WO2014004625A1

WO2014004625A1 - Microorganisms for producing ethylene glycol using synthesis gas

Info

Publication number: WO2014004625A1
Application number: PCT/US2013/047821
Authority: WO
Inventors: Robin E. Osterhout; Anthony P. Burgard; Mark J. Burk
Original assignee: Genomatica Inc
Current assignee: Genomatica Inc
Priority date: 2012-06-26
Filing date: 2013-06-26
Publication date: 2014-01-03
Anticipated expiration: 2014-12-26

Description

MICROORGANISMS FOR PRODUCING ETHYLENE GLYCOL

USING SYNTHESIS GAS

BACKGROUND OF THE INVENTION

[0001] This application claims the benefit of priority of United States Provisional application serial No. 61/664,653, filed June 26, 2012, the entire contents of which is incorporated herein by reference.

[0002] 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. [0003] 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. [0004] 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. Around 82% of 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.

[0005] Typically, in the manufacture of ethylene glycol, 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.

[0006] Synthesis gas (syngas) is a mixture of primarily H₂ and CO that can be obtained via gasification of any organic feedstock, such as coal, coal oil, natural gas, biomass, or waste organic matter. Numerous gasification processes have been developed, and most designs are based on partial oxidation, where limiting oxygen avoids full combustion, of organic materials at high temperatures (500-1500°C) to provide syngas as, for example, 0.5: 1-3 :1 H₂/CO mixture. Steam is sometimes added to increase the hydrogen content, typically with increased C0₂ production through the water gas shift reaction.

[0007] Today, coal is the main substrate used for industrial production of syngas, which is usually used for heating and power and as a feedstock for Fischer-Tropsch synthesis of methanol and liquid hydrocarbons. Many large chemical and energy companies employ coal gasification processes on large scale and there is experience in the industry using this technology.

[0008] Overall, technology now exists for cost-effective production of syngas from a plethora of materials, including coal, biomass, wastes, polymers, and the like, at virtually any location in the world. Biomass gasification technologies are being practiced commercially, particularly for heat and energy generation. [0009] Despite the availability of organisms that utilize syngas, such organisms are generally poorly characterized and are not well-suited for commercial development. For example, Clostridium and related bacteria are strict anaerobes that are intolerant to high concentrations of certain products such as butanol, thus limiting titers and

commercialization potential. The Clostridia also produce multiple products, which presents separations issues in isolating a desired product. Finally, development of facile genetic tools to manipulate clostridial genes is in its infancy, therefore, they are not currently amenable to rapid genetic engineering to improve yield or production characteristics of a desired product.

[0010] Thus, there exists a need to develop microorganisms and methods of their use to utilize syngas or other gaseous carbon sources for the production of desired chemicals, such as ethylene glycol. More specifically, there exists a need to develop microorganisms for syngas utilization that also have existing and efficient genetic tools to enable their rapid engineering to produce valuable products at useful rates and quantities. Microbial organisms and methods for effectively producing commercial quantities of ethylene glycol are described herein and include related advantages. SUMMARY OF INVENTION

[0011] 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

microorganisms of the invention can express exogenous nucleic acids that catalyze the fixation of C0₂ 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. In another embodiment, 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.

BRIEF DESCRIPTION OF THE DRAWINGS [0012] Figure 1 shows exemplary pathways for converting CO and/or C0₂ 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. hydroxypyruvate reductase; and P. glycerate decarboxylase. Formyl- THF represents 10-formyl-tetrahydro folate. Methenyl-THF represents 5,10- methenyltetrahydrofolate. Methylene -THF represents 5, 10-methylenetetrahydro folate. [0013] Figure 2 shows an exemplary flux distribution for achieving the maximum theoretical ethylene glycol yield from glucose and C0₂. The exemplary enzymes for converting C0₂ 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. [0014] Figure 3 shows exemplary pathways for converting CO and/or C0₂ 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

oxidoreductase; M. glycolaldehyde reductase; N. hydroxypyruvate aminotransferase or dehydrogenase; O. serine decarboxylase; P. hydroxypyruvate reductase; Q. glycerate decarboxylase; R. tartronate semi aldehyde reductase; and S. ethanolamine

aminotransferase, amine oxidase or dehydrogenase. Formyl-THF represents 10-formyl- tetrahydrofolate. Methenyl-THF represents 5, 10-methenyltetrahydro folate. Methylene- THF is 5, 10-methylenetetrahydro folate.

[0015] 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.

hydroxypyruvate decarboxylase; O. hydroxypyruvate reductase; P. glycerate

decarboxylase; and Q. methanol dehydrogenase. Formyl-THF represents 10-formyl- tetrahydrofolate. Methenyl-THF represents 5, 10-methenyltetrahydro folate. Methylene- THF represents 5, 10-methylenetetrahydro folate. The exemplary pathways shown in Figure 3 for conversion of formate to ethylene glycol can also be used in combination with steps 4Q and 4A for production of ethylene glycol.

[0016] 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).

[0017] 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) were grown and extracts prepared. 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.

DETAILED DESCRIPTION OF THE INVENTION

[0018] 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 C0₂, 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, C0₂ and/or methanol into glycine which can subsequently be converted into serine, glyoxylate, acetyl-CoA, cell mass, and useful products such as ethylene glycol.

[0019] The glycine synthase pathway is one of several known biological routes for fixing C0₂. In this pathway, C0₂ is reduced and subsequently attached to a

tetrahydrofolate (THF) carrier molecule. Following the isomerisation and reduction of the formyl-THF intermediate to methylene-THF, glycine synthase (also called the glycine cleavage complex or glycine cleavage system) joins methylene-THF with C0₂ to form glycine. Glycine is further converted to metabolic precursors such as acetyl-phosphate, serine, pyruvate, and acetyl-CoA. Although this C0₂ 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)). [0020] The glycine synthase pathway can serve as a secondary carbon assimilation pathway during growth on other substrates such as carbohydrates. In this capacity, the glycine synthase pathway enzymes harness excess reducing equivalents generated in glycolysis and/or methanol oxidation to fix C0₂, contributing to improved yields of products such as ethylene glycol.

[0021] Several pathways for synthesis of ethylene glycol from C0₂, CO and/or H₂ are shown in Figure 1. In these pathways, C0₂ 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 C0₂ to glycine is catalyzed by the glycine cleavage complex in step IE.

[0022] The conversion of glycine to EG via serine can accomplished by one of several pathways shown in Figure 1. Glycine is converted to serine by serine

hydroxymethyltransferase (step IF). In one pathway, 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). In an alternate pathway, 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.

[0023] Pathways from glycine to ethylene glycol that proceed through glyoxylate are shown in Figure 3. In 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.

Alternately, 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. [0024] 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 (G/H/I, M/N/I, M/O/P) 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.

[0025] The fixation of C0₂ to glycine and further to biomass and products such as ethylene glycol in the absence of carbohydrates (shown in Figures 1 and 3) requires energy and reducing equivalents. For the pathway shown in Figure 1, each molecule of EG synthesized from C0₂ requires 10 reducing equivalents and two molecules of ATP. These reducing equivalents can be supplied from one or more reduced components of syngas, H₂ or CO. For example, reducing equivalents (e.g., 2 [H]) are obtained by the conversion of CO and water to C0₂ via carbon monoxide dehydrogenase or from the activity of a hydrogen-utilizing hydrogenase which transfers electrons from H₂ 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). Alternately, 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)).

[0026] In one embodiment, microorganisms of the invention can fix carbon from exogenous CO and/or C0₂ 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) can grow on the syngas-generated acetyl-CoA in the presence of a suitable external electron acceptor such as nitrate. This electron acceptor is required to accept electrons from the reduced quinone formed via succinate dehydrogenase. A further advantage of adding an external electron acceptor is that additional energy for cell growth, maintenance, and product formation can be generated from respiration of acetyl-CoA. [0027] In one embodiment, 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 C0₂ 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.

[0028] 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.

[0029] For fermentative processes, 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.

[0030] 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. In one embodiment, 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. In such microorganisms, 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. In other embodiments, 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. Thus, 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. [0031] 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. In the first step of the pathways depicted in Figure 4, 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.

[0032] Accordingly, 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. Such an organism is capable of converting methanol, a relatively inexpensive organic feedstock that can be derived from synthesis gas, and gases comprising CO, C0₂, and/or H₂ into ethylene glycol and/or cell mass.

[0033] As used herein, the term "non-naturally occurring" when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within an ethylene glycol biosynthetic pathway.

[0034] 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.

[0035] As used herein, the term "isolated" when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non- naturally occurring environments. Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.

[0036] 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.

[0037] As used herein, the term "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. [0038] As used herein, the term "CoA" or "coenzyme A" is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.

[0039] As used herein, the term "substantially anaerobic" when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.

[0040] "Exogenous" as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non- chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term "endogenous" refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species whereas "homologous" refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid. [0041] It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where 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. Similarly, it is understood that more than two 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. Thus, 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.

[0042] 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. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.

[0043] Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

[0044] An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

[0045] 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. [0046] In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.

[0047] A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species.

Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.

[0048] Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having ethylene glycol biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes.

[0049] 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.

[0050] Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1;

x dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept- 16- 1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x dropoff: 50; expect: 10.0;

wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

In one embodiment, 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. In some aspects, 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, 3D, 3E, 3F, 3G, 3H, 3N, 30, 3S and 3M (see Figure 3 and Examples I-III); (8) 4Q, 4A, IB, IC, ID, IE, IF, 1G, 1H and II (see Figures 1 and 4 and Examples I, II and VII); (9) 4Q, 4A, IB, IC, ID, IE, IF, 1M, IN and II (see Figures 1 and 4 and Examples I, II and VII); (10) 4Q, 4A, IB, IC, ID, IE, IF, 1M, 10 and IP (see Figures 1 and 4 and Examples I, II and VII); (11) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3R and 3Q (see Figures 3 and 4 and Examples I-III and VII); (12) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3P and 3Q (see Figures 3 and 4 and Examples I-III and VII); (13) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 31 and 3M (see Figures 3 and 4 and Examples I-III and VII); and (14) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3N, 30, 3S and 3M (see Figures 3 and 4 and Examples I-III and VII), wherein 1A is a formate dehydrogenase, wherein IB is a formyltetrahydro folate synthetase, wherein IC is a methenyltetrahydro folate cyclohydrolase, ID is a methylenetetrahydro folate dehydrogenase, wherein IE is a glycine cleavage complex, wherein IF is a serine hydroxymethyltransferase, wherein 1G is a serine decarboxylase, wherein 1H is an ethanolamine aminotransferase, an amine oxidase or a dehydrogenase, wherein II is a glycolaldehyde reductase, wherein 1M is a serine aminotransferase, amine oxidase or dehydrogenase, wherein IN is a hydroxypyruvate decarboxylase, wherein 10 is a hydroxypyruvate reductase, wherein IP a glycerate decarboxylase, wherein 3A is a formate dehydrogenase, wherein 3B is a

formyltetrahydrofolate synthetase, wherein 3C is a methenyltetrahydrofolate

cyclohydrolase, wherein 3D is a methylenetetrahydrofolate dehydrogenase, wherein 3E is a glycine cleavage complex, wherein 3F is a glycine aminotransferase, amine oxidase or dehydrogenase, wherein 3G is a glyoxylate carboxyligase, wherein 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

semialdehyde reductase, wherein 3 S is an ethanolamine aminotransferase, amine oxidase or dehydrogenase, wherein 4A is a formaldehyde dehydrogenase, and wherein 4Q is a methanol dehydrogenase.

[0051] In another embodiment, 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. For example, 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.

[0052] In yet another embodiment, the non-naturally occurring microbial organism can further include a CO dehydrogenase, a hydrogenase or a ferredoxin oxidoreductase. For example, in some aspects, 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). In another aspect, the microbial organism can include two exogenous nucleic acids, wherein each exogenous nucleic acid encodes an enzyme selected from a CO

dehydrogenase, a hydrogenase and a ferredoxin oxidoreductase. Or alternatively, 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. [0053] In one embodiment, 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. For example, 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.

Accordingly, in one aspect, 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. Non-limiting examples of 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. [0054] 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.

[0055] In one aspect of the invention, the at least one exogenous nucleic acid included in the non-naturally occurring microbial organism is a heterologous nucleic acid. In another aspect, the non-naturally occurring microbial organism of the invention is in a substantially anaerobic culture medium. [0056] In another embodiment, 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 H₂ to ethylene glycol, wherein the microorganism lacks the ability to convert CO and H₂ to ethylene glycol in the absence of the at least one exogenous nucleic acid. Such a synthesis gas or other gas can further include C0₂. Thus, a non-naturally occurring microorganism of the invention can include a pathway that increases the efficiency of converting C0₂, CO and/or H₂ to ethylene glycol. In addition, 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 C0₂ and H₂ to ethylene glycol, wherein the microorganism lacks the ability to convert C0₂ and H₂ to ethylene glycol in the absence of the at least one exogenous nucleic acid. The gas can further include CO.

[0057] 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 C0₂ 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 C0₂. Further, 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. In yet another embodiment, 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 C0₂, in

combination with H₂, 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 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 H₂ and C0₂, 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. Such 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).

[0058] 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. Additionally 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. [0059] In yet another embodiment, 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 C0₂, in combination with H₂, 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.

Additionally provided is a non-naturally occurring microorganism including at least one exogenous nucleic acid encoding an enzyme that confers utilization of CO, in combination with H₂ and C0₂, 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. Such a microorganism can be used to produce a desired product such as ethylene glycol from the carbon source, as disclosed herein.

[0060] The invention also provides a non-naturally occurring microbial organism capable of producing ethylene glycol utilizing methanol and/or syngas. Thus, the microbial organism of the invention is capable of utilizing methanol, methanol and CO, C0₂ and/or H₂, for example, C0₂, C0₂ and H₂, CO, CO and H₂, C0₂ and CO, or C0₂, CO and H₂, to produce ethylene glycol. In one embodiment, the microbial organism is engineered to utilize methanol and/or syngas to produce ethylene glycol (see Examples I- III and VII). In one embodiment, 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. In such a non-naturally occurring microbial organism, the ethylene glycol pathway can confer the ability to convert methanol, C0₂, CO and/or H₂, or a combination thereof, to ethylene glycol.

[0061] In an additional embodiment, 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], H₂ to 2[H], 2[H] to NAD(P)H, C0₂ 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 tartronate semialdehyde, tartronate semialdehyde to glycerate, tartronate semialdehyde to hydroxypyruvate, and hydroxypyruvate to serine. One skilled in the art will understand that these are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. Thus, 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.

[0062] 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. For example, as disclosed herein, an ethylene glycol pathway is exemplified in Figures 1-4. Therefore, in addition to a microbial organism containing an ethylene glycol pathway that produces ethylene glycol, 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.

[0063] It is understood that 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. As disclosed herein, such 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.

However, it is understood that a non-naturally occurring microbial organism that produces an ethylene glycol pathway intermediate can be utilized to produce the intermediate as a desired product. [0064] The invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.

[0065] As disclosed herein, 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

deprotonated form, in particular since the ionized form is known to depend on the pH in which the compound is found. It is understood that 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. Some such 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

unsaturation, providing for example, propenyl, butenyl, pentyl, and hexenyl O- or S- carboxylates. 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. Other biosynthetically accessible 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

transesterification of an O- or S-carboxylate. S-carboxylates are exemplified by CoA S- esters, cysteinyl S-esters, alkylthioesters, and various aryl and heteroaryl thioesters. [0066] 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. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve ethylene glycol biosynthesis. Thus, a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as ethylene glycol.

[0067] 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; and 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.

[0068] Similarly, 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

Eurotiales, family Trichocomaceae, including the genus Aspergillus; and the order Mucorales, family Mucoraceae, including the genus Rhizopus. Non-limiting species of host yeast or fungi include Saccharomyces cerevisiae, Schizosaccharomyces pombe,

Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, and the like. 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.

[0069] Depending on the ethylene glycol biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed ethylene glycol pathway- encoding nucleic acid and up to all encoding nucleic acids for one or more ethylene glycol biosynthetic pathways. For example, 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. In a host deficient in all enzymes or proteins of an ethylene glycol pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of 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. [0070] Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the ethylene glycol pathway deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism of the invention can have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen, up to all nucleic acids encoding the enzymes or proteins constituting an ethylene glycol biosynthetic pathway disclosed herein. In some embodiments, 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. In some embodiments, 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

ethanolamine, which increases the overall yield of ethylene glycol produced by the microorganism. As one non- limiting example, utilizing the pathway of Figure 2, which shows the production of ethylene glycol from C0₂ and glucose, 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. [0071] Generally, 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. For example, 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. In addition, 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. [0072] In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize ethylene glycol. In this specific embodiment 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. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the ethylene glycol biosynthetic pathway.

[0073] In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism. [0074] It is understood that, in methods of the invention, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non- naturally occurring microbial organism of the invention. The nucleic acids can be introduced so as to confer, for example, an ethylene glycol biosynthetic pathway onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer ethylene glycol biosynthetic capability. For example, 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. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, 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. Similarly, 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.

[0075] In addition to the biosynthesis of ethylene glycol as described herein, the non- naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and/or with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, 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. [0076] In other embodiments, the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, ethylene glycol. In these embodiments, biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of 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. Alternatively, 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.

[0077] Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non- naturally occurring microbial organisms and methods of the invention together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce ethylene glycol. [0078] 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. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and

mammalian genomes, the identification of genes encoding the requisite ethylene glycol biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of ethylene glycol described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.

[0079] In some instances, such as when an alternative ethylene glycol biosynthetic pathway exists in an unrelated species, 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. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize ethylene glycol. [0080] 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. Such methods can be found described in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

[0081] 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. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al, J. Biol. Chem.

280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

[0082] 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. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein. [0083] In some embodiments, 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. Accordingly, in some embodiments, 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. In some aspects, 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

Figure 1 and Example I and II); (3) 1A, IB, IC, ID, IE, IF, IM, 10 and IP (see Figure 1 and Example 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, 3D, 3E, 3F, 3G, 3H, 3N, 30, 3S and 3M (see Figure 3 and Examples I-III); (8) 4Q, 4A, IB, IC, ID, IE, IF, 1G, 1H and II (see Figures 1 and 4 and Examples I, II and VII); (9) 4Q, 4A, IB, IC, ID, IE, IF, IM, IN and II (see Figures 1 and 4 and Examples I, II and VII); (10) 4Q, 4A, IB, IC, ID, IE, IF, IM, 10 and IP (see Figures 1 and 4 and Examples I, II and VII); (11) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3R and 3Q (see Figures 3 and 4 and Examples I-III and VII); (12) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3P and 3Q (see Figures 3 and 4 and Examples I-III and VII); (13) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 31 and 3M (see Figures 3 and 4 and Examples I-III and VII); and (14) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3N, 30, 3S and 3M (see Figures 3 and 4 and Examples I-III and VII),, wherein 1A is a formate dehydrogenase, wherein IB is a formyltetrahydro folate synthetase, wherein IC is a methenyltetrahydro folate

cyclohydrolase, ID is a methylenetetrahydro folate dehydrogenase, wherein IE is a glycine cleavage complex, wherein IF is a serine hydroxymethyltransferase, wherein 1G is a serine decarboxylase, wherein 1H is an ethanolamine aminotransferase, an amine oxidase or a dehydrogenase, wherein II is a glycolaldehyde reductase, wherein IM is a serine aminotransferase, amine oxidase or dehydrogenase, wherein IN is a hydroxypyruvate decarboxylase, wherein 10 is a hydroxypyruvate reductase, wherein IP a glycerate decarboxylase, wherein 3A is a formate dehydrogenase, wherein 3B is a

formyltetrahydrofolate synthetase, wherein 3C is a methenyltetrahydrofolate

semialdehyde reductase, wherein 3 S is an ethanolamine aminotransferase, amine oxidase or dehydrogenase, wherein 4A is a formaldehyde dehydrogenase, and wherein 4Q is a methanol dehydrogenase. [0084] In another embodiment, 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. For example, 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.

[0085] In yet another embodiment, the non-naturally occurring microbial organism used in the method of the invention can further include a CO dehydrogenase, a

hydrogenase or a ferredoxin oxidoreductase. For example, in some aspects, 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). In another aspect, 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

dehydrogenase, a hydrogenase and a ferredoxin oxidoreductase. Or alternatively, 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

oxidoreductase.

[0086] In one embodiment, 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. For example, 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. Accordingly, in one aspect, 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. Non-limiting examples of 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. [0087] In one aspect of the invention, 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. In another aspect, the non-naturally occurring microbial organism used in the method of the invention is in a substantially anaerobic culture medium. [0088] Suitable purification and/or assays to test for the production of ethylene glycol can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas

Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al, Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art. For example, glycolaldehyde reductase activity can be measured by its NADH- dependent glycolaldehyde reduction to ethylene glycol using a molar absorption coefficient of 6.22X10-3 M-l at 340 nm.

[0089] The ethylene glycol can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.

[0090] Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention. For example, the ethylene glycol producers can be cultured for the biosynthetic production of ethylene glycol. [0091] For the 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. For strains where growth is not observed anaerobically, 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.

[0092] If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.

[0093] The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass,

hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of 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.

[0094] In addition to renewable feedstocks such as those exemplified above, the ethylene glycol microbial organisms of the invention also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the ethylene glycol producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.

[0095] Synthesis gas, also known as syngas or producer gas, 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 H₂ 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 H₂ and CO, syngas can also include C0₂ and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, C0₂.

[0096] As disclosed herein, gaseous carbon sources such as syngas including CO and/or C0₂ can be utilized by non-naturally occurring microorganisms of the invention to produce ethylene glycol. Although generally exemplified herein as syngas, it is understood that any source of gaseous carbon including CO and/or C0₂ can be utilized by the non-naturally occurring microorganisms of the invention. Thus, the invention relates to non-naturally occurring microorganisms that are capable of utilizing CO and/or C0₂ as a carbon source.

[0097] Thus, the non-naturally occurring microorganisms of the invention can use syngas or other gaseous carbon sources providing CO and/or C0₂ to produce ethylene glycol. In the case of C0₂, additional sources include, but are not limited to, production of C0₂ as a byproduct in ammonia and hydrogen plants, where methane is converted to C0₂; combustion of wood and fossil fuels; production of C0₂ 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, CaC0₃, in the manufacture of lime, CaO; production of C0₂ 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.

[0098] Accordingly, given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as 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. Accordingly, the invention provides a non-naturally occurring microbial organism that produces and/or secretes ethylene glycol when gro_wn 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.

[0099] 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. Generally, 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.

[00100] In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S.

publication 2009/0047719, filed August 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the ethylene glycol producers can synthesize ethylene glycol at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular

concentrations, ethylene glycol producing microbial organisms can produce ethylene glycol intracellularly and/or secrete the product into the culture medium. [00101] In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of ethylene glycol can include the addition of an osmoprotectant to the culturing conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, 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. In one aspect, 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. [00102] In some embodiments, 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. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources." 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.

[00103] In some embodiments, 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.

[00104] In some embodiments, 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. In some embodiments, 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. For example, as discussed herein, 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. In some such embodiments, 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 C0₂, which can possess a larger amount of carbon- 14 than its petroleum-derived counterpart. [00105] The unstable carbon isotope carbon- 14 or radiocarbon makes up for roughly 1 in 10¹² 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 (¹⁴N). 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".

[00106] Methods of determining the isotopic ratios of atoms in a compound are well known to those skilled in the art. 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). Such 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.

[00107] In the case of carbon, 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.

[00108] The biobased content of a compound is estimated by the ratio of carbon- 14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm = (S-B)/(M-B), where B, S and M represent the ¹⁴C/¹²C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the ¹⁴C/¹²C 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

19 per mil (Olsson, The use of Oxalic acid as a Standard, in, Radiocarbon Variations and Absolute Chronology, Nobel Symposium, 12th Proa, John Wiley & Sons, New York (1970)). Mass spectrometry results, for example, measured by ASM, are calculated using the internationally agreed upon definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to 5¹³C_VPDB=-19 per mil. This is equivalent to an absolute (AD 1950) ¹⁴C/¹²C ratio of 1.176 ± 0.010 x 10^~12 (Karlen et al, Arkiv Geofysik, 4:465-471 (1968)). The standard calculations take into account the differential uptake of one isotope with respect to another, for example, the preferential uptake in biological systems of C¹² over C¹³ over C¹⁴, and these corrections are reflected as a Fm corrected for δ¹³.

[00109] 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.

[00110] As described in ASTM D6866, the percent modern carbon (pMC) 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.

[00111] ASTM D6866 quantifies the biobased content relative to the material's total organic content and does not consider the inorganic carbon and other non-carbon containing substances present. For example, a product that is 50% starch-based material and 50%) water would be considered to have a Biobased Content = 100% (50%> organic content that is 100% biobased) based on ASTM D6866. In another example, a product that is 50%) starch-based material, 25% petroleum-based, and 25% water would have a Biobased Content = 66.7%> (75% organic content but only 50%> of the product is biobased). In another example, a product that is 50% organic carbon and is a petroleum- based product would be considered to have a Biobased Content = 0%> (50%> organic carbon but from fossil sources). Thus, based on the well known methods and known standards for determining the biobased content of a compound or material, one skilled in the art can readily determine the biobased content and/or prepared downstream products that utilize of the invention having a desired biobased content.

[00112] Applications of carbon-14 dating techniques to quantify bio-based content of materials are known in the art (Currie et al., Nuclear Instruments and Methods in Physics Research B, 172:281-287 (2000)). For example, carbon-14 dating has been used to quantify bio-based content in terephthalate-containing materials (Colonna et al., Green Chemistry, 13:2543-2548 (2011)). Notably, 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). In contrast, 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).

[00113] Accordingly, in some embodiments, 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. For example, in some aspects 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%. In some such embodiments, the uptake source is C0₂. In some embodiments, 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. In this aspect, 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%. In some embodiments, 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. Using such 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. [00114] Further, 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 C0₂ that occurs in the environment. For example, in some aspects 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 C0₂ that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as disclosed herein, that a product can have a carbon- 12 versus carbon-13 versus carbon- 14 isotope ratio of about the same value as the C0₂ 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 C0₂ 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.

[00115] 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. Accordingly, in some embodiments, 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.

[00116] As used herein, the term "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. Such 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. Alternatively, the biological organism can utilize atmospheric carbon. As used herein, the term "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.

[00117] In some embodiments, 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. Thus, in some aspects, 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. For example, 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. [00118] 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.

[00119] As described herein, one exemplary growth condition for achieving biosynthesis of ethylene glycol includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, 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 N₂/C0₂ mixture or other suitable non-oxygen gas or gases.

[00120] 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. Generally, and as with non-continuous culture procedures, 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.

[00121] 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.

[00122] In addition to the above fermentation procedures using the ethylene glycol producers of the invention for continuous production of substantial quantities of ethylene glycol, the ethylene glycol producers 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.

[00123] To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US

2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US

2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of ethylene glycol.

[00124] One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework (Burgard et al, Biotechnol. Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable microorganisms which overproduce the target product.

Specifically, the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild- type states because the genes selected by OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring microbial organisms for further optimization of biosynthesis of a desired product.

[00125] Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the

performance limits of metabolic networks in the presence of gene additions or deletions. OptKnock computational framework allows the construction of model formulations that 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.

[00126] 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. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.

[00127] These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted.

[00128] Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host microbial organisms. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration of the invention, some methods are described herein with reference to the OptKnock

computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art.

[00129] The methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism.

Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes.

[00130] Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set. One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter

aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur.

[00131] To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al, Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock

computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®.

[00132] 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.

[00133] As discussed above, the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum-growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism's ability to self-optimize under selective pressures. The OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry. The identification of optimal gene/reaction knockouts requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the resulting network

overproduces the biochemical of interest (Burgard et al, Biotechnol. Bioeng. 84:647-657 (2003)).

[00134] An in silico stoichiometric model of E. coli metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US

2004/0009466, and in U.S. Patent No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth- coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of deletions. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above.

[00135] As disclosed herein, a nucleic acid encoding a desired activity of an ethylene glycol pathway can be introduced into a host organism. In some cases, it can be desirable to modify an activity of an ethylene glycol pathway enzyme or protein to increase production of ethylene glycol. For example, known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule.

Additionally, 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.

[00136] One such optimization method is directed evolution. 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, >10⁴). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al, Biomol.Eng 22: 11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax. Biomol.Eng 22:1-9 (2005).; and Sen et al, Appl

Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes. Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example:

selectivity/specificity, for conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (K_m), including broadening substrate binding to include non-natural substrates; inhibition (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; and anaerobic activity, for operation of an aerobic enzyme in the absence of oxygen.

[00137] Described below in more detail are exemplary methods that have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of an ethylene glycol pathway enzyme or protein.

[00138] 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 Mn²⁺ 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.

[00139] 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 Mn²⁺ concentration can vary the mutation rate somewhat. This technique uses a simple error-prone, single-step method to create a full copy of the plasmid with 3 - 4 mutations/kbp. No restriction enzyme digestion or specific primers are required. Additionally, this method is typically available as a commercially available kit. [00140] 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. Fragments prime each other and recombination occurs when one copy primes another copy (template switch). This method can be used with >lkbp DNA sequences. In addition to mutational recombinants created by fragment reassembly, 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.

[00141] 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 (Taq and Mutazyme) reduce error-prone biases because of opposite mutational spectra.

[00142] In Random Priming Recombination (RPR) 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.

[00143] In Heteroduplex Recombination linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (Volkov et al, Nucleic Acids Res. 27:el8 (1999); and Volkov et al, Methods Enzymol. 328:456-463 (2000)). The mismatch repair step is at least somewhat mutagenic. Heteroduplexes transform more efficiently than linear homoduplexes. This method is suitable for large genes and whole operons. [00144] Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al., Nat. Biotechnol. 19:354-359 (2001)) employs Dnase I fragmentation and size fractionation of single stranded DNA (ssDNA). Homologous fragments are hybridized in the absence of polymerase to a complementary ssDNA scaffold. Any overlapping unhybridized fragment ends are trimmed down by an 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.

[00145] Recombined Extension on Truncated templates (RETT) entails template switching of unidirectionally growing strands from primers in the presence of

unidirectional ssDNA fragments used as a pool of templates (Lee et al, J. Molec.

Catalysis 26: 119-129 (2003)). No DNA endonucleases are used. Unidirectional ssDNA is made by DNA polymerase with random primers or serial deletion with exonuclease. Unidirectional ssDNA are only templates and not primers. Random priming and exonucleases do not introduce sequence bias as true of enzymatic cleavage of DNA shuffling/RACHITT. RETT can be easier to optimize than StEP because it uses normal PCR conditions instead of very short extensions. Recombination occurs as a component of the PCR steps, that is, no direct shuffling. This method can also be more random than StEP due to the absence of pauses.

[00146] In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol.Biol 352: 191-204 (2007); Bergquist et al, Biomol.Eng 22:63-72 (2005); Gibbs et al, Gene 271 : 13-20 (2001)) this can be used to control the tendency of other methods such as DNA shuffling to regenerate parental genes. This method can be combined with random mutagenesis (epPCR) of selected gene segments. This can be a good method to block the reformation of parental sequences. No endonucleases are needed. By adjusting input concentrations of segments made, one can bias towards a desired backbone. This method allows DNA shuffling from unrelated parents without restriction enzyme digests and allows a choice of random mutagenesis methods.

[00147] Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY) creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest

(Ostermeier et al, Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al, Nat. Biotechnol. 17: 1205-1209 (1999)). Truncations are introduced in opposite direction on pieces of 2 different genes. These are ligated together and the fusions are cloned. This technique does not require homology between the 2 parental genes. When ITCHY is combined with DNA shuffling, the system is called SCRATCHY (see below). A major advantage of both is no need for homology between parental genes; for example, functional fusions between an E. coli and a human gene were created via ITCHY. When ITCHY libraries are made, all possible crossovers are captured.

[00148] Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO- ITCHY) is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (Lutz et al, Nucleic Acids Res 29:E16 (2001)). Relative to ITCHY, THIO- ITCHY can be easier to optimize, provide more reproducibility, and adjustability.

[00149] 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%. [00150] In Random Drift Mutagenesis (RNDM) mutations are made via epPCR followed by screening/selection for those retaining usable activity (Bergquist et al, Biomol. Eng. 22:63-72 (2005)). Then, these are used in DOGS to generate recombinants with fusions between multiple active mutants or between active mutants and some other desirable parent. Designed to promote isolation of neutral mutations; its purpose is to screen for retained catalytic activity whether or not this activity is higher or lower than in the original gene. RNDM is usable in high throughput assays when screening is capable of detecting activity above background. RNDM has been used as a front end to DOGS in generating diversity. The technique imposes a requirement for activity prior to shuffling or other subsequent steps; neutral drift libraries are indicated to result in higher/quicker improvements in activity from smaller libraries. Though published using epPCR, this could be applied to other large-scale mutagenesis methods. [00151] Sequence Saturation Mutagenesis (SeSaM) 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

Wong et al, Anal. Biochem. 341 : 187-189 (2005)). Using this technique it can be possible to generate a large library of mutants within 2 to 3 days using simple methods. This technique is non-directed in comparison to the mutational bias of DNA polymerases. Differences in this approach makes this technique complementary (or an alternative) to epPCR.

[00152] In Synthetic Shuffling, 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)). In this technique, one can design the fragments to be shuffled. This aids in increasing the resulting diversity of the progeny. One can design 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.

[00153] 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. Additionally, the technique works well with very short fragments (86 bp) and has a low error rate. The chemical cleavage of DNA used in this technique results in very few unshuffled clones. [00154] In Sequence Homology-Independent Protein Recombination (SHIPREC), a linker is used to facilitate fusion between two distantly related or unrelated genes.

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.

19:456-460 (2001)). This produces a limited type of shuffling and a separate process is required for mutagenesis. In addition, since no homology is needed, this technique can create a library of chimeras with varying fractions of each of the two unrelated parent genes. SHIPREC was tested with a heme-binding domain of a bacterial CP450 fused to N-terminal regions of a mammalian CP450; this produced mammalian activity in a more soluble enzyme.

[00155] In Gene Site Saturation Mutagenesis™ (GSSM™) 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. The sequence in the primer is NNN or NNK (coding) and MNN (noncoding) (N = all 4, K = G, T, M = A, C). After extension, 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

Reassembly, 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.

[00156] Combinatorial Cassette Mutagenesis (CCM) 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.

[00157] Combinatorial Multiple Cassette Mutagenesis (CMCM) 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.

40:3589-3591 (2001)). As with CCM, this method can test virtually all possible alterations over a target region. If used along with methods to create random mutations and shuffled genes, it provides an excellent means of generating diverse, shuffled proteins. This approach was successful in increasing, by 51 -fold, the enantioselectivity of an enzyme.

[00158] In the Mutator Strains technique, 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. In order for effective use, 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.

[00159] Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids (Rajpal et al., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)). Rather than saturating each site with all possible amino acid changes, a set of nine is chosen to cover the range of amino acid R- group chemistry. Fewer changes per site allows multiple sites to be subjected to this type of mutagenesis. A >800-fold increase in binding affinity for an antibody from low nanomolar to picomolar has been achieved through this method. This is a rational approach to minimize the number of random combinations and can increase the ability to find improved traits by greatly decreasing the numbers of clones to be screened. This has been applied to antibody engineering, specifically to increase the binding affinity and/or reduce dissociation. The technique can be combined with either screens or selections. [00160] 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 GeneReassembly™ (TGR™) Technology supplied by Verenium

Corporation). Typically this technology is used in combination with ultra-high-throughput screening to query the represented sequence space for desired improvements. This technique allows multiple gene recombination independent of homology. The exact number and position of cross-over events can be pre-determined using fragments designed via bioinformatic analysis. This technology leads to a very high level of diversity with virtually no parental gene reformation and a low level of inactive genes. Combined with GSSM™, a large range of mutations can be tested for improved activity. The method allows "blending" and "fine tuning" of DNA shuffling, for example, codon usage can be optimized.

[00161] In Silico Protein Design Automation (PDA) is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics (Hayes et al, Proc. Natl. Acad. Sci. USA 99: 15926-15931 (2002)). This technology uses in silico structure -based entropy predictions in order to search for structural tolerance toward protein amino acid variations. Statistical mechanics is applied to calculate coupling interactions at each position. Structural tolerance toward amino acid substitution is a measure of coupling. Ultimately, this technology is designed to yield desired modifications of protein properties while maintaining the integrity of structural characteristics. The method computationally assesses and allows filtering of a very large number of possible sequence variants (10⁵⁰). The choice of sequence variants to test is related to predictions based on the most favorable thermodynamics. Ostensibly only stability or properties that are linked to stability can be effectively addressed with this technology. The method has been successfully used in some therapeutic proteins, especially in engineering immunoglobulins. In silico predictions avoid testing

extraordinarily large numbers of potential variants. Predictions based on existing three- dimensional structures are more likely to succeed than predictions based on hypothetical structures. This technology can readily predict and allow targeted screening of multiple simultaneous mutations, something not possible with purely experimental technologies due to exponential increases in numbers.

[00162] Iterative Saturation Mutagenesis (ISM) 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.

[00163] Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques, as described herein. [00164] It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

EXAMPLE I

Enzyme candidates for conversion of syngas to glycine

[00165] Formate dehydrogenase (FDH, Step 1 A) is a two subunit selenocysteine- containing protein that catalyzes the transfer of electrons from a reduced carrier to C0₂, 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,

Environ Microbiol (2008)). Another set of genes encoding formate dehydrogenase activity with a propensity for C0₂ reduction is encoded by Sfum_2703 through Sfum_2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur J Biochem. 270:2476-2485 (2003)); Reda et al, PNAS 105: 10654-10658 (2008)). A similar set of genes presumed to carry out the same function are encoded by CHY 0731, CHY 0732, and CHY 0733 in C.

hydrogenoformans (Wu et al, PLoS Genet 1 :e65 (2005)). Homologs are also found in C. carboxidivorans P7.

[00166] 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,

Arch.Microbiol 149:280-285 (1988); Lovell et al, Biochemistry 29:5687-5694 (1990); O'brien et al., Experientia.Suppl. 26:249-262 (1976), FHS in Clostridium acidurici (Whitehead and Rabinowitz, J Bacteriol 167:205-209(1986); Whitehead and Rabinowitz, J.Bacteriol. 170:3255-3261 (1988)), and CHY_2385 in C hydrogenoformans (Wu et al, PLoS Genet. I:e65 (2005)). Homologs exist in C. carboxidivorans P7.

Protein ( ,cn Bank ID GI Number Organism

Moth 0109 YP 428991.1 83588982 Moorella thermoacetica

CHY 2385 YP 361182.1 78045024 Carboxydothermus

hydrogenoformans

FHS P13419.1 120562 Clostridium acidurici

CcarbDRAFT 1913 ZP 05391913.1 255524966 Clostridium carboxidivorans P7 CcarbDRAFT 2946 ZP 05392946.1 255526022 Clostridium carboxidivorans P7

[00167] In thermoacetica, E. coli, and C. hydrogenoformans,

methenyltetrahydrofolate cyclohydrolase (Step 1C, EC 3.5.4.9) and

methylenetetrahydrofolate dehydrogenase (Step ID, EC 1.5.1.5) are carried out by the bi- functional gene products of Moth l 516,folD, and CHY l 878, respectively (D'Ari and Rabinowitz, J Biol Chem. 266:23953-23958 (1991); Pierce et al, Environ Microbiol (2008); Wu et al, PLoS Genet l :e65 (2005)). A homolog exists in C carboxidivorans P7.

[00168] The reversible NAD(P)H-dependent conversion of 5,10- methylenetetrahydrofolate and C0₂ to glycine is catalyzed by the glycine cleavage complex (Step IE, also called glycine cleavage system), composed of four protein components; 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)). The glycine cleavage system of E. 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.

[00169] 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, H₂, 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. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteins form a membrane-bound enzyme complex that is proposed to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO to C0₂ and H₂ (Fox et al, J Bacteriol 178:6200-6208 (1996)). The CODH-I of C.

hydrogenoformans and its adjacent genes have been proposed to catalyze a similar functional role based on their similarity to the R. rubrum CODH/hydrogenase gene cluster (Wu et al, PLoS Genet. I :e65 (2005)). The C. hydrogenoformans CODH-I was also shown to exhibit intense CO oxidation and C0₂ reduction activities when linked to an electrode (Parkin et al, J Am.Chem.Soc. 129: 10328-10329 (2007)). The genes encoding the C. hydrogenoformans CODH-II and CooF, a neighboring protein, were cloned and sequenced (Gonzalez and Robb, FEMS Microbiol Lett. 191 :243-247 (2000)). The resulting complex was membrane -bound, although cytoplasmic fractions of CODH-II were shown to catalyze the formation of NADPH suggesting an anabolic role (Svetlitchnyi et al, J Bacteriol. 183:5134-5144 (2001)). The crystal structure of the CODH-II is also available (Dobbek et al, Science 293: 1281-1285 (2001)). Additional CODH enzymes can be found in a diverse array of organisms including Clostridium ljungdahli, Geobacter metallireducens GS-15, Chlorobium phaeobacteroides DSM 266, Clostridium

cellulolyticum H10, Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774, Pelobacter carbinolicus DSM 2380, and Campylobacter curvus 525.92.

Protein ( ,cn Bank ID Organism

Number

CODH (putative) YP 430813 83590804 Moorella thermoacetica

CODH-I (CooS- YP 360644 78043418 Carboxydothermus

I) hydrogenoformans

CooF YP 360645 78044791 Carboxydothermus

hydrogenoformans

HypA YP 360646 78044340 Carboxydothermus

hydrogenoformans

CooH YP 360647 78043871 Carboxydothermus

hydrogenoformans

CooU YP 360648 78044023 Carboxydothermus

hydrogenoformans CooX YP 360649 78043124 Carboxydothermus

hydrogenoformans

CooL YP 360650 78043938 Carboxydothermus

hydrogenoformans

CooK YP 360651 78044700 Carboxydothermus

hydrogenoformans

CooM YP 360652 78043942 Carboxydothermus

hydrogenoformans

CooM AAC45116 1515466 Rhodospirillum rubrum

CooK AAC45117 1515467 Rhodospirillum rubrum

CooL AAC45118 1515468 Rhodospirillum rubrum

CooX AAC45119 1515469 Rhodospirillum rubrum

CooU AAC45120 1515470 Rhodospirillum rubrum

CooH AAC45121 1498746 Rhodospirillum rubrum

CooF AAC45122 1498747 Rhodospirillum rubrum

COOH (CooS) AAC45123 1498748 Rhodospirillum rubrum

CooC AAC45124 1498749 Rhodospirillum rubrum

CooT AAC45125 1498750 Rhodospirillum rubrum

CooJ AAC45126 1498751 Rhodospirillum rubrum

CODH-II (CooS- YP_358957 78044574 Carboxydothermus

Π) hydrogenoformans

CooF YP_358958 78045112 Carboxydothermus

hydrogenoformans

CODH (putative) ZP 05390164.1 255523193 Clostridium carboxidivorans P7

ZP 05390341.1 255523371 Clostridium carboxidivorans P7

ZP 05391756.1 255524806 Clostridium carboxidivorans P7

ZP 05392944.1 255526020 Clostridium carboxidivorans P7

CooF ZP 05394386.1 255527518 Clostridium carboxidivorans P7

CooF ZP 05394384.1 255527516 Clostridium carboxidivorans P7

HypA ZP 05392031.1 255525086 Clostridium carboxidivorans P7

CooH ZP 05392429.1 255525492 Clostridium carboxidivorans P7

CooL ZP 05392428.1 255525491 Clostridium carboxidivorans P7

CooM ZP 05392434.1 255525497 Clostridium carboxidivorans P7

CLJU c09110 ADK13979.1 300434212 Clostridium ljungdahli

CLJU c09100 ADK13978.1 300434211 Clostridium ljungdahli

CLJU c09090 ADK13977.1 300434210 Clostridium ljungdahli

Cpha266 0148 YP 910642.1 119355998 Chlorobium phaeobacteroides

Cpha266 0149 YP 910643.1 119355999 Chlorobium phaeobacteroides

Ccel 0438 YP 002504800.1 220927891 Clostridium cellulolyticum H10

Ddes_0382 YP 002478973.1 220903661 Desulfovibrio desulfuricans subsp.

desulfuricans

Ddes_0381 YP_002478972.1 220903660 Desulfovibrio desulfuricans subsp.

desulfuricans

Pear 0057 YP 355490.1 7791767 Pelobacter carbinolicus

Pear 0058 YP 355491.1 7791766 Pelobacter carbinolicus

Pear 0058 YP 355492.1 7791765 Pelobacter carbinolicus

CooS YP 001407343.1 154175407 Campylobacter curvus [00170] Hydrogenase enzymes useful in the invention uptake molecular hydrogen and transfer electrons to acceptors such as ferredoxins. In some cases, hydrogenase encoding genes are located adjacent to a CODH. In Rhodospirillum rubrum, the encoded

CODH/hydrogenase proteins form a membrane-bound enzyme complex that has been indicated to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO and H₂0 to C0₂ and H₂ (Fox et al, J Bacteriol. 178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and its adjacent genes have been proposed to catalyze a similar functional role based on their similarity to the R. rubrum

CODH/hydrogenase gene cluster (Wu et al, PLoS Genet. I :e65 (2005)). The C.

hydrogenoformans CODH-I was also shown to exhibit intense CO oxidation and C0₂ reduction activities when linked to an electrode (Parkin et al., J Am.Chem.Soc. 129: 10328- 10329 (2007)).

Protein ( ,cn Bank ID GI Number Organism

CooL AAC45118 1515468 Rhodospirillum rubrum

CooX AAC45119 1515469 Rhodospirillum rubrum

CooU AAC45120 1515470 Rhodospirillum rubrum

CooH AAC45121 1498746 Rhodospirillum rubrum

CooF AAC45122 1498747 Rhodospirillum rubrum

CODH (CooS) AAC45123 1498748 Rhodospirillum rubrum

CooC AAC45124 1498749 Rhodospirillum rubrum

CooT AAC45125 1498750 Rhodospirillum rubrum

CooJ AAC45126 1498751 Rhodospirillum rubrum

CODH-I (CooS-I) YP 360644 7804347S Carboxydothermus

hydrogenoformans

CooF YP 360645 78044791 Carboxydothermus

hydrogenoformans

HypA YP 360646 78044340 Carboxydothermus

hydrogenoformans

CooH YP 360647 78043871 Carboxydothermus

hydrogenoformans

CooU YP 360648 78044023 Carboxydothermus

hydrogenoformans

CooX YP 360649 78043124 Carboxydothermus

hydrogenoformans

CooL YP 360650 78043938 Carboxydothermus

hydrogenoformans

CooK YP 360651 78044700 Carboxydothermus

hydrogenoformans

CooM YP 360652 78043942 Carboxydothermus

hydrogenoformans

CooC YP 360654.1 78043296 Carboxydothermus

hydrogenoformans

CooA-1 YP 360655.1 78044021 Carboxydothermus

hydrogenoformans [00171] The genomes of E. coli and other enteric bacteria encode up to four

hydrogenase enzymes (Sawers, G., Antonie Van Leeuwenhoek 66:57-88 (1994); Sawers et al, J Bacteriol. 164: 1324-1331 (1985); Sawers and Boxer, Eur.J Biochem. 156:265-275 (1986); Sawers et al, J Bacteriol. 168:398-404 (1986)). Given the multiplicity of enzyme activities E. coli or another host organism can provide sufficient hydrogenase activity to split incoming molecular hydrogen and reduce the corresponding acceptor. Endogenous hydrogen- lyase enzymes of E. 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.

190: 1447-1458 (2008)). 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 C0₂ as the exclusive carbon source indicating that reducing equivalents are extracted from H₂ to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L., J Bacteriol. 150:702-709 (1982); Drake and Daniel, Res Microbiol 155:869-883 (2004); Kellum and Drake, J Bacteriol. 160:466- 469 (1984)). 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).

Protein GenBank ID GI Number Organism

HypA NP 417206 16130633 Escherichia coli

HypB NP 417207 16130634 Escherichia coli

HypC NP 417208 16130635 Escherichia coli

HypD NP 417209 16130636 Escherichia coli

HypE NP 417210 226524740 Escherichia coli

HypF NP 417192 16130619 Escherichia coli

HycA NP 417205 16130632 Escherichia coli

HycB NP 417204 16130631 Escherichia coli

HycC NP 417203 16130630 Escherichia coli

HycD NP 417202 16130629 Escherichia coli

HycE NP 417201 16130628 Escherichia coli

HycF NP 417200 16130627 Escherichia coli HycG NP 417199 16130626 Escherichia coli

HycH NP 417198 16130625 Escherichia coli

Hycl NP 417197 16130624 Escherichia coli

HyfA NP 416976 90111444 Escherichia coli

HyfB NP 416977 16130407 Escherichia coli

HyfC NP 416978 90111445 Escherichia coli

HyfD NP 416979 16130409 Escherichia coli

HyfE NP 416980 16130410 Escherichia coli

HyfF NP 416981 16130411 Escherichia coli

HyfG NP 416982 16130412 Escherichia coli

HyfH NP 416983 16130413 Escherichia coli

Hyfl NP 416984 16130414 Escherichia coli

Hyft NP 416985 90111446 Escherichia coli

HyfR NP 416986 90111447 Escherichia coli

[00172] Proteins in M. thermoacetica whose genes are homologous to the E. coli hydrogenase genes are shown below.

Protein ( ,cn Bank ID GI Number Organism

Moth 2175 YP 431007 83590998 Moorella thermoacetica

Moth 2176 YP 431008 83590999 Moorella thermoacetica

Moth 2177 YP 431009 83591000 Moorella thermoacetica

Moth 2178 YP 431010 83591001 Moorella thermoacetica

Moth 2179 YP 431011 83591002 Moorella thermoacetica

Moth 2180 YP 431012 83591003 Moorella thermoacetica

Moth 2181 YP 431013 83591004 Moorella thermoacetica

Moth 2182 YP 431014 83591005 Moorella thermoacetica

Moth 2183 YP 431015 83591006 Moorella thermoacetica

Moth 2184 YP 431016 83591007 Moorella thermoacetica

Moth 2185 YP 431017 83591008 Moorella thermoacetica

Moth 2186 YP 431018 83591009 Moorella thermoacetica

Moth 2187 YP 431019 83591010 Moorella thermoacetica

Moth 2188 YP 431020 83591011 Moorella thermoacetica

Moth 2189 YP 431021 83591012 Moorella thermoacetica

Moth 2190 YP 431022 83591013 Moorella thermoacetica

Moth 2191 YP 431023 83591014 Moorella thermoacetica

Moth 2192 YP 431024 83591015 Moorella thermoacetica

Moth 0439 YP 429313 83589304 Moorella thermoacetica

Moth 0440 YP 429314 83589305 Moorella thermoacetica

Moth 0441 YP 429315 83589306 Moorella thermoacetica

Moth 0442 YP 429316 83589307 Moorella thermoacetica

Moth 0809 YP 429670 83589661 Moorella thermoacetica

Moth 0810 YP 429671 83589662 Moorella thermoacetica

Moth 0811 YP 429672 83589663 Moorella thermoacetica

Moth 0812 YP 429673 83589664 Moorella thermoacetica

Moth 0814 YP 429674 83589665 Moorella thermoacetica

Moth 0815 YP 429675 83589666 Moorella thermoacetica Moth 0816 YP 429676 83589667 Moorella thermoacetica

Moth 1193 YP 430050 83590041 Moorella thermoacetica

Moth 1194 YP 430051 83590042 Moorella thermoacetica

Moth 1195 YP 430052 83590043 Moorella thermoacetica

Moth 1196 YP 430053 83590044 Moorella thermoacetica

Moth 1717 YP 430562 83590553 Moorella thermoacetica

Moth 1718 YP 430563 83590554 Moorella thermoacetica

Moth 1719 YP 430564 83590555 Moorella thermoacetica

Moth 1883 YP 430726 83590717 Moorella thermoacetica

Moth 1884 YP 430727 83590718 Moorella thermoacetica

Moth 1885 YP 430728 83590719 Moorella thermoacetica

Moth 1886 YP 430729 83590720 Moorella thermoacetica

Moth 1887 YP 430730 83590721 Moorella thermoacetica

Moth 1888 YP 430731 83590722 Moorella thermoacetica

Moth 1452 YP 430305 83590296 Moorella thermoacetica

Moth 1453 YP 430306 83590297 Moorella thermoacetica

Moth 1454 YP 430307 83590298 Moorella thermoacetica

[00173] Genes encoding hydrogenase enzymes from C. ljungdahli are shown below.

[00174] 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 0₂-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. Chem., 284(52), 36462-36472 (2009)), and Thiocapsa roseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728 (2004)). The Synechocystis enzyme is capable of generating NADPH from hydrogen. Overexpression of both the Hox operon from Synechocystis str. PCC 6803 and the accessory genes encoded by the Hyp operon from Nostoc sp. PCC 7120 led to increased hydrogenase activity compared to expression of the Hox genes alone (Germer, J. Biol. Chem. 284(52), 36462-36472 (2009)).

Protein ( ,cn Bank ID GI Number Organism

HoxF NP 942727.1 38637753 Ralstonia eutropha HI 6

HoxU NP 942728.1 38637754 Ralstonia eutropha HI 6

HoxY NP 942729.1 38637755 Ralstonia eutropha HI 6

HoxH NP 942730.1 38637756 Ralstonia eutropha HI 6

HoxW NP 942731.1 38637757 Ralstonia eutropha HI 6

Hoxl NP 942732.1 38637758 Ralstonia eutropha HI 6

HoxE NP 953767.1 39997816 Geobacter sulfurreducens

HoxF NP 953766.1 39997815 Geobacter sulfurreducens

HoxU NP 953765.1 39997814 Geobacter sulfurreducens

HoxY NP 953764.1 39997813 Geobacter sulfurreducens

HoxH NP 953763.1 39997812 Geobacter sulfurreducens

GSU2717 NP 953762.1 39997811 Geobacter sulfurreducens

HoxE NP 441418.1 16330690 Synechocystis str. PCC 6803

HoxF NP 441417.1 16330689 Synechocystis str. PCC 6803

Unknown NP_441416.1 16330688 Synechocystis str. PCC 6803 function

HoxU NP 441415.1 16330687 Synechocystis str. PCC 6803

HoxY NP 441414.1 16330686 Synechocystis str. PCC 6803

Unknown NP_441413.1 16330685 Synechocystis str. PCC 6803 function

Unknown NP_441412.1 16330684 Synechocystis str. PCC 6803 function

HoxH NP 441411.1 16330683 Synechocystis str. PCC 6803

HypF NP 484737.1 17228189 Nostoc sp. PCC 7120

HypC NP 484738.1 17228190 Nostoc sp. PCC 7120

HypD NP 484739.1 17228191 Nostoc sp. PCC 7120

Unknown NP_484740.1 17228192 Nostoc sp. PCC 7120

function

HypE NP 484741.1 17228193 Nostoc sp. PCC 7120

HypA NP 484742.1 17228194 Nostoc sp. PCC 7120

HypB NP 484743.1 17228195 Nostoc sp. PCC 7120

Hox IE AAP50519.1 37787351 Thiocapsa roseopersicina Protein ( ,cn Bank ID GI Number Organism

HoxlF AAP50520.1 37787352 Thiocapsa roseopersicina

HoxlU AAP50521.1 37787353 Thiocapsa roseopersicina

HoxlY AAP50522.1 37787354 Thiocapsa roseopersicina

HoxlH AAP50523.1 37787355 Thiocapsa roseopersicina

[00175] Hydrogenase and CODH enzymes transfer electrons to acceptors such as ferridoxins. 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). The H. 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,

Extremophiles 14:79-85 (2010)). The 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). Additional 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.

Protein ( ,cn Bank ID GI Number Organism

fdxl BAE02673.1 68163284 Hydrogenobacter thermophilus

M11214.1 AAA83524.1 144806 Clostridium pasteurianum

Zfx AAY79867.1 68566938 Sulfolobus acidocalarius

Fdx AAC75578.1 1788874 Escherichia coli

hp 0277 AAD07340.1 2313367 Helicobacter pylori fdxA CAL34484.1 112359698 Campylobacter jejuni

Moth 0061 ABC 18400.1 83571848 Moorella thermoacetica

Moth 1200 ABC19514.1 83572962 Moorella thermoacetica Moth 1888 ABC20188.1 83573636 Moorella thermoacetica

Moth 2112 ABC20404.1 83573852 Moorella thermoacetica

Moth 1037 ABC19351.1 83572799 Moorella thermoacetica

CcarbDRAFT 4383 ZP 05394383.1 255527515 Clostridium carboxidivorans P7

CcarbDRAFT 2958 ZP 05392958.1 255526034 Clostridium carboxidivorans P7

CcarbDRAFT 2281 ZP 05392281.1 255525342 Clostridium carboxidivorans P7

CcarbDRAFT 5296 ZP 05395295.1 255528511 Clostridium carboxidivorans P7

CcarbDRAFT 1615 ZP 05391615.1 255524662 Clostridium carboxidivorans P7

CcarbDRAFT 1304 ZP 05391304.1 255524347 Clostridium carboxidivorans P7 cooF AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxN CAA35699.1 46143 Rhodobacter capsulatus

Rru A2264 ABC23064.1 83576513 Rhodospirillum rubrum

Rru A1916 ABC22716.1 83576165 Rhodospirillum rubrum

Rru A2026 ABC22826.1 83576275 Rhodospirillum rubrum

cooF AAC45122.1 1498747 Rhodospirillum rubrum

fdxN AAA26460.1 152605 Rhodospirillum rubrum

Alvin 2884 ADC63789.1 288897953 Allochromatium vinosum DSM 180 fdx YP 002801146.1 226946073 Azotobacter vinelandii DJ

CKL 3790 YP 001397146.1 153956381 Clostridium kluyveri DSM 555 ferl NP 949965.1 39937689 Rhodopseudomonas palustris CGA009 fdx CAA12251.1 3724172 Thauera aromatica

CHY 2405 YP 361202.1 78044690 Carboxydothermus hydrogenoformans fer YP 359966.1 78045103 Carboxydothermus hydrogenoformans fer AAC83945.1 1146198 Bacillus subtilis

fdxl NP 249053.1 15595559 Pseudomonas aeruginosa PA01 fhL AP 003148.1 89109368 Escherichia coli K-12

CLJU c00930 ADK13195.1 300433428 Clostridium ljungdahli

CLJU cOOOlO ADK13115.1 300433348 Clostridium ljungdahli

CLJU c01820 ADK13272.1 300433505 Clostridium ljungdahli

CLJU c 17980 ADK14861.1 300435094 Clostridium ljungdahli

CLJU c 17970 ADK14860.1 300435093 Clostridium ljungdahli

CLJU c22510 ADK15311.1 300435544 Clostridium ljungdahli

CLJU c26680 ADK15726.1 300435959 Clostridium ljungdahli

CLJU c29400 ADK15988.1 300436221 Clostridium ljungdahli

[00176] 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). Ferredoxin:NADP+

oxidoreductase (FNR, EC 1.18.1.2) has a noncovalently bound FAD co factor that

facilitates the reversible transfer of electrons from NADPH to low-potential acceptors such as ferredoxins or flavodoxins (Blaschkowski et al, Eur. J. Biochem. 123:563-569 (1982);

Fujii et al, 1977). 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). 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

Clostridium carboxydivorans P7. The 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)). Finally, 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.

Protein ( ,cn Bank ID GI Number Organism

fqrB NP 207955.1 15645778 Helicobacter pylori fqrB YP 001482096.1 157414840 Campylobacter jejuni

RPA3954 CAE29395.1 39650872 Rhodopseudomonas palustris fpr BAH29712.1 225320633 Hydrogenobacter

thermophilus

yumC NP 391091.2 255767736 Bacillus subtilis

fpr P28861.4 399486 Escherichia coli

hcaD AAC75595.1 1788892 Escherichia coli

LOCI 00282643 NP 001149023.1 226497434 Zea mays

NfnA YP 001393861.1 153953096 Clostridium kluyveri

NfnB YP 001393862.1 153953097 Clostridium kluyveri

CcarbDRAFT 2639 ZP 05392639.1 255525707 Clostridium carboxidivorans

P7

CcarbDRAFT 2638 ZP 05392638.1 255525706 Clostridium carboxidivorans

P7

CcarbDRAFT 2636 ZP 05392636.1 255525704 Clostridium carboxidivorans

P7

CcarbDRAFT 5060 ZP 05395060.1 255528241 Clostridium carboxidivorans

P7

CcarbDRAFT 2450 ZP 05392450.1 255525514 Clostridium carboxidivorans

P7

CcarbDRAFT 1084 ZP 05391084.1 255524124 Clostridium carboxidivorans

P7 RnfC EDK33306.1 146346770 Clostridium kluyveri

RnfD EDK33307.1 146346771 Clostridium kluyveri

RnfG EDK33308.1 146346772 Clostridium kluyveri

RnfE EDK33309.1 146346773 Clostridium kluyveri

RnfA EDK33310.1 146346774 Clostridium kluyveri

RnfB EDK33311.1 146346775 Clostridium kluyveri

CLJU cll410 (RnfB) ADK14209.1 300434442 Clostridium ljungdahli

CLJU cll400 (RnfA) ADK14208.1 300434441 Clostridium ljungdahli

CLJU cll390 (RnfE) ADK14207.1 300434440 Clostridium ljungdahli

CLJU cll380 (RnfG) ADK14206.1 300434439 Clostridium ljungdahli

CLJU cll370 (RnfD) ADK14205.1 300434438 Clostridium ljungdahli

CLJU cll360 (RnfC) ADK14204.1 300434437 Clostridium ljungdahli

EXAMPLE II

Enzyme candidates for glycine to EG pathways via serine

[00177] Conversion of glycine to serine is catalyzed by serine

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

269:9155-65 (1994)). Similar enzymes have been studied in Corynebacterium glutamicum and Methylobacterium extorquens (Chistoserdova et al, J Bacteriol 176:6759-62 (1994); Schweitzer et al, J Biotechnol 139:214-21 (2009)).

[00178] The conversion of serine to hydroxypyruvate (Step 1M) is catalyzed by an enzyme with serine aminotransferase, amine oxidase or dehydrogenase activity.

Exemplary 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. 4:333-340 (2000)). 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.,

Biochem.Biophys.Res.Commun. 228:341-346 (1996)). The human enzyme, encoded by agxt, functions as a serine :pyruvate aminotransferase, an alanine: 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. In addition to the alanine: glyoxylate acitivty, 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)). In several organisms 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)).

[00179] The conversion of serine to hydroxypyruvate (Figure 1, Step 1) is alternately catalyzed by serine dehydrogenase, also called serine oxidoreductase (deaminating).

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). An enzyme with serine dehydrogenase activity from

Petroselinum crispum was purified and characterized although the gene associated with the enzyme has not been identified to date (Kretovich et al., Izv.Akad.Nauk SSSR Ser.Biol. 2:295-301 (1966)). Serine dehydrogenase activity attributed to L-amino-acid

dehydrogenase was identified in soil bacteria isolates, but specific genes were not identified (Mohammadi et al., Iran Biomed.J 11 :131-135 (2007)). The glutamate dehydrogenase from Vigna unguiculata accepts serine as an alternate substrate. The gene associated with this enzyme has not been identified to date. Other glutamate

dehydrogenase enzymes are encoded by gdhA in Escherichia coli (Korber et al, J

Mol.Biol. 234:1270-1273 (1993); McPherson et al, Nucleic Acids Res. 11 :5257-5266 (1983)), gdh from Thermotoga maritime (Kort et al, Extremophiles. 1 :52-60 (1997); Lebbink et al, J Mol.Biol. 280:287-296 (1998); Lebbink et al, J Mol.Biol. 289:357-369 (1999)), and gdhAl from Halobacterium salinarum (Ingoldsby et al, Gene 349:237-244 (2005)).

[00180] Serine oxidase enzymes convert also convert serine to hydroxypyruvate. Serine oxidase converts serine, 0₂ 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)).

Stereoisomers of serine are interconverted by serine racemace enzymes (Miranda, et al., Gene, 256:183-188 (2000); Arias et al Mol Microbiol 31 : 1653-64 (1999)). Exemplary serine oxidase, activator enzymes and serine racemase enzymes are shown below.

Protein ( ,cn Bank ID GI Number Organism

Srr NP 068766.1 11345492 Homo sapiens

DAOA NP 758958.3 126362975 Homo sapiens

DAOA NP 001155284.1 240120173 Homo sapiens

DAOA NP 001155286.1 240120029 Homo sapiens Protein ( ,οιι Bank ID GI Number Organism

vanT AAD22403.1 4545123 Enterococcus gallinarum

[00181] Decarboxylation of hydroxypyruvate to glycolaldehyde (Figure 1, Step 3 and Figure 2, Step 3) is catalyzed by hydroxypyruvate decarboxylase (Step IN, EC 4.1.1.40), an enzyme found in many mammals (Hendrick et al., Arch.Biochem.Biophys. 105:261-269 (1964)). The enzyme activity has been studied in the context of hydroxypyruvate metabolism to oxalate in rat mitochondria, although the activity is not associated with a gene to date (Rofe et al, Biochem.Med.Metab Biol. 36: 141-150 (1986)). Other keto-acid decarboxylases include pyruvate decarboxylase (EC 4.1.1.1), benzoylformate

decarboxylase (EC 4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chain alpha- ketoacid decarboxylase. Several 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.

268: 1698-1704 (2001); Li et al, Biochemistry. 38: 10004-10012 (1999); ter Schure et al, Appl.Environ.Microbiol. 64:1303-1307 (1998)). The PDC from Zymomonas mobilus, encoded by pdc, also has a broad substrate range and has been a subject of directed engineering studies to alter the affinity for different substrates (Siegert et al, Protein Eng Des Sel 18:345-357 (2005)). An additional candidate is the kdcA gene product of

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)).

[00182] The reduction of glycolaldehyde to ethylene glycol (Step II) is catalyzed by glycolaldehyde reductase. The iron-activated 1,2-PDO oxidoreductase (EC 1.1.1.77) E. coli encoded by fucO efficiently catalyzes the reduction of glycolaldehyde (Obradors et al, Eur J Biochem. 258:207-213 (1998); Boronat et al, J Bacteriol. 153: 134-139 (1983)). Other aldehyde reductase enzyme candidates include air A from Acinetobacter sp. Strain M-l encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al.,

Appl.Environ.Microbiol. 66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et al, Nature 451 : 86-89 (2008)) and the adhA gene product from Zymomonas mobilis, which was demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al, Appl Microbiol Biotechnol 22:249-254 (1985)).

[00183] Serine decarboxylase (EC 4.1.1.-) 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.

[00184] The conversion of ethanolamine to glycolaldehyde is catalyzed by an enzyme with ethanolamine aminotransferase, amine oxidase or dehydrogenase activity. An enzyme with ethanolamine aminotransferase activity has not been demonstrated to date.

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.

172:7035-7042 (1990)) and puuE (Kurihara et al, J.Biol.Chem. 280:4602-4608 (2005)). 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. The E. 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)). In addition, 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)).

[00185] The oxidative deamination of ethanolamine to glycolaldehyde is catalyzed by 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, 0₂ 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. Alternately, 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

Bacteriol. 150:398-401 (1982)). Additional enzyme candidates are found in Geobacillus stearothermophilus (Heydari et al, Appl Environ.Microbiol 70:937-942 (2004)),

Agrobacterium tumefaciens (Hashimoto et al, J Biochem. 106:76-80 (1989); Misono and Nagasaki, J Bacteriol. 150:398-401 (1982)), and Achromobacter denitrificans

(Ruldeekulthamrong et al, BMB.Rep. 41 :790-795 (2008)).

[00186] 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

(Chistoserdova et al, J Bacteriol. 185:2980-2987 (2003)). Hydroxypyruvate reductase enzymes from Hyphomicrobium methylovorum and Methylobacterium sp. MB200 have been cloned and heterologously expressed in E. coli (Yoshida et al, Eur. J Biochem.

223:727-732 (1994)). 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 (GRHPPv) are found in mammals including Homo sapiens and Mus musculus.

Recombinant NADPH-dependent GRHPR enzymes from these organisms were heterologously expressed in E. coli (Booth et al, J Mol.Biol. 360: 178-189 (2006)).

Protein GenBank ID GI Number Organism

ghrA NP 415551.2 90111205 Escherichia coli

ghrB NP 418009.2 90111614 Escherichia coli

D31857.1 :286..1254 BAA06662.1 1304133 Hyphomicrobium methylovorum Protein ( ,cn Bank ID GI Number Organism

hprA ACS39571.1 240008345 Methylobacterium extorquens

GRHPR NP 036335.1 6912396 Homo sapiens

GRHPR NP 525028.1 17933768 Mus musculus

[00187] 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.

[00188] An additional candidate 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. In 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)). This enzyme has been overexpressed and characterized in E. coli (Phalip et al., FEBS Lett. 351 :95-99 (1994)). In other organisms the enzyme is a dimer, encoded by aldC in Streptococcus thermophilus (Monnet et al, Lett. Appl. Microbiol. 36:399-405 (2003)), aldB in Bacillus brevis (Najmudin et al, Acta Cry stallogr. D.Biol. Cry stallogr. 59: 1073-1075 (2003); Diderichsen et al, J.Bacteriol. 172:4315-4321 (1990)) and budA from Enterobacter aerogenes (Diderichsen et al, J.Bacteriol. 172:4315-4321 (1990)). The enzyme from Bacillus brevis was cloned and overexpressed in Bacillus subtilis and structurally characterized (Najmudin et al, Acta Cry stallogr. D.Biol. Cry stallogr. 59: 1073-1075 (2003)). A similar enzyme from

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)).

Protein ( ,cn Bank ID GI Number Organism

aldB NP 267384.1 15673210 Lactococcus lactis Protein ( ,cn Bank ID GI Number Organism

aldC Q8L208 75401480 Streptococcus thermophilus aldB P23616.1 113592 Bacillus brevis

budA P05361.1 113593 Enterobacter aerogenes

EXAMPLE III

Enzyme candidates for glycine to EG pathways via glyoxylate

[00189] 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. 125:29-34 (1980)) and Pseudonocardia dioxanivorans (Grostern et al, AEM 78:3298-3308 (2012)). 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.

[00190] Hydroxypyruvate isomerase (Step H) 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

(Ashiuchi et al, Biochim.Biophys.Acta 1435: 153-159 (1999); Cusa et al, J Bacteriol. 181 :7479-7484 (1999)). This enzyme has also been purified and characterized in Bacillus fastidiosus, although the associated gene is not known (de Windt et al,

Biochim.Biophys.Acta 613:556-562 (1980)). 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.

[00191] 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

Microbiol 120:523-27 (1980)). Enzyme candidates are described above. The aspartate transaminase from Trichoderma virie also accepts glycine as a substrate (Eze et al, supra). An enzyme with glycine dehydrogenase activity was characterized in Nitrobacter agilis (Sanders et al, J Biol Chem 247:2015-25 (1972)). A gene associated with this enzyme has not been identified to date. Enzymes with glycine oxidase activity have been characterized in Bacillus subtilis (Jamil et al, Biochem 49(34):7377-83 (2010)) and Geobacillus kaustophilus (Takami et al, Nucleic Acids Res. 32 (21), 6292-6303 (2004)).

[00192] 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);

Monterrubio et al, J Bacteriol. 182:2672-2674 (2000); Njau et al, J Biol Chem

275:38780-38786 (2000)). The glycerate dehydrogenase encoded by garR of Salmonella typhimurium was recently crystallized (Osipiuk et al, J Struct.Funct.Genomics 10:249-253 (2009)). Gene GenBank Accession No. GI o. Organism

glxR YP 004333910.1 331697671 Pseudonocardia dioxanivorans garR AAC76159.3 145693186 Escherichia coli

glxR AAC73611.1 1786719 Escherichia coli

garR NP 462161.1 16766546 Salmonella typhimurium

EXAMPLE IV

Methods for Handling CO and Anaerobic Cultures [00193] This example describes methods used in handling CO and anaerobic cultures. [00194] A. Handling of CO in small quantities for assays and small cultures. CO is an odorless, colorless and tasteless gas that is a poison. Therefore, cultures and assays that utilized CO required special handling. Several assays, including CO oxidation, acetyl-CoA synthesis, CO concentration using myoglobin, and CO tolerance/utilization in small batch cultures, called for small quantities of the CO gas that were dispensed and handled within a fume hood. Biochemical assays called for saturating very small quantities (<2 mL) of the biochemical assay medium or buffer with CO and then performing the assay. All of the CO handling steps were performed in a fume hood with the sash set at the proper height and blower turned on; CO was dispensed from a compressed gas cylinder and the regulator connected to a Schlenk line. The latter ensures that equal concentrations of CO were dispensed to each of several possible cuvettes or vials. The Schlenk line was set up containing an oxygen scrubber on the input side and an oil pressure release bubbler and vent on the other side. Assay cuvettes were both anaerobic and CO-containing. Threfore, the assay cuvettes were tightly sealed with a rubber stopper and reagents were added or removed using gas-tight needles and syringes. Secondly, small (-50 mL) cultures were grown with saturating CO in tightly stoppered serum bottles. As with the biochemical assays, the CO-saturated microbial cultures were equilibrated in the fume hood using the Schlenk line setup. Both the biochemical assays and microbial cultures were in portable, sealed containers and in small volumes making for safe handling outside of the fume hood. The compressed CO tank was adjacent to the fume hood.

[00195] Typically, 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 H₂ 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.

[00197] 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. While there are oxygen-tolerant acetogens, the repertoire of enzymes in the Wood-Ljungdahl pathway might be incompatible in the presence of oxygen because most are metallo-enzymes, key components are ferredoxins, and regulation can divert metabolism away from the Wood-Ljungdahl pathway to maximize energy acquisition. At the same time, cells in culture act as oxygen scavengers that moderate the need for extreme measures in the presence of large cell growth.

[00198] 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 0₂ concentration of 1 ppm or less and 1 atm pure N₂. In one example, 3 oxygen scrubbers/catalyst regenerators were used, and the chamber included an 0₂ 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 N₂ 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.

[00199] The anaerobic chambers achieved levels of 0₂ 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. In an alternative anaerobic chamber configuration, 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 0₂ monitoring, test strips can be used instead.

[00200] D. Anaerobic microbiology. Small cultures were handled as described above for CO handling. In particular, serum or media bottles are fitted with thick rubber stoppers and aluminum crimps are employed to seal the bottle. Medium, such as Terrific Broth, is made in a conventional manner and dispensed to an appropriately sized serum bottle. The bottles are sparged with nitrogen for ~30 min of moderate bubbling. This removes most of the oxygen from the medium and, after this step, each bottle is capped with a rubber stopper (such as Bellco 20 mm septum stoppers; Bellco, Vineland, NJ) and crimp-sealed (Bellco 20 mm). Then the bottles of medium are autoclaved using a slow (liquid) exhaust cycle. At least sometimes 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. 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. This is made by weighing the sodium sulfide into a dry beaker and the cysteine into a serum bottle, bringing both into the anaerobic chamber, dissolving the sodium sulfide into anaerobic water, then adding this to the cysteine in the serum bottle. The bottle is stoppered immediately as the sodium sulfide solution generates hydrogen sulfide gas upon contact with the cysteine. When injecting into the culture, a syringe filter is used to sterilize the solution. Other components are added through syringe needles, such as B12 (10 μΜ cyanocobalamin), nickel chloride (NiCl₂, 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 μΜ— 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). To facilitate faster growth under anaerobic conditions, the 1 liter bottles were inoculated with 50 mL of a preculture grown anaerobically. Induction of the pAl-lacOl promoter in the vectors was performed by addition of isopropyl β-D-l- thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM and was carried out for about 3 hrs. [00201] Large cultures can be grown in larger bottles using continuous gas addition while bubbling. A rubber stopper with a metal bubbler is placed in the bottle after medium addition and sparged with nitrogen for 30 minutes or more prior to setting up the rest of the bottle. Each bottle is put together such that a sterile filter will sterilize the gas bubbled in and the hoses on the bottles are compressible with small C clamps. Medium and cells are stirred with magnetic stir bars. Once all medium components and cells are added, the bottles are incubated in an incubator in room air but with continuous nitrogen sparging into the bottles.

EXAMPLE V

CO oxidation (CODH) Assay [00202] This example describes assay methods for measuring CO oxidation (CO dehydrogenase; CODH).

[00203] The 7 gene CODH/ ACS operon of Moorella thermoacetica was cloned into E. coli expression vectors. The intact ~10 kbp DNA fragment was cloned, and it is likely that some of the genes in this region are expressed from their own endogenous promoters and all contain endogenous ribosomal binding sites. These clones were assayed for CO oxidation, using an assay that quantitatively measures CODH activity. Antisera to the M. thermoacetica gene products was used for Western blots to estimate specific activity. M. thermoacetica is Gram positive, and ribosome binding site elements are expected to work well in E. coli. This activity, described below in more detail, was estimated to be -1/50th of the M. thermoacetica specific activity. It is possible that CODH activity of

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.

[00204] 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.

[00205] Initially, 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. [00206] 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.

[00207] In more detail, 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 (CH₃ viologen) stock was 1 M in water. Each assay used 20 microliters for 20 mM final concentration. When methyl viologen was added, an 18 Ga needle (partial) was used as a jacket to facilitate use of a Hamilton syringe to withdraw the CH₃ viologen. 4 -5 aliquots were drawn up and discarded to wash and gas equilibrate the syringe. A small amount of sodium dithionite (0.1 M stock) was added when making up the CH₃ viologen stock to slightly reduce the CH₃ viologen. The temperature was equilibrated to 55°C in a heated Olis spectrophotometer (Bogart GA). A blank reaction (CH₃ viologen + buffer) was run first to measure the base rate of CH₃ viologen reduction. Crude E. coli cell extracts of ACS90 and ACS91 (CODH-ACS operon of M. thermoacetica with and without, respectively, the first cooC). 10 microliters of extract were added at a time, mixed and assayed. Reduced CH₃ viologen turns purple. The results of an assay are shown in Table I.

Table I. Crude extract CO Oxidation Activities.

i ACS90 Ί .1 mg/ml ; ACS91 1 11.8 mg/m l

\ Mta98 : 9.8 mg/ml : Mta99 1 11.2 mg/m l

Extract Vol OD/ ! U/ml ! U/me

ACS90 ! 10 mi crol ite rs ; 0.073 ; 0.376 ; 0.049 ;

ACS91 : 10 mi crol ite rs \ 0.096 \ 0.494 : 0.042 ;

Mta99 ! 10 mi crol ite rs ; 0.0031 ; 0.016 ; 0.0014 ;

ACS90 : 10 mi crol ite rs : 0.099 0.51 0.066 ;

Mta99 ! 25 mi crol ite rs ; 0.012 ; 0.025 ; 0.0022 ;

ACS91 \ 25 mi crol ite rs : 0.215 \ 0.443 \ 0.037 ;

Mta98 ; 25 mi crol ite rs ! 0.019 \ 0.039 ! 0.004 :

ACS91 \ 10 mi crol ite rs \ 0.129 0.66 0.056 ;

Ave rages

; ACS90 : 0.057 U/mg

i ACS91 ; 0.045 U/mg

Mta99 : 0.0018 U/mg

[00208] Mta98/Mta99 are E. coli MG1655 strains that express methanol

methyltransferase genes from M thermoacetia and, therefore, are negative controls for the ACS90 ACS91 E. coli strains that contain M. thermoacetica CODH operons.

[00209] If ~ 1% of the cellular protein is CODH, then these figures would be approximately 100X less than the 500 U/mg activity of pure M. thermoacetica CODH. Actual estimates based on Western blots are 0.5% of the cellular protein, so the activity is about 50X less than for thermoacetica CODH. Nevertheless, this experiment demonstrates CO oxidation activity in recombinant E. coli with a much smaller amount in the negative controls. The small amount of CO oxidation (CH₃ viologen reduction) seen in the negative controls indicates that E. coli may have a limited ability to reduce CH₃ viologen. [00210] To estimate the final concentrations of CODH and Mtr proteins, SDS-PAGE followed by Western blot analyses were performed on the same cell extracts used in the CO oxidation, ACS, methyltransferase, and corrinoid Fe-S assays. The antisera used were polyclonal to purified M. thermoacetica CODH-ACS and Mtr proteins and were visualized using an alkaline phosphatase-linked goat-anti-rabbit secondary antibody. The Westerns were performed and results are shown in Figure 5. The amounts of CODH in ACS90 and ACS91 were estimated at 50 ng by comparison to the control lanes.

Expression of 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.

[00211] The CO oxidation assays were repeated using extracts of Moorella

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.

[00212] These results describe the CO oxidation (CODH) assay and results.

Recombinant E. coli cells expressed CO oxidation activity as measured by the methyl viologen reduction assay.

EXAMPLE VI

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, NiCl₂, Fe(II)NH₄S0₄, 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. An empty vector (pZA33) was used as a control, and cultures containing the pZA33 empty vector as well as both ACS90 and ACS91 were tested with both N₂ and CO. All were inoculated and grown for 36 hrs with shaking (250 rpm) at 37°C. At the end of the 36 hour period, examination of the flasks showed high amounts of growth in all. The bulk of the observed growth occurred overnight with a long lag. [00215] Given that all cultures appeared to grow well in the presence of CO, the final CO concentrations were confirmed. This was performed using an assay of the spectral shift of myoglobin upon exposure to CO. Myoglobin reduced with sodium dithionite has an absorbance peak at 435 nm; this peak is shifted to 423 nm with CO. Due to the low wavelength and need to record a whole spectrum from 300 nm on upwards, quartz cuvettes must be used. CO concentration is measured against a standard curve and depends upon the Henry's Law constant for CO of maximum water solubility = 970 micromolar at 20°C and 1 atm.

[00216] For the myoglobin test of CO concentration, cuvettes were washed 10X with water, IX with acetone, and then stoppered as with the CODH assay. N₂ was blown into the cuvettes for ~10 min. A volume of 1 ml of anaerobic buffer (HEPES, pH 8.0, 2mM DTT) was added to the blank (not equilibrated with CO) with a Hamilton syringe. A volume of 10 microliter myoglobin (~1 mM— can be varied, just need a fairly large amount) and 1 microliter dithionite (20 mM stock) were added. A CO standard curve was made using CO saturated buffer added at 1 microliter increments. Peak height and shift was recorded for each increment. 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.

Table II. Carbon Monoxide Concentrations, 36 hrs.

Strain and Growth Conditions ; Final CO concentration (micromolar) ;

pZA33-CO 930

ACS90-CO 638

494

734

883

ave 687

SD 164

ACS91-CO 728

812 ;

760

611

ave. 728

SD 85

[00217] The results shown in Table II indicate that the cultures grew whether or not a strain was cultured in the presence of CO or not. These results indicate that E. coli can tolerate exposure to CO under anaerobic conditions and that E. coli cells expressing the CODH-ACS operon can metabolize some of the CO.

[00218] These results demonstrate that E. coli cells, whether expressing CODH/ACS or not, were able to grow in the presence of saturating amounts of CO. Furthermore, these grew equally well as the controls in nitrogen in place of CO. This experiment

demonstrated that laboratory strains of E. coli are insensitive to CO at the levels achievable in a syngas project performed at normal atmospheric pressure. In addition, preliminary experiments indicated that the recombinant E. coli cells expressing

CODH/ACS actually consumed some CO, probably by oxidation to carbon dioxide.

Example VII.

Enzymes for Converting Methanol to Ethylene Glycol

[00219] This example describes gene candidates for converting methanol to ethylene glycol.

[00220] 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. [00221] 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 (EC 1.1.1.244) 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)).

[00222] 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)).

[00223] In addition to the formaldehyde dehydrogenase enzymes listed above, alternate enzymes and pathways for converting formaldehyde to formate are known in the art. For example, many organisms employ glutathione-dependent formaldehyde oxidation pathways, in which formaldehyde is converted to formate in three steps via the intermediates S-hydroxymethylglutathione and S-formylglutathione (Vorholt et al, J Bacteriol 182:6645-50 (2000)). 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). The conversion of formaldehyde to S-hydroxymethylglutathione can also occur spontaneously in the presence of glutathione.

[00224] Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.

Claims

What is claimed is:

1. A non-naturally occurring microbial organism, said microbial organism having an ethylene glycol pathway and comprising at least one exogenous nucleic acid encoding an ethylene glycol pathway enzyme expressed in a sufficient amount to produce ethylene glycol, wherein said ethylene glycol pathway comprises a pathway selected from:

(1) 1A, IB, 1C, ID, IE, IF, 1G, 1H and II;

(2) 1A, IB, 1C, ID, IE, IF, 1M, IN and II;

(3) 1A, IB, 1C, ID, IE, IF, 1M, 10 and IP;

(4) 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3R and 3Q;

(5) 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3P and 3Q;

(6) 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 31 and 3M;

(7) 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3N, 30, 3S and 3M;

(8) 4Q, 4A, IB, 1C, ID, IE, IF, 1G, 1H and II;

(9) 4Q, 4A, IB, 1C, ID, IE, IF, 1M, IN and II;

(10) 4Q, 4A, IB, 1C, ID, IE, IF, 1M, 10 and IP;

(11) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3R and 3Q;

(12) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3P and 3Q;

(13) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 31 and 3M; and

(14) 4Q, 4A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3N, 30, 3S and 3M, wherein 1 A is a formate dehydrogenase, wherein IB is a formyltetrahydro folate synthetase, wherein 1C is a methenyltetrahydro folate cyclohydrolase, ID is a

methylenetetrahydro folate dehydrogenase, wherein IE is a glycine cleavage complex, wherein IF is a serine hydroxymethyltransferase, wherein 1G is a serine decarboxylase, wherein 1H is an ethanolamine aminotransferase, an amine oxidase or a dehydrogenase, wherein II is a glycolaldehyde reductase, wherein 1M is a serine aminotransferase, amine oxidase or dehydrogenase, wherein IN is a hydroxypyruvate decarboxylase, wherein 10 is a hydroxypyruvate reductase, wherein IP a glycerate decarboxylase, wherein 3 A is a formate dehydrogenase, wherein 3B is a formyltetrahydrofolate synthetase, wherein 3C is a methenyltetrahydrofolate cyclohydrolase, wherein 3D is a methylenetetrahydrofolate dehydrogenase, wherein 3E is a glycine cleavage complex, wherein 3F is a glycine aminotransferase, amine oxidase or dehydrogenase, wherein 3G is a glyoxylate carboxyligase, wherein 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 semialdehyde reductase, wherein 3 S is an ethanolamine aminotransferase, amine oxidase or dehydrogenase, wherein 4A is a formaldehyde dehydrogenase, and wherein 4Q is a methanol dehydrogenase.

2. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen exogenous nucleic acids each encoding an ethylene glycol pathway enzyme.

3. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(14).

4. The non-naturally occuring microbial organism of claim 1, wherein said microbial organism further comprises at least one exogenous nucleic acid encoding a CO dehydrogenase, a hydrogenase or a ferredoxin oxidoreductase.

5. The non-naturally occuring microbial organism of claim 1, wherein said microbial organism further comprises a pathway that converts glucose to an ethylene glycol pathway intermediate, wherein said ethylene glycol pathway intermediate is selected from the group consisting of glycine, glyoxylate, tartronate semialdehyde, hydroxypyruvate, serine and glycerate.

6. The non-naturally occuring microbial organism of claim 5, wherein said serine pathway comprises one or more glycolytic enzyme, a 3-phosphoglycerate dehydrogenase and a phosphoserine aminotransferase.

7. The non-naturally occurring microbial organism of claim 1, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

8. The non-naturally occurring microbial organism of claim 1, wherein said non- naturally occurring microbial organism is in a substantially anaerobic culture medium.

9. A method for producing ethylene glycol, comprising culturing the non- naturally occurring microbial organism of any one of claims 1 -8 under conditions and for a sufficient period of time to produce ethylene glycol.

10. The method of claim 9, wherein said method further comprises separating the ethylene glycol from other components in the culture.

11. The method of claim 10, wherein the separating comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, absorption chromatography, or ultrafiltration.

12. Culture medium comprising bioderived ethylene glycol, wherein said bioderived ethylene glycol was produced by a non-naturally occurring microbial organism of any one of claims 1-8.

13. The culture medium of claim 12, wherein said culture medium is separated from said non-naturally occurring microbial organism.

14. Bioderived ethylene glycol produced according to the method of any one of claims 9-11.

15. A composition comprising said bioderived ethylene glycol of claim 14 and a compound other than said bioderived ethylene glycol.

16. A biobased product comprising said bioderived ethylene glycol of claims 14, wherein said biobased product is an antifreeze, a coolant, a polyester fiber, a fiberglass, a resin or a film.

17. The biobased product of claim 16 comprising at least 5%, at least 10%, at least 20%, at least 30%>, at least 40%> or at least 50%> bioderived ethylene glycol.

18. A process for producing a biobased product of claim 16 or 17 comprising chemically reacting said bioderived ethylene glycol with itself or another compound in a reaction that produces said biobased product.