WO2014004625A1 - Microorganismes destinés à la production d'éthylèneglycol utilisant un gaz de synthèse - Google Patents

Microorganismes destinés à la production d'éthylèneglycol utilisant un gaz de synthèse Download PDF

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WO2014004625A1
WO2014004625A1 PCT/US2013/047821 US2013047821W WO2014004625A1 WO 2014004625 A1 WO2014004625 A1 WO 2014004625A1 US 2013047821 W US2013047821 W US 2013047821W WO 2014004625 A1 WO2014004625 A1 WO 2014004625A1
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ethylene glycol
microbial organism
dehydrogenase
pathway
naturally occurring
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PCT/US2013/047821
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English (en)
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Robin E. Osterhout
Anthony P. Burgard
Mark J. Burk
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Genomatica, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

  • the present invention relates generally to biosynthetic processes, and more specifically to organisms having biosynthetic capability of converting synthesis gas or other gaseous carbon sources to ethylene glycol.
  • 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.
  • Ethylene glycol is a chemical commonly used in many commercial and industrial applications including production of antifreezes and coolants.
  • Ethylene glycol is also used as a raw material in the production of a wide range of products including polyester fibers for clothes, upholstery, carpet and pillows; fiberglass used in products such as jet skis, bathtubs, and bowling balls; and polyethylene terephthalate resin used in packaging film and bottles.
  • polyester fibers for clothes, upholstery, carpet and pillows
  • fiberglass used in products such as jet skis, bathtubs, and bowling balls
  • polyethylene terephthalate resin used in packaging film and bottles.
  • ethylene glycol consumed worldwide is used in the production of polyester fibers, resins and films. Strong growth in polyester demand has led to global growth rates of 5-6%/year for ethylene glycol.
  • the second largest market for ethylene is antifreeze formulations.
  • ethylene oxide is first produced by the oxidation of ethylene in the presence of oxygen or air and a silver oxide catalyst.
  • a crude ethylene glycol mixture is then produced by the hydrolysis of ethylene oxide with water under pressure. Fractional distillation under vacuum is used to separate the ethylene glycol from the higher glycols.
  • Ethylene glycol was previously manufactured by the hydrolysis of ethylene oxide, which was produced via ethylene chlorohydrin but this method has been superseded by the direct oxidation route.
  • Ethylene glycol is a colorless, odorless, viscous, hygroscopic sweet-tasting liquid and is classified as harmful by the EC Dangerous Substances Directive.
  • Synthesis gas is a mixture of primarily H 2 and CO that can be obtained via gasification of any organic feedstock, such as coal, coal oil, natural gas, biomass, or waste organic matter.
  • Any organic feedstock such as coal, coal oil, natural gas, biomass, or waste organic matter.
  • Numerous gasification processes have been developed, and most designs are based on partial oxidation, where limiting oxygen avoids full combustion, of organic materials at high temperatures (500-1500°C) to provide syngas as, for example, 0.5: 1-3 :1 H 2 /CO mixture.
  • Steam is sometimes added to increase the hydrogen content, typically with increased C0 2 production through the water gas shift reaction.
  • 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.
  • 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.
  • Clostridia also produce multiple products, which presents separations issues in isolating a desired product.
  • 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.
  • the invention provides a non-naturally occurring microbial organism containing an ethylene glycol pathway, wherein the ethylene glycol pathway includes at least one exogenous nucleic acid encoding an ethylene glycol pathway enzyme expressed in a sufficient amount to produce ethylene glycol.
  • the non-naturally occurring microbial organism containing an ethylene glycol pathway, wherein the ethylene glycol pathway includes at least one exogenous nucleic acid encoding an ethylene glycol pathway enzyme expressed in a sufficient amount to produce ethylene glycol.
  • microorganisms of the invention can express exogenous nucleic acids that catalyze the fixation of C0 2 or CO to ethylene glycol.
  • the microorganisms of the invention can further include enzymes to generate energy and reducing equivalents from syngas and other gaseous sources.
  • the microorganisms of the invention can include enzymes to convert methanol and/or syngas or other gaseous sources into ethylene glycol.
  • the invention additionally provides methods of using such microbial organisms to produce ethylene glycol, by culturing a non-naturally occurring microbial organism containing an ethylene glycol pathway as described herein under conditions and for a sufficient period of time to produce ethylene glycol.
  • Figure 1 shows exemplary pathways for converting CO and/or C0 2 to ethylene glycol via serine.
  • Exemplary enzymes for converting the depicted compounds include the following: A. formate dehydrogenase; B. formyltetrahydro folate synthetase; C.
  • methenyltetrahydrofolate cyclohydrolase D. methylenetetrahydrofolate dehydrogenase; E. glycine cleavage complex; F. serine hydroxymethyltransferase; G. serine decarboxylase; H. ethanolamine aminotransferase, amine oxidase or dehydrogenase; I. glycolaldehyde reductase; J. CO dehydrogenase; K. hydrogenase; L. Ferredoxin:NADPH oxidoreductase; M. serine aminotransferase, amine oxidase or dehydrogenase; N. hydroxypyruvate decarboxylase; O.
  • FIG. 1 shows an exemplary flux distribution for achieving the maximum theoretical ethylene glycol yield from glucose and C0 2 .
  • the exemplary enzymes for converting C0 2 to ethylene glycol are shown in Figure 1.
  • 3PG represents 3- phosphoglycerate.
  • 3PHP represents 3-phosphohydroxypyruvate.
  • 3PS represents 3- phosphoserine.
  • Glucose is converted to 3PG by glycolytic enzymes.
  • 3PG is converted to 3PHP by 3-phosphoglycerate dehydrogenase.
  • 3PHP is converted to 3PS by phosphoserine aminotransferase.
  • 3PS is converted to serine by phosphoserine phosphatase.
  • Formyl-THF represents 10-formyl-tetrahydro folate.
  • Methenyl-THF represents 5,10- methenyltetrahydro folate.
  • Methylene-THF is 5, 10-methylenetetrahydro folate.
  • Figure 3 shows exemplary pathways for converting CO and/or C0 2 to ethylene glycol via glyoxylate.
  • Exemplary enzymes for converting the depicted compounds include the following: A. formate dehydrogenase; B. formyltetrahydro folate synthetase; C.
  • methenyltetrahydrofolate cyclohydrolase D. methylenetetrahydrofolate dehydrogenase; E. glycine cleavage complex; F. glycine aminotransferase, amine oxidase or dehydrogenase; G. glyoxylate carboxyligase; H. hydroxypyruvate isomerase; I. hydroxypyruvate decarboxylase; J. CO dehydrogenase; K. hydrogenase; L. Ferredoxin:NADPH
  • M glycolaldehyde reductase
  • N hydroxypyruvate aminotransferase or dehydrogenase
  • O serine decarboxylase
  • P hydroxypyruvate reductase
  • Q glycerate decarboxylase
  • R tartronate semi aldehyde reductase
  • S ethanolamine
  • Formyl-THF represents 10-formyl- tetrahydrofolate.
  • Methenyl-THF represents 5, 10-methenyltetrahydro folate.
  • Methylene- THF is 5, 10-methylenetetrahydro folate.
  • Figure 4 shows exemplary pathways for converting methanol (MeOH) to ethylene glycol.
  • Exemplary enzymes for converting the depicted compounds include the following: A. formaldehyde dehydrogenase; B. formyltetrahydrofolate synthetase; C. methenyltetrahydrofolate cyclohydrolase; D. methylenetetrahydrofolate dehydrogenase; E. glycine cleavage complex; F. serine hydroxymethyltransferase; G. serine decarboxylase; H. ethanolamine aminotransferase, amine oxidase or dehydrogenase; I. glycolaldehyde reductase; M. serine aminotransferase, amine oxidase or dehydrogenase; N.
  • Formyl-THF represents 10-formyl- tetrahydrofolate.
  • Methenyl-THF represents 5, 10-methenyltetrahydro folate.
  • Methylene- THF represents 5, 10-methylenetetrahydro folate.
  • Figure 5 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91 (lane2), Mta98/99 (lanes 3 and 4) cell extracts with size standards (lane 5) and controls of M. thermoacetica CODH (Moth_1202/1203) or Mtr (Moth l 197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and 1000 ng).
  • Figure 6 shows CO oxidation assay results.
  • Cells M. thermoacetica or E. coli with the CODH/ACS operon; ACS90 or ACS91 or empty vector: pZA33S
  • Assays were performed at 55°C at various times on the day the extracts were prepared. Reduction of methylviologen was followed at 578 nm over a 120 sec time course.
  • This invention is directed, at least in part, to non-naturally occurring microorganisms that express exogenous nucleic acids encoding glycine synthase pathway enzymes, which catalyze the fixation of C0 2 , CO, or methanol to glycine in conjunction with a pathway to further convert glycine to ethylene glycol.
  • the microorganisms of the invention can further include enzymes to generate energy and reducing equivalents from syngas.
  • Microorganisms with a glycine synthase pathway are capable of assimilating carbon in the form of CO, C0 2 and/or methanol into glycine which can subsequently be converted into serine, glyoxylate, acetyl-CoA, cell mass, and useful products such as ethylene glycol.
  • the glycine synthase pathway is one of several known biological routes for fixing C0 2 .
  • C0 2 is reduced and subsequently attached to a
  • glycine synthase also called the glycine cleavage complex or glycine cleavage system
  • C0 2 glycine cleavage complex
  • C0 2 fixation pathway has been demonstrated in several Clostridial species, it has not been demonstrated as the sole pathway of autotrophic growth to date (Bar-Even et al, J Exp Botany 1-18 (2011)).
  • the glycine synthase pathway can serve as a secondary carbon assimilation pathway during growth on other substrates such as carbohydrates.
  • the glycine synthase pathway enzymes harness excess reducing equivalents generated in glycolysis and/or methanol oxidation to fix C0 2 , contributing to improved yields of products such as ethylene glycol.
  • C0 2 is first converted to formate in step 1 A by an enzyme with formate dehydrogenase activity. Formate is then ligated to THF by formyltetrahydro folate synthase in step IB. The product, formyl-THF, is then converted to methenyl-THF and subsequently to methylene-THF in steps 1C and ID. Subsequent NAD(P)H-dependent conversion of methylene-THF and C0 2 to glycine is catalyzed by the glycine cleavage complex in step IE.
  • hydroxymethyltransferase step IF.
  • serine is decarboxylated to ethanolamine (step 1G). Ethanolamine is then converted to its corresponding aldehyde by an aminotransferase, amine oxidase or dehydrogenase (step 1H). Reduction of glycolaldehyde yields EG (step II).
  • serine is converted to hydroxypyruvate by an aminotransferase, amine oxidase or dehydrogenase (step 1M). Hydroxypyruvate is then decarboxylated to glycolaldehyde and subsequently reduced to EG (steps IN, II). Alternately, hydroxypyruvate is first reduced to glycerate, which is then decarboxylated to EG (steps 10, IP).
  • Exemplary enzyme candidates for pathway enzymes (Steps 1-9 of Figure 1) are described in Example II.
  • step 3G glyoxylate carboligase converts two equivalents of glyoxylate to tartronate semialdehyde (step G). Reduction of tartronate semialdehyde forms hydroxypyruvate (step H). Hydroxypyruvate can be converted to ethylene glycol by the enzymes of the pathways described herein, such as the pathways of Figure 1.
  • hydroxypyruvate is converted to serine via reductive or transamination (step N).
  • Serine is then converted to ethylene glycol by the enzymes of the pathways described herein, such as the pathways of Figure 1.
  • the maximum theoretical yield of ethylene glycol from glucose is 2.4 mol/mol (0.835 g/g).
  • the maximum yield of ethylene glycol from serine via any of the serine to EG pathways shown in Figure 1 is 2 mol/mol glucose.
  • Non-naturally occurring organisms of the present invention employing an ethylene glycol biosynthetic pathway in conjunction with a glycine synthase pathway can achieve the maximum theoretical EG yield. This yield is improved over that of organisms that do not have an active glycine synthase pathway.
  • reducing equivalents are obtained by the conversion of CO and water to C0 2 via carbon monoxide dehydrogenase or from the activity of a hydrogen-utilizing hydrogenase which transfers electrons from H 2 to an acceptor such as ferredoxin, flavodoxin, FAD + , NAD + , or NADP + .
  • Energy is obtained by transferring electrons to an acceptor such as oxygen or nitrate (for example, via oxidative phosphorylation).
  • the organism of the invention can generate energy via an Na + - or H + - dependant ATP synthase, which utilizes Na + or H + ion gradients, respectively, to drive ATP synthesis (Muller, V. Appl Environ Microbiol 69:6345-6353 (2003)).
  • microorganisms of the invention can fix carbon from exogenous CO and/or C0 2 and/or methanol to synthesize acetyl-CoA, cell mass, and products such as ethylene glycol.
  • a host organism engineered with these capabilities that also naturally possesses the capability for anapleurosis e.g., E. coli
  • 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.
  • microorganisms disclosed herein can be grown under strictly anaerobically conditions and provided with exogenous glucose as a carbon and energy source. Metabolizing glucose or other carbohydrates provides one potential source of C0 2 that can be fixed via the pathways disclosed herein. Alternatively, or in addition to glucose, nitrate can be added to the fermentation broth to serve as an electron acceptor and initiator of growth. Anaerobic growth of E. 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 enzymes disclosed herein.
  • the great potential of syngas as a feedstock resides in its ability to be efficiently and cost-effectively converted into chemicals and fuels of interest.
  • Two main technologies for syngas conversion are Fischer-Tropsch processes and fermentative processes.
  • the Fischer-Tropsch (F-T) technology has been developed since World War II and involves inorganic and metal-based catalysts that allow efficient production of methanol or mixed hydrocarbons as fuels.
  • the drawbacks of F-T processes are: 1) a lack of product selectivity, which results in difficulties separating desired products; 2) catalyst sensitivity to poisoning; 3) high energy costs due to high temperatures and pressures required; and 4) the limited range of products available at commercially competitive costs.
  • syngas has been shown to serve as a carbon and energy source for many anaerobic microorganisms that can convert this material into products such as ethanol, acetate and hydrogen.
  • the main benefits of fermentative conversion of syngas are the selectivity of organisms for production of single products, greater tolerance to syngas impurities, lower operating temperatures and pressures, and potential for a large portfolio of products from syngas.
  • the main drawbacks of fermentative processes are that organisms known to convert syngas tend to generate only a limited range of chemicals, such as ethanol and acetate, and are not efficient producers of other chemicals, the organisms lack established tools for genetic manipulation, and the organisms are sensitive to end products at high concentrations.
  • the present invention relates to the generation of microorganisms that are effective at producing ethylene glycol from syngas or other gaseous carbon sources.
  • the organisms and methods of the present invention allow production of ethylene glycol at costs that are significantly advantaged over both traditional petroleum-based products and products derived directly from glucose, sucrose or lignocellulosic sugars.
  • the invention provides a non-naturally occurring microorganism capable of utilizing syngas or other gaseous carbon sources to produce ethylene glycol in which the parent microorganism lacks the natural ability to utilize syngas.
  • one or more proteins or enzymes are expressed in the microorganism, thereby conferring a pathway to utilize syngas or other gaseous carbon source to produce ethylene glycol.
  • the invention provides a non-naturally occurring microorganism that has been genetically modified, for example, by expressing one or more exogenous proteins or enzymes that confer an increased efficiency of production of ethylene glycol, where the parent microorganism has the ability to utilize syngas or other gaseous carbon source.
  • the invention relates to generating a microorganism with a new metabolic pathway capable of utilizing syngas as well as generating a microorganism with improved efficiency of utilizing syngas or other gaseous carbon source to produce ethylene glycol.
  • Methanol can also be utilized as a carbon source to form ethylene glycol.
  • Figure 4 depicts pathways for the conversion of methanol to ethylene glycol.
  • methanol is oxidized to formaldehyde by a methanol dehydrogenase enzyme.
  • Formaldehyde dehydrogenase oxidizes formaldehyde to formate.
  • Formate is then converted to ethylene glycol by one or more of the pathways shown in Figures 1-3.
  • the net conversion of two equivalents of methanol to one equivalent of ethylene glycol generates two excess reducing equivalents.
  • These reducing equivalents can be utilized to generate energy for the ethylene glycol pathway, biomass formation and/or cell maintenance. Alternately, the excess reducing equivalents can be utilized to fix additional carbon.
  • Methanol can be utilized as the sole carbon substrate, or can be co-utilized with syngas, glucose, or other feedstocks disclosed herein.
  • the present invention additionally provides a non-naturally occurring microorganism expressing an exogenous nucleic acid encoding an enzyme that catalyzes the conversion of methanol to ethylene glycol.
  • a non-naturally occurring microorganism expressing an exogenous nucleic acid encoding an enzyme that catalyzes the conversion of methanol to ethylene glycol.
  • Such an organism is capable of converting methanol, a relatively inexpensive organic feedstock that can be derived from synthesis gas, and gases comprising CO, C0 2 , and/or H 2 into ethylene glycol and/or cell mass.
  • non-naturally occurring when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species.
  • Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material.
  • 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 ethylene glycol 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.
  • 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.
  • microbial As used herein, the terms "microbial,” “microbial organism” or
  • microorganism are 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.
  • ethylene glycol refers to a compound having the molecular formula C2H6O2, a molecular mass of 62.068 g/mol and the IUPAC name of ethane- 1,2-diol. In its pure form, ethylene glycol is an odorless, colorless, syrupy, sweet- tasting liquid.
  • 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.
  • 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.
  • heterologous refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the host microbial organism.
  • exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.
  • the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid.
  • a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein.
  • two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids.
  • exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids.
  • the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.
  • the non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration.
  • stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.
  • An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms.
  • mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides.
  • Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor.
  • Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable.
  • Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.
  • Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species.
  • a specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase.
  • a second example is the separation of mycoplasma 5 '-3' exonuclease and Drosophila DNA polymerase III activity.
  • the DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.
  • paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions.
  • Paralogs can originate or derive from, for example, the same species or from a different species.
  • microsomal epoxide hydrolase epoxide hydrolase I
  • soluble epoxide hydrolase epoxide hydrolase II
  • Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor.
  • Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.
  • a nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species.
  • Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species.
  • a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein.
  • Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.
  • Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score.
  • Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.
  • Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1;
  • Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept- 16- 1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x dropoff: 50; expect: 10.0;
  • the invention provides a non-naturally occurring microbial organism, wherein the microbial organism has an ethylene glycol pathway and includes at least one exogenous nucleic acid encoding an ethylene glycol pathway enzyme expressed in a sufficient amount to produce ethylene glycol.
  • the ethylene glycol pathway of the microbial organisms of the invention is selected from: (1) 1A, IB, IC, ID, IE, IF, 1G, 1H and II (see Figure 1 and Example I and II); (2) 1A, IB, IC, ID, IE, IF, 1M, IN and II (see Figure 1 and Examples I and II); (3) 1A, IB, IC, ID, IE, IF, 1M, 10 and IP (see Figure 1 and Examples I and II); (4) 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3R and 3Q (see Figure 3 and Examples I-III); (5) 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3P and 3Q (see Figure 3 and Examples I-III); (6) 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 31 and 3M (see Figure 3 and Examples I-III); (7) 3A, 3B, 3C, 3
  • 3D is a methylenetetrahydrofolate dehydrogenase
  • 3E is a glycine cleavage complex
  • 3F is a glycine aminotransferase, amine oxidase or dehydrogenase
  • 3G is a glyoxylate carboxyligase
  • 3H is a
  • hydroxypyruvate isomerase wherein 31 is a hydroxypyruvate decarboxylase, wherein 3M is a glycolaldehyde reductase, wherein 3N is a hydroxypyruvate aminotransferase or dehydrogenase, wherein 30 is a serine decarboxylase, wherein 3P is a hydroxypyruvate reductase, wherein 3Q is a glycerate decarboxylase, wherein 3R is a tartronate
  • the non-naturally occurring microbial organism of the invention can include two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen exogenous nucleic acids each encoding an ethylene glycol pathway enzyme as disclosed herein.
  • the microbial organism can include exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(14) as disclosed above.
  • the non-naturally occurring microbial organism can further include a CO dehydrogenase, a hydrogenase or a ferredoxin oxidoreductase.
  • the non-naturally occurring microbial organism of the invention can include at least one exogenous nucleic acid encoding a CO dehydrogenase, a hydrogenase or a ferredoxin:NADPH oxidoreductase (see Figures 1 and 3 and Example I).
  • the microbial organism can include two exogenous nucleic acids, wherein each exogenous nucleic acid encodes an enzyme selected from a CO
  • the microbial organism of the invention can include three exogenous nucleic acids, wherein each of the three exogenous nucleic acids each encode one of the enzymes selected from a CO dehydrogenase, a hydrogenase and a ferredoxin oxidoreductase.
  • the invention provides a non-naturally occurring microbial organism as disclosed here, wherein the microbial organism further includes a pathway that converts glucose or another carbon source disclosed herein to an ethylene glycol pathway intermediate.
  • the microbial organism of the invention can include a pathway that converts glucose to glycine, glucose to glyoxylate, glucose to tartronate semialdehyde, glucose to hydroxypyruvate, glucose to serine or glucose to glycerate.
  • the microbial organism of the invention can include a serine pathway in combination with an ethylene glycol pathway as disclosed herein.
  • the serine pathway in some aspects, can include one or more glycolytic enzyme, a 3- phosphoglycerate dehydrogenase and a phosphoserine aminotransferase.
  • the one or more glycolytic enzyme includes the glucose PTS transport system enzymes, phosphoglucose isomerase, 6-phosphofructokinase, fructose bisphosphate aldolase, triosephosphate isomerase, or glyceraldehyde-3 -phosphate dehydrogenase.
  • the pathways for production of ethylene glycol intermediates can be endogenous to the host microorganism or engineered utilizing the compositions and methods disclosed herein. It is also understood that other compositions and methods that are well known in the art for increasing the production of an ethylene glycol intermediate can also be used in combination with the pathways disclosed herein.
  • the at least one exogenous nucleic acid included in the non-naturally occurring microbial organism is a heterologous nucleic acid.
  • the non-naturally occurring microbial organism of the invention is in a substantially anaerobic culture medium.
  • the invention provides a non-naturally occurring microorganism having at least one exogenous nucleic acid encoding an enzyme that confers to the microorganism a pathway to convert synthesis gas, also known as syngas, or other gaseous carbon source, having CO and H 2 to ethylene glycol, wherein the microorganism lacks the ability to convert CO and H 2 to ethylene glycol in the absence of the at least one exogenous nucleic acid.
  • a synthesis gas or other gas can further include C0 2 .
  • a non-naturally occurring microorganism of the invention can include a pathway that increases the efficiency of converting C0 2 , CO and/or H 2 to ethylene glycol.
  • the invention provides a non-naturally occurring microorganism including at least one exogenous nucleic acid encoding an enzyme that confers to the microorganism a pathway to convert a gaseous carbon source having C0 2 and H 2 to ethylene glycol, wherein the microorganism lacks the ability to convert C0 2 and H 2 to ethylene glycol in the absence of the at least one exogenous nucleic acid.
  • the gas can further include CO.
  • the invention also relates to a non-naturally occurring microorganism including at least one exogenous nucleic acid encoding an enzyme that confers utilization of syngas or other gas including CO and/or C0 2 as a carbon source to the microorganism, wherein the microorganism lacks the ability to utilize the carbon source in the absence of the at least one exogenous nucleic acid encoding an enzyme that confers utilization of CO and/or C0 2 .
  • the invention provides a non-naturally occurring microorganism including at least one exogenous nucleic acid encoding an enzyme that confers utilization of carbon monoxide and/or carbon dioxide as a carbon source to the microorganism, wherein the microorganism lacks the ability to utilize the carbon source in the absence of the at least one exogenous nucleic acid.
  • the invention provides a non-naturally occurring microorganism, including at least one exogenous nucleic acid encoding an enzyme that confers utilization of CO and/or C0 2 , in
  • microorganism lacks the ability to utilize the carbon source in the absence of the at least one exogenous nucleic acid.
  • the invention additionally provides a non-naturally occurring microorganism including at least one exogenous nucleic acid encoding an enzyme that confers utilization of CO, in combination with H 2 and C0 2 , as a carbon source to the microorganism, wherein the microorganism lacks the ability to utilize the carbon source in the absence of the at least one exogenous nucleic acid.
  • a microorganism can be used to produce ethylene glycol as disclosed herein.
  • Such a non- naturally occurring microorganism can express at least one exogenous nucleic acid that increases production of the product, as disclosed herein (see Figures 1-4).
  • the invention further provides a non-naturally occurring microorganism including at least one exogenous nucleic acid encoding an enzyme that confers utilization of syngas or other gaseous carbon source to the microorganism, wherein the
  • microorganism has the ability to utilize the carbon source in the absence of the at least one exogenous nucleic acid, whereby expression of the at least one exogenous proteins increases the efficiency of utilization of the carbon source.
  • the invention provides a non-naturally occurring microorganism including at least one exogenous nucleic acid encoding an enzyme that confers utilization of carbon monoxide as a carbon source to the microorganism, wherein the microorganism has the ability to utilize the carbon source in the absence of the at least one exogenous nucleic acid, whereby expression of the at least one exogenous nucleic acid increases the efficiency of utilization of the carbon source.
  • the invention provides a non-naturally occurring microorganism including at least one exogenous nucleic acid encoding an enzyme that confers utilization of CO and/or C0 2 , in combination with H 2 , as a carbon source to the microorganism, wherein the microorganism has the ability to utilize the carbon source in the absence of the at least one exogenous nucleic acid, whereby expression of the at least one exogenous proteins increases the efficiency of utilization of the carbon source.
  • a non-naturally occurring microorganism including at least one exogenous nucleic acid encoding an enzyme that confers utilization of CO, in combination with H 2 and C0 2 , as a carbon source to the microorganism, wherein the microorganism has the ability to utilize the carbon source in the absence of the at least one exogenous exogenous nucleic acid, whereby expression of the at least one exogenous nucleic acid increases the efficiency of utilization of the carbon source.
  • a microorganism can be used to produce a desired product such as ethylene glycol from the carbon source, as disclosed herein.
  • the invention also provides a non-naturally occurring microbial organism capable of producing ethylene glycol utilizing methanol and/or syngas.
  • the microbial organism of the invention is capable of utilizing methanol, methanol and CO, C0 2 and/or H 2 , for example, C0 2 , C0 2 and H 2 , CO, CO and H 2 , C0 2 and CO, or C0 2 , CO and H 2 , to produce ethylene glycol.
  • the microbial organism is engineered to utilize methanol and/or syngas to produce ethylene glycol (see Examples I- III and VII).
  • the invention provides a non-naturally occurring microbial organism having an ethylene glycol pathway comprising at least one exogenous nucleic acid encoding an ethylene glycol pathway enzyme or protein expressed in a sufficient amount to produce ethylene glycol, the ethylene glycol pathway including a formaldehyde dehydrogenase and a methanol dehydrogenase.
  • the ethylene glycol pathway can confer the ability to convert methanol, C0 2 , CO and/or H 2 , or a combination thereof, to ethylene glycol.
  • the invention provides a non-naturally occurring microbial organism having an ethylene glycol pathway, wherein the non-naturally occurring microbial organism includes at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of CO to 2[H], H 2 to 2[H], 2[H] to NAD(P)H, C0 2 to formate, formate to formyl-THF, formyl-THF to methylene -THF, methylene-THF to glycine, glycine to serine, serine to ethanolamine, ethanolamine to glycolaldehyde, glycolaldehyde to ethylene glycol, serine to hydroxypyruvate, hydroxypyruvate to glycolaldehyde, hydroxypyruvate to glycerate, glycerate to ethylene glycol, glycine to glyoxylate, glyoxylate to tar
  • the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of an ethylene glycol pathway, such as that shown in Figures 1-4.
  • ethylene glycol pathway While generally described herein as a microbial organism that contains an ethylene glycol pathway, it is understood that the invention additionally provides a non- naturally occurring microbial organism including at least one exogenous nucleic acid encoding an ethylene glycol pathway enzyme expressed in a sufficient amount to produce an intermediate of an ethylene glycol pathway.
  • an ethylene glycol pathway is exemplified in Figures 1-4.
  • the invention additionally provides a non-naturally occurring microbial organism including at least one exogenous nucleic acid encoding an ethylene glycol pathway enzyme, where the microbial organism produces an ethylene glycol pathway intermediate, for example, formate, formyl-THF, methenyl-THF, methylene-THF, serine, ethanolamine, glycolaldehyde, hydroxypyruvate, glycerate, glyoxylate or tartronate semialdehyde.
  • a non-naturally occurring microbial organism including at least one exogenous nucleic acid encoding an ethylene glycol pathway enzyme, where the microbial organism produces an ethylene glycol pathway intermediate, for example, formate, formyl-THF, methenyl-THF, methylene-THF, serine, ethanolamine, glycolaldehyde, hydroxypyruvate, glycerate, glyoxylate or tartronate semialdehyde.
  • any of the pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of Figures 1-4, can be utilized to generate a non-naturally occurring microbial organism that produces any pathway intermediate or product, as desired.
  • a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to produce a desired product.
  • a non-naturally occurring microbial organism that produces an ethylene glycol pathway intermediate can be utilized to produce the intermediate as a desired product.
  • the invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction.
  • reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product.
  • reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.
  • the ethylene glycol pathway intermediates glycine, glyoxylate, hydroxypyruvate and glycerate, as well as other intermediates are carboxylic acids, which can occur in various ionized forms, including fully protonated, partially protonated, and fully deprotonated forms. Accordingly, the suffix "-ate,” or the acid form, can be used interchangeably to describe both the free acid form as well as any
  • carboxylate products or intermediates includes ester forms of carboxylate products or pathway intermediates, such as O- carboxylate and S-carboxylate esters.
  • O- and S-carboxylates can include lower alkyl, that is CI to C6, branched or straight chain carboxylates.
  • O- or S-carboxylates include, without limitation, methyl, ethyl, n-propyl, n-butyl, i-propyl, sec-butyl, and tert- butyl, pentyl, hexyl O- or S-carboxylates, any of which can further possess an
  • O-carboxylates can be the product of a biosynthetic pathway.
  • Exemplary O-carboxylates accessed via biosynthetic pathways can include, without limitation, methyl glyoxylate, ethyl glyoxylate, and n-propyl glyoxylate.
  • O-carboxylates can include medium to long chain groups, that is C7-C22, O-carboxylate esters derived from fatty alcohols, such heptyl, octyl, nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, and behenyl alcohols, any one of which can be optionally branched and/or contain unsaturations.
  • O-carboxylate esters can also be accessed via a biochemical or chemical process, such as esterification of a free carboxylic acid product or
  • 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 ethylene glycol biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular ethylene glycol biosynthetic pathway can be expressed.
  • a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression.
  • the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve ethylene glycol biosynthesis.
  • a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as ethylene glycol.
  • Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable or suitable to fermentation processes.
  • Exemplary bacteria include any species selected from the order Enterobacteriales, family Enter obacteriaceae, including the genera Escherichia and Klebsiella; the order
  • Aeromonadales family Succinivibrionaceae, including the genus Anaerobio spirillum; the order Pasteur ellales, family Pasteur ellaceae, including the genera Actinobacillus and Mannheimia; the order Rhizobiales, family Bradyrhizobiaceae, including the genus Rhizobium; the order Bacillales, family Bacillaceae, including the genus Bacillus; the order Actinomycetales, families Corynebacteriaceae and Streptomycetaceae, including the genus Corynebacterium and the genus Streptomyces, respectively; order Rhodospirillales, family Acetobacteraceae, including the genus Gluconobacter; the order
  • Sphingomonadales family Sphingomonadaceae, including the genus Zymomonas
  • the order Lactobacillales families Lactobacillaceae and Streptococcaceae, including the genus Lactobacillus and the genus Lactococcus, respectively
  • the order Clostridiales family Clostridiaceae, genus Clostridium
  • the order Pseudomonadales family Pseudomonadaceae, including the genus Pseudomonas.
  • Non-limiting species of host bacteria include 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 species of yeast or fungi species include any species selected from the order Saccharomycetales, family Saccaromycetaceae, including the genera Saccharomyces, Kluyveromyces and Pichia; the order Saccharomycetales, family Dipodascaceae, including the genus Yarrowia; the order Schizosaccharomycetales, family Schizosaccaromycetaceae, including the genus Schizosaccharomyces; the order
  • Non-limiting species of host yeast or fungi include Saccharomyces cerevisiae, Schizosaccharomyces pombe,
  • 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.
  • the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed ethylene glycol pathway- encoding nucleic acid and up to all encoding nucleic acids for one or more ethylene glycol biosynthetic pathways.
  • ethylene glycol biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid.
  • exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins.
  • exogenous expression of all enzymes or proteins in a pathway for production of ethylene glycol can be included, such as a formate dehydrogenase; a
  • formyltetrahydrofolate synthetase a methenyltetrahydrofolate cyclohydrolase; a methylenetetrahydrofolate dehydrogenase; a glycine cleavage complex; a serine hydroxymethyltransferase; a serine decarboxylase; an ethanolamine aminotransferase, amine oxidase or dehydrogenase; and a glycolaldehyde reductase.
  • a non-naturally occurring microbial organism of the invention can have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen, up to all nucleic acids encoding the enzymes or proteins constituting an ethylene glycol biosynthetic pathway disclosed herein.
  • the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize ethylene glycol 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 ethylene glycol pathway intermediates such as glycine, glyoxylate, tartonate semialdehyde, hydroxypyruvate, serine, glycerate, glycolaldehyde or ethanolamine.
  • the non-naturally occurring microbial organism of the invention can include one or more exogenous nucleic acids encoding an enzyme that facilitates or optimizes the production of glycine, glyoxylate, tartonate semialdehyde, hydroxypyruvate, serine, glycerate, glycolaldehyde or
  • the microorganisms of the invention can include an exogenous nucleic acid encoding one or more glycolytic enzymes, such as the glucose PTS transport system enzymes, phosphoglucose isomerase, 6-phosphofructokinase, fructose bisphosphate aldolase, triosephosphate isomerase, or glyceraldehyde-3 -phosphate dehydrogenase, a 3-phosphoglycerate dehydrogenase, a phosphoserine aminotransferase or a phosphoserine phosphatase.
  • glycolytic enzymes such as the glucose PTS transport system enzymes, phosphoglucose isomerase, 6-phosphofructokinase, fructose bisphosphate aldolase, triosephosphate isomerase, or glyceraldehyde-3 -phosphate dehydrogenase, a 3-phosphoglycerate dehydrogenase, a phosphoserine aminotransferase
  • a host microbial organism is selected such that it produces a precursor or an intermediate of an ethylene glycol pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or intermediate or increased production of a precursor or an intermediate naturally produced by the host microbial organism.
  • glycine, glyoxylate, tartonate semialdehyde, hydroxypyruvate and serine are produced naturally in a host organism such as E. coli.
  • a host organism can be engineered to increase production of a precursor, as disclosed herein.
  • a microbial organism that has been engineered to produce a desired precursor or intermediate can be used as a host organism and further engineered to express enzymes or proteins of an ethylene glycol pathway.
  • a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize ethylene glycol.
  • it can be useful to increase the synthesis or accumulation of an ethylene glycol pathway product to, for example, drive ethylene glycol pathway reactions toward ethylene glycol production. Increased synthesis or
  • accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described ethylene glycol pathway enzymes or proteins.
  • Overexpression of the enzyme or enzymes and/or protein or proteins of the ethylene glycol 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 ethylene glycol, through overexpression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, that is, up to all nucleic acids encoding ethylene glycol biosynthetic pathway enzymes or proteins.
  • a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the ethylene glycol biosynthetic pathway.
  • exogenous expression of the encoding nucleic acids is employed.
  • Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user.
  • endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element.
  • an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time.
  • an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.
  • any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non- naturally occurring microbial organism of the invention.
  • the nucleic acids can be introduced so as to confer, for example, an ethylene glycol biosynthetic pathway onto the microbial organism.
  • encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer ethylene glycol biosynthetic capability.
  • a non-naturally occurring microbial organism having an ethylene glycol biosynthetic pathway can include at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the combination of a formate dehydrogenase and a serine
  • hydroxymethyltransferase or alternatively a hydroxypyruvate decarboxylase and a glycolaldehyde reductase, or alternatively a hydroxypyruvate isomerase and a ferredoxin oxidoreductase, and the like.
  • a hydroxymethyltransferase or alternatively a hydroxypyruvate decarboxylase and a glycolaldehyde reductase, or alternatively a hydroxypyruvate isomerase and a ferredoxin oxidoreductase, and the like.
  • any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, a serine decarboxylase, an ethanolamine aminotransferase and a glycolaldehyde reductase, or alternatively a glycine aminotransferase, a glyoxylate carboxyligase and a tartronate semialdehyde reductase, or alternatively a glycolaldehyde reductase, CO dehydrogenase or a ferredoxin oxidoreductase, 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.
  • a serine decarboxylase an ethanolamine aminotransferase and a glycolaldehyde reductase
  • a glycine aminotransferase
  • any combination of four such as a glycine cleavage complex, a serine aminotransferase, a hydroxypyruvate reductase and a glycerate decarboxylase, or alternatively a formate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase, a hydroxypyruvate isomerase and a glycerate decarboxylase, or alternatively a serine decarboxylase, an ethanolamine aminotransferase, CO
  • dehydrogenase and a hydrogenase 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.
  • the non- naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and/or with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes.
  • one alternative to produce ethylene glycol other than use of the ethylene glycol producers is through addition of another microbial organism capable of converting an ethylene glycol pathway intermediate to ethylene glycol.
  • One such procedure includes, for example, the fermentation of a microbial organism that produces an ethylene glycol pathway intermediate.
  • the ethylene glycol pathway intermediate can then be used as a substrate for a second microbial organism that converts the ethylene glycol pathway intermediate to ethylene glycol.
  • the ethylene glycol pathway intermediate can be added directly to another culture of the second organism or the original culture of the ethylene glycol pathway intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps.
  • the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, ethylene glycol.
  • biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product.
  • the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized.
  • the biosynthesis of ethylene glycol 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.
  • ethylene glycol 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 ethylene glycol intermediate and the second microbial organism converts the intermediate to ethylene glycol.
  • Sources of encoding nucleic acids for an ethylene glycol 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, Achromobacter denitrificans, Acinetobacter sp.
  • Strain M-l Agrobacterium tumefaciens, Allochromatium vinosum DSM 180, Arabidopsis thaliana, Azotobacter vinelandii DJ, Bacillus brevis, Bacillus methanolicus, Bacillus subtilis, Beta vulgaris, Brassica napus, Burkholderia ambifaria, Campylobacter curvus, Campylobacter jejuni, Candida boidinii, Carboxydothermus hydrogenoformans, Chlorobium phaeobacteroides, Clostridium acidurici, Clostridium carboxidivorans P7, Clostridium cellulolyticum H10, Clostridium kluyveri DSM 555, Clostridium ljungdahli, Clostridium pasteurianum, Desulfovibrio desulfuricans subsp.
  • desulfuricans Drosophila melanogaster, Enterobacter aerogenes, Enterococcus gallinarum, Escherichia coli K-12, Geobacillus kaustophilus, Geobacillus stearothermophilus, Geobacter sulfurreducens, Halobacterium salinarum, Helicobacter pylori, Homo sapiens, Hydrogenobacter thermophilus, Hyphomicrobium methylovorum, Hyphomicrobium zavarzinii, Lactococcus lactis, Lysinibacillus fusiformis, Lysinibacillus sphaericus, Methylobacter marinus, Methylobacterium extorquens, Moorella
  • thermoacetica Mus musculus, Nostoc sp. PCC 7120, Pelobacter carbinolicus, Pichia pastoris, Pseudomonas aeruginosa PA01, Pseudonocardia dioxanivorans, Pseudomonas putida, Ralstonia eutropha, Ralstonia eutropha HI 6, Rattus norvegicus, Rhodobacter capsulatus, Rhodopseudomonas palustris, Rhodopseudomonas palustris CGA009, Rhodospirillum rubrum, Saccharomyces cerevisiae, Salmonella enterica, Salmonella typhimurium, Streptococcus thermophilus, Sulfolobus acidocalarius, Sus scrofa,
  • Synechocystis str. PCC 6803 Syntrophobacter fumaroxidans, Thauera aromatica, Thermotoga maritima, Thiocapsa roseopersicina, Zymomonas mobilis, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes.
  • PCC 6803 Syntrophobacter fumaroxidans, Thauera aromatica, Thermotoga maritima, Thiocapsa roseopersicina, Zymomonas mobilis, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes.
  • the complete genome sequence available for now more than 550 species including 395 microorganism genomes and a variety of yeast, fungi, plant, and
  • ethylene glycol biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ.
  • teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize ethylene glycol.
  • Methods for constructing and testing the expression levels of a non-naturally occurring ethylene glycol-producing host can be performed, for example, by recombinant and detection methods well known in the art.
  • Exogenous nucleic acid sequences involved in a pathway for production of ethylene glycol 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.
  • 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.
  • targeting signals such as an N-terminal mitochondrial or other targeting signal
  • genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells.
  • a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells.
  • An expression vector or vectors can be constructed to include one or more ethylene glycol biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism.
  • Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.
  • both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • the transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art.
  • 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.
  • PCR polymerase chain reaction
  • 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 ethylene glycol that includes culturing a non-naturally occurring microbial organism as disclosed herein under conditions and for a sufficient period of time to produce ethylene glycol.
  • the invention provide a method for producing ethylene glycol that includes culturing a non-naturally occurring microbial organism having an ethylene glycol pathway and at least one exogenous nucleic acid encoding an ethylene glycol pathway enzyme expressed in a sufficient amount to produce ethylene glycol.
  • the ethylene glycol pathway of the microbial organism used in the method of the invention is selected from: (1) 1A, IB, IC, ID, IE, IF, 1G, 1H and II (see Figure 1 and Example I and II); (2) 1A, IB, IC, ID, IE, IF, IM, IN and II (see
  • ID is a methylenetetrahydro folate dehydrogenase
  • IE is a glycine cleavage complex
  • IF is a serine hydroxymethyltransferase
  • 1G is a serine decarboxylase
  • 1H is an ethanolamine aminotransferase, an amine oxidase or a dehydrogenase
  • II is a glycolaldehyde reductase
  • IM is a serine aminotransferase, amine oxidase or dehydrogenase
  • IN is a hydroxypyruvate decarboxylase
  • 10 is a hydroxypyruvate reductase
  • IP a glycerate decarboxylase
  • 3A is a formate dehydrogenase
  • 3B is a glycerate decarboxylase
  • 3D is a methylenetetrahydrofolate dehydrogenase
  • 3E is a glycine cleavage complex
  • 3F is a glycine aminotransferase, amine oxidase or dehydrogenase
  • 3G is a glyoxylate carboxyligase
  • 3H is a
  • hydroxypyruvate isomerase wherein 31 is a hydroxypyruvate decarboxylase, wherein 3M is a glycolaldehyde reductase, wherein 3N is a hydroxypyruvate aminotransferase or dehydrogenase, wherein 30 is a serine decarboxylase, wherein 3P is a hydroxypyruvate reductase, wherein 3Q is a glycerate decarboxylase, wherein 3R is a tartronate
  • the non-naturally occurring microbial organism used in the method of the invention includes two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen exogenous nucleic acids each encoding an ethylene glycol pathway enzyme as disclosed herein.
  • the microbial organism can include exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(14) as disclosed above.
  • the non-naturally occurring microbial organism used in the method of the invention can further include a CO dehydrogenase, a
  • the non- naturally occurring microbial organism of the invention can include at least one exogenous nucleic acid encoding a CO dehydrogenase, a hydrogenase or a ferredoxin :NADPH oxidoreductase (see Figures 1 and 3 and Example I).
  • the microbial organism used in the method of the invention can include two exogenous nucleic acids, wherein each exogenous nucleic acid encodes an enzyme selected from a CO
  • the microbial organism used in the method of the invention can include three exogenous nucleic acids, wherein each of the three exogenous nucleic acids each encode one of the enzymes selected from a CO dehydrogenase, a hydrogenase and a ferredoxin
  • the invention provides a method for producing ethylene glycol that includes culturing a non-naturally occurring microbial organism, wherein the microbial organism further includes a pathway that converts glucose or another carbon source disclosed herein to an ethylene glycol pathway intermediate.
  • the microbial organism used in the method of the invention can include a pathway that converts glucose to glycine, glucose to glyoxylate, glucose to tartronate semialdehyde, glucose to hydroxypyruvate, glucose to serine or glucose to glycerate.
  • the microbial organism used in the method of the invention can include a serine pathway in combination with an ethylene glycol pathway as disclosed herein.
  • the serine pathway in some aspects, can include one or more glycolytic enzyme, a 3- phosphoglycerate dehydrogenase and a phosphoserine aminotransferase.
  • the one or more glycolytic enzyme includes the glucose PTS transport system enzymes, phosphoglucose isomerase, 6-phosphofructokinase, fructose bisphosphate aldolase, triosephosphate isomerase, or glyceraldehyde-3 -phosphate dehydrogenase.
  • the at least one exogenous nucleic acid included in the non-naturally occurring microbial organism used in the method of the invention is a heterologous nucleic acid.
  • the non-naturally occurring microbial organism used in the method of the invention is in a substantially anaerobic culture medium.
  • the ethylene glycol can be separated from other components in the culture using a variety of methods well known in the art.
  • separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.
  • any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention.
  • the ethylene glycol producers can be cultured for the biosynthetic production of ethylene glycol.
  • the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp- cap.
  • microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration.
  • Exemplary anaerobic conditions have been described previously and are well-known in the art.
  • Exemplary aerobic and anaerobic conditions are described, for example, in United State publication 2009/0047719, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein.
  • the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH.
  • the growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.
  • the growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism.
  • Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch.
  • Other sources of carbohydrate include, for example, renewable feedstocks and biomass.
  • Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass,
  • biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
  • carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
  • renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of ethylene glycol.
  • Yet another carbon source that can be included in the growth medium is glycerol. It is also understood that a carbon source can be used alone as the sole source of carbon or in combination with other carbon sources described herein or known in the art.
  • the ethylene glycol microbial organisms of the invention also can be modified for growth on syngas as its source of carbon.
  • one or more proteins or enzymes are expressed in the ethylene glycol producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.
  • Synthesis gas also known as syngas or producer gas
  • syngas is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues.
  • Syngas is a mixture primarily of H 2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H 2 and CO, syngas can also include C0 2 and other gases in smaller quantities.
  • synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, C0 2 .
  • gaseous carbon sources such as syngas including CO and/or C0 2 can be utilized by non-naturally occurring microorganisms of the invention to produce ethylene glycol.
  • syngas gaseous carbon sources
  • any source of gaseous carbon including CO and/or C0 2 can be utilized by the non-naturally occurring microorganisms of the invention.
  • the invention relates to non-naturally occurring microorganisms that are capable of utilizing CO and/or C0 2 as a carbon source.
  • the non-naturally occurring microorganisms of the invention can use syngas or other gaseous carbon sources providing CO and/or C0 2 to produce ethylene glycol.
  • additional sources include, but are not limited to, production of C0 2 as a byproduct in ammonia and hydrogen plants, where methane is converted to C0 2 ; combustion of wood and fossil fuels; production of C0 2 as a byproduct of fermentation of sugar in the brewing of beer, whisky and other alcoholic beverages, or other fermentative processes; thermal decomposition of limestone, CaC0 3 , in the manufacture of lime, CaO; production of C0 2 as byproduct of sodium phosphate manufacture; and directly from natural carbon dioxide springs, where it is produced by the action of acidified water on limestone or dolomite.
  • a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate.
  • Such compounds include, for example, ethylene glycol and any of the intermediate metabolites in the ethylene glycol 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 ethylene glycol biosynthetic pathways.
  • the invention provides a non-naturally occurring microbial organism that produces and/or secretes ethylene glycol when gro w n on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the ethylene glycol pathway when grown on a carbohydrate or other carbon source.
  • the ethylene glycol producing microbial organisms of the invention can initiate synthesis from an intermediate, for example, formate, formyl-THF, methenyl-THF, methylene-THF, glycine, glyoxylate, tartonate semialdehyde, hydroxypyruvate, serine, glycerate, glycolaldehyde or ethanolamine.
  • 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 ethylene glycol pathway enzyme or protein in sufficient amounts to produce ethylene glycol. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce ethylene glycol. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of ethylene glycol resulting in intracellular concentrations between about 0.1-2000 mM or more.
  • the intracellular concentration of ethylene glycol is between about 3-1500 mM, particularly between about 5-1250 mM and more particularly between about 8-1000 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.
  • 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.
  • ethylene glycol producing microbial organisms can produce ethylene glycol intracellularly and/or secrete the product into the culture medium.
  • growth condition for achieving biosynthesis of ethylene glycol can include the addition of an osmoprotectant to the culturing conditions.
  • the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant.
  • an osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress.
  • Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose.
  • Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2- methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine.
  • the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used.
  • the amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about lOmM, no more than about 50mM, no more than about lOOmM or no more than about 500mM.
  • the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in ethylene glycol or any ethylene glycol pathway intermediate.
  • uptake sources can provide isotopic enrichment for any atom present in the product ethylene glycol or ethylene glycol pathway intermediate, or for side products generated in reactions diverging away from an ethylene glycol pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.
  • the uptake sources can be selected to alter the carbon- 12, carbon-13, and carbon-14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can be selected to alter the nitrogen- 14 and nitrogen- 15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31 , phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios.
  • the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources.
  • An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom.
  • An example of a man-made uptake source includes, for example, an uptake source that is at least partially derived from a chemical synthetic reaction.
  • Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to achieve a desired isotopic ratio.
  • a target atom isotopic ratio of an uptake source can be achieved by selecting a desired origin of the uptake source as found in nature.
  • a natural source can be a biobased derived from or synthesized by a biological organism or a source such as petroleum-based products or the atmosphere.
  • a source of carbon for example, can be selected from a fossil fuel-derived carbon source, which can be relatively depleted of carbon- 14, or an environmental or atmospheric carbon source, such as C0 2 , which can possess a larger amount of carbon- 14 than its petroleum-derived counterpart.
  • the unstable carbon isotope carbon- 14 or radiocarbon makes up for roughly 1 in 10 12 carbon atoms in the earth's atmosphere and has a half-life of about 5700 years.
  • the stock of carbon is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen ( 14 N).
  • Fossil fuels contain no carbon- 14, as it decayed long ago. Burning of fossil fuels lowers the atmospheric carbon- 14 fraction, the so-called "Suess effect".
  • Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as accelerated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR).
  • AMS accelerated mass spectrometry
  • SIRMS Stable Isotope Ratio Mass Spectrometry
  • SNIF-NMR Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance
  • mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC), high performance liquid chromatography (HPLC) and/or gas chromatography, and the like.
  • ASTM D6866 was developed in the United States as a standardized analytical method for determining the biobased content of solid, liquid, and gaseous samples using radiocarbon dating by the American Society for Testing and Materials (ASTM) International. The standard is based on the use of radiocarbon dating for the determination of a product's biobased content. ASTM D6866 was first published in 2004, and the current active version of the standard is ASTM D6866-11 (effective April 1, 2011). Radiocarbon dating techniques are well known to those skilled in the art, including those described herein.
  • the biobased content of a compound is estimated by the ratio of carbon- 14 ( 14 C) to carbon-12 ( 12 C).
  • Fraction Modern is a measurement of the deviation of the 14 C/ 12 C ratio of a sample from "Modern.” Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard reference materials (SRM) 4990b) normalized to
  • An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially available.
  • the Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933 ⁇ 0.001 (the weighted mean). The isotopic ratio of HOx II is - 17.8 per mil.
  • ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)).
  • a Fm 0% represents the entire lack of carbon- 14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source.
  • a Fm 100%, after correction for the post- 1950 injection of carbon- 14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a "modern" source includes biobased sources.
  • the percent modern carbon can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon- 14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon- 14 activities are referenced to a "pre-bomb" standard, and because nearly all new biobased products are produced in a post-bomb environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old.
  • polypropylene terephthalate (PPT) polymers derived from renewable 1,3-propanediol and petroleum-derived terephthalic acid resulted in Fm values near 30% (i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) (Currie et al., supra, 2000).
  • polybutylene terephthalate polymer derived from both renewable 1 ,4-butanediol and renewable terephthalic acid resulted in bio-based content exceeding 90%> (Colonna et al, supra, 2011).
  • the present invention provides ethylene glycol or an ethylene glycol intermediate that has a carbon- 12, carbon- 13, and carbon-14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source.
  • the ethylene glycol or an ethylene glycol intermediate can have an Fm value of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%> or as much as 100%.
  • the uptake source is C0 2 .
  • the present invention provides ethylene glycol or an ethylene glycol intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that reflects petroleum-based carbon uptake source.
  • the ethylene glycol or an ethylene glycol intermediate can have an Fm value of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%), less than 25%, less than 20%>, less than 15%, less than 10%, less than 5%, less than 2% or less than 1%.
  • the present invention provides ethylene glycol or an ethylene glycol intermediate that has a carbon- 12, carbon-13, and carbon- 14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source.
  • a combination of uptake sources is one way by which the carbon- 12, carbon-13, and carbon- 14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources.
  • the present invention relates to the biologically produced ethylene glycol or ethylene glycol intermediate as disclosed herein, and to the products derived therefrom, wherein the ethylene glycol or an ethylene glycol intermediate has a carbon- 12, carbon-13, and carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment.
  • the invention provides bioderived ethylene glycol or a bioderived ethylene glycol intermediate having a carbon- 12 versus carbon-13 versus carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment, or any of the other ratios disclosed herein.
  • a product can have a carbon- 12 versus carbon-13 versus carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived ethylene glycol or a bioderived ethylene glycol intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product.
  • Methods of chemically modifying a bioderived product of ethylene glycol, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein.
  • the invention further provides antifreezes, coolants, polyester fibers, fiberglass, resins or films having a carbon- 12 versus carbon-13 versus carbon- 14 isotope ratio of about the same value as the C0 2 that occurs in the environment, wherein the antifreezes, coolants, polyester fibers, fiberglass, resins or films are generated directly from or in combination with bioderived ethylene glycol or a bioderived ethylene glycol intermediate as disclosed herein.
  • Ethylene glycol is a chemical commonly used in many commercial and industrial applications. Non-limiting examples of such applications include production of antifreezes and coolants. Moreover, ethylene glycol is also used as a raw material in the production of a wide range of products including polyester fibers for clothes, upholstery, carpet and pillows; fiberglass used in products such as jet skis, bathtubs, and bowling balls; and polyethylene terephthalate resin used in packaging film and bottles. Around 82% of ethylene glycol consumed worldwide is used in the production of polyester fibers, resins and films. The second largest market for ethylene glycol is in the production of antifreeze formulations.
  • the invention provides biobased antifreezes, coolants, polyester fibers, fiberglass, resins or films including one or more bioderived ethylene glycol or bioderived ethylene glycol intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.
  • bioderived means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism.
  • a biological organism in particular the microbial organisms of the invention disclosed herein, can utilize feedstock or biomass, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source.
  • the biological organism can utilize atmospheric carbon.
  • biobased means a product as described above that is composed, in whole or in part, of a bioderived compound of the invention.
  • a biobased or bioderived product is in contrast to a petroleum derived product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock.
  • the invention provides an antifreeze, coolant, polyester fiber, fiberglass, resin or film including bioderived ethylene glycol or bioderived ethylene glycol intermediate, wherein the bioderived ethylene glycol or bioderived ethylene glycol intermediate includes all or part of the ethylene glycol or ethylene glycol intermediate used in the production of the antifreeze, coolant, polyester fiber, fiberglass, resin or film.
  • the invention provides a biobased antifreeze, coolant, polyester fiber, fiberglass, resin or film including at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100%) bioderived ethylene glycol or bioderived ethylene glycol intermediate as disclosed herein. Additionally, in some aspects, the invention provides a biobased antifreeze, coolant, polyester fiber, fiberglass, resin or film wherein the ethylene glycol or ethylene glycol intermediate used in its production is a combination of bioderived and petroleum derived ethylene glycol or ethylene glycol intermediate.
  • a biobased antifreeze, coolant, polyester fiber, fiberglass, resin or film can be produced using 50%> bioderived ethylene glycol and 50%> petroleum derived ethylene glycol or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product includes a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing antifreeze, coolant, polyester fiber, fiberglass, resin or film using the bioderived ethylene glycol or bioderived ethylene glycol intermediate of the invention are well known in the art.
  • the culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.
  • one exemplary growth condition for achieving biosynthesis of ethylene glycol includes anaerobic culture or fermentation conditions.
  • the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions.
  • an anaerobic condition 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 N 2 /C0 2 mixture or other suitable non-oxygen gas or gases.
  • the culture conditions described herein can be scaled up and grown continuously for manufacturing of ethylene glycol.
  • 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 ethylene glycol.
  • the continuous and/or near-continuous production of ethylene glycol will include culturing a non-naturally occurring ethylene glycol producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase.
  • Continuous culture under such conditions can include, for example, growth or culturing for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.
  • Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of ethylene glycol can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.
  • the ethylene glycol producers of the invention also can be, for example, simultaneously subjected to chemical synthesis and/or enzymatic procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical and/or enzymatic conversion to convert the product to other compounds, if desired.
  • metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US
  • Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of ethylene glycol.
  • OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable microorganisms which overproduce the target product.
  • the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth.
  • biochemical production 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.
  • 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.
  • 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.
  • FBA flux balance analysis
  • OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the
  • OptKnock computational framework allows the construction of model formulations that allow an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems.
  • the metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. publication 2002/0168654, filed January 10, 2002, in International Patent No. PCT/US02/00660, filed January 10, 2002, and U.S. publication 2009/0047719, filed August 10, 2007.
  • SimPheny® 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 June 14, 2002, and in International Patent Application No. PCT/US03/18838, filed June 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.
  • constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions.
  • the space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.
  • metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration of the invention, some methods are described herein with reference to the OptKnock
  • a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes.
  • the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set.
  • One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene.
  • 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.
  • 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.
  • integer cuts an optimization method, termed integer cuts. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions.
  • the methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®.
  • the set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.
  • the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum-growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism's ability to self-optimize under selective pressures.
  • the OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry.
  • the identification of optimal gene/reaction knockouts requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the resulting network
  • 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
  • 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.
  • a nucleic acid encoding a desired activity of an ethylene glycol pathway can be introduced into a host organism.
  • it can be desirable to modify an activity of an ethylene glycol pathway enzyme or protein to increase production of ethylene glycol.
  • known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule.
  • optimization methods can be applied to increase the activity of an enzyme or protein and/or decrease an inhibitory activity, for example, decrease the activity of a negative regulator.
  • Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identife ⁇ i through the development and implementation of sensitive high- throughput screening assays that allow the automated screening of many enzyme variants (for example, >10 4 ). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened.
  • selectivity/specificity for conversion of non-natural substrates
  • temperature stability for robust high temperature processing
  • pH stability for bioprocessing under lower or higher pH conditions
  • substrate or product tolerance so that high product titers can be achieved
  • binding (K m ) including broadening substrate binding to include non-natural substrates
  • inhibition (K;) to remove inhibition by products, substrates, or key intermediates
  • activity (kcat) to increases enzymatic reaction rates to achieve desired flux
  • expression levels to increase protein yields and overall pathway flux
  • oxygen stability for operation of air sensitive enzymes under aerobic conditions
  • anaerobic activity for operation of an aerobic enzyme in the absence of oxygen.
  • EpPCR (Pritchard et al, J Theor.Biol. 234:497-509 (2005)) introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions by the addition of Mn 2+ ions, by biasing dNTP concentrations, or by other conditional variations.
  • the five step cloning process to confine the mutagenesis to the target gene of interest involves: 1) error-prone PCR amplification of the gene of interest; 2) restriction enzyme digestion; 3) gel purification of the desired DNA fragment; 4) ligation into a vector; 5) transformation of the gene variants into a suitable host and screening of the library for improved performance.
  • This method can generate multiple mutations in a single gene simultaneously, which can be useful to screen a larger number of potential variants having a desired activity.
  • a high number of mutants can be generated by EpPCR, so a high- throughput screening assay or a selection method, for example, using robotics, is useful to identify those with desirable characteristics.
  • Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., Nucleic Acids Res. 32:el45 (2004); and Fujii et al, Nat. Protoc. 1 :2493-2497 (2006)) has many of the same elements as epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats. Adjusting the Mn 2+ concentration can vary the mutation rate somewhat.
  • DNA or Family Shuffling (Stemmer, Proc Natl Acad Sci USA 91 : 10747- 10751 (1994)); and Stemmer, Nature 370:389-391 (1994)) typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes.
  • nucleases such as Dnase I or EndoV
  • Fragments prime each other and recombination occurs when one copy primes another copy (template switch).
  • This method can be used with >lkbp DNA sequences.
  • this method introduces point mutations in the extension steps at a rate similar to error-prone PCR. The method can be used to remove deleterious, random and neutral mutations.
  • Staggered Extension (StEP) (Zhao et al, Nat. Biotechnol. 16:258-261 (1998)) entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec). Growing fragments anneal to different templates and extend further, which is repeated until full-length sequences are made. Template switching means most resulting fragments have multiple parents.
  • Combinations of low- fidelity polymerases reduce error-prone biases because of opposite mutational spectra.
  • Random Priming Recombination random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al, Nucleic Acids Res 26:681-683 (1998)). Base misincorporation and mispriming via epPCR give point mutations. Short DNA fragments prime one another based on homology and are recombined and reassembled into full-length by repeated thermocycling. Removal of templates prior to this step assures low parental recombinants. This method, like most others, can be performed over multiple iterations to evolve distinct properties. This technology avoids sequence bias, is independent of gene length, and requires very little parent DNA for the application.
  • ssDNA single stranded DNA
  • ssDNA scaffold Any overlapping unhybridized fragment ends are trimmed down by an exonuclease. Gaps between fragments are filled in and then ligated to give a pool of full-length diverse strands hybridized to the scaffold, which contains U to preclude amplification. The scaffold then is destroyed and is replaced by a new strand complementary to the diverse strand by PCR amplification.
  • the method involves one strand (scaffold) that is from only one parent while the priming fragments derive from other genes, and the parent scaffold is selected against. Thus, no reannealing with parental fragments occurs. Overlapping fragments are trimmed with an exonuclease. Otherwise, this is conceptually similar to DNA shuffling and StEP. Therefore, there should be no siblings, few inactives, and no unshuffled parentals. This technique has advantages in that few or no parental genes are created and many more crossovers can result relative to standard DNA shuffling.
  • Incremental Truncation for the Creation of Hybrid Enzymes creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest
  • THIO- ITCHY Thio-Incremental Truncation for the Creation of Hybrid Enzymes
  • ITCHY Thio-Incremental Truncation for the Creation of Hybrid Enzymes
  • SCRATCHY combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al, Proc. Natl. Acad. Sci. USA 98: 11248-11253 (2001)). SCRATCHY combines the best features of ITCHY and DNA shuffling. First, ITCHY is used to create a comprehensive set of fusions between fragments of genes in a DNA homology- independent fashion. This artificial family is then subjected to a DNA-shuffling step to augment the number of crossovers. Computational predictions can be used in optimization. SCRATCHY is more effective than DNA shuffling when sequence identity is below 80%.
  • RNDM Random Drift Mutagenesis
  • Sequence Saturation Mutagenesis is a random mutagenesis method that: 1) generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage; this pool is used as a template to 2) extend in the presence of "universal" bases such as inosine; 3) replication of an inosine-containing complement gives random base incorporation and, consequently, mutagenesis (Wong et al, Biotechnol. J. 3:74-82 (2008); Wong et al, Nucleic Acids Res. 32:e26 (2004); and
  • overlapping oligonucleotides are designed to encode "all genetic diversity in targets" and allow a very high diversity for the shuffled progeny (Ness et al, Nat. Biotechnol. 20: 1251-1255 (2002)).
  • this technique one can design the fragments to be shuffled. This aids in increasing the resulting diversity of the progeny.
  • sequence/codon biases to make more distantly related sequences recombine at rates approaching those observed with more closely related sequences. Additionally, the technique does not require physically possessing the template genes.
  • Nucleotide Exchange and Excision Technology NexT exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (Muller et al, Nucleic Acids Res. 33:el 17 (2005)).
  • the gene is reassembled using internal PCR primer extension with proofreading polymerase.
  • the sizes for shuffling are directly controllable using varying dUPT::dTTP ratios. This is an end point reaction using simple methods for uracil incorporation and cleavage.
  • Other nucleotide analogs, such as 8-oxo-guanine, can be used with this method.
  • Nuclease treatment is used to generate a range of chimeras between the two genes. These fusions result in libraries of single-crossover hybrids (Sieber et al, Nat. Biotechnol.
  • GSSMTM Gene Site Saturation Mutagenesis
  • the starting materials are a supercoiled dsDNA plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004)).
  • Primers carrying the mutation of interest, anneal to the same sequence on opposite strands of DNA.
  • the mutation is typically in the middle of the primer and flanked on each side by approximately 20 nucleotides of correct sequence.
  • Dpnl is used to digest dam-methylated DNA to eliminate the wild-type template.
  • This technique explores all possible amino acid substitutions at a given locus (that is, one codon). The technique facilitates the generation of all possible replacements at a single- site with no nonsense codons and results in equal to near-equal representation of most possible alleles. This technique does not require prior knowledge of the structure, mechanism, or domains of the target enzyme. If followed by shuffling or Gene
  • this technology creates a diverse library of recombinants containing all possible combinations of single-site up-mutations.
  • the usefulness of this technology combination has been demonstrated for the successful evolution of over 50 different enzymes, and also for more than one property in a given enzyme.
  • Combinatorial Cassette Mutagenesis involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations (Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science 241 :53-57 (1988)). Simultaneous substitutions at two or three sites are possible using this technique. Additionally, the method tests a large multiplicity of possible sequence changes at a limited range of sites. This technique has been used to explore the information content of the lambda repressor DNA-binding domain.
  • Combinatorial Multiple Cassette Mutagenesis is essentially similar to CCM except it is employed as part of a larger program: 1) use of epPCR at high mutation rate to 2) identify hot spots and hot regions and then 3) extension by CMCM to cover a defined region of protein sequence space (Reetz et al, Angew. Chem. Int. Ed Engl.
  • conditional ts mutator plasmids allow increases of 20 to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required (Selifonova et al, Appl. Environ. Microbiol. 67:3645-3649 (2001)).
  • This technology is based on a plasmid-derived mutD5 gene, which encodes a mutant subunit of DNA polymerase III. This subunit binds to endogenous DNA polymerase III and compromises the proofreading ability of polymerase III in any strain that harbors the plasmid. A broad-spectrum of base substitutions and frameshift mutations occur.
  • the mutator plasmid should be removed once the desired phenotype is achieved; this is accomplished through a temperature sensitive (ts) origin of replication, which allows for plasmid curing at 41°C. It should be noted that mutator strains have been explored for quite some time (see Low et al, J. Mol. Biol. 260:359-3680 (1996)). In this technique, very high spontaneous mutation rates are observed. The conditional property minimizes non-desired background mutations. This technology could be combined with adaptive evolution to enhance mutagenesis rates and more rapidly achieve desired phenotypes.
  • ts temperature sensitive
  • LTM Look-Through Mutagenesis
  • Gene Reassembly is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene (Tunable GeneReassemblyTM (TGRTM) Technology supplied by Verenium
  • the method computationally assesses and allows filtering of a very large number of possible sequence variants (10 50 ).
  • the choice of sequence variants to test is related to predictions based on the most favorable thermodynamics. Ostensibly only stability or properties that are linked to stability can be effectively addressed with this technology.
  • the method has been successfully used in some therapeutic proteins, especially in engineering immunoglobulins. In silico predictions avoid testing
  • ISM Iterative Saturation Mutagenesis involves: 1) using knowledge of structure/function to choose a likely site for enzyme improvement; 2) performing saturation mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego CA); 3) screening/selecting for desired properties; and 4) using improved clone(s), start over at another site and continue repeating until a desired activity is achieved (Reetz et al, Nat. Protoc. 2:891-903 (2007); and Reetz et al, Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)). This is a proven methodology, which assures all possible replacements at a given position are made for screening/selection.
  • Formate dehydrogenase is a two subunit selenocysteine- containing protein that catalyzes the transfer of electrons from a reduced carrier to C0 2 , forming formate.
  • Enzymes with FDH activity utilize various electron carriers such as, for example, NADH (EC 1.2.1.2), NADPH (EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) and hydrogenases (EC 1.1.99.33).
  • FDH enzymes have been characterized from 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,
  • Formyltetrahydrofolate synthetase (EC 6.3.4.3, Step IB), also called formate- THF ligase, 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,
  • thermoacetica E. coli, and C. hydrogenoformans
  • Step ID methylenetetrahydrofolate dehydrogenase
  • Step ID EC 1.5.1.5
  • Step ID EC 1.5.1.5
  • Moth l 516,folD, and CHY l 878 bi- functional gene products of Moth l 516,folD, and CHY l 878, respectively
  • Step IE also called glycine cleavage system
  • P, H, T and L The glycine cleavage complex is involved in glycine catabolism in organisms such as E. coli and glycine biosynthesis in eukaryotes (Kikuchi et al, Proc Jpn Acad Ser 84:246 (2008)).
  • coli is encoded by four genes: gcvPHT and IpdA (Okamura et al, Eur J Biochem 216:539-48 (1993);Heil et al, Microbiol 148:2203-14 (2002)).
  • Activity of the glycine cleavage system in the direction of glycine biosynthesis has been demonstrated in vivo in Saccharomyces cerevisiae (Maaheimo et al, Eur J Biochem 268:2464-79 (2001)).
  • the yeast GCV is encoded by GCV1, GCV2, GCV3 and LPD1.
  • CO dehydrogenase enymes 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, H 2 , or water, respectively.
  • CODH enzymes are found in M. thermoacetica, C. hydrogenoformans and C. carboxidivorans P7. In some cases, CODH encoding genes are found adjacent to hydrogenase encoding genes.
  • 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 C0 2 and H 2 (Fox et al, J Bacteriol 178:6200-6208 (1996)).
  • Hydrogenase enzymes useful in the invention uptake molecular hydrogen and transfer electrons to acceptors such as ferredoxins.
  • hydrogenase encoding genes are located adjacent to a CODH. In Rhodospirillum rubrum, the encoded
  • CODH/hydrogenase proteins form a membrane-bound enzyme complex that has been indicated to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO and H 2 0 to C0 2 and H 2 (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. I :e65 (2005)). The C.
  • CODH (CooS) AAC45123 1498748 Rhodospirillum rubrum
  • coli include 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.
  • the M thermoacetica and Clostridium ljungdahli hydrogenases are suitable for a host that lacks sufficient endogenous hydrogenase activity. M.
  • thermoacetica and C. ljungdahli can grow with C0 2 as the exclusive carbon source indicating that reducing equivalents are extracted from H 2 to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L., J Bacteriol. 150:702-709 (1982); Drake and Daniel, Res Microbiol 155:869-883 (2004); Kellum and Drake, J Bacteriol. 160:466- 469 (1984)).
  • 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 C. ljungdahli (see for example US 2012/0003652).
  • Ralstonia eutropha HI 6 uses hydrogen as an energy source with oxygen as a terminal electron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an "02- tolerant" hydrogenase (Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that is periplasmically-oriented and connected to the respiratory chain via a b-type cytochrome (Schink and Schlegel, Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J. Biochem. 248, 179-186 (1997)). R.
  • eutropha also contains an 0 2 -tolerant soluble hydrogenase encoded by the Hox operon which is cytoplasmic and directly reduces NAD+ at the expense of hydrogen (Schneider and Schlegel, Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J. Bact. 187(9) 3122-3132(2005)). Soluble hydrogenase enzymes are additionally present in several other organisms including Geobacter sulfurreducens (Coppi, Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803 (Germer, J. Biol.
  • the Synechocystis enzyme is capable of generating NADPH from hydrogen.
  • Ferredoxins are small acidic proteins containing one or more iron-sulfur clusters that function as intracellular electron carriers with a low reduction potential. Reduced ferredoxins donate electrons to Fe-dependent enzymes such as ferredoxin- NADP + oxidoreductase, pyruvate :ferredoxin oxidoreductase (PFOR) and 2- oxoglutarate:ferredoxin oxidoreductase (OFOR).
  • ferredoxin- NADP + oxidoreductase pyruvate :ferredoxin oxidoreductase (PFOR) and 2- oxoglutarate:ferredoxin oxidoreductase (OFOR).
  • ferredoxin- NADP + oxidoreductase pyruvate :ferredoxin oxidoreductase (PFOR) and 2- oxoglutarate:ferredoxin oxidoreductase (O
  • thermophilus gene fdxl encodes a [4Fe-4S]-type ferredoxin that is required for the reversible carboxylation of 2- oxoglutarate and pyruvate by OFOR and PFOR, respectively (Yamamoto et al,
  • ferredoxin associated with the Sulfolobus solfataricus 2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S] type ferredoxin (Park et al. 2006). While the gene associated with this protein has not been fully sequenced, the N-terminal domain shares 93% homology with the zfx ferredoxin from S. acidocaldarius. The E. coli genome encodes a soluble ferredoxin of unknown physiological function, fdx. Some evidence indicates that this protein can function in iron- sulfur cluster assembly (Takahashi and Nakamura, 1999).
  • ferredoxin proteins have been characterized in Helicobacter pylori (Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et al. 2001).
  • a 2Fe-2S ferredoxin from Clostridium pasteurianum has been cloned and expressed in E. coli (Fujinaga and Meyer, Biochemical and Biophysical Research Communications, 192(3): (1993)).
  • Acetogenic bacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7, Clostridium ljungdahli and Rhodospirillum rubrum are predicted to encode several ferredoxins, listed below.
  • thermophilus fdxl BAE02673.1 68163284 Hydrogenobacter thermophilus
  • Ferredoxin oxidoreductase enzymes transfer electrons from ferredoxins or flavodoxins to NAD(P)H.
  • Two enzymes catalyzing the reversible transfer of electrons from reduced ferredoxins to NAD(P)+ are ferredoxin :NAD+ oxidoreductase (EC 1.18.1.3) and ferredoxin :NADP+ oxidoreductase (FNR, EC 1.18.1.2).
  • oxidoreductase (FNR, EC 1.18.1.2) has a noncovalently bound FAD co factor that
  • the Helicobacter pylori FNR encoded by HP1164 (fqrB) is coupled to the activity of pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the pyruvate-dependent production of NADPH (St et al. 2007).
  • PFOR ferredoxin oxidoreductase
  • An analogous enzyme is found in Campylobacter jejuni (St Maurice et al, J Bacteriol. 189:4764-4773 (2007)).
  • a ferredoxin:NADP+ oxidoreductase enzyme is encoded in the E. coli genome by fpr (Bianchi et al. 1993).
  • Ferredoxin:NAD+ oxidoreductase utilizes reduced ferredoxin to generate NADH from NAD+. In several organisms, including E. coli, this enzyme is a component of multifunctional dioxygenase enzyme complexes.
  • the ferredoxin :NAD+ oxidoreductase of E. coli, encoded by hcaD, is a component of the 3-phenylproppionate dioxygenase system involved in involved in aromatic acid utilization (Diaz et al. 1998).
  • NADH: ferredoxin reductase activity was detected in cell extracts of Hydrogenobacter thermophilus, although a gene with this activity has not yet been indicated (Yoon et al. 2006). Additional ferredoxin:NAD(P)+ oxidoreductases have been annotated in
  • NADH-dependent reduced ferredoxin NADP oxidoreductase of C kluyveri, encoded by nfnAB, catalyzes the concomitant reduction of ferredoxin and NAD+ with two equivalents of NADPH (Wang et al, J Bacteriol 192: 5115-5123 (2010)).
  • the energy-conserving membrane-associated Rnf-type proteins Seedorf et al, PNAS 105:2128-2133 (2008); and Herrmann, J. Bacteriol 190:784- 791 (2008) provide a means to generate NADH or NADPH from reduced ferredoxin.
  • glycine hydroxymethyltransferase also called glycine hydroxymethyltranferase (EC 2.1.2.1, Step IF).
  • This enzyme reversibly converts glycine and 5, 10-methylenetetrahydro folate to serine and THF.
  • Serine methyltransferase has several side reactions including the reversible cleavage of 3 -hydroxy acids to glycine and an aldehyde, and the hydrolysis of 5,10- methenyl-THF to 5-formyl-THF.
  • This enzyme is encoded by glyA of E. coli (Plamann et al, Gene 22:9-18 (1983)).
  • Serine hydroxymethyltranferase enzymes of S. cerevisiae include SHM1 (mitochondrial) and SHM2 (cytosolic) (McNeil et al, J Biol Chem
  • Step 1M The conversion of serine to hydroxypyruvate (Step 1M) is catalyzed by an enzyme with serine aminotransferase, amine oxidase or dehydrogenase activity.
  • serine aminotransferase enzymes include serine :pyruvate aminotransferase (EC 2.6.1.510), alanine: glyoxylate aminotransferase (EC 2.6.1.44) and serine: glyoxylate aminotransferase (EC 2.6.1.45).
  • Serine:pyruvate aminotransferase participates in serine metabolism and glyoxylate detoxification in mammals. These enzymes have been shown to utilize a variety of alternate oxo donors such as pyruvate, phenylpyruvate and glyoxylate; and amino acceptors including alanine, glycine and phenylalanine (Ichiyama et al., Mol. Urol.
  • the rat mitochondria serine :pyruvate aminotransferase encoded by agxt, is also active as an alanine-glyoxylate aminotransferase.
  • This enzyme was heterologously expressed in E. coli (Oda et al, J Biochem. 106:460-467 (1989)). Similar enzymes have been characterized in humans and flies (Oda et al.,
  • the human enzyme encoded by agxt, functions as a serine :pyruvate aminotransferase, an alanine: glyoxylate
  • glyoxylate aminotransferase and a serine glyoxylate aminotransferase (Nagata et al., Biomed.Res. 30:295-301 (2009)).
  • the fly enzyme is encoded by spat (Han et al, FEBS Lett. 527: 199- 204 (2002)).
  • An exemplary alanine: glyoxylate aminotransferases is encoded by AGT1 of Arabidopsis thaliana.
  • the purified, recombinant AGT1 expressed in E. coli also catalyzed serine: glyoxylate and
  • serine :pyruvate aminotransferase activities (Liepman et al., Plant J 25:487-498 (2001)).
  • serine glyoxylate aminotransferase enzymes (EC 2.6.1.45) also exhibit reduced but detectable serine :pyruvate aminotransferase activity.
  • Exemplary enzymes are found in Phaseolus vulgaris, Pisum sativum, Secale cereal and Spinacia oleracea.
  • Serine glyoxylate aminotransferase enzymes interconvert serine and hydroxypyruvate and utilize glyoxylate as an amino acceptor.
  • the serine glyoxylate aminotransferase from the obligate methylotroph Hyphomicrobium methylovorum GM2 has been functionally expressed in E. coli and characterized (Hagishita et al., Eur. J Biochem. 241 : 1-5 (1996)).
  • Enzymes in the EC class 1.4.1 catalyze the oxidative deamination of alpha-amino acids with NAD+, NADP+ or FAD as acceptor, and the reactions are typically reversible.
  • Exemplary enzymes with serine oxidoreductase (deaminating) activity include serine dehydrogenase (EC 1.4.1.7), L-amino acid dehydrogenase (EC 1.4.1.5) and glutamate dehydrogenase (EC 1.4.1.2).
  • dehydrogenase enzymes are encoded by gdhA in Escherichia coli (Korber et al, J
  • Serine oxidase enzymes convert also convert serine to hydroxypyruvate. Serine oxidase converts serine, 0 2 and water to ammonia, hydrogen peroxide and
  • hydroxypyruvate (Chumakov, et al, Proc. Nat. Acad. Sci., 99(21):13675-13680; Verral et al, Eur J Neurosci., 26(6) 1657-1669 (2007)).
  • Some amine oxidases are specific for the D- or L-amino acid (Dixon and Kleppe, Biochim Biophys Acta, 96: 368-382 (1965)).
  • keto-acid decarboxylase (EC 4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chain alpha- ketoacid decarboxylase.
  • keto-acid decarboxylase enzymes have been shown to accept hydroxypyruvate as an alternate substrate, including the kivd gene product of Lactococcus lactis (de la Plaza et al, F EMS Microbiol Lett. 238:367-374 (2004)) and the pdcl gene product of Saccharomyces cerevisiae (Cusa et al., J Bacteriol. 181 :7479-7484 (1999)).
  • the S. cerevisiae enzyme has been extensively studied, engineered for altered activity, and functionally expressed in E. coli (Killenberg-Jabs et al., Eur.J.Biochem.
  • Lactococcus lactis which decarboxylates a variety of branched and linear ketoacid substrates including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate, 3-methyl-2- oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate (Smit et al, Appl Environ Microbiol 71 :303-311 (2005)).
  • Serine decarboxylase catalyzes the decarboxylation of serine to ethanolamine ( Figure 1, Step 5). Enzymes with this activity have been characterized in plants such as Spinacia oleracea, Arabidopsis thaliana and Brassica napus in the context of choline biosynthesis.
  • the A. thaliana serine decarboxylase encoded by AtSDC is a soluble tetramer and was characterized by heterologous expression in E. coli and ability to complement a yeast mutant deficient in ethanolamine biosynthesis (Rontein et al, J Biol.Chem. 276:35523-35529 (2001)).
  • the Brassica napus serine decarboxylase was identified and characterized in the same study. A similar enzyme is found in Spinacia oleracea although the gene has not been identified to date (Summers et al, Plant Physiol 103: 1269-1276 (1993)).
  • Other serine decarboxylase candidates can be identified by sequence homology to the Arabidopsis or Brassica enzymes. A candidate with high homology is the putative serine decarboxylase from Beta vulgaris.
  • Exemplary candidates are aminotransferases with broad substrate specificity that convert terminal amines to aldehydes, such as gamma-aminobutyrate GABA transaminase (EC 2.6.1.19), diamine aminotransferase (EC 2.6.1.29) and putrescine aminotransferase (EC 2.6.1.82).
  • GABA aminotransferase naturally interconverts succinic semialdehyde and glutamate to 4-aminobutyrate and alpha-ketoglutarate and is known to have a broad substrate range (Schulz et al, 56:1-6 (1990); Liu et al, 43: 10896-10905 (2004)).
  • the two GABA transaminases in E. coli are encoded by gabT (Bartsch et al., J Bacteriol.
  • GABA transaminases in Mus musculus and Sus scrofa have also been shown to react with a range of alternate substrates (Cooper, Methods Enzymol. 113:80-82 (1985)). Additional enzyme candidates for interconverting ethanolamine and glycolaldehyde are putrescine aminotransferases and other diamine aminotransferases.
  • coli putrescine aminotransferase is encoded by the ygjG gene and the purified enzyme also was able to transaminate cadaverine and spermidine (Samsonova et al., BMC.Microbiol 3:2 (2003)).
  • activity of this enzyme on 1 ,7-diaminoheptane and with amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Samsonova et al, BMCMicrobiol 3:2 (2003); Kim, J Biol.Chem. 239:783-786 (1964)).
  • ethanolamine dehydrogenase or ethanolamine oxidoreductase deaminating
  • One enzyme with this functionality is ethanolamine oxidase (EC 1.4.3.8), which utilizes oxygen as an electron acceptor, converting ethanolamine, 0 2 and water to ammonia, hydrogen peroxide and glycolaldehyde (Schomburg et al, Springer Handbook of Enzymes. 320-323 (2005)).
  • Ethanolamine oxidase has been characterized in Pseudomonas sp and Phormia regina; however, the enzyme activity has not been associated with a gene to date.
  • the oxidative deamination of ethanolamine can be catalyzed by a deaminating oxidoreductase that utilizes NAD+, NADP+ or FAD as acceptor.
  • An exemplary enzyme for catalyzing the conversion of a primary amine to an aldehyde is lysine 6-dehydrogenase (EC 1.4.1.18), encoded by the lysDH genes. This enzyme catalyzes the oxidative deamination of the 6- amino group of L-lysine to form 2-aminoadipate-6-semialdehyde (Misono et al., J
  • Hydroxypyruvate reductase (EC 1.1.1.29 and EC 1.1.1.81), also called glycerate dehydrogenase, catalyzes the reversible NAD(P)H-dependent reduction of hydroxypyruvate to glycerate ( Figure 1, Step 8).
  • the ghrA and ghrB genes of E. coli encode enzymes with hydroxypyruvate reductase activity (Nunez et al, Biochem. J 354:707-715 (2001)). Both gene products also catalyze the reduction of glyoxylate to glycolate and the ghrB gene product prefers hydroxypyruvate as a substrate.
  • Hydroxypyruvate reductase participates in the serine cycle in methylotrophic bacterium such as Methylobacterium extorquens AMI and Hyphomicrobium methylovorum
  • the Methylobacterium sp. MB200 HPR has not been assigned a GenBank identifier to date but the sequence is available in the literature and bears 98% identity to the sequence of the M. extorquens hprA gene product, which uses both NADH and NADPH as cofactors (Chistoserdova et al, J Bacteriol. mnil ⁇ -l l (1991)).
  • Bifunctional enzymes with hydroxypyruvate reductase and glyoxylate reductase activities are found in mammals including Homo sapiens and Mus musculus.
  • An enzyme with glycerate decarboxylase activity can be used to convert glycerate to ethylene glycol ( Figure 1, Step 9). Such an enzyme has not been characterized to date. However, a similar alpha,beta-hydroxyacid decarboxylation reaction is catalyzed by tartrate decarboxylase (EC 4.1.1.73). The enzyme, characterized in Pseudomonas sp. group Ve-2, is NAD + dependent and catalyzes a coupled oxidation-reduction reaction that proceeds through an oxaloglycolate intermediate (Furuyoshi et al., JBiochem. 110:520- 525 (1991)).
  • a side reaction catalyzed by this enzyme is the NAD + dependent oxidation of tartrate (1% of activity).
  • Glycerate was not reactive as a substrate for this enzyme and was instead an inhibitor, so enzyme engineering or directed evolution can be used for this enzyme to function in the desired context.
  • a gene has not been associated with this enzyme activity to date.
  • glycerate decarboxylase is acetolactate decarboxylase (EC 4.1.1.5) which participates in citrate catabolism and branched-chain amino acid biosynthesis, converting the 2-hydroxyacid 2-acetolactate to acetoin.
  • Lactococcus lactis the enzyme is a hexamer encoded by gene aldB, and is activated by valine, leucine and isoleucine (Goupil-Feuillerat et al, J.Bacteriol. 182:5399-5408 (2000); Goupil et al, Appl.Environ.Microbiol. 62:2636-2640 (1996)).
  • Leuconostoc lactis has been purified and characterized but the gene has not been isolated to date (O'Sullivan et al, FEMS Microbiol. Lett. 194:245-249 (2001)).
  • Glyoxylate carboligase (EC 4.1.1.47), also known as tartrate semialdehyde synthase, catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde (Step 3G).
  • the E. coli enzyme, encoded by gel, is active under anaerobic conditions and requires FAD for activity although no net redox reaction takes place (Chang et al, JBiol.Chem. 268:3911-3919 (1993)).
  • Glyoxylate carboligase activity has also been detected in Ralstonia eutropha (Eschmann et al., Arch.Microbiol.
  • Additional candidate glyoxylate carboligase enzymes can be identified by sequence homology. Two exemplary candidates with high homology to the E. coli enzyme are found in Salmonella enterica and Burkholderia ambifaria.
  • Step H Hydroxypyruvate isomerase catalyzes the reversible isomerization of hydroxypyruvate and tartronate semialdehyde.
  • the E. coli enzyme, encoded by hyi, is cotranscribed with glyoxylate carboligase (gel) in a glyoxylate utilization operon
  • Hydroxypyruvate isomerase enzyme candidates in other organisms such as Ralstonia eutropha and Burkholderia ambifaria can be identified by sequence homology to the E. coli gene product. Note that the predicted hydroxypyruvate isomerase gene candidates in these organisms are also co-localized with genes predicted to encode glyoxylate carboligase.
  • Step 3F The conversion of glycine to glyoxylate in Step 3F is catalyzed by a glycine aminotransferase, amine oxidase or dehydrogenase.
  • Aminotransferase enzymes that utilize glycine as an amino donor include serine: glyoxylate aminotransferase alanine: glyoxylate transaminase and serine :pyruvate aminotransferase (Smith, Biochem Biophys Acta 321 : 156-64 (1973); Noguchi et al, Biochem J 159:607-13 (1976); Eze et al, J Gen
  • Tartronate semialdehyde reductase (EC 1.1.1.60, step 3R) catalyzes the reduction of tartronate semialdehyde to glycerate.
  • a tartronate semialdehyde reductase enzyme is encoded by glxR (psed_3889) of Pseudonocardia dioxanivorans (Grostern et al, AEM 78:3298-3308 (2012)).
  • Two tartronate semialdehyde reductase isozymes by the genes garR and glxR ofE. coli (Cusa et al, J Bacteriol. 181 :7479-7484 (1999);
  • a Schlenk line was used to dispense CO to cuvettes, each vented. Rubber stoppers on the cuvettes were pierced with 19 or 20 gage disposable syringe needles and were vented with the same. An oil bubbler was used with a CO tank and oxygen scrubber. The glass or quartz spectrophotometer cuvettes have a circular hole on top into which a Kontes stopper sleeve, Sz7 774250-0007 was fitted. The CO detector unit was positioned proximal to the fume hood. [00196] B. Handling of CO in larger quantities fed to large-scale cultures.
  • Fermentation cultures are fed either CO or a mixture of CO and H 2 to simulate syngas as a feedstock in fermentative production. Therefore, quantities of cells ranging from 1 liter to several liters can include the addition of CO gas to increase the dissolved concentration of CO in the medium. In these circumstances, fairly large and continuously administered quantities of CO gas are added to the cultures. At different points, the cultures are harvested or samples removed. Alternatively, cells are harvested with an integrated continuous flow centrifuge that is part of the fermenter.
  • the fermentative processes are carried out under anaerobic conditions. In some cases, it is uneconomical to pump oxygen or air into fermenters to ensure adequate oxygen saturation to provide a respiratory environment. In addition, the reducing power generated during anaerobic fermentation may be needed in product formation rather than respiration. Furthermore, many of the enzymes for various pathways are oxygen-sensitive to varying degrees. Classic acetogens such as M. thermoacetica are obligate anaerobes and the enzymes in the Wood-Ljungdahl pathway are highly sensitive to irreversible inactivation by molecular oxygen.
  • C Anaerobic chamber and conditions.
  • Exemplary anaerobic chambers are available commercially (see, for example, Vacuum Atmospheres Company, Hawthorne CA; MBraun, Newburyport MA).
  • Conditions included an 0 2 concentration of 1 ppm or less and 1 atm pure N 2 .
  • 3 oxygen scrubbers/catalyst regenerators were used, and the chamber included an 0 2 electrode (such as Teledyne; City of Industry CA). Nearly all items and reagents were cycled four times in the airlock of the chamber prior to opening the inner chamber door. Reagents with a volume >5mL were sparged with pure N 2 prior to introduction into the chamber.
  • Gloves are changed twice/yr and the catalyst containers were regenerated periodically when the chamber displays increasingly sluggish response to changes in oxygen levels.
  • the chamber's pressure was controlled through one-way valves activated by solenoids. This feature allowed setting the chamber pressure at a level higher than the surroundings to allow transfer of very small tubes through the purge valve.
  • the anaerobic chambers achieved levels of 0 2 that were consistently very low and were needed for highly oxygen sensitive anaerobic conditions. However, growth and handling of cells does not usually require such precautions.
  • platinum or palladium can be used as a catalyst that requires some hydrogen gas in the mix. Instead of using solenoid valves, pressure release can be controlled by a bubbler. Instead of using instrument-based 0 2 monitoring, test strips can be used instead.
  • a needle can be poked through the stopper to provide exhaust during autoclaving; the needle needs to be removed immediately upon removal from the autoclave.
  • the sterile medium has the remaining medium components, for example buffer or antibiotics, added via syringe and needle.
  • the bottles Prior to addition of reducing agents, the bottles are equilibrated for 30 - 60 minutes with nitrogen (or CO depending upon use).
  • a reducing agent such as a 100 x 150 mM sodium sulfide, 200 mM cysteine-HCl is added.
  • the bottle is stoppered immediately as the sodium sulfide solution generates hydrogen sulfide gas upon contact with the cysteine.
  • a syringe filter is used to sterilize the solution.
  • syringe needles such as B12 (10 ⁇ cyanocobalamin), nickel chloride (NiCl 2 , 20 microM final concentration from a 40 mM stock made in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture), and ferrous ammonium sulfate (final concentration needed is 100 ⁇ — made as 100-lOOOx stock solution in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture).
  • B12 10 ⁇ cyanocobalamin
  • NiCl 2 20 microM final concentration from a 40 mM stock made in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture
  • ferrous ammonium sulfate final concentration needed is 100 ⁇ — made as 100-lOOOx stock solution in anaerobic water in the chamber and
  • IPTG isopropyl ⁇ -D-l- thiogalactopyranoside
  • CO oxidation (CODH) Assay [00202] This example describes assay methods for measuring CO oxidation (CO dehydrogenase; CODH).
  • recombinant E. coli cells could be limited by the fact that M. thermoacetica enzymes have temperature optima around 55°C. Therefore, a mesophilic CODH/ACS pathway could be advantageous such as the close relative of Moorella that is mesophilic and does have an apparently intact CODH/ACS operon and a Wood-Ljungdahl pathway, Desulfitobacterium hafniense.
  • Acetogens as potential host organisms include, but are not limited to,
  • Rhodospirillum rubrum Moorella thermoacetica and Desulfitobacterium hafniense.
  • CO oxidation is both the most sensitive and most robust of the CODH/ACS assays. It is likely that an E. co/z ' -based syngas using system will ultimately need to be about as anaerobic as Clostridial (i.e., Moorella) systems, especially for maximal activity. Improvement in CODH should be possible but will ultimately be limited by the solubility of CO gas in water.
  • each of the genes was cloned individually into expression vectors. Combined expression units for multiple subunits/1 complex were generated. Expression in E. coli at the protein level was determined. Both combined M. thermoacetica
  • CODH/ACS operons and individual expression clones were made.
  • CO oxidation assay This assay is one of the simpler, reliable, and more versatile assays of enzymatic activities within the Wood-Ljungdahl pathway and tests CODH (Seravalli et al, Biochemistry 43:3944-3955 (2004)).
  • a typical activity of M thermoacetica CODH specific activity is 500 U at 55°C or ⁇ 60U at 25°C.
  • This assay employs reduction of methyl viologen in the presence of CO. This is measured at 578 nm in stoppered, anaerobic, glass cuvettes.
  • glass rubber stoppered cuvettes were prepared after first washing the cuvette four times in deionized water and one time with acetone. A small amount of vacuum grease was smeared on the top of the rubber gasket. The cuvette was gassed with CO, dried 10 min with a 22 Ga. needle plus an exhaust needle. A volume of 0.98 mlL of reaction buffer (50 mM Hepes, pH 8.5, 2mM dithiothreitol (DTT) was added using a 22 Ga. needle, with exhaust needled, and 100%CO. Methyl viologen (CH 3 viologen) stock was 1 M in water. Each assay used 20 microliters for 20 mM final concentration.
  • reaction buffer 50 mM Hepes, pH 8.5, 2mM dithiothreitol (DTT) was added using a 22 Ga. needle, with exhaust needled, and 100%CO.
  • Methyl viologen (CH 3 viologen) stock was 1 M in water. Each assay used 20 microliters
  • Mta98/Mta99 are E. coli MG1655 strains that express methanol
  • thermoacetia methyltransferase genes from M thermoacetia and, therefore, are negative controls for the ACS90 ACS91 E. coli strains that contain M. thermoacetica CODH operons.
  • CODH-ACS operon genes including 2 CODH subunits and the
  • methyltransferase were confirmed via Western blot analysis. Therefore, the recombinant E. coli cells express multiple components of a 7 gene operon. In addition, both the methyltransferase and corrinoid iron sulfur protein were active in the same recombinant E. coli cells. These proteins are part of the same operon cloned into the same cells.
  • thermoacetica cells for the positive controls. Though CODH activity in E. coli ACS90 and ACS91 was measurable, it was at about 130 - 150 X lower than the M. thermoacetica control. The results of the assay are shown in Figure 6. Briefly, cells (M. thermoacetica or E. coli with the CODH/ACS operon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extracts prepared as described above. Assays were performed as described above at 55°C at various times on the day the extracts were prepared. Reduction of methylviologen was followed at 578 nm over a 120 sec time course.
  • E. coli CO Tolerance Experiment and CO Concentration Assay (myoglobin assay) [00213] This example describes the tolerance of E. coli for high concentrations of CO. [00214] To test whether or not E. coli can grow anaerobically in the presence of saturating amounts of CO, cultures were set up in 120 ml serum bottles with 50 ml of Terrific Broth medium (plus reducing solution, NiCl 2 , Fe(II)NH 4 S0 4 , cyanocobalamin, IPTG, and chloramphenicol) as described above for anaerobic microbiology in small volumes. One half of these bottles were equilibrated with nitrogen gas for 30 min. and one half was equilibrated with CO gas for 30 min.
  • the cultures tested were pZA33/CO, ACS90/CO, and ACS91/CO. Each of these was added in 1 microliter increments to the same cuvette. Midway through the experiment a second cuvette was set up and used. The results are shown in Table II.
  • CODH/ACS actually consumed some CO, probably by oxidation to carbon dioxide.
  • This example describes gene candidates for converting methanol to ethylene glycol.
  • FIG. 4 Pathways for converting methanol to glycine, and further to ethylene glycol are shown in Figure 4.
  • the glycine to ethylene glycol pathways shown in Figure 3 can also be utilized in conjunction with a methanol to glycine pathway.
  • Enzyme candididates for converting the intermediate formate to ethylene glycol are described above in Examples I through III.
  • Enyzme candidates for converting methanol to formate are described in this example.
  • Methanol can be converted to formate in two enzymatic steps (Step 4Q and 4A). In the first step, methanol is oxidized to formaldehyde by methanol dehydrogenase.
  • NAD+ dependent methanol dehydrogenase enzymes catalyze the conversion of methanol and NAD+ to formaldehyde and NADH.
  • An enzyme with this activity was first characterized in Bacillus methanolicus. This enzyme is zinc and magnesium dependent, and activity of the enzyme is enhanced by the activating enzyme encoded by act (Kloosterman et al, J Biol Chem 277:34785-92 (2002)). Additional NAD(P)+ dependent enzymes can be identified by sequence homology. Methanol dehydrogenase enzymes utilizing different electron acceptors are also known in the art.
  • Examples include cytochrome dependent enzymes such as mxalF of the methylotroph Methylobacterium extorquens (Nunn et al, Nucl Acid Res 16:7722 (1988)).
  • Methanol dehydrogenase enzymes of methanotrophs such as Methylococcus capsulatis function in a complex with methane monooxygenase (MMO) (Myronova et al, Biochem 45: 11905-14 (2006)).
  • Methanol can also be oxidized to formaldehyde by alcohol oxidase enzymes such as methanol oxidase (EC 1.1.3.13) of Candida boidinii (Sakai et al, Gene 114: 67-73 (1992)).
  • Oxidation of formaldehyde to formate is catalyzed by formaldehyde dehydrogenase.
  • An NAD+ dependent formaldehyde dehydrogenase enzyme is encoded by fdhA of Pseudomonas putida (Ito et al, J Bacteriol 176: 2483-2491 (1994)).
  • Additional formaldehyde dehydrogenase enzymes include the NAD+ and glutathione independent formaldehyde dehydrogenase from Hyphomicrobium zavarzinii (Jerome et al, Appl Microbiol Biotechnol 77:779-88 (2007)), the glutathione dependent formaldehyde dehydrogenase of Pichia pastoris (Sunga et al, Gene 330:39-47 (2004)) and the NAD(P)+ dependent formaldehyde dehydrogenase of Methylobacter marinus (Speer et al, FEMS Microbiol Lett, 121(3):349-55 (1994)).
  • the enzymes of this pathway are S- (hydroxymethyl)glutathione synthase (EC 4.4.1.22), glutathione-dependent formaldehyde dehydrogenase (EC 1.1.1.284) and S-formylglutathione hydrolase (EC 3.1.2.12).
  • S- (hydroxymethyl)glutathione synthase EC 4.4.1.22
  • glutathione-dependent formaldehyde dehydrogenase EC 1.1.1.284
  • S-formylglutathione hydrolase EC 3.1.2.12

Abstract

L'invention concerne un organisme microbien non naturel, contenant une voie de l'éthylèneglycol, la voie comprenant au moins un acide nucléique exogène codant pour une enzyme de la voie de l'éthylèneglycol exprimée en quantité suffisante pour produire de l'éthylèneglycol. Les microorganismes non naturels de l'invention peuvent exprimer des acides nucléiques exogènes qui catalysent la fixation de CO2 ou de CO en éthylèneglycol. Les microorganismes de l'invention peuvent en outre comporter des enzymes pour générer de l'énergie et des équivalents réducteurs à partir de méthanol, de gaz de synthèse et d'autres sources gazeuses. L'invention concerne, de plus, des procédés d'utilisation de tels organismes microbiens pour produire de l'éthylèneglycol, par culture d'un organisme microbien non naturel, contenant une voie de l'éthylèneglycol, dans des conditions et pendant un laps de temps suffisant pour produire de l'éthylèneglycol.
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EP3765598A4 (fr) * 2018-03-12 2021-12-15 Braskem S.A. Procédés de co-production d'éthylène glycol et de composés à trois carbones
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WO2019126400A1 (fr) * 2017-12-19 2019-06-27 Lanzatech, Inc. Micro-organismes et procédés de production biologique d'éthylène glycol
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EP3765598A4 (fr) * 2018-03-12 2021-12-15 Braskem S.A. Procédés de co-production d'éthylène glycol et de composés à trois carbones
US11384369B2 (en) 2019-02-15 2022-07-12 Braskem S.A. Microorganisms and methods for the production of glycolic acid and glycine via reverse glyoxylate shunt
WO2021116330A1 (fr) * 2019-12-10 2021-06-17 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Nouveau micro-organisme génétiquement modifié capable de croître sur du formiate, du méthanol, du méthane ou du co2
WO2022045481A1 (fr) * 2020-08-26 2022-03-03 전남대학교산학협력단 Composition pour la production de glycolaldéhyde à partir de formaldéhyde, et procédé de production d'acide glycolique, d'éthylène glycol ou d'éthanolamine à partir de formaldéhyde à l'aide de la composition
WO2023015285A1 (fr) * 2021-08-06 2023-02-09 Lanzatech, Inc. Micro-organismes et procédés de production biologique d'éthylène glycol
US11952607B2 (en) 2021-08-06 2024-04-09 Lanzatech, Inc. Microorganisms and methods for improved biological production of ethylene glycol

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