, 4-hydroxybutyrate, or 1,4-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.
As used herein, the term “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.
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 biochemical.
As used herein, the term “CoA” 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.
As used herein, the term “substantially anaerobic” when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.
“Exogenous” as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. 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. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.
The non-naturally occurring 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. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.
Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.
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. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.
Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5′-3′ exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.
In contrast, 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. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. 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. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.
Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes.
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, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan.-5-1999) 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 (Sep.-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.
Escherichia coli is a common organism with a well-studied set of available genetic tools. Engineering the capability to convert CO2, CO and/or H2 into acetyl-CoA, the central metabolite from which 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 active in acetogenic organisms such as Morella 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. 43:701-715 (1942)). The Wood-Ljungdahl pathway includes two branches: the Eastern (or methyl) branch that enables the conversion of CO2 to methyltetrahydrofolate (Me-THF) and the Western (or carbonyl) branch that enables the conversion of methyl-THF, CO, and Coenzyme-A into acetyl-CoA as shown in FIG. 2A. Such an organism is capable of converting gases comprising CO, CO2, and/or H2 into acetyl-CoA, cell mass, and products such as isopropanol, 4-hydroxybutyrate and 1,4-butanediol. Such an organism is also capable of producing isopropanol, 4-hydroxybutyrate or 1,4-butanediol from carbohydrates at the stoichiometric optimum yield. For example, in combination with the acetyl-CoA to isopropanol pathway, the Wood-Ljungdahl enzymes provide the means to produce 4 moles of isopropanol for every 3 moles of glucose as opposed to 1 mol isopropanol/1 mol glucose which would be attainable in the absence of the Wood-Ljungdahl pathway enzymes. In some embodiments, the present invention 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 methyltransferase system. Such an organism is capable converting methanol, a relatively inexpensive organic feedstock that can be derived from synthesis gas, and gases including CO, CO2, and/or H2 into acetyl-CoA, cell mass, and products. In addition to gaseous substrates, the organism can utilize methanol exclusively or in combination with carbohydrate feedstocks such as glucose to produce products such as isopropanol, 4-hydroxybutyrate or 1,4-butanediol in a high yield. Additional product molecules that can be produced by the teachings of this invention include but are not limited to ethanol, butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, adipic acid, 6-aminocaproic acid, hexamethylenediamine, 3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, acrylic acid, and long chain hydrocarbons, alcohols, acids, and esters.
In some embodiments organisms of the present invention have the following capabilities as depicted in FIG. 2B: 1) a functional methyltransferase system enabling 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 IPA from acetyl-CoA. In other embodiments, organisms of the present invention have a functional methyltransferase system, the ability to synthesize acetyl-CoA, and the ability to synthesize 4-HB from acetyl-CoA as depicted in FIG. 2C. Still other organisms described herein have a functional methyltransferase system, the ability to synthesize acetyl-CoA, and the ability to synthesize BDO from acetyl-CoA depicted in FIG. 2D.
In some embodiments, organisms of the present invention are able to ‘fix’ carbon from exogenous CO and/or CO2 and methanol to synthesize acetyl-CoA, cell mass, and products. The direct conversion of synthesis gas to acetate is an energetically neutral process (see FIGS. 1 and 2A). 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. ATP consumption is circumvented by ensuring that the methyl group on the methyl branch product, methyl-THF, is obtained from methanol rather than CO2. This thereby 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 naturally possesses the capability for anapleurosis, such as 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 dehydrogenase. One 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. In other embodiments, a pyruvate ferredoxin oxidoreductase (PFOR) enzyme can be inserted into the strain to provide the synthesis of biomass precursors in the absence of an external electron acceptor. A further characteristic of organisms of the present invention is the capability of extracting reducing equivalents from molecular hydrogen. This enables a high yield of reduced products such as ethanol, butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, adipic acid, 6-aminocaproic acid, hexamethylenediamine, 3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, and acrylic acid.
Organisms of the present invention can produce acetyl-CoA, cell mass, and targeted chemicals, more specifically IPA, 4-HB, or BDO, from: 1) methanol and CO, 2) methanol, CO2, and H2, 3) methanol, CO, CO2, and H2, 4) methanol and synthesis gas comprising CO and H2, 5) methanol and synthesis gas comprising CO, CO2, and H2, 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 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 catalyze each step of the pathways for the conversion of synthesis gas and methanol to acetyl-CoA, and further to isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. To engineer a production host for the utilization of syngas and methanol, one or more exogenous DNA sequence(s) encoding the enzymes can be expressed in the microorganism.
In some embodiments, the present invention provides a non-naturally occurring microbial organism having an isopropanol pathway that includes at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol. The isopropanol pathway enzyme includes an acetoacetyl-CoA thiolase, an acetoacetyl-CoA transferase, an acetoacetate decarboxylase, and an isopropanol dehydrogenase. In some embodiments, an isopropanol pathway of the present invention includes a succinyl-CoA:3-ketoacid-CoA transferase (SCOT), an acetoacetate decarboxylase, and an isopropanol dehydrogenase. SCOT is a type of acetoacetyl-CoA transferase.
The use of a succinyl-CoA:3-ketoacid-CoA transferase confers a multitude of benefits when converting acetoacetyl-CoA to acetoacetate as part of an isopropanol synthesis pathway: 1) Succinyl-CoA:3-ketoacid-CoA transferase can function as part of a pathway from acetyl-CoA to isopropanol that also generates an energetic equivalent (e.g., ATP, GTP); 2) Succinyl-CoA:3-ketoacid-CoA will not compete with the product pathway for acetyl-CoA substrate; 3) Succinyl-CoA:3-ketoacid-CoA relies on a CoA donor/acceptor molecule, succinate, that is naturally synthesized in most organisms for industrial production.
The non-naturally occurring organisms of the invention are more efficient from an energetic standpoint compared to organisms that utilize acetoacetyl-CoA hydrolase for isopropanol production. Acetoacetyl-CoA hydrolase removes the CoA group from acetoacetyl-CoA without the generation of any energetic equivalents. Succinyl-CoA:3-ketoacid-CoA transferase in combination with, for example, succinyl-CoA synthetase enables the net generation of one energetic equivalent (e.g., GTP, ATP). Succinyl-CoA synthetase is a nearly ubiquitously present enzyme and is an integral component of the well-known tricarboxylic acid cycle.
Acetyl-CoA:acetoacetyl-CoA transferase can be used in combination with acetate kinase and phosphotransacetylase or an ADP-forming acetyl-CoA synthetase to generate an energetic equivalent. Such combinations are more advantageous from an energetic standpoint than using acetoacetyl-CoA hydrolase. However, acetate kinase, phosphotransacetylase, and ADP-forming acetyl-CoA synthetase are reversible which can lead to the formation of substantial amounts of acetate byproduct. Succinyl-CoA synthetase has a lower activity or no activity at all on acetyl-CoA compared to phosphotransacetylase or an ADP-forming acetyl-CoA synthetase.
It is thus desirable in the case of isopropanol to use a CoA donor/acceptor molecule that is completely disconnected from the product pathway. Butyryl-CoA:acetoacetyl-CoA transferase is one such enzyme and is taught by Subbian et al., US 2008/0293125. However, butyrate is not naturally present at substantial levels in most organisms including the industrial workhouse organisms Escherichia coli and Saccharomyces cerevisiae. Thus the capability of synthesizing a catalytic amount of butyrate would have to engineered into such organisms to enable butyryl-CoA:acetoacetyl-CoA transferase to function as part of an isopropanol synthesis pathway. Butyrate is also a foul smelling compound and substantial leakage of butyrate out of the organism could require special process engineering steps to ensure that it is not released into the air. On the other hand, succinate is non-odorous and is synthesized in a plethora of organisms including E. coli and S. cerevisiae. Typical succinate forming enzymes include succinyl-CoA synthetase and alpha-ketoglutarate dehydrogenase, both components of the well-known tricarboxylic acid cycle.
Such organisms may also include at least one enzyme or polypeptide such as a corrinoid protein, a methyltetrahydrofolate:corrinoid protein methyltransferase, a corrinoid iron-sulfur protein, a nickel-protein assembly protein, a ferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and a hydrogenase.
In some embodiments, organisms having an isopropanol pathway have a methanol methyltransferase. In such embodiments, the organisms utilize a feedstock such as 1) methanol and CO, 2) methanol, CO2, and H2, 3) methanol, CO, CO2, and H2, 4) methanol and synthesis gas comprising CO and H2, 5) methanol and synthesis gas comprising CO, CO2, and H2, 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.
In other embodiments, organisms of the present invention have a formate dehydrogenase, a formyltetrahydrofolate synthetase, a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolate dehydrogenase, and a methylenetetrahydrofolate reductase. Such organisms utilize a feedstock selected from the group consisting of 1) CO, 2) CO2 and H2, 3) CO and CO2, 4) synthesis gas comprising CO and H2, 5) synthesis gas comprising CO, CO2, and H2, and 6) one or more carbohydrates. Exemplary carbohydrates include but are not limited to glucose, sucrose, xylose, arabinose, and glycerol.
The invention also provides a non-naturally occurring microbial organism having a 4-hydroxybutryate pathway that includes at least one exogenous nucleic acid encoding an 4-hydroxybutryate pathway enzyme expressed in a sufficient amount to produce 4-hydroxybutryate. The 4-hydroxybutryate pathway enzyme includes an acetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, a phosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyrate kinase.
Such organisms can also include at least one enzyme or polypeptide such as a corrinoid protein, a methyltetrahydrofolate:corrinoid protein methyltransferase, a corrinoid iron-sulfur protein, a nickel-protein assembly protein, a ferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and a hydrogenase.
In some embodiments, organisms that have a 4-hydroxybutyrate pathway can include a methanol methyltransferase. Such organisms utilize a feedstock such as 1) methanol and CO, 2) methanol, CO2, and H2, 3) methanol, CO, CO2, and H2, 4) methanol and synthesis gas comprising CO and H2, 5) methanol and synthesis gas comprising CO, CO2, and H2, 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.
Other organisms that have a 4-hydroxybutyrate pathway can have a formate dehydrogenase, a formyltetrahydrofolate synthetase, a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolate dehydrogenase, and a methylenetetrahydrofolate reductase. Such organisms utilize a feedstock such as 1) CO, 2) CO2 and H2, 3) CO and CO2, 4) synthesis gas comprising CO and H2, 5) synthesis gas comprising CO, CO2, and H2, and 6) one or more carbohydrates. Exemplary carbohydrates include but are not limited to glucose, sucrose, xylose, arabinose, and glycerol.
The present invention also provides a non-naturally occurring microbial organism having a 1,4-butanediol pathway that includes at least one exogenous nucleic acid encoding a 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce 1,4-butanediol. The 1,4-butanediol pathway enzyme include, for example, an acetoacetyl-CoA thiolase, a 3-Hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyrate reductase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyrate kinase, and a 1,4-butanediol dehydrogenase.
Such organisms can also include at least one enzyme or polypeptide such as a corrinoid protein, a methyltetrahydrofolate:corrinoid protein methyltransferase, a corrinoid iron-sulfur protein, a nickel-protein assembly protein, a ferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and a hydrogenase.
In some embodiments, an organism having a 1,4-butanediol pathway can include a methanol methyltransferase. Such organisms utilize a feedstock such as 1) methanol and CO, 2) methanol, CO2, and H2, 3) methanol, CO, CO2, and H2, 4) methanol and synthesis gas comprising CO and H2, 5) methanol and synthesis gas comprising CO, CO2, and H2, 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.
In other embodiments, an organism having a 1,4-butanediol pathway can include a formate dehydrogenase, a formyltetrahydrofolate synthetase, a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolate dehydrogenase, and a methylenetetrahydrofolate reductase. Such organisms utilize a feedstock selected from the group consisting of: 1) CO, 2) CO, and H2, 3) CO and CO2, 4) synthesis gas comprising CO and H2, 5) synthesis gas comprising CO, CO2, and H2, and 6) one or more carbohydrates. Exemplary carbohydrates include but are not limited to glucose, sucrose, xylose, arabinose, and glycerol.
Also disclosed herein is a non-naturally occurring microbial organism having an acetyl-CoA pathway that includes at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA. The acetyl-CoA pathway enzyme includes a methanol methyltransferase and an acetyl-CoA synthase.
In still further embodiments, the present invention provides a non-naturally occurring microbial organism having an isopropanol pathway comprising at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol, said isopropanol pathway enzyme comprising a methanol methyltransferase, a corrinoid protein, a methyltetrahydrofolate:corrinoid protein methyltransferase, a corrinoid iron-sulfur protein, a nickel-protein assembly protein, a ferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and a hydrogenase.
In some embodiments, such an organism can include an exogenous polypeptide or enzyme such as an acetoacetyl-CoA thiolase, an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, a phosphotransacetoacetylase, an acetoacetate kinase, an acetoacetate decarboxylase, and an isopropanol dehydrogenase. In additional embodiments, such an organism can include an exogenous polypeptide or enzyme such as a succinyl-CoA:3-ketoacid-CoA transferase (SCOT), an acetoacetate decarboxylase, and an isopropanol dehydrogenase.
Expression of the modified Wood-Ljungdahl pathway in a foreign host (see FIG. 2B) requires a set of methyltransferases to utilize the carbon and hydrogen provided by methanol and the carbon provided by CO and/or CO2. A complex of 3 methyltransferase proteins, denoted MtaA, MtaB, and MtaC, perform the desired methanol methyltransferase activity (Naidu and Ragsdale, J. Bacteriol. 183:3276-3281 (2001); Ragsdale, S. W., Crit. Rev. Biochem. Mol. Biol. 39:165-195 (2004); Sauer et al., Eur. J. Biochem. 243:670-677 (1997); Tallant and Krzycki, J. Bacteriol. 178:1295-1301 (1996); Tallant and Krzycki, J. Bacteriol. 179:6902-6911 (1997); Tallant et al., J Biol. Chem. 276:4485-4493 (2001)).
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., J. Bacteriol. 188:7922-7931 (2006)) and Methanosarcina acetivorans (Galagan et al., Genome Res 12:532-542 (2002)), as well as the acetogen, Moorella thermoacetica (Das et al., Proteins 67:167-176 (2007)). In general, the 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







MtaB1
YP_304299
73668284
Methanosarcina barkeri


MtaC1
YP_304298
73668283
Methanosarcina barkeri


MtaB2
YP_307082
73671067
Methanosarcina barkeri


MtaC2
YP_307081
73671066
Methanosarcina barkeri


MtaB3
YP_304612
73668597
Methanosarcina barkeri


MtaC3
YP_304611
73668596
Methanosarcina barkeri


MtaB1
NP_615421
20089346
Methanosarcina acetivorans


MtaB1
NP_615422
20089347
Methanosarcina acetivorans


MtaB2
NP_619254
20093179
Methanosarcina acetivorans


MtaC2
NP_619253
20093178
Methanosarcina acetivorans


MtaB3
NP_616549
20090474
Methanosarcina acetivorans


MtaC3
NP_616550
20090475
Methanosarcina acetivorans


MtaB
YP_430066
83590057
Moorella thermoacetica


MtaC
YP_430065
83590056
Moorella thermoacetica









The MtaB1 and MtaC1 genes, YP—304299 and YP—304298, from M. barkeri were cloned into E. coli and sequenced (Sauer et al., Eur. J. Biochem. 243:670-677 (1997)). The crystal structure of this methanol-cobalamin methyltransferase complex is also available (Hagemeier et al., Proc Natl Acad Sci US.A 103:18917-18922 (2006)). The MtaB genes, YP—307082 and YP—304612, in M. barkeri were identified by sequence homology to YP—304299. In general, homology searches are an effective means of identifying methanol methyltransferases because 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 56:1183-1194 (2005)). Mutant strains lacking two of the sets were able to grow on methanol, whereas a strain lacking all three sets of MtaB and MtaC genes sets could not grow on methanol. This indicates that each set of genes plays a role in methanol utilization. The 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. 61:537-540 (2005)) and further characterized by Northern hybridization and Western Blotting (Das et al., Proteins 67:167-176 (2007)).
MtaA is zinc protein that catalyzes the transfer of the methyl group from MtaC to either 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., J. Bacteriol. 188:7922-7931 (2006)) and Methanosarcina acetivorans (Galagan et al., Genome Res 12:532-542 (2002)), as well as the acetogen, Moorella thermoacetica (Das et al., Proteins 67:167-176 (2007)). In general, MtaA proteins that catalyze the transfer of the methyl group from CH3-MtaC are difficult to identify bioinformatically as they share similarity to other corrinoid protein methyltransferases 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.










TABLE 2





Protein
GenBank ID
GI Number
Organism







MtaA
YP_304602
73668587
Methanosarcina barkeri


MtaA1
NP_619241
20093166
Methanosarcina acetivorans


MtaA2
NP_616548
20090473
Methanosarcina acetivorans









The MtaA gene, YP—304602, from M. barkeri was cloned, sequenced, and functionally overexpressed in E. coli (Halms and Thauer, Eur. J. Biochem. 235:653-659 (1996)). In M. acetivorans, MtaA1 is required for growth on methanol, whereas MtaA2 is dispensable even though methane production from methanol is reduced in MtaA2 mutants (Bose et al., J. Bacteriol. 190:4017-4026 (2008)). There are multiple additional MtaA homologs in M. barkeri and M. 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 CH3-MtaC to Coenzyme M, an M. thermoacetica MtaA is likely to transfer the methyl group to methyltetrahydrofolate given the similar roles of methyltetrahydrofolate and Coenzyme M in methanogens and acetogens, respectively. The protein sequences of putative MtaA encoding genes from M. thermoacetica can be identified by the following GenBank accession numbers.










TABLE 3





Protein
GenBank ID
GI Number
Organism







MtaA
YP_430937
83590928
Moorella thermoacetica


MtaA
YP_431175
83591166
Moorella thermoacetica


MtaA
YP_430935
83590926
Moorella thermoacetica









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 corrinoide-iron-sulfur-protein is methylated by methyltetrahydrofolate via a methyltransferase. Expression of ACS/CODH in a foreign host can be done by introducing one or more of the following proteins and their corresponding activities: Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD), Nickel-protein assembly protein (AcsF), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB and AcsC), Carbon monoxide dehydrogenase (AcsA) or 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 (Morton et al., J Biol. Chem. 266:23824-23828 (1991); Ragsdale, S. W., Crit. Rev. Biochem. Mol. Biol. 39:165-195 (2004); Roberts et al., Proc Natl Acad Sci US.A 86:32-36 (1989)). Each of the genes in this operon from the acetogen, M. thermoacetica, has already been cloned and expressed actively in E. coli (Lu et al., J Biol. Chem. 268:5605-5614 (1993); Roberts et al., Proc Natl Acad Sci US.A 86:32-36 (1989)). The protein sequences of these genes can be identified by the following GenBank accession numbers.










TABLE 4





Protein
GenBank ID
GI Number
Organism







AcsE
YP_430054
83590045
Moorella thermoacetica


AcsD
YP_430055
83590046
Moorella thermoacetica


AcsF
YP_430056
83590047
Moorella thermoacetica


Orf7
YP_430057
83590048
Moorella thermoacetica


AcsC
YP_430058
83590049
Moorella thermoacetica


AcsB
YP_430059
83590050
Moorella thermoacetica


AcsA
YP_430060
83590051
Moorella thermoacetica


CooC
YP_430061
83590052
Moorella thermoacetica









The hydrogenogenic bacterium, Carboxydothermus hydrogenoformans, can utilize carbon monoxide as a growth substrate by means of acetyl-CoA synthase (Wu et al., PLoS Genet. 1:e65 (1995)). In strain Z-2901, the acetyl-CoA synthase enzyme complex lacks carbon monoxide dehydrogenase due to a frameshift mutation (Wu et al., PLoS Genet. 1:e65 (1995)), 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 101:446-451 (2004)). The protein sequences of the C. hydrogenoformans genes from strain Z-2901 are identified by the following GenBank accession numbers. Sequences for Carboxydothermus hydrogenoformans DSM 6008 are not yet accessible in publicly available databases.












TABLE 5







Protein
GenBank ID
GI Number
Organism









AcsE
YP_360065
78044202
Carboxydothermus






hydrogenoformans



AcsD
YP_360064
78042962
Carboxydothermus






hydrogenoformans



AcsF
YP_360063
78044060
Carboxydothermus






hydrogenoformans



Orf7
YP_360062
78044449
Carboxydothermus






hydrogenoformans



AcsC
YP_360061
78043584
Carboxydothermus






hydrogenoformans



AcsB
YP_360060
78042742
Carboxydothermus






hydrogenoformans



CooC
YP_360059
78044249
Carboxydothermus






hydrogenoformans










Homologous ACS/CODH genes can also be found in the draft genome assembly of Clostridium carboxidivorans P7.










TABLE 6





Protein
GenBank ID
GI Number
Organism







AcsA
ZP_05392944.1
255526020
Clostridium carboxidivorans





P7


CooC
ZP_05392945.1
255526021
Clostridium carboxidivorans





P7


AcsF
ZP_05392952.1
255526028
Clostridium carboxidivorans





P7


AcsD
ZP_05392953.1
255526029
Clostridium carboxidivorans





P7


AcsC
ZP_05392954.1
255526030
Clostridium carboxidivorans





P7


AcsE
ZP_05392955.1
255526031
Clostridium carboxidivorans





P7


AcsB
ZP_05392956.1
255526032
Clostridium carboxidivorans





P7


Orf7
ZP_05392958.1
255526034
Clostridium carboxidivorans





P7









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 US.A 103:17921-17926 (2006)). This organism contains two sets of genes that encode ACS/CODH activity (Rother and Metcalf Proc Natl Acad Sci US.A 101:16929-16934 (2004)). The protein sequences of both sets of M. acetivorans genes can be identified by the following GenBank accession numbers.










TABLE 7





Protein
GenBank ID
GI Number
Organism







AcsC
NP_618736
20092661
Methanosarcina acetivorans


AcsD
NP_618735
20092660
Methanosarcina acetivorans


AcsF, CooC
NP_618734
20092659
Methanosarcina acetivorans


AcsB
NP_618733
20092658
Methanosarcina acetivorans


AcsEps
NP_618732
20092657
Methanosarcina acetivorans


AcsA
NP_618731
20092656
Methanosarcina acetivorans


AcsC
NP_615961
20089886
Methanosarcina acetivorans


AcsD
NP_615962
20089887
Methanosarcina acetivorans


AcsF, CooC
NP_615963
20089888
Methanosarcina acetivorans


AcsB
NP_615964
20089889
Methanosarcina acetivorans


AcsEps
NP_615965
20089890
Methanosarcina acetivorans


AcsA
NP_615966
20089891
Methanosarcina acetivorans









The AcsC, AcsD, AcsB, AcsEps, 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 hydrogenogenic bacteria such as C. hydrogenoformans. Hypotheses for the existence of two active CODH/ACS operons in M. acetivorans include catalytic properties (i.e., Km, Vmax, kcat) that favor carboxidotrophic or aceticlastic growth or differential gene regulation enabling various stimuli to induce CODH/ACS expression (Rother et al., Arch. Microbiol 188:463-472 (2007)).
In M. thermoacetica, C. hydrogenoformans and C. carboxidivorans P7, additional 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 accepters such as oxidized ferredoxin, NADP+, water, or hydrogen peroxide to form reduced ferredoxin, NADPH, H2, or water, respectively. In some cases, hydrogenase encoding genes are located adjacent to a CODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteins form a membrane-bound enzyme complex that is proposed to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO to CO2 and H2 (Fox et al., J. Bacteriol. 178:6200-6208 (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:e65 (2005)). The C. hydrogenoformans CODH-I was also shown to exhibit intense CO oxidation and CO2 reduction activities when linked to an electrode (Parkin et al., J. Am. Chem. Soc. 129:10328-10329 (2007)). The genes encoding the C. hydrogenoformans CODH-II and CooF, a neighboring protein, were cloned and sequenced (Gonzalez and Robb, FEMS Microbiol Lett. 191:243-247 (2000)). The resulting complex was membrane-bound, although cytoplasmic fractions of CODH-II were shown to catalyze the formation of NADPH suggesting an anabolic role (Svetlitchnyi et al., J. Bacteriol. 183:5134-5144 (2001)). The crystal structure of the CODH-II is also available (Dobbek et al., Science 293:1281-1285 (2001)). The protein sequences of exemplary CODH and hydrogenase genes can be identified by the following GenBank accession numbers.










TABLE 8





Protein
GenBank ID
GI Number
Organism







CODH (putative)
YP_430813
83590804
Moorella thermoacetica


CODH-I (CooS-I)
YP_360644
78043418
Carboxydothermus hydrogenoformans


CooF
YP_360645
78044791
Carboxydothermus hydrogenoformans


HypA
YP_360646
78044340
Carboxydothermus hydrogenoformans


CooH
YP_360647
78043871
Carboxydothermus hydrogenoformans


CooU
YP_360648
78044023
Carboxydothermus hydrogenoformans


CooX
YP_360649
78043124
Carboxydothermus hydrogenoformans


CooL
YP_360650
78043938
Carboxydothermus hydrogenoformans


CooK
YP_360651
78044700
Carboxydothermus hydrogenoformans


CooM
YP_360652
78043942
Carboxydothermus hydrogenoformans


CooM
AAC45116
1515466
Rhodospirillum rubrum


CooK
AAC45117
1515467
Rhodospirillum rubrum


CooL
AAC45118
1515468
Rhodospirillum rubrum


CooX
AAC45119
1515469
Rhodospirillum rubrum


CooU
AAC45120
1515470
Rhodospirillum rubrum


CooH
AAC45121
1498746
Rhodospirillum rubrum


CooF
AAC45122
1498747
Rhodospirillum rubrum


CODH (CooS)
AAC45123
1498748
Rhodospirillum rubrum


CooC
AAC45124
1498749
Rhodospirillum rubrum


CooT
AAC45125
1498750
Rhodospirillum rubrum


CooJ
AAC45126
1498751
Rhodospirillum rubrum


CODH-II (CooS-II)
YP_358957
78044574
Carboxydothermus hydrogenoformans


CooF
YP_358958
78045112
Carboxydothermus hydrogenoformans


CODH (putative)
ZP_05390164.1
255523193
Clostridium carboxidivorans P7



ZP_05390341.1
255523371
Clostridium carboxidivorans P7



ZP_05391756.1
255524806
Clostridium carboxidivorans P7



ZP_05392944.1
255526020
Clostridium carboxidivorans P7


CooF
ZP_05394386.1
255527518
Clostridium carboxidivorans P7


CooF
ZP_05394384.1
255527516
Clostridium carboxidivorans P7


HypA
ZP_05392031.1
255525086
Clostridium carboxidivorans P7


CooH
ZP_05392429.1
255525492
Clostridium carboxidivorans P7


CooL
ZP_05392428.1
255525491
Clostridium carboxidivorans P7


CooM
ZP_05392434.1
255525497
Clostridium carboxidivorans P7









Anaerobic growth on synthesis gas and methanol in the absence of an external electron acceptor is conferred upon the host organism with MTR and ACS/CODH activity by providing pyruvate synthesis via pyruvate ferredoxin oxidoreductase (PFOR). The PFOR from Desulfovibrio africanus has been cloned and expressed in E. coli resulting in an active recombinant enzyme that was stable for several days in the presence of oxygen (Pieulle et al., J. Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is reported to be conferred by a 60 residue extension in the polypeptide chain of the D. africanus enzyme. The M. thermoacetica PFOR is also well characterized (Menon and Ragsdale, Biochemistry 36:8484-8494 (1997)) and was shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui and Ragsdale, J Biol. Chem. 275:28494-28499 (2000)). Further, E. coli possesses an uncharacterized open reading frame, ydbK, that encodes a protein that is 51% identical to the M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E. coli has been described (Blaschkowski et al., Eur. J. Biochem. 123:563-569 (1982)). Homologs also exist in C. carboxidivorans P7. The protein sequences of these exemplary PFOR enzymes are identified by the following GenBank accession numbers shown in Table 9 below. Several additional PFOR enzymes are described in Ragsdale, S. W., Chem. Rev. 103:2333-2346 (2003). Finally, flavodoxin reductases (e.g., fqrB from Helicobacter pylori or Campylobacter jejuni) (St Maurice et al., J. Bacteriol. 189:4764-4773 (2007)) or Rnf-type proteins (Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); and Herrmann, J. Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPH from the reduced ferredoxin generated by PFOR. These proteins are identified below in Table 9.










TABLE 9





Protein
GenBank ID
GI Number
Organism







Por
CAA70873.1
1770208
Desulfovibrio africanus


Por
YP_428946.1
83588937
Moorella thermoacetica


YdbK
NP_415896.1
16129339
Escherichia coli


Por
ZP_05392150.1
255525208
Clostridium carboxidivorans





P7


Por
ZP_05390930.1
255523967
Clostridium carboxidivorans





P7


Por
ZP_05394396.1
255527529
Clostridium carboxidivorans





P7


Por
ZP_05394397.1
255527530
Clostridium carboxidivorans





P7


FqrB
NP_207955.1
15645778
Helicobacter pylori


FqrB
YP_001482096.1
157414840
Campylobacter jejuni


RnfC
EDK33306.1
146346770
Clostridium kluyveri


RnfD
EDK33307.1
146346771
Clostridium kluyveri


RnfG
EDK33308.1
146346772
Clostridium kluyveri


RnfE
EDK33309.1
146346773
Clostridium kluyveri


RnfA
EDK33310.1
146346774
Clostridium kluyveri


RnfB
EDK33311.1
146346775
Clostridium kluyveri









The conversion of pyruvate into acetyl-CoA can be catalyzed by several other enzymes or their combinations thereof. For example, 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 decarboxylase (E1), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). This enzyme is naturally present in several organisms, including E. coli and S. cerevisiae. In the E. coli enzyme, specific residues in the E1 component are responsible for substrate specificity (Bisswanger, H., J. Biol. Chem. 256:815-82 (1981); Bremer, J., Eur. J. Biochem. 8:535-540 (1969); Gong et al., J. Biol. Chem. 275:13645-13653 (2000)). Enzyme engineering efforts have improved the E. coli PDH enzyme activity under anaerobic conditions (Kim et al., J. Bacteriol. 190:3851-3858 (2008); Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtilis complex is active and required for growth under anaerobic conditions (Nakano et al., J. Bacteriol. 179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterized during growth on glycerol, is also active under anaerobic conditions (Menzel et al., J. Biotechnol. 56:135-142 (1997)). Crystal structures of the enzyme complex from bovine kidney (Zhou et al., Proc. Natl. Acad. Sci. U.S.A. 98:14802-14807 (2001)) and the E2 catalytic domain from 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. 6:383-398 (1990)), Lactococcus lactis (Melchiorsen et al., Appl. Microbiol. Biotechnol. 58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al., Oral. Microbiol Immunol. 18:293-297 (2003)). 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. Microbiol. 27:477-492 (1998)). Both pflB and tdcE from E. coli require the presence of pyruvate formate lyase activating enzyme, encoded by pflA. Further, a short protein encoded by yfiD in E. coli can associate with and restore activity to oxygen-cleaved pyruvate formate lyase (Vey et al., Proc. Natl. Acad. Sci. U.S.A. 105:16137-16141 (2008). Note that pflA and pflB from E. coli were expressed in S. cerevisiae as a means to increase cytosolic acetyl-CoA for butanol production as described in WO/2008/080124]. Additional pyruvate formate lyase and activating enzyme candidates, encoded by pfl and act, respectively, are found in Clostridium pasteurianum (Weidner et al., J. Bacteriol. 178:2440-2444 (1996)).
Further, different enzymes can be used in combination to convert pyruvate into acetyl-CoA. For example, in S. cerevisiae, 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., J. Bacteriol. 177:2878-2886 (1995)), Salmonella enterica (Starai et al., Microbiology 151:3793-3801 (2005); Starai et al., J. Biol. Chem. 280:26200-26205 (2005)), and Moorella thermoacetica (described already). Alternatively, 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 phosphotransacetylyase 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 Zymomonas mobilis. 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, J. 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.
Unlike the redox neutral conversion of CO and MeOH to acetyl-CoA or acetate, the production of more highly reduced products such as ethanol, butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, adipic acid, 6-aminocaproic acid, hexamethylenediamine, 3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, and acrylic acid at the highest possible yield requires the extraction of additional reducing equivalents from both CO and H2 (for example, see ethanol formation in FIG. 2A). Specifically, reducing equivalents (e.g., 2 [H] in FIG. 2) are obtained by the conversion of CO and water to CO2 via carbon monoxide dehydrogenase or directly from the activity of a hydrogen-utilizing hydrogenase which transfers electrons from H2 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 66:57-88 (1994); Sawers et al., J. Bacteriol. 164:1324-1331 (1985); Sawers and Boxer, Eur. J. Biochem. 156:265-275 (1986); Sawers et al., J. Bacteriol. 168:398-404 (1986)). Given the multiplicity of enzyme activities E. coli or another host organism can provide sufficient hydrogenase activity to split incoming molecular hydrogen and reduce the corresponding acceptor. Among 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. coli is also dependent upon the expression of the hyp genes whose corresponding proteins are involved in the assembly of the hydrogenase complexes (Jacobi et al., Arch. Microbiol 158:444-451 (1992); Rangarajan et al., J. Bacteriol. 190:1447-1458 (2008)). The M. thermoacetica hydrogenases are suitable for a host that lacks sufficient endogenous hydrogenase activity. M. thermoacetica can grow with CO2 as the exclusive carbon source indicating that reducing equivalents are extracted from H2 to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L., J. Bacteriol. 150:702-709 (1982); Drake and Daniel, Res Microbiol 155:869-883 (2004); Kellum and Drake, J. Bacteriol. 160:466-469 (1984)) (see FIG. 2A). M. thermoacetica has homologs to several hyp, hyc, and hyf genes from E. coli. These protein sequences encoded for by these genes are 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 in Table 10.












TABLE 10







Protein
GenBank ID
GI Number
Organism









HypA
NP_417206
16130633
Escherichia coli



HypB
NP_417207
16130634
Escherichia coli



HypC
NP_417208
16130635
Escherichia coli



HypD
NP_417209
16130636
Escherichia coli



HypE
NP_417210
226524740
Escherichia coli



HypF
NP_417192
16130619
Escherichia coli










Proteins in M. thermoacetica whose genes are homologous to the E. coli hyp genes are shown below in Table 11. Hydrogenase 3 proteins are listed in Table 12. Hydrogenase 4 proteins are shown in Table 13.










TABLE 11





Protein
GenBank ID
GI Number
Organism







Moth_2175
YP_431007
83590998
Moorella thermoacetica


Moth_2176
YP_431008
83590999
Moorella thermoacetica


Moth_2177
YP_431009
83591000
Moorella thermoacetica


Moth_2178
YP_431010
83591001
Moorella thermoacetica


Moth_2179
YP_431011
83591002
Moorella thermoacetica


Moth_2180
YP_431012
83591003
Moorella thermoacetica


Moth_2181
YP_431013
83591004
Moorella thermoacetica





















TABLE 12







Protein
GenBank ID
GI Number
Organism









HycA
NP_417205
16130632
Escherichia coli



HycB
NP_417204
16130631
Escherichia coli



HycC
NP_417203
16130630
Escherichia coli



HycD
NP_417202
16130629
Escherichia coli



HycE
NP_417201
16130628
Escherichia coli



HycF
NP_417200
16130627
Escherichia coli



HycG
NP_417199
16130626
Escherichia coli



HycH
NP_417198
16130625
Escherichia coli



HycI
NP_417197
16130624
Escherichia coli






















TABLE 13







Protein
GenBank ID
GI Number
Organism









HyfA
NP_416976
90111444
Escherichia coli



HyfB
NP_416977
16130407
Escherichia coli



HyfC
NP_416978
90111445
Escherichia coli



HyfD
NP_416979
16130409
Escherichia coli



HyfE
NP_416980
16130410
Escherichia coli



HyfF
NP_416981
16130411
Escherichia coli



HyfG
NP_416982
16130412
Escherichia coli



HyfH
NP_416983
16130413
Escherichia coli



HyfI
NP_416984
16130414
Escherichia coli



HyfJ
NP_416985
90111446
Escherichia coli



HyfR
NP_416986
90111447
Escherichia coli










Proteins in M. thermoacetica whose genes are homologous to the E. coli hyc and/or hyf genes are shown in Table 14 below. Additional hydrogenase-encoding gene clusters in M. thermoacetica are shown in Table 15.










TABLE 14





Protein
GenBank ID
GI Number
Organism







Moth_2182
YP_431014
83591005
Moorella thermoacetica


Moth_2183
YP_431015
83591006
Moorella thermoacetica


Moth_2184
YP_431016
83591007
Moorella thermoacetica


Moth_2185
YP_431017
83591008
Moorella thermoacetica


Moth_2186
YP_431018
83591009
Moorella thermoacetica


Moth_2187
YP_431019
83591010
Moorella thermoacetica


Moth_2188
YP_431020
83591011
Moorella thermoacetica


Moth_2189
YP_431021
83591012
Moorella thermoacetica


Moth_2190
YP_431022
83591013
Moorella thermoacetica


Moth_2191
YP_431023
83591014
Moorella thermoacetica


Moth_2192
YP_431024
83591015
Moorella thermoacetica



















TABLE 15





Protein
GenBank ID
GI Number
Organism







Moth_0439
YP_429313
83589304
Moorella thermoacetica


Moth_0440
YP_429314
83589305
Moorella thermoacetica


Moth_0441
YP_429315
83589306
Moorella thermoacetica


Moth_0442
YP_429316
83589307
Moorella thermoacetica


Moth_0809
YP_429670
83589661
Moorella thermoacetica


Moth_0810
YP_429671
83589662
Moorella thermoacetica


Moth_0811
YP_429672
83589663
Moorella thermoacetica


Moth_0812
YP_429673
83589664
Moorella thermoacetica


Moth_0814
YP_429674
83589665
Moorella thermoacetica


Moth_0815
YP_429675
83589666
Moorella thermoacetica


Moth_0816
YP_429676
83589667
Moorella thermoacetica


Moth_1193
YP_430050
83590041
Moorella thermoacetica


Moth_1194
YP_430051
83590042
Moorella thermoacetica


Moth_1195
YP_430052
83590043
Moorella thermoacetica


Moth_1196
YP_430053
83590044
Moorella thermoacetica


Moth_1717
YP_430562
83590553
Moorella thermoacetica


Moth_1718
YP_430563
83590554
Moorella thermoacetica


Moth_1719
YP_430564
83590555
Moorella thermoacetica


Moth_1883
YP_430726
83590717
Moorella thermoacetica


Moth_1884
YP_430727
83590718
Moorella thermoacetica


Moth_1885
YP_430728
83590719
Moorella thermoacetica


Moth_1886
YP_430729
83590720
Moorella thermoacetica


Moth_1887
YP_430730
83590721
Moorella thermoacetica


Moth_1888
YP_430731
83590722
Moorella thermoacetica


Moth_1452
YP_430305
83590296
Moorella thermoacetica


Moth_1453
YP_430306
83590297
Moorella thermoacetica


Moth_1454
YP_430307
83590298
Moorella thermoacetica









Isopropanol production is achieved in recombinant E. coli following expression of two heterologous genes from C. acetobutylicum (thl and adc encoding acetoacetyl-CoA thiolase and acetoacetate decarboxylase, respectively) and one from C. beijerinckii (adh encoding a secondary alcohol dehydrogenase), along with the increased expression of the native atoA and atoD genes which encode acetoacetyl-CoA:acetate:CoA transferase activity (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007)).
Acetoacetyl-CoA thiolase converts two molecules of acetyl-CoA into one molecule each of acetoacetyl-CoA and CoA. Such a conversion is the first step for isopropanol, 4-hydroxybutyrate, and 1,4-butanediol synthesis from acetyl-CoA, as shown in FIGS. 2 and 3. Exemplary acetoacetyl-CoA thiolase enzymes include the gene products of atoB from E. coli (Martin et al., Nat. Biotechnol 21:796-802 (2003)), thlA and thlB from C. acetobutylicum (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007); Winzer et al., J. Mol. Microbiol Biotechnol 2:531-541 (2000), and ERG10 from S. cerevisiae Hiser et al., J. Biol. Chem. 269:31383-31389 (1994)).










TABLE 16





Protein
GenBank ID
GI Number
Organism







AtoB
NP_416728
16130161
Escherichia coli


ThlA
NP_349476.1
15896127
Clostridium acetobutylicum


ThlB
NP_149242.1
15004782
Clostridium acetobutylicum


ERG10
NP_015297
6325229
Saccharomyces cerevisiae









The conversion of acetoacetyl-CoA to acetoacetate or of 4-hydroxybutyryl-CoA to 4-hydroxybutyrate can be carried out by an acetoacetyl-CoA transferase or 4-hydroxybutyryl-CoA transferase, respectively. These enzymes conserve the energy stored in the CoA-ester bonds of acetoacetyl-CoA and 4-hydroxybutyryl-CoA. Many transferases have broad specificity and thus can utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3-mercaptopropionate, propionate, vinylacetate, butyrate, among others. Acetoacetyl-CoA transferase catalyzes the conversion of acetoacetyl-CoA to acetoacetate while transferring the CoA moiety to a CoA acceptor molecule. Several exemplary transferase enzymes capable of catalyzing this transformation are provided below. These enzymes either naturally exhibit the desired acetoacetyl-CoA transferase activity or they can be engineered via directed evolution to accept acetoacetyl-CoA as a substrate with increased efficiency. Such enzymes, either naturally or following directed evolution, are also suitable for catalyzing the conversion of 4-hydroxybutyryl-CoA to 4-hydroxybutyrate via a transferase mechanism.
In one embodiment an exemplary acetoacetyl-CoA transferase is acetoacetyl-CoA:acetate-CoA transferase. This enzyme naturally converts acetate to acetyl-CoA while converting acetoacetyl-CoA to acetoacetate. In another embodiment, a succinyl-CoA:3-ketoacid CoA transferase (SCOT) catalyzes the conversion of the 3-ketoacyl-CoA, acetoacetyl-CoA, to the 3-ketoacid, acetoacetate.
Acetoacetyl-CoA:acetyl-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. acetobutylicum (Jojima et al., Appl Microbiol Biotechnol. 77:1219-1224 (2008)), and ctfAB from Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)). Information related to these proteins and genes is shown below:










TABLE 17





Protein
GENBANK ID
GI NUMBER
ORGANISM







AtoA
P76459.1
2492994
Escherichia coli


AtoD
P76458.1
2492990
Escherichia coli


CtfA
NP_149326.1
15004866
Clostridium acetobutylicum


CtfB
NP_149327.1
15004867
Clostridium acetobutylicum


CtfA
AAP42564.1
31075384
Clostridium





saccharoperbutylacetonicum


CtfB
AAP42565.1
31075385
Clostridium





saccharoperbutylacetonicum









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. 272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein. Expr. Purif. 53:396-403 (2007)), and Homo sapiens (Fukao et al. Genomics 68:144-151 (2000); Tanaka et al., Mol. Hum. Reprod. 8:16-23 (2002)). Information related to these proteins and genes is shown below:










TABLE 18





Protein
GENBANK ID
GI NUMBER
ORGANISM







HPAG1_0676
YP_627417
108563101
Helicobacter pylori


HPAG1_0677
YP_627418
108563102
Helicobacter pylori


ScoA
NP_391778
16080950
Bacillus subtilis


ScoB
NP_391777
16080949
Bacillus subtilis


OXCT1
NP_000427
4557817
Homo sapiens


OXCT2
NP_071403
11545841
Homo sapiens









Additional suitable acetoacetyl-CoA and 4-hydroxybutyryl-CoA transferases are encoded by the gene products of cat1, cat2, and cat3 of Clostridium kluyveri. These enzymes have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively (Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 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., J. Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem. 279:45337-45346 (2004)). Yet another transferase capable of the desired conversions is butyryl-CoA:acetoacetate CoA-transferase. Exemplary enzymes can be found in Fusobacterium nucleatum (Barker et al., J. Bacteriol. 152(1):201-7 (1982)), Clostridium SB4 (Barker et al., J. Biol. Chem. 253(4):1219-25 (1978)), and Clostridium acetobutylicum (Wiesenbom et al., Appl. Environ. Microbiol. 55(2):323-9 (1989)). Although specific gene sequences were not provided for butyryl-CoA:acetoacetate CoA-transferase in these references, the genes FN0272 and FN0273 have been annotated as a butyrate-acetoacetate CoA-transferase (Kapatral et al., J. Bact. 184(7) 2005-2018 (2002)). Homologs in Fusobacterium nucleatum such as FN1857 and FN1856 also likely have the desired acetoacetyl-CoA transferase activity. FN1857 and FN1856 are located adjacent to many other genes involved in lysine fermentation and are thus very likely to encode an acetoacetate:butyrate CoA transferase (Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197 (2007)). Additional candidates from Porphyrmonas gingivalis and Thermoanaerobacter tengcongensis can be identified in a similar fashion (Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197 (2007)). Information related to these proteins and genes is shown below:










TABLE 19





Protein
GENBANK ID
GI NUMBER
ORGANISM







Cat1
P38946.1
729048
Clostridium kluyveri


Cat2
P38942.2
1705614
Clostridium kluyveri


Cat3
EDK35586.1
146349050
Clostridium kluyveri


TVAG_395550
XP_001330176
123975034
Trichomonas vaginalis G3


Tb11.02.0290
XP_828352
71754875
Trypanosoma brucei


FN0272
NP_603179.1
19703617
Fusobacterium nucleatum


FN0273
NP_603180.1
19703618
Fusobacterium nucleatum


FN1857
NP_602657.1
19705162
Fusobacterium nucleatum


FN1856
NP_602656.1
19705161
Fusobacterium nucleatum


PG1066
NP_905281.1
34540802
Porphyromonas gingivalis W83


PG1075
NP_905290.1
34540811
Porphyromonas gingivalis W83


TTE0720
NP_622378.1
20807207
Thermoanaerobacter tengcongensis MB4


TTE0721
NP_622379.1
20807208
Thermoanaerobacter tengcongensis MB4









Acetoacetyl-CoA can be hydrolyzed to acetoacetate by acetoacetyl-CoA hydrolase. Similarly, 4-hydroxybutyryl-CoA can be hydrolyzed to 4-hydroxybutyate by 4-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, J. Biol. Chem. 258(8):4725-4733 (1983)). Additionally, an enzyme from Rattus norvegicus brain (Robinson et al., Biochem. Biophys. Res. Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The acot12 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)). Additionally, 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 acetoacetyl-CoA transferases and 4-hydroxybutyryl-CoA transferases can also be used as hydrolases with certain mutations to change their function. The acetyl-CoA hydrolase, ACH1, from S. cerevisiae represents another candidate hydrolase (Buu et al., J. Biol. Chem. 278:17203-17209 (2003)). Information related to these proteins and genes is shown below:










TABLE 20





Protein
GENBANK ID
GI NUMBER
ORGANISM







Acot12
NP_570103.1
18543355
Rattus norvegicus


GctA
CAA57199
559392
Acidaminococcus fermentans


GctB
CAA57200
559393
Acidaminococcus fermentans


ACH1
NP_009538
6319456
Saccharomyces cerevisiae









Another candidate hydrolase is the human dicarboxylic acid thioesterase, acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J. Biol. Chem. 280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which can also hydrolyze a broad range of CoA thioesters (Naggert et al., J. Biol. Chem. 266:11044-11050 (1991)) including 3-hydroxybutyryl-CoA (Tseng et al., Appl. Environ. Microbiol. 75(10):3137-3145 (2009)). A similar enzyme has also been characterized in the rat liver (Deana, Biochem. Int. 26:767-773 (1992)). Other potential E. coli thioester hydrolases include the gene products of tesA (Bonner and Bloch, J. Biol. Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol. Rev. 29:263-279 (2005); Zhuang et al., FEBS Lett. 516:161-163 (2002)), paaI (Song et al., J. Biol. Chem. 281:11028-11038 (2006)), and ybdB (Leduc et al., J. Bacteriol. 189:7112-7126 (2007)). Information related to these proteins and genes is shown below:










TABLE 21





Protein
GENBANK ID
GI NUMBER
ORGANISM







Acot8
CAA15502
3191970
Homo sapiens


TesB
NP_414986
16128437
Escherichia coli


Acot8
NP_570112
51036669
Rattus norvegicus


TesA
NP_415027
16128478
Escherichia coli


YbgC
NP_415264
16128711
Escherichia coli


PaaI
NP_415914
16129357
Escherichia coli


YbdB
NP_415129
16128580
Escherichia coli









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., J. 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:










TABLE 22





Protein
GENBANK ID
GI NUMBER
ORGANISM







Hibch
Q5XIE6.2
146324906
Rattus norvegicus


Hibch
Q6NVY1.2
146324905
Homo sapiens


Hibch
P28817.2
2506374
Saccharomyces cerevisiae


BC_2292
AP09256
29895975
Bacillus cereus





ATCC 14579









The hydrolysis of acetoacetyl-CoA or 4-hydroxybutyryl-CoA can alternatively be carried out by a single enzyme or enzyme complex that exhibits acetoacetyl-CoA or 4-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. For example, the product of the LSC1 and LSC2 genes of S. cerevisiae and the sucC and sucD genes of E. coli naturally form a succinyl-CoA synthetase complex that catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Gruys et al., U.S. Pat. No. 5,958,745, filed Sep. 28, 1999). Information related to these proteins and genes is shown below:










TABLE 23





Protein
GENBANK ID
GI NUMBER
ORGANISM







SucC
NP_415256.1
16128703
Escherichia coli


SucD
AAC73823.1
1786949
Escherichia coli


LSC1
NP_014785
6324716
Saccharomyces cerevisiae


LSC2
NP_011760
6321683
Saccharomyces cerevisiae









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-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)), and the 6-carboxyhexanoate-CoA ligase from Bacilis subtilis (Bower et al., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidate enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)), which naturally catalyze the ATP-dependant conversion of acetoacetate into acetoacetyl-CoA. 4-Hydroxybutyryl-CoA synthetase activity has been demonstrated in Metallosphaera sedula (Berg et al., Science 318:1782-1786 (2007)). This function has been tentatively assigned to the Msed—1422 gene. Information related to these proteins and genes is shown below:










TABLE 24







GI



Protein
GENBANK ID
NUMBER
ORGANISM


















Phl
CAJ15517.1
77019264
Penicillium chrysogenum


PhlB
ABS19624.1
152002983
Penicillium chrysogenum


PaaF
AAC24333.2
22711873
Pseudomonas putida


BioW
NP_390902.2
50812281
Bacillus subtilis


AACS
NP_084486.1
21313520
Mus musculus


AACS
NP_076417.2
31982927
Homo sapiens


Msed_1422
YP_001191504
146304188
Metallosphaera sedula









ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another candidate enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP. Several enzymes with broad substrate specificities have been described in the literature. ACD I from Archaeoglobus fulgidus, encoded by AF 1211, was shown to operate on a variety of linear and branched-chain substrates including acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate, indoleacetate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). The enzyme from 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:










TABLE 25





Protein
GENBANK ID
GI NUMBER
ORGANISM







AF1211
NP_070039.1
11498810
Archaeoglobus fulgidus





DSM 4304


Scs
YP_135572.1
55377722
Haloarcula marismortui





ATCC 43049


PAE3250
NP_560604.1
18313937
Pyrobaculum aerophilum





str. IM2









An alternative method for removing the CoA moiety from acetoacetyl-CoA or 4-hydroxybutyryl-CoA is to apply a pair of enzymes such as a phosphate-transferring acyltransferase and a kinase to impart acetoacetyl-CoA or 4-hydroxybutyryl-CoA synthetase activity. Exemplary names for these enzymes include phosphotrans-4-hydroxybutyrylase/4-hydroxybutyrate kinase, which can remove the CoA moiety from 4-hydroxybutyryl-CoA, and phosphotransacetoacetylase/acetoacetate kinase which can remove the CoA moiety from acetoacetyl-CoA. This general activity enables the net hydrolysis of the CoA-ester of either molecule with the simultaneous generation of ATP. For example, the butyrate kinase (buk)/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)). Specifically, 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. 33-38 (2000). Additional ptb genes can be found in butyrate-producing bacterium L2-50 (Louis et al. J. Bacteriol. 186:2099-2106 (2004)) and Bacillus megaterium (Vazquez et al. Curr. Microbiol 42:345-349 (2001)). 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:










TABLE 26





Protein
GENBANK ID
GI NUMBER
ORGANISM







Pta
NP_416800.1
16130232
Escherichia coli


Ptb
NP_349676
15896327
Clostridium acetobutylicum


Ptb
AAR19757.1
38425288
butyrate-producing bacterium





L2-50


Ptb
CAC07932.1
10046659
Bacillus megaterium









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 buk1 and buk2 ((Walter et al. Gene 134(1):107-111 (1993); Huang et al. J. Mol. Microbiol. Biotechnol. 2(1):33-38 (2000)), and the E. coli gamma-glutamyl kinase, encoded by proB (Smith et al. J. Bacteriol. 157:545-551 (1984)). These enzymes phosphorylate acetate, butyrate, and glutamate, respectively. The ackA gene product from E. coli also phosphorylates propionate (Hesslinger et al. Mol. Microbiol. 27:477-492 (1998)). Information related to these proteins and genes is shown below:










TABLE 27





Protein
GENBANK ID
GI NUMBER
ORGANISM







AckA
NP_416799.1
16130231
Escherichia coli


Buk1
NP_349675
15896326
Clostridium acetobutylicum


Buk2
Q97II1
20137415
Clostridium acetobutylicum


ProB
NP_414777.1
16128228
Escherichia coli









Acetoacetate decarboxylase converts acetoacetate into carbon dioxide and acetone. Exemplary acetoacetate decarboxylase enzymes are encoded by the gene products of adc from C. acetobutylicum (Petersen and Bennett, Appl Environ. Microbiol 56:3491-3498 (1990)) and adc from Clostridium saccharoperbutylacetonicum (Kosaka, et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)). The enzyme from C. beijerinkii can be inferred from sequence similarity. These proteins are identified below in Table 28.










TABLE 28





Protein
GenBank ID
GI Number
Organism


















Adc
NP_149328.1
15004868
Clostridium acetobutylicum


Adc
AAP42566.1
31075386
Clostridium





saccharoperbutylacetonicum


Adc
YP_001310906.1
150018652
Clostridium beijerinckii









The final step in the isopropanol synthesis pathway involves the reduction of acetone to isopropanol. Exemplary alcohol dehydrogenase enzymes capable of this transformation include adh from C. beijerinckii (Hanai et al., Appl. Environ. Microbiol. 73:7814-7818 (2007); Jojima et al., Appl. Microbiol. Biotechnol. 77:1219-1224 (2008)) and adh from Thermoanaerobacter brockii (Hanai et al., Appl. Environ. Microbiol. 73:7814-7818 (2007); Peretz et al., Anaerobe 3:259-270 (1997)). Additional characterized enzymes include alcohol dehydrogenases from Ralstonia eutropha (formerly Alcaligenes eutrophus) (Steinbuchel and Schlegel et al., Eur. J. Biochem. 141:555-564 (1984)) and Phytomonas species (Uttaro and Opperdoes et al., Mol. Biochem. Parasitol. 85:213-219 (1997)).










TABLE 29





Protein
GenBank ID
GI Number
Organism


















Adh
P14941.1
113443
Thermoanaerobobacter brockii


Adh
AAA23199.2
60592974
Clostridium beijerinckii


Adh
YP_299391.1
73539024
Ralstonia eutropha


iPDH
AAP39869.1
31322946
Phtomonas sp.









Exemplary 3-hydroxyacyl dehydrogenases which convert acetoacetyl-CoA to 3-hydroxybutyryl-CoA include hbd from C. acetobutylicum (Boynton et al., J. Bacteriol. 178:3015-3024 (1996)), hbd from C. beijerinckii (Colby and Chen et al., Appl. Environ. Microbiol. 58:3297-3302 (1992)), and a number of similar enzymes from Metallosphaera sedula (Berg et al., Science 318:1782-1786 (2007)).










TABLE 29





Protein
GenBank ID
GI Number
Organism


















hbd
NP_349314.1
15895965
Clostridium





acetobutylicum


hbd
AAM14586.1
20162442
Clostridium beijerinckii


Msed_1423
YP_001191505
146304189
Metallosphaera sedula


Msed_0399
YP_001190500
146303184
Metallosphaera sedula


Msed_0389
YP_001190490
146303174
Metallosphaera sedula


Msed_1993
YP_001192057
146304741
Metallosphaera sedula









The gene product of crt from C. acetobutylicum catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA (Atsumi et al., Metab. Eng. (2007); Boynton et al., J. Bacteriol. 178:3015-3024 (1996)). Further, enoyl-CoA hydratases are reversible enzymes and thus suitable candidates for catalyzing the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA. The enoyl-CoA hydratases, phaA and phaB, of P. putida are believed to carry out the hydroxylation of double bonds during phenylacetate catabolism (Olivera et al., Proc. Nat. Acad. Sci. U.S.A. 95:6419-6424 (1998)). The paaA and paaB from P. fluorescens catalyze analogous transformations (Olivera et al., Proc. Nat. Acad. Sci. U.S.A. 95:6419-6424 (1998)). Lastly, a number of Escherichia coli genes have been shown to demonstrate enoyl-CoA hydratase functionality including maoC, paaF, and paaG (Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003); Park and Lee, J. Bacteriol. 185:5391-5397 (2003); Park and Lee, Appl. Biochem. Biotechnol 113-116:335-346 (2004); Park and Yup, Biotechnol. Bioeng. 86:681-686 (2004)).










TABLE 30





Protein
GenBank ID
GI Number
Organism


















crt
NP_349318.1
15895969
Clostridium acetobutylicum


paaA
NP_745427.1
26990002
Pseudomonas putida


paaB
NP_745426.1
26990001
Pseudomonas putida


phaA
ABF82233.1
106636093
Pseudomonas fluorescens


phaB
ABF82234.1
106636094
Pseudomonas fluorescens


maoC
NP_415905.1
16129348
Escherichia coli


paaF
NP_415911.1
16129354
Escherichia coli


paaG
NP_415912.1
16129355
Escherichia coli









Several enzymes that naturally catalyze the reverse reaction (i.e., the dehydration of 4-hydroxybutyryl-CoA to crotonoyl-CoA) in vivo have been identified in numerous species. This transformation is used for 4-aminobutyrate fermentation by Clostridium aminobutyricum (Scherf and Buckel, Eur. J. Biochem. 215:421-429 (1993)) and succinate-ethanol fermentation by Clostridium kluyveri (Scherf et al., Arch. Microbiol. 161:239-245 (1994)). The transformation is also a step in Archaea, for example, Metallosphaera sedula, as part of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway (Berg et al., Science 318:1782-1786 (2007)). This pathway uses the hydration of crotonoyl-CoA to form 4-hydroxybutyryl-CoA. The reversibility of 4-hydroxybutyryl-CoA dehydratase is well-documented (Friedrich et al., Angew. Chem. Int. Ed. Engl. 47:3254-3257 (2008); Muh et al., Eur. J. Biochem. 248:380-384 (1997); Muh et al., Biochemistry 35:11710-11718 (1996)) and the equilibrium constant has been reported to be about 4 on the side of crotonoyl-CoA (Scherf and Buckel, Eur. J. Biochem. 215:421-429 (1993)). This indicates that the downstream 4-hydroxybutyryl-CoA dehydrogenase keeps the 4-hydroxybutyryl-CoA concentration low so as to not create a thermodynamic bottleneck at crotonyl-CoA.










TABLE 31





Protein
GenBank ID
GI Number
Organism


















AbID
CAB60035
70910046
Clostridium





aminobutyricum


AbfD
YP_001396399
153955634
Clostridium kluyveri


Msed_1321
YP_001191403
146304087
Metallosphaera sedula


Msed_1220
YP_001191305
146303989
Metallosphaera sedula









Alcohol-forming 4-hydroxybutyryl-CoA reductase enzymes catalyze the 2 reduction steps required to form 1,4-butanediol from 4-hydroxybutyryl-CoA. Exemplary 2-step oxidoreductases that convert an acyl-CoA to alcohol include those that transform substrates such as acetyl-CoA to ethanol (e.g., adhE from E. coli (Kessler et al., FEBS. Lett. 281:59-63 (1991)) and butyryl-CoA to butanol (e.g. adhE2 from C. acetobutylicum (Fontaine et al., J. Bacteriol. 184:821-830 (2002)). The adhE2 enzyme from C. acetobutylicum was specifically shown in ref. Burk et al., WO/2008/115840 (2008). to produce BDO from 4-hydroxybutyryl-CoA. In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenterokles has been shown to oxide the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol. Lett. 27:505-510 (2005)).










TABLE 32





Protein
GenBank ID
GI Number
Organism







adhE
NP_415757.1
16129202
Escherichia coli


adhE2
AAK09379.1
12958626
Clostridium acetobutylicum


adhE
AAV66076.1
55818563
Leuconostoc mesenteroides









Another exemplary enzyme can convert malonyl-CoA to 3-HP. An NADPH-dependent enzyme with this activity has characterized in Chloroflexus aurantiacus where it participates in the 3-hydroxypropionate cycle (Hugler et al., J. Bacteriol. 184:2404-2410 (2000); Strauss and Fuchs, Eur. J. Biochem. 215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly substrate-specific and shows little sequence similarity to other known oxidoreductases (Hugler et al., J. Bacteriol. 184:2404-2410 (2002)). No enzymes in other organisms have been shown to catalyze this specific reaction; however there is bioinformatic evidence that other organisms may have similar pathways (Klatt et al., Environ. Microbiol. 9:2067-2078 (2007)). Enzyme candidates in other organisms including Roseiflexus castenholzii, Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity.










TABLE 33





Protein
GenBank ID
GI Number
Organism


















mcr
AAS20429.1
42561982
Chloroflexus





aurantiacus


Rcas_2929
YP_001433009.1
156742880
Roseiflexus





castenholzii


NAP1_02720
ZP_01039179.1
85708113
Erythrobacter sp.





NAP1


MGP2080_00535
ZP_01626393.1
119504313
marine gamma





proteobacterium





HTCC2080









An alternative route to BDO from 4-hydroxybutyryl-CoA involves first reducing this compound to 4-hydroxybutanal. Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA to its corresponding aldehyde. Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acr1 encoding a fatty acyl-CoA reductase (Reiser and Somerville, J. Bacteriol. 179:2969-2975 (1997)), the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)), and a CoA- and NADP-dependent succinate semialdehyde dehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). SucD of P. gingivalis is another succinate semialdehyde dehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). These succinate semialdehyde dehydrogenases were specifically shown in ref. Burk et al., WO/2008/115840 (2008) to convert 4-hydroxybutyryl-CoA to 4-hydroxybutanal as part of a pathway to produce 1,4-butanediol. The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another capable enzyme as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., J. Bacteriol. 175:377-385 (1993)).










TABLE 34





Protein
GenBank ID
GI Number
Organism


















acr1
YP_047869.1
50086359
Acinetobacter calcoaceticus


acr1
AAC45217
1684886
Acinetobacter baylyi


acr1
BAB85476.1
18857901
Acinetobacter sp. Strain M-1


sucD
P38947.1
172046062
Clostridium kluyveri


sucD
NP_904963.1
34540484
Porphyromonas gingivalis


bphG
BAA03892.1
425213
Pseudomonas sp









An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is 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 theimoacidophilic archael bacteria (Berg et al., Science 318:1782-1786 (2007); Thauer, R. K. Science 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Hugler et al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed—0709 in Metallosphaera sedula (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Berg et al., Science 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius. Yet another gene for CoA-acylating aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). These proteins are identified below in Table 35.










TABLE 35





Protein
GenBank ID
GI Number
Organism


















Msed_0709
YP_001190808.1
146303492
Metallosphaera sedula


Mcr
NP_378167.1
15922498
Sulfolobus tokodaii


asd-2
NP_343563.1
15898958
Sulfolobus solfataricus


Saci_2370
YP_256941.1
70608071
Sulfolobus





acidocaldarius


Ald
AAT66436
49473535
Clostridium beijerinckii


eutE
AAA80209
687645
Salmonella typhimurium


eutE
P77445
2498347
Escherichia coli









4-Hydroxybutyryl-CoA can also be converted to 4-hydroxybutanal in several enzymatic steps, though the intermediate 4-hydroxybutyrate. First, 4-hydroxybutyryl-CoA can be converted to 4-hydroxybutyrate by a CoA transferase, hydrolase or synthetase. Alternately, 4-hydroxybutyryl-CoA can be converted to 4-hydroxybutyrate via a phosphonated intermediate by enzymes with phosphotrans-4-hydroxybutyrylase and 4-hydroxybutyrate kinase. Exemplary candidates for these enzymes are described above.
Subsequent conversion of 4-hydroxybutyrate to 4-hydroxybutanal is catalyzed by an aryl-aldehyde dehydrogenase, or equivalently a carboxylic acid reductase. Such an enzyme is found in Nocardia iowensis. Carboxylic acid reductase catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)) and is capable of catalyzing the conversion of 4-hydroxybutyrate to 4-hydroxybutanal. This enzyme, encoded by car, was cloned and functionally expressed in E. coli (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)). Expression of the 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. 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, Fla. (2006)).















GenBank



Gene name
GI No.
Accession No.
Organism


















Car
40796035
AAR91681.1
Nocardia iowensis





(sp. NRRL 5646)


Npt
114848891
ABI83656.1
Nocardia iowensis





(sp. NRRL 5646)









Additional car and npt genes can be identified based on sequence homology.















GenBank



Gene name
GI No.
Accession No.
Organism







fadD9
121638475
YP_978699.1
Mycobacterium bovis BCG


BCG_2812c
121638674
YP_978898.1
Mycobacterium bovis BCG


nfa20150
54023983
YP_118225.1
Nocardia farcinica IFM





10152


nfa40540
54026024
YP_120266.1
Nocardia farcinica IFM





10152


SGR_6790
182440583
YP_001828302.1
Streptomyces griseus subsp.





griseus NBRC 13350


SGR_665
182434458
YP_001822177.1
Streptomyces griseus subsp.





griseus NBRC 13350


MSMEG_2956
YP_887275.1
YP_887275.1
Mycobacterium smegmatis





MC2 155


MSMEG_5739
YP_889972.1
118469671
Mycobacterium smegmatis





MC2 155


MSMEG_2648
YP_886985.1
118471293
Mycobacterium smegmatis





MC2 155


MAP1040c
NP_959974.1
41407138
Mycobacterium avium subsp.





paratuberculosis K-10


MAP2899c
NP_961833.1
41408997
Mycobacterium avium subsp.





paratuberculosis K-10


MMAR_2117
YP_001850422.1
183982131
Mycobacterium marinum M


MMAR_2936
YP_001851230.1
183982939
Mycobacterium marinum M


MMAR_1916
YP_001850220.1
183981929
Mycobacterium marinum M


TpauDRAFT_33060
ZP_04027864.1
227980601
Tsukamurella paurometabola





DSM 20162


TpauDRAFT_20920
ZP_04026660.1
ZP_04026660.1
Tsukamurella paurometabola





DSM 20162


CPCC7001_1320
ZP_05045132.1
254431429
Cyanobium PCC7001


DDBDRAFT_0187729
XP_636931.1
66806417
Dictyostelium discoideum





AX4









An additional enzyme candidate found in Streptomyces griseus is encoded by the griC and griD genes. This enzyme is believed to convert 3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde as deletion of either griC or griD led to accumulation of extracellular 3-acetylamino-4-hydroxybenzoic acid, a shunt product of 3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot. 60(6):380-387 (2007)). Co-expression of griC and griD with SGR—665, an enzyme similar in sequence to the Nocardia iowensis npt, can be beneficial.













Gene

GENBANK



name
GI No.
Accession No.
Organism







griC
182438036
YP_001825755.1
Streptomyces griseus subsp.





griseus NBRC 13350


Grid
182438037
YP_001825756.1
Streptomyces griseus subsp.





griseus NBRC 13350









An enzyme with similar characteristics, 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 NAD(P)H to yield the aldehyde and AMP. Like CAR, 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. coli (Guo et al., Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate, but did not react with adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J. Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTase has not been identified to date.













Gene

GENBANK



name
GI No.
Accession No.
Organism







LYS2
171867
AAA34747.1
Saccharomyces cerevisiae


LYS5
1708896
P50113.1
Saccharomyces cerevisiae


LYS2
2853226
AAC02241.1
Candida albicans


LYS5
28136195
AAO26020.1
Candida albicans


Lys1p
13124791
P40976.3
Schizosaccharomyces pombe


Lys7p
1723561
Q10474.1
Schizosaccharomyces pombe


Lys2
3282044
CAA74300.1
Penicillium chrysogenum









Enzymes exhibiting 1,4-butanediol dehydrogenase activity are capable of forming 1,4-butanediol from 4-hydroxybutanal. Exemplary genes encoding enzymes that catalyze the conversion of an aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalently aldehyde reductase) include alrA encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol. 66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae Atsumi et al. Nature 451:86-89 (2008)), yahD from E. coli which has preference for molecules longer than C(3) (Sulzenbacher et al., J. Mol. Biol. 342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum which converts butyryaldehyde into butanol (Walter et al., J. Bacteriol. 174:7149-7158 (1992)). ADH1 from Zymomonas mobilis has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita, Appl. Microbiol. Biotechnol. 22:249-254 (1985)).










TABLE 36





Protein
GenBank ID
GI Number
Organism







alrA
BAB12273.1
9967138
Acinetobacter sp. Strain M-1


ADH2
NP_014032.1
6323961
Saccharomyces cerevisiae


yqhD
NP_417484.1
16130909
Escherichia coli


bdh I
NP_349892.1
15896543
Clostridium acetobutylicum


bdh II
NP_349891.1
15896542
Clostridium acetobutylicum


adhA
YP_162971.1
56552132
Zymomonas mobilis









Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC 1.1.1.61) also fall into this category. Such enzymes have been characterized in Ralstonia eutropha (Bravo et al., J. Forensic Sci. 49:379-387 (2004)), Clostridium kluyveri (Wolff and Kenealy, Protein Expr. Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al., J. Biol. Chem. 278:41552-41556 (2003)).










TABLE 37





Protein
GenBank ID
GI Number
Organism







4hbd
YP_726053.1
113867564
Ralstonia eutropha H16


4hbd
L21902.1
146348486
Clostridium kluyveri





DSM 555


4hbd
Q94B07
75249805
Arabidopsis thaliana









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 use Coenzyme B12 (cobalamin) as a cofactor. In Morella thermoacetica, a cascade of methyltransferase proteins mediate incorporation of methanol derived methyl groups into the acetyl-CoA synthase pathway. Recent work (Das et al., Proteins 67:167-176 (2007)) indicates that MtaABC are encoded by Moth—1208-09 and Moth—2346. These genes are cloned via proof-reading PCR and linked together for expression in a high-copy number vector such as pZE22-S under control of the repressible PA1-lacO1 promoter (Lutz and Bujard, Nucleic Acids Res. 25:1203-1210 (1997)). Cloned genes are verified by PCR and or restriction enzyme mapping to demonstrate construction and insertion of the 3-gene set into the expression vector. DNA sequencing of the presumptive clones is carried out to confirm the expected sequences of each gene. Once confirmed, the final construct is expressed in E. coli K-12 (MG1655) cells by addition of IPTG inducer between 0.05 and 1 mM final concentration. Expression of the cloned genes is monitored using SDS-PAGE of whole cell extracts. To optimize levels of soluble vs. pellet (potentially inclusion body origin) protein, the affect of titration of the promoter on these levels can be examined. If no acceptable expression is obtained, higher or lower copy number vectors or variants in promoter strength are tested.
To determine if expression of the 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, J. Bacteriol. 183:3276-3281 (2001)) or for Methanosarcina barkeri methanol methyltransferase (Sauer et al., Eur. J. Biochem. 243:670-677 (1997); Tallant and Krzycki, J. Bacteriol. 178:1295-1301 (1996); Tallant and Krzycki, J. Bacteriol. 179:6902-6911 (1997); Tallant et al., J. Biol. Chem. 276:4485-4493 (2001)). For a positive control, M. thermoacetica cells are cultured in parallel and assayed anaerobically to confirm endogenous methyltransferase activity. Demonstration of dependence on exogenously added coenzyme B12 confirms methanol: corrinoid methyltransferase activity in E. coli. 
Once 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 required in single-copy, or enables 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:e16 (1999); Muyrers et al. Nucleic Acids Res. 27:1555-1557 (1999); Zhang et al., 1998 Nat. Genet. 20:123-128 (1998)). A potential issue with RecET-based integration of a cassette and removal of a FRT or loxP-bounded selectable marker by FLP or Cre is the production of a recombination scar at each integration site. While problems caused by this can be minimized by a number of methods, other means that do not leave genomic scars are available. The standard alternative, is to introduce the desired genes using integrative ‘suicide’ plasmids coupled to counter-selection such as that allowed by the Bacillus sacB gene (Link et al., J. Bacteriol. 179:6228-6237 (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).
Using standard PCR methods, entire ACS/CODH operons are assembled into low or medium copy number vectors such as pZA33-S(P15A-based) or pZS13-S (pSC101-based). As described for the methyltransferase genes, 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 B12 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. 22:1287-1295 (1994); Ringquist et al., Mol. Microbiol. 6:1219-1229 (1992)). However, 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 CO, 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 CH3-THF is added to the medium.
The ACS/CODH genes are cloned and expressed in cells also expressing the methanol-methyltransferase system. This is 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 will be added to the fermentation broth to serve as an electron acceptor and initiator of growth. Anaerobic growth of E. coli on fatty acids, which are ultimately metabolized to acetyl-CoA, has been demonstrated in the presence of nitrate (Campbell et al., Mol. Microbiol. 47:793-805 (2003)). Oxygen can also be provided as long as its intracellular levels are maintained below any inhibition threshold of the engineered enzymes. ‘Synthetic syngas’ of a composition suitable for these experiments is employed along with methanol. 13C-labeled methanol or 13C-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).
The pyruvate ferredoxin oxidoreductase genes from M. thermoacetica, D. africanus, and E. coli are cloned and expressed in strains exhibiting MTR and 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 will be performed using low or single-copy plasmid vectors or single-copy chromosomal integrations. Activity assays described in ref. (Furdui and Ragsdale, J. Biol. Chem. 275:28494-28499 (2000)) will 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 is 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 are cloned and expressed in strains exhibiting MTR, ACS/CODH, and PFOR activities. Conditions, promoters, etc., are described above.
The normative genes needed for isopropanol 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. At this point, these (CODH/ACS, etc.) genes are integrated into the genome and expressed from promoters that can be used constitutively or with inducers (i.e., PA1-lacO1 is inducible in cells containing lacI or is otherwise constitutive). Once expression and yields of isopropanol are optimized, the base strain is further modified by integration of a single copy of these genes at a neutral locus. Given the relatively limited number of genes (at minimum, 3, and at most, 6), Applicants construct an artificial operon encoding the required genes. This operon is introduced using integrative plasmids and is coupled to counter-selection methods such as that allowed by the Bacillus sacB gene (Link et al., J. Bacteriol. 179:6228-6237 (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.
To over express any native genes, for example, 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 isopropanol production, RecET-based methods are applied to integrate a stronger upstream promoter. In the case of atoB, this gene is the last in an operon and the next gene downstream (yfaP) 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). Once the correct clone is obtained and validated, using qRT-PCR, FLP or Cre expression is used to select for removal of the FRT- or loxP-bounded marker.
The normative genes needed for 4-hydroxybutyrate 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. At this point, these (CODH/ACS, etc.) genes are integrated into the genome and expressed from promoters that can be used constitutively or with inducers (i.e., PA1-lacO1 is inducible in cells containing lacI or is otherwise constitutive). Once expression and yields of 4-hydroxybutyrate are optimized, the base strain is further modified by integration of a single copy of these genes at a neutral locus. Given the relatively limited number of genes (at minimum, 5, and at most, 6), an artificial operon encoding the required genes can be constructed. This operon is introduced using integrative plasmids and is coupled to counter-selection methods such as that allowed by the Bacillus sacB gene (Link et al., J. Bacteriol. 179:6228-6237 (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 normative genes needed for 1,4-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. At this point, these (CODH/ACS, etc.) genes are integrated into the genome and expressed from promoters that can be used constitutively or with inducers (i.e., PA1-lacO1 is inducible in cells containing lad or is otherwise constitutive). Once expression and yields of 1,4-butanediol are optimized, the base strain is further modified by integration of a single copy of these genes at a neutral locus. Given the relatively limited number of genes (at minimum, 5, and at most, 6), an artificial operon encoding the required genes can be constructed. This operon is introduced using integrative plasmids and is coupled to counter-selection methods such as that allowed by the Bacillus sacB gene (Link et al., J. Bacteriol. 179:6228-6237 (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.
Engineering the capability to convert synthesis gas 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. 43:701-715 (1942)). The Wood-Ljungdahl pathway comprises of two branches: the Eastern (or methyl) branch that enables the conversion of CO2 to methyltetrahydrofolate (Me-THF) and the Western (or carbonyl) branch that enables the conversion of methyl-THF, CO, and Coenzyme-A into acetyl-CoA (FIG. 3). In some embodiments, the present invention provides 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, CO2, and/or H2 into acetyl-CoA, cell mass, and products.
Another organism of the present invention contains three capabilities which are depicted in FIG. 3A: 1) a functional methyl branch of the Wood-Ljungdahl pathway which enables the conversion of THF and CO2 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 isopropanol from acetyl-CoA. The fifth organism described in this invention, depicted in FIG. 3B, contains a functional methyl branch of the Wood-Ljungdahl pathway, the ability to synthesize acetyl-CoA, and the ability to synthesize 4-hydroxybutyrate from acetyl-CoA. The sixth organism described in this invention, depicted in FIG. 3C, contains a functional methyl branch of the Wood-Ljungdahl pathway, the ability to synthesize acetyl-CoA, and the ability to synthesize 1,4-butanediol from acetyl-CoA.
These three organisms are able to ‘fix’ carbon from exogenous CO and/or CO2 to synthesize acetyl-CoA, cell mass, and products. A host organism engineered with these capabilities that also naturally possesses the capability for anapleurosis (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 into the strain to enable synthesis of biomass precursors in the absence of an external electron acceptor. A further characteristic of the engineered organism is the capability for extracting reducing equivalents from molecular hydrogen. This enables a high yield of reduced products such as ethanol, butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, adipic acid, 6-aminocaproic acid, hexamethylenediamine, 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid, methacrylic acid, and acrylic acid.
The organisms provided herein can produce acetyl-CoA, cell mass, and targeted chemicals, more specifically isopropanol, 4-hydroxybutyrate, and 1,4-butanediol, from: 1) CO, 2) CO2 and H2, 3) CO, CO2, and H2, 4) synthesis gas comprising CO and H2, 5) synthesis gas comprising CO, CO2, and H2, 6) one or more carbohydrates, 7) methanol and one or more carbohyrates, and 8) methanol. Exemplary carbohydrates include but are not limited to glucose, sucrose, xylose, arabinose and glycerol.
Successfully engineering any of 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. Below are described enzymes that catalyze steps 1 through 5 of the pathways depicted in FIGS. 3A through 3C. These enzymes are required to enable the conversion of synthesis gas to acetyl-CoA. Enzymes for steps 6 through 17 in FIG. 3A and steps 6 through 20 in FIGS. 3B and 3C were described above. To engineer a production host for the utilization of syngas, one or more exogenous DNA sequence(s) encoding the requisite enzymes can be expressed in the microorganism.
Formate dehydrogenase is a two subunit selenocysteine-containing protein that catalyzes the incorporation of CO2 into formate in Moorella thermoacetica (Andreesen and Ljungdahl, J. Bacteriol. 116:867-873 (1973); Li et al., J. Bacteriol. 92:405-412 (1966); Yamamoto et al., J. Biol. Chem. 258:1826-1832 (1983). The loci, Moth—2312 is responsible for encoding the alpha subunit of formate dehydrogenase while the beta subunit is encoded by Moth 2314 (Pierce et al., Environ. Microbiol. (2008)). Another set of genes encoding formate dehydrogenase activity with a propensity for CO2 reduction is encoded by Sfum—2703 through Sfum—2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur. J. Biochem. 270:2476-2485 (2003)); Reda et al., Proc. Natl. Acad. Sci. U.S.A. 105:10654-10658 (2008)). A similar set of genes presumed to carry out the same function are encoded by CHY—0731, CHY—0732, and CHY—0733 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005)). Homologs are also found in C. carboxidivorans P7.










TABLE 38





Protein
GenBank ID
GI Number
Organism







Moth_2312
YP_431142
148283121
Moorella thermoacetica


Moth_2314
YP_431144
83591135
Moorella thermoacetica


Sfum_2703
YP_846816.1
116750129
Syntrophobacter fumaroxidans


Sfum_2704
YP_846817.1
116750130
Syntrophobacter fumaroxidans


Sfum_2705
YP_846818.1
116750131
Syntrophobacter fumaroxidans


Srum_2706
YP_846819.1
116750132
Syntrophobacter fumaroxidans


CHY_0731
YP_359585.1
78044572
Carboxydothermus hydrogenoformans


CHY_0732
YP_359586.1
78044500
Carboxydothermus hydrogenoformans


CHY_0733
YP_359587.1
78044647
Carboxydothermus hydrogenoformans


CcarbDRAFT_0901
ZP_05390901.1
255523938
Clostridium carboxidivorans P7


CcarbDRAFT_4380
ZP_05394380.1
255527512
Clostridium carboxidivorans P7









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 (Lovell et al., Arch. Microbiol. 149:280-285 (1988); Lovell et al., Biochemistry 29:5687-5694 (1990); O'brien et al., Experientia. Suppl. 26:249-262 (1976), FHS in Clostridium acidurici (Whitehead and Rabinowitz, J. Bacteriol. 167:205-209 (1986); Whitehead and Rabinowitz, J. Bacteriol. 170:3255-3261 (1988)), and CHY 2385 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005)). Homologs exist in C. carboxidivorans P7.










TABLE 39





Protein
GenBank ID
GI Number
Organism







Moth_0109
YP_428991.1
83588982
Moorella thermoacetica


CHY_2385
YP_361182.1
78045024
Carboxydothermus hydrogenoformans


FHS
P13419.1
120562
Clostridium acidurici


CcarbDRAFT_1913
ZP_05391913.1
255524966
Clostridium carboxidivorans P7


CcarbDRAFT_2946
ZP_05392946.1
255526022
Clostridium carboxidivorans P7









In M. thermoacetica, E. coli, and C. hydrogenoformans, methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolate dehydrogenase are carried out by the bi-functional gene products of Moth—1516, folD, and CHY—1878, respectively (D'Ari and Rabinowitz, J. Biol. Chem. 266:23953-23958 (1991); Pierce et al., Environ. Microbiol. (2008); Wu et al., PLoS Genet. 1:e65 (2005)). A homolog exists in C. carboxidivorans P7.










TABLE 40





Protein
GenBank ID
GI Number
Organism







Moth_1516
YP_430368.1
83590359
Moorella thermoacetica


folD
NP_415062.1
16128513
Escherichia coli


CHY_1878
YP_360698.1
78044829
Carboxydothermus hydrogenoformans


CcarbDRAFT_2948
ZP_05392948.1
255526024
Clostridium carboxidivorans P7









The final step of the methyl branch of the Wood-Ljungdahl pathway is catalyzed by methylenetetrahydrofolate reductase. In M. thermoacetica, this enzyme is oxygen-sensitive and contains an iron-sulfur cluster (Clark and Ljungdahl, J. Biol. Chem. 259:10845-10849 (1984)). This enzyme is encoded by metF in E. coli (Sheppard et al., J. Bacteriol. 181:718-725 (1999)) and CHY—1233 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (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.










TABLE 41





Protein
GenBank ID
GI Number
Organism







metF
NP_418376.1
16131779
Escherichia coli


CHY_1233
YP_360071.1
78044792
Carboxydothermus





hydrogenoformans









While E. coli naturally possesses the capability for some of the required transformations (i.e., methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate dehydrogenase, methylenetetrahydrofolate reductase), it is thought that the methyl branch enzymes from acetogens may have significantly higher (50-100×) specific activities than those from non-acetogens (Morton et al., Genetics and molecular biology of anaerobic bacteria, p. 389-406, Springer Verlag, New York (1992.)). The formate dehydrogenase also appears to be specialized for anaerobic conditions (Ljungdahl and Andreesen, FEBS Lett. 54:279-282 (1975)). Therefore, various non-native versions of each of these are expressed in the strain of E. coli capable of methanol and syngas utilization. Specifically, these genes are cloned and combined into an expression vector designed to express them as a set. Initially, a high or medium copy number vector is chosen (using ColE1 or P15A replicons). One promoter that can be used is a strongly constitutive promoter such as lambda pL or an IPTG-inducible version of this, pL-lacO (Lutz and Bujard, Nucleic Acids Res. 25:1203-1210 (1997)). To make an artificial operon, 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. Ultimately, the genes are integrated into the E. coli chromosome. Enzyme assays are performed as described in (Clark and Ljungdahl, J. Biol. Chem. 259:10845-10849 (1984); Clark and Ljungdahl, Meth. Enzymol. 122:392-399 (1986); D'Ari and Rabinowitz, J. Biol. Chem. 266:23953-23958 (1991); de Mata and Rabinowitz, J. Biol. Chem. 255:2569-2577 (1980); Ljungdahl and Andreesen, Meth. Enzymol. 53:360-372 (1978); Lovell et al., 1988 Arch. Microbiol 149:280-285 (1988); Yamamoto et al., J. Biol. Chem. 258:1826-1832 (1983)).
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 syngas consisting of CO, CO2, and/or H2, for incorporation into acetyl-CoA, cell mass, and isopropanol or 1,4-butanediol. Initial conditions employ strictly anaerobically grown cells provided with exogenous glucose as a carbon and energy source. Metabolizing glucose or other carbohydrates to acetyl-CoA provides one potential source of CO2 that can be fixed via the Wood-Ljungdahl pathway. Alternatively, or in addition to glucose, nitrate will be added to the fermentation broth to serve as an electron acceptor and initiator of growth. Anaerobic growth of E. coli on fatty acids, which are ultimately metabolized to acetyl-CoA, has been demonstrated in the presence of nitrate (Campbell et al., Mol. Microbiol. 47:793-805 (2003)). Oxygen can also be provided as long as its intracellular levels are maintained below any inhibition threshold of the engineered enzymes. ‘Synthetic syngas’ of a composition suitable for these experiments can also be employed. 13C-labeled CO and/or CO2 are provided to the cells and analytical mass spectrometry is employed to measure incorporation of the labeled carbon into acetate, isopropanol, 4-hydroxybutyrate, 1,4-butanediol, and cell mass (e.g., proteinogenic amino acids).
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. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, 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 isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathway can be expressed. For example, if 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. Alternatively, if 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 isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthesis. Thus, 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 isopropanol, 4-hydroxybutyrate, or 1,4-butanediol.
Depending on the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathways. For example, isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway, 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. For example, exogenous expression of all enzymes or proteins in a pathway for production of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol can be included.
Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway deficiencies of the selected host microbial organism. Therefore, 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, up to all nucleic acids encoding the enzymes or proteins constituting an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize isopropanol, 4-hydroxybutyrate, or 1,4-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 isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway precursors such as acetyl-CoA.
Generally, a host microbial organism is selected such that it produces the precursor of an isopropanol, 4-hydroxybutyrate, or 1,4-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. For example, 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. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway.
In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. In this specific embodiment it can be useful to increase the synthesis or accumulation of an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway product to, for example, drive isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway reactions toward isopropanol, 4-hydroxybutyrate, or 1,4-butanediol production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway enzymes or proteins. Over expression the enzyme or enzymes and/or protein or proteins of the isopropanol, 4-hydroxybutyrate, or 1,4-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 isopropanol, 4-hydroxybutyrate, or 1,4-butanediol, through overexpression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, that is, up to all nucleic acids encoding isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathway enzymes or proteins. In addition, 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 isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathway.
In particularly useful embodiments, 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. However, 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. Thus, 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. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.
It is understood that, in methods of the invention, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention. The nucleic acids can be introduced so as to confer, for example, an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathway onto the microbial organism. Alternatively, 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 isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic capability. For example, a non-naturally occurring microbial organism having an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins. Thus, it is understood that 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. Similarly, it is understood that 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 and so forth, 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. Similarly, 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.
In addition to the biosynthesis of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol as described herein, 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. For example, one alternative to produce isopropanol, 4-hydroxybutyrate, or 1,4-butanediol other than use of the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol producers is through addition of another microbial organism capable of converting an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway intermediate to isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. One such procedure includes, for example, the fermentation of a microbial organism that produces an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway intermediate. The isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway intermediate can then be used as a substrate for a second microbial organism that converts the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway intermediate to isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. The isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway intermediate can be added directly to another culture of the second organism or the original culture of the isopropanol, 4-hydroxybutyrate, or 1,4-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.
In other embodiments, 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, isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. In these embodiments, biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of isopropanol, 4-hydroxybutyrate, or 1,4-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. Alternatively, isopropanol, 4-hydroxybutyrate, or 1,4-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 an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol intermediate and the second microbial organism converts the intermediate to isopropanol, 4-hydroxybutyrate, or 1,4-butanediol.
Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring microbial organisms and methods of the invention together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce isopropanol, 4-hydroxybutyrate, or 1,4-butanediol.
Sources of encoding nucleic acids for an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations enabling biosynthesis of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.
In some instances, such as when an alternative isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathway exists in an unrelated species, isopropanol, 4-hydroxybutyrate, or 1,4-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. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize isopropanol, 4-hydroxybutyrate, or 1,4-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, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger and Pichia pastoris. Exemplary cyanobacteria include Acaryochloris marina MBIC11017, Anabaena sp. PCC 7120, Anabaena variabilis ATCC 29413, Agmenellum quadruplicatum, Chlorobium tepidum TLS, Cyanothece sp. ATCC 51142, Gloeobacter violaceus PCC 7421, Microcystis aeruginosa NIES-843, Nostoc punctiforme ATCC 29133, Prochlorococcus marinus MED4, Prochlorococcus marinus MIT9313, Prochlorococcus marinus SS120, Prochlorococcus marinus str. AS9601, Prochlorococcus marinus str. MIT 9211, Prochlorococcus marinus str. MIT 9215, Prochlorococcus marinus str. MIT 9301, Prochlorococcus marinus str. MIT 9303, Prochlorococcus marinus str. MIT 9312, Prochlorococcus marinus str. MIT 9515, Prochlorococcus marinus str. NATL1A, Prochlorococcus marinus str. NATL2A, Rhodopseudomonas palustris CGA009, Synechococcus elongatus PCC 6301, Synechococcus elongatus PCC 7942, Synechococcus sp. CC9311, Synechococcus sp. CC9605, Synechococcus sp. CC9902, Synechococcus sp. JA-2-3BVa(2-13), Synechococcus sp. JA-3-3Ab, Synechococcus sp. PCC 7002, Synechococcus sp. RCC307, Synechococcus sp. WH 7803, Synechococcus sp. WH8102, Synechocystis sp. PCC 6803, Thermosynechococcus elongatus BP-1, Trichodesmium erythraeum IMS101. Exemplary algae include Botryococcus braunii, Chlamydomonas reinhardii, Chlorella sp., Crypthecodinium cohnii, Cylindrotheca sp., Dunaliella primolecta, Isochrysis sp., Monallanthus salina, Nannochloris sp., Nannochloropsis sp., Neochloris oleoabundans, Nitzschia sp., Phaeodactylum tricornutum, Schizochytrium sp., and Tetraselmis sueica. 
E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce a desired product.
Methods for constructing and testing the expression levels of a non-naturally occurring isopropanol, 4-hydroxybutyrate, or 1,4-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, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).
Exogenous nucleic acid sequences involved in a pathway for production of isopropanol, 4-hydroxybutyrate, or 1,4-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, transduction, transfection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some 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. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, 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. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.
An expression vector or vectors can be constructed to include one or more isopropanol, 4-hydroxybutyrate, or 1,4-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. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.
The invention provides a method for producing isopropanol that includes culturing a non-naturally occurring microbial organism having an isopropanol pathway. The pathway includes at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol under conditions and for a sufficient period of time to produce isopropanol. The isopropanol pathway comprising an acetoacetyl-CoA thiolase, an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, a phosphotransacetoacetylase, an acetoacetate kinase, an acetoacetate decarboxylase, and an isopropanol dehydrogenase. An alternative isopropanol pathway comprises acetoacetyl-CoA thiolase, succinyl-CoA:3-ketoacid CoA transferase, acetoacetate decarboxylase, and an isopropanol dehydrogenase.
In embodiments where an organism has a methanol methyltransferase, culturing can be carried out utilizing a feedstock such as 1) methanol and CO, 2) methanol, CO2, and H2, 3) methanol, CO, CO2, and H2, 4) methanol and synthesis gas comprising CO and H2, 5) methanol and synthesis gas comprising CO, CO2, and H2, 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.
In embodiments where an organism has a formate dehydrogenase, a formyltetrahydrofolate synthetase, a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolate dehydrogenase, and a methylenetetrahydrofolate reductase, the organism can utilize a feedstock such as 1) CO, 2) CO2 and H2, 3) CO and CO2, 4) synthesis gas comprising CO and H2, 5) synthesis gas comprising CO, CO2, and H2, and 6) one or more carbohydrates.
Similarly, 4-hydroxybutyrate or 1,4-butanediol can also be produced by culturing the appropriate organisms as described herein above.
Suitable purification and/or assays to test for the production of isopropanol, 4-hydroxybutyrate, or 1,4-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 Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art (see, for example, WO/2008/115840 and Hanai et al., Appl. Environ. Microbiol. 73:7814-7818 (2007)).
The isopropanol, 4-hydroxybutyrate, or 1,4-butanediol can be separated from other components in the culture using a variety of methods well known in the art. Such 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. For example, the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol producers can be cultured for the biosynthetic production of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol.
For the production of isopropanol, 4-hydroxybutyrate, or 1,4-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. For strains where growth is not observed anaerobically, microaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in U.S. patent application Ser. No. 11/891,602, filed Aug. 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein.
If desired, 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.
In addition to renewable feedstocks such as those exemplified above, the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol microbial organisms of the invention also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.
Although organisms of the present invention are designed to utilize syngas and/or methanol as a growth source, they may also utilize, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol.
Accordingly, given the teachings and guidance provided herein, those skilled in the art will understand that 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, CO2, hydrogen, and the like. Such compounds include, for example, isopropanol, 4-hydroxybutyrate, or 1,4-butanediol and any of the intermediate metabolites in the isopropanol, 4-hydroxybutyrate, or 1,4-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 isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathways. Accordingly, the invention provides a non-naturally occurring microbial organism that produces and/or secretes isopropanol, 4-hydroxybutyrate, or 1,4-butanediol when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway when grown on a carbohydrate or other carbon source. The isopropanol, 4-hydroxybutyrate, or 1,4-butanediol producing microbial organisms of the invention can initiate synthesis from an intermediate, for example, acetyl-CoA.
The non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway enzyme or protein in sufficient amounts to produce isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol resulting in intracellular concentrations between about 0.1-2000 mM or more. Generally, the intracellular concentration of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol is between about 3-1800 mM, particularly between about 5-1700 mM and more particularly between about 8-1600 mM, including about 100 mM, 200 mM, 500 mM, 800 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.
In some embodiments, 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. patent application Ser. No. 11/891,602, filed Aug. 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. Under such anaerobic conditions, the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol producers can synthesize isopropanol, 4-hydroxybutyrate, or 1,4-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, isopropanol, 4-hydroxybutyrate, or 1,4-butanediol producing microbial organisms can produce isopropanol, 4-hydroxybutyrate, or 1,4-butanediol intracellularly and/or secrete the product into the culture medium.
The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.
As described herein, one exemplary growth condition for achieving biosynthesis of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refers to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N2/CO2 mixture or other suitable non-oxygen gas or gases.
The culture conditions described herein can be scaled up and grown continuously for manufacturing of isopropanol, 4-hydroxybutyrate, or 1,4-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 isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol will include culturing a non-naturally occurring isopropanol, 4-hydroxybutyrate, or 1,4-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 be include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include 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 isopropanol, 4-hydroxybutyrate, or 1,4-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.
In addition to the above fermentation procedures using the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol producers of the invention for continuous production of substantial quantities of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol, the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol producers also can be, for example, simultaneously subjected to chemical synthesis procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical conversion to convert the product to other compounds, if desired.
Important process considerations for a syngas fermentation are high biomass concentration and good gas-liquid mass transfer (Bredwell et al., Biotechnol. Prog. 15:834-844 (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 Calif.), 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 is performed with glass fits 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.
In order to achieve the overall target productivity, methods of cell retention or recycle are employed. 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., J. Biosci. Bioeng. 99:252-258 (2005)). Various other methods can also be used (Bredwell et al., Biotechnol. Prog. 15:834-844 (1999); Datar et al., Biotechnol. Bioeng. 86:587-594 (2004)). Additional optimization can be tested such as overpressure at 1.5 atm to improve mass transfer (Najafpour and Younesi, Enzyme Microb. Technol. 38[1-2], 223-228 (2006)).
Once satisfactory performance is achieved using pure H2/CO as the feed, synthetic gas mixtures are generated containing inhibitors likely to be present in commercial syngas. For example, a typical impurity profile is 4.5% CH4, 0.1% C2H2, 0.35% C2H6, 1.4% C2H4, and 150 ppm nitric oxide (Datar et al., Biotechnol. Bioeng. 86:587-594 (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. For example, it has been shown that 40 ppm NO is inhibitory to C. carboxidivorans (Ahmed and Lewis, Biotechnol. Bioeng. 97:1080-1086 (2007)). Cultures are tested in shake-flask cultures before moving to a fermentor. Also, different levels of these potential inhibitory compounds are tested to quantify the effect they have on cell growth. This knowledge is used to develop specifications for syngas purity, which is utilized for scale up studies and production. If any particular component is found to be difficult to decrease or remove from syngas used for scale up, an adaptive evolution procedure is utilized to adapt cells to tolerate one or more impurities.
To generate better producers, 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. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of isopropanol, 4-hydroxybutyrate, or 1,4-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. 84:647-657 (2003)). OptKnock is a metabolic modeling and simulation program that suggests gene deletion strategies that result in genetically stable microorganisms which overproduce the target product. Specifically, 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. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring microbial organisms for further optimization of biosynthesis of a desired product.
Briefly, 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 enable 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 Jan. 10, 2002, in International Patent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. patent application Ser. No. 11/891,602, filed Aug. 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. publication 2003/0233218, filed Jun. 14, 2002, and in International Patent Application No. PCT/US03/18838, filed Jun. 13, 2003. 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.
These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted.
Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host microbial organisms. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration of the invention, some methods are described herein with reference to the 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.
Once identified, 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. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. 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.
To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®.
The methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.
As discussed above, 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. Pat. No. 7,127,379. As disclosed herein, 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. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. 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.
It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.
Example I
ACS/CODH Gene Insertions in E. coli 
This example describes the creation of E. coli plasmids that express the M. thermoacetica ACS/CODH operon genes including those required 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. 25:1203-1210 (1997)); these come with compatible replicons that cover a range of copy numbers. Additionally, each contains prA1-laco1; 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. These were constructed in both p15A and ColE1-based vectors for medium to high copy numbers.
To estimate the final concentrations recombinant proteins, SDS-PAGE followed by Western blot analyses were performed on the same cell extracts used in the CO oxidation, ACS, methyltransferase, and corrinoid Fe—S assays. The antisera used were polyclonal to purified M. thermoacetica ACS/CODH and Mtr proteins and were visualized using an alkaline phosphatase-linked goat-anti-rabbit secondary antibody. The Westerns Blots are shown in FIGS. 4A and 4B. 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 43:3944-3955 (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 55 C or  ̃60 U 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 FIG. 5). About 0.5% of the cellular protein was CODH as estimated by Western blotting; therefore, the data in Table 42 are approximately 50× 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 (CH3 viologen reduction) seen in the negative controls indicates that E. coli may have a limited ability to reduce CH3 viologen.











TABLE 42








ACS90
7.7 mg/ml
ACS91
11.8 mg/ml



Mta98
9.8 mg/ml
Mta99
11.2 mg/ml
















Extract
Vol
OD/
U/ml
U/mg






ACS90
10 microliters
0.073
0.376
0.049



ACS91
10 microliters
0.096
0.494
0.042



Mta99
10 microliters
0.0031
0.016
0.0014



ACS90
10 microliters
0.099
0.51
0.066



Mta99
25 microliters
0.012
0.025
0.0022



ACS91
25 microliters
0.215
0.443
0.037



Mta98
25 microliters
0.019
0.039
0.004



ACS91
10 microliters
0.129
0.66
0.056





Averages


ACS90 0.057 U/mg


ACS91 0.045 U/mg


Mta99 0.0018 U/mg






This assay is an in vitro reaction that synthesizes acetyl-CoA from methyl-tetrahydrofolate, CO, and CoA using ACS/CODH, methyltransferase, and CFeSP (Raybuck et al., Biochemistry 27:7698-7702 (1988)). By adding or leaving out each of the enzymes involved, 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 HCl, 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 will be determined by the specific substrates used in the reaction.
This assay was run in order to determine if the ACS/CODH operon expressed in E. coli expresses the Fe—S corrinoid protein activity. Therefore, 14C-labeled methyl-THF was used as a labeled substrate to measure acetate synthesis by radioactivity incorporation into isolated acetate samples. Six different conditions were tested:

    
    
        1. Purified ACS/CODH, MeTr, and CFeSP as a positive control
        2. Purified ACS/CODH with ACS90 cell extract
        3. Purified ACS/CODH with ACS91 cell extract
        4. Purified ACS/CODH, MeTr with ACS90 cell extract
        5. Purified ACS/CODH, MeTr with ACS91 cell extract
        6. Purified ACS/CODH, MeTr with as much ACS91 cell extract as possible (excluding the MES buffer)
    
    


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 ( ̃60 ul 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°. The final reagent added was the 14C-methyl-tetrahydrofolate, which started the reaction (t=0s). 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 ul samples were added to 15 ul 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.
Within the ACS/CODH operon is encoded an essential methyltransferase activity that catalyzes the transfer of CH3 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). Within the operon in M. thermoacetica, the Mtr-encoding gene is Moth 1197 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  ̃450 nm, while non-methylated CFeSP, with Co(I), has a large peak at 390 nm. This spectrum is due to both the cobalt and iron-sulfur cluster chromophores. Additionally, it should be noted that the CFeSP can spontaneously oxidize to Co(II), which creates a broad absorption peak at  ̃470 nm (Seravalli et al., Biochem. 38:5728-5735 (1999)). See FIG. 6 for the results from E. coli cells containing ACS90.
To test whether or not E. coli can grow anaerobically in the presence of saturating amounts of CO, 120 ml serum bottles with 50 ml of Terrific Broth medium (plus NiCl2, Fe(II)NH4SO4, and cyanocobalamin) were made in anaerobic conditions. One half of these bottles were equilibrated with nitrogen gas for 30 min. and one half was equilibrated with CO gas for 30 min. An empty vector (pZA33) was used as a control and that and both ACS90 and ACS91 were tested with both N2 and CO. All were grown for 36 hrs with shaking (250 rpm) at 37 C. At the end of the 36 period, examination of the flasks showed high amounts of growth in all (FIG. 7). The bulk of the observed growth occurred overnight with a long lag of some (low but visible) density. Inocula sizes were  ̃0.5 ml from E. coli stocks.
The final CO concentrations are measured using an assay of the spectral shift of myoglobin upon exposure to CO. Myoglobin reduced with sodium dithionite has an absorbance peak at 435 nm; this peak is shifted to 423 nm with CO. Due to the low wavelength (and need to record a whole spectrum from 300 nm on upwards) quartz cuvettes must be used. CO concentration is measured against a standard curve and depends upon the Henry's Law constant for CO of maximum water solubility=970 micromolar at 20 C and 1 atm.
The results shown in Table 43 are very encouraging. Growth reached similar levels (by visual inspection) whether or not a strain was cultured in the presence of CO or not. Furthermore, the negative control had a final CO concentration of 930 micromolar vs. 688 and 728 micromolar for the ACS/CODH operon expressing strains. Clearly, the error in these measurements is high given the large standard deviations. Nevertheless, this test does allow two tentative conclusions: 1) E. coli can tolerate exposure to CO under anaerobic conditions, and 2) E. coli cells expressing the ACS/CODH operon might be metabolizing some of the CO. The second conclusion is significantly less certain than the first.









TABLE 43






Stain and Growth Conditions
Final CO concentration



pZA33-CO
(micromolar)


















ACS90-CO
638




494




734




883



ave
687



SD
164



ACS91-CO
728




812




760




611



ave.
728



SD
85









Example II
Enhancing the Yield of Fermentation Products from Sugars with Wood-Ljungdahl Pathway Enzymes
In this example, we describe a non-naturally occurring microorganism expressing genes encoding enzymes that catalyze the carbonyl-branch of the Wood-Ljungdahl pathway. Wood-Ljungdahl pathway enzymes assimilate carbon in the form of CO and/or CO2 into acetyl-CoA, which can subsequently be converted to useful chemical products such as isopropanol, 4-hydroxybutyrate (4-HB) and 1,4-butanediol (14-BDO). The Wood-Ljungdahl pathway can also serve as a secondary carbon assimilation pathway during growth on other substrates such as glucose. In this capacity, WL pathway enzymes harness reducing equivalents generated in glycolysis to convert CO2 to acetyl-CoA, cell mass and downstream products at high yield.
Isopropanol: Engineering a Wood-Ljungdahl pathway into a glucose-utilizing and isopropanol-producing organism improves the maximum isopropanol yield up to 33%. The production of isopropanol has been demonstrated in E. coli (Nahvi et al., Chem. Biol. 9:1043 (2002)). Specifically, acetyl CoA can be converted into acetoacetyl CoA, transformed into acetoacetate, decarboxylated to form acetone and then reduced to form isopropanol (see FIG. 1). This pathway is redox-imbalanced and yields only 1 mol IPA/mol glc or 0.333 g/g. If the IPA pathway is supplemented with CO2-fixation by the Wood-Ljungdahl pathway, the theoretical stoichiometric yield of IPA (1.33 mol/mol, 0.444 lb/lb) is achievable, the pathway is redox-balanced, and can generate energy.
FIG. 8A shows a flux distribution for the conversion of one mole of glucose into 1.33 moles of isopropanol. The conversion of one mole of glucose to two moles of pyruvate generates four reducing equivalents and two ATPs. Pyruvate is subsequently converted to molar quantities of acetyl-CoA by one or more enzymes including pyruvate formate lyase (PFL), pyruvate dehydrogenase (PDH) or pyruvate ferredoxin oxidoreductase (PFOR). PDH and PFOR generate molar quantities of CO2 and two reducing equivalents per pyruvate consumed. Pyruvate formate lyase forms formate as a byproduct, which can be converted to CO, and two reducing equivalents by formate dehydrogenase. In the absence of WL pathway enzymes, the two moles of acetyl-CoA are converted to one mole of isopropanol. Assuming reducing equivalents are generated from the pyruvate to acetyl-CoA conversion, this pathway generates an excess of 3 reducing equivalents per glucose consumed. When an active WL pathway is present 0.66 moles of CO2 or formate (per mole glucose consumed) are assimilated into acetyl-CoA, which is subsequently converted to 0.33 moles of isopropanol. This combined glycolysis/Wood Ljungdahl pathway is exactly balanced from a redox standpoint and is also capable of generating energy. Two ATPs are generated in the conversion of glucose to pyruvate. In this example, an additional 1.33 ATPs are recovered from conversion of acetyl-CoA to acetate, yielding a total supply of 3.33 ATPs per glucose consumed. The Wood-Ljungdahl pathway consumes 0.67 ATPs leaving 2.67 ATPs available for cell growth and metabolism.
4-Hydroxybutyrate: A similar improvement in yield is achievable for the production of 4-hydroxybutyrate. The maximum 4-HB yield from glucose alone is 1 mole 4-HB per mole glucose consumed (0.578 g/g). The pathway, shown in FIG. 8B, is redox-imbalanced and requires aeration and/or the formation of fermentation byproducts. An organism with a WL pathway can achieve the maximum stoichiometric yield of 4-HB (1.33 mol/mol or 0.768 g/g). A flux distribution of the maximum theoretical yield is shown in FIG. 8B. Like the isopropanol pathway described previously, the pathway is redox-balanced and yields a maximum availability of 2.67 ATP for cell growth and maintenance.
1,4-Butanediol: 1,4-Butanediol can also be synthesized from acetyl-CoA via the intermediate 4-hydroxybutyryl-CoA. In this pathway, acetoacetyl-CoA is converted to 4-hydroxybutyryl-CoA as described previously in the 4-hydroxybutyrate pathway. 4-Hydroxybutyryl-CoA is subsequently converted to 14-BDO by a bifunctional aldehyde dehydrogenase or a CoA-dependent aldehyde dehydrogenase and alcohol dehydrogenase. Alternatively, it can be converted to 4-hydroxybutyrate which can be first reduced directly to 4-hydroxybutyraldehyde and subsequently reduced to 1,4-butanediol. As this pathway is not redox-balanced, the maximum 14-BDO yield of this pathway from sugars is 1 mol/mol (0.5 g/g). Additional assimilation of CO) via Wood-Ljungdahl pathway further improves the yield to the theoretical maximum of 1.09 mol/mol (0.545 g/g). A predicted flux distribution for achieving the maximum theoretical yield is shown in FIG. 8C. In this pathway, 2 ATPs are generated in glycolysis and 0.18 ATPs are consumed in the WL pathway, leaving a maximum of 1.82 ATPs available for cell growth and maintenance.
Example III
Enhancing the Yield of Fermentation Products from Sugars with Wood-Ljungdahl Pathway Enzymes and a MtaABC-Type Methyltransferase System
An organism engineered to express genes encoding an ACS/CODH enzyme and a MtaABC-type methyltransferase system is capable of producing acetyl-CoA and downstream products at high yields from glucose and methanol. The methyltransferase system enables the assimilation of methanol into 5-methyl-tetrahydrofolate (Me-THF). The CODH/ACS enzyme confers the functionality to convert CO2 to CO and CO, CoA and Me-THF to acetyl-CoA. The recombinant organism can be further engineered to transform acetyl-CoA to products of interest such as isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. Microorganisms with these features are capable of metabolizing sugars in conjunction with methanol to achieve high yields of chemical products. The ratio of glucose to methanol consumed impacts the overall product yield.
An organism engineered to produce isopropanol from glucose via acetyl-CoA achieves a maximum isopropanol yield of 1 mole product per mole glucose utilized. Co-feeding methanol to this organism at a ratio of 2 methanol per one glucose further increases the yield to a maximum of 2 moles isopropanol per mole of glucose consumed. In the maximum yield scenario 2 moles of methanol are utilized per glucose consumed, producing 2 moles of IPA. In addition, 2 moles of CO2 are assimilated into acetyl-CoA via ACS/CODH. The reduction of CO2 to CO by ACS/CODH consumes reducing equivalents, enabling redox balance and improved product yield. The energetic yield of this pathway is also favorable, with 4 ATP equivalents generated per glucose consumed. An exemplary flux distribution of the conversion of methanol and glucose to isopropanol is shown in FIG. 9. The relationship between the glucose/methanol feed ratio and the maximum product yield in FIG. 10.
A similar improvement in yield is observed for a 4-HB-producing organism utilizing the 4-HB pathway from acetyl-CoA in FIG. 2. One mole of glucose and two moles each of methanol and CO2 are assimilated in a redox-balanced pathway to form two moles of 4-hydroxybutyrate. Similarly, the yield of 1,4-BDO from carbohydrates can be improved by co-utilizing methanol.
Example IV
Engineering Cobalamin Synthesis into an Organism
The key enzyme of the Wood-Ljungdahl pathway, ACS/CODH, requires cobalamin (vitamin B12) to function. B12 is synthesized de novo in some organisms but must be supplied exogenously to others. Still other organisms such as S. cerevisiae lack the ability to efficiently uptake B12. This example describes a strategy for engineering de novo B12 synthetic capability into an organism.
B12 biosynthetic pathways have been characterized in several organisms including Salmonella typhimurium LT2 (Roth et al., J. Bacteriol. 175:3303-3316 (1993)), Lactobacillus reuteri CRL1098 (Sennett et al., Annu. Rev. Biochem. 50:1053-1086 (1981)) and Bacillus megaterium (Brey et al., J. Bacteriol. 167:623-630 (1986)). Bacterial B12 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., J. Org. Chem. 68:2529-2539 (2003)). The final products of the biosynthesis of vitamin B12 are 5′-deoxyadenosylcobalamin (coenzyme B12) and methylcobalamin (MeCbl). Vitamin B12 is defined as cyanocobalamin (CNCbl) which is the form commonly prepared in industry. In this example, B12 refers to all three analogous molecules.
The anaerobic cobalamin biosynthesis pathway has been well-characterized in Salmonella typhimurium LT2 (Roth et al., J. Bacteriol. 175:3303-3316 (1993)). 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., J. Bacteriol. 178:753-767 (1996)). To further improve yield of the cobyric acid precursor, the authors removed the known regulatory elements of cbiA and altered the RBS. The genes and corresponding GenBank identifiers and gi numbers are listed below.










TABLE 44





Protein
GenBank ID
GI Number
Organism







cysG
NP_462380.1
16766765
Salmonella typhimurium


cbiK
NP_460970.1
16765355
Salmonella typhimurium


cbiL
NP_460969.1
16765354
Salmonella typhimurium


cbiH
NP_460972.1
16765357
Salmonella typhimurium


cbiF
NP_460974.1
16765359
Salmonella typhimurium


cbiG
NP_460973.1
16765358
Salmonella typhimurium


cbiD
NP_460977.1
16765362
Salmonella typhimurium


cbiJ
NP_460971.1
16765356
Salmonella typhimurium


cbiE
NP_460976.1
16765361
Salmonella typhimurium


cbiT
NP_460975.1
16765360
Salmonella typhimurium


cbiC
NP_460978.1
16765363
Salmonella typhimurium


cbiA
NP_460980.1
16765365
Salmonella typhimurium


fldA
NP_459679.1
16764064
Salmonella typhimurium


cobA
P31570.1
399274
Salmonella typhimurium


cbiP
AAA27268.1
154436
Salmonella typhimurium


cbiB
Q05600.1
543942
Salmonella typhimurium


cobU
NP_460963.1
16765348
Salmonella typhimurium


cobT
NP_460961.1
16765346
Salmonella typhimurium


cobs
AAA27270.1
154438
Salmonella typhimurium


cobC
NP_459635.1
16764020
Salmonella typhimurium


cysG
NP_462380.1
16766765
Salmonella typhimurium









Some organisms unable to synthesize B12 de novo are able to catalyze some steps of the pathway. 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., J. 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., J. Bacteriol. 184:2404-2410 (2002); Ishige et 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., J. Bacteriol. 178:753-767 (1996)).










TABLE 45





Protein
GenBank ID
GI Number
Organism







cobT
NP_416495.1
16129932
Escherichia coli K12 sp.





MG1655


cobS
NP_416496.1
16129933
Escherichia coli K12 sp.





MG1655


cobU
NP_416497.1
16129934
Escherichia coli K12 sp.





MG1655


cysG
NP_417827.1
16131246
Escherichia coli K12 sp.





MG1655









S. cerevisiae is not able to synthesize B12 de novo, nor is it able to uptake the vitamin at detectable levels. However, the S. cerevisiae genome encodes two proteins, Met1p and Met8p, that catalyze several B12 pathway reactions. Met1p 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., Biochem. J. 338 (Pt 3):701-708 (1999)).










TABLE 46





Protein
GenBank ID
GI Number
Organism







Met1p
NP_012995.1
6322922
Saccharomyces cerevisiae


Met8p
NP_009772.1
6319690
Saccharomyces cerevisiae









Any or all of these genes can be introduced into an organism deficient or inefficient in one or more components of cobalamin synthesis to enable or increase the efficiency of cobalamin synthesis.
Example V
Engineering Enhanced Cobalamin Uptake Capability in an Organism
This example describes engineering B12 uptake capability into a host organism. B12 uptake requires a specific transport system (Sennett et al., Annu. Rev. Biochem. 50:1053-1086 (1981)). The B12 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., J. 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 (Heller et al., J. Bacteriol. 161:896-903 (1985); Raux et al., J. Bacteriol. 178:753-767 (1996)). Transport across the inner membrane is facilitated by an ABC type uptake system composed of BtuF, BtuD (ATP binding component) and BtuC (permease) (Raux et al., Biochem. J. 338 (Pt 3):701-708 (1999)). Crystal structures of the BtuCDF complex are available (Raux et al., J. Bacteriol. 178:753-767 (1996); Raux et al., Biochem. J. 338 (Pt 3):701-708 (1999)). An additional protein, BtuE, is coexpressed in the btuCED operon, but this protein is not required for B12 transport and its function is unknown (Rioux and Kadner, Mol. Gen. Genet. 217:301-308.(1989)). The btuCED operon is constitutively expressed. The GenBank identifiers and GI numbers of the genes associated with B12 transport are listed below.










TABLE 47





Protein
GenBank ID
GI Number
Organism







btuB
NP_418401.1
16131804
Escherichia coli K12 sp.





MG1655


btuC
NP_416226.1
16129667
Escherichia coli K12 sp.





MG1655


btuD
NP_416224.1
16129665
Escherichia coli K12 sp.





MG1655


btuF
NP_414700.1
16128151
Escherichia coli K12 sp.





MG1655


tonB
NP_415768.1
16129213
Escherichia coli K12 sp.





MG1655


exbB
NP_417479.1
16130904
Escherichia coli K12 sp.





MG1655


exbD
NP_417478.1
16130903
Escherichia coli K12 sp.





MG1655









The B12 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 B12 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 B12 binding site removes inhibition and improves the efficiency of B12 uptake (Bulthuis et al., U.S. Pat. No. 6,432,686). These strategies were successfully employed to improve B12 uptake capability in 1,3-PDO producing microorganisms (WO/1999/058686) and (Bulthuis et al., U.S. Pat. No. 6,432,686). A recent patent application describes improving the efficiency of B12 uptake (WO/2008/152016) by deleting negative regulatory proteins such as C. glutamicum btuR2.
S. typhimurium possesses both high and low affinity transporters for B12. The high affinity transporter is encoded by btuB (Rioux and Kadner, J. Bacteriol. 171:2986-2993 (1989)). Like 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 B12 binding protein is encoded by btuD and btuE, and btuC is predicted to encode the permease.










TABLE 48





Protein
GenBank ID
GI Number
Organism







btuB
AAA27031.1
153891
Salmonella typhimurium LT2


btuC
NP_460306.1
16764691
Salmonella typhimurium LT2


btuD
NP_460308.1
16764693
Salmonella typhimurium LT2


btuE
AAL20266.1
16419860
Salmonella typhimurium LT2









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 efficiency cobalamin uptake.
Method for Quantifying B12 in the Culture Medium.
To quantify the amount of B12 in the culture medium, cell free samples are run on HPLC. Cobalamin quantification is achieved by comparing peak area ratios at 278 nm and 361 num with standards, then applying peak areas to standard curves of cobalamin.
Throughout this application various publications have been referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
Although the invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific examples and studies detailed above are only illustrative of the invention. It should understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

## Claims

1. A non-naturally occurring microbial organism comprising:
(i) a 1,4-butanediol pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce 1,4-butanediol, wherein said 1,4-butanediol pathway enzyme is selected from the group consisting of an acetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming), and a 1,4-butanediol dehydrogenase; and
(ii) an acetyl-CoA pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, wherein said acetyl-CoA pathway enzyme is selected from the group consisting of a corrinoid protein, a methyltetrahydrofolate:corrinoid protein methyltransferase, a corrinoid iron-sulfur protein, a nickel-protein assembly protein, a ferredoxin, a carbon monoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and a hydrogenase.

2. The organism of claim 1, wherein said acetoacetyl-CoA thiolase is encoded by a gene selected from the group consisting of atoB, thlA, thlB, and erg10.

3. The organism of claim 1, wherein said 3-Hydroxybutyryl-CoA dehydrogenase is encoded by a gene selected from the group consisting of hbd, msed—1423, msed—0399, msed—0389, and msed—1933.

4. The organism of claim 1, wherein said crotonase is encoded by a gene selected from the group consisting of crt, paaA, paaB, phaA, phaB, maoC, paaF, and paaG.

5. The organism of claim 1, wherein said crotonyl-CoA hydratase is encoded by a gene selected from the group consisting of abfD, msed—1321, and msed—1220.

6. The organism of claim 1, wherein said 4-hydroxybutyryl-CoA reductase (alcohol forming) is encoded by a gene selected from the group consisting of adhE, adhE2, mcr, rcas—2929, nap1—02720, and mgp2080—00535.

7. The organism of claim 1, wherein said 4-hydroxybutyryl-CoA reductase (aldehyde forming) is encoded by a gene selected from the group consisting of acr1, sucD, bphG, msed—0709, mcr, asd-2, and saci—2370.

8. The organism of claim 1, wherein said 1,4-butanediol dehydrogenase is encoded by a gene selected from the group consisting of alrA, adh2, yqhD, bdh I, bdh II, and 4hbd.

9. The organism of claim 1, wherein said corrinoid protein is encoded by a gene selected from the group consisting of mtaC, mtaC1, mtaC2, and mtaC3.

10. The organism of claim 1, wherein said methyltetrahydrofolate:corrinoid protein methyltransferase is encoded by a gene selected from the group consisting of mtaA, and mtaA1, mtaA2.

11. The organism of claim 1, wherein said methyltetrahydrofolate:corrinoid protein methyltransferase is encoded by the gene acsE.

12. The organism of claim 1, wherein said corrinoid iron-sulfur protein is encoded by the gene acsD.

13. The organism of claim 1, wherein said nickel-protein assembly protein is encoded by at least one gene selected from the group consisting of acsF and cooC.

14. The organism of claim 1, wherein said ferredoxin is encoded by the gene orf7.

15. The organism of claim 1, wherein said carbon monoxide dehydrogenase is encoded by the gene acsA.

16. The organism of claim 1, wherein said pyruvate ferredoxin oxidoreductase is encoded by a gene selected from the group consisting of por and ydbK.

17. The organism of claim 1, wherein said hydrogenase is encoded by at least one gene selected from the group consisting of hypA, hypB, hypC, hypD, hypE, hypF, moth—2175, moth—2176, moth—2177, moth—2178, moth—2179, moth—2180, moth—2181, hycA, hycB, hycC, hycD, hycE, hycF, hycG, hycH, hycI, hyfA, hyfB, hyfC, hyfD, hyfE, hyfF, hyfG, hyfH, hyfI, hyfJ, hyfR, moth—2182, moth—2183, moth—2184, moth—2185, moth—2186, moth—2187, moth—2188, moth—2189, moth—2190, moth—2191, moth—2192, moth—0439, moth—0440, moth—0441, moth—0442, moth—0809, moth—0810, moth—0811, moth—0812, moth—0813, moth—0814, moth—0815, moth—0816, moth—1193, moth—1194, moth—1195, moth—1196, moth—1717, moth—1718, moth—1719, moth—1883, moth—1884, moth—1885, moth—1886, moth—1887, moth—1888, moth—1452, moth—1453, and moth—1454.

18. The organism of claim 1, further comprising the polypeptide_AcsEps encoded by the gene acsEps.

19. The organism of claim 1, further comprising at least one enzyme or polypeptide encoded by a gene selected from the group consisting of codh, codh-I, cooF, hypA, cooH, cooU, cooX, cooL, cooK, cooM, cooT, cooJ, and codh-II.

20. The organism of claim 1, further comprising a methanol methyltransferase.

21. The organism of claim 20, wherein said methanol methyltransferase is encoded by a gene selected from the group consisting of mtaB, mtaB1, mtaB2, and mtaB3.

22. The organism of claim 20, wherein said organism utilizes a feedstock selected from the group consisting of: 1) methanol and CO, 2) methanol, CO2, and H2, 3) methanol, CO, CO2, and H2, 4) methanol and synthesis gas comprising CO and H2, 5) methanol and synthesis gas comprising CO, CO2, and H2, 6) one or more carbohydrates, 7) methanol and one or more carbohydrates, and 8) methanol.

23. A method for producing 1,4-butanediol, comprising culturing the non-naturally occurring microbial organism of claim 1.