EA042922B1 - MICROORGANISMS AND METHODS FOR THE BIOLOGICAL PRODUCTION OF ETHYLENE GLYCOL - Google Patents

Description

The invention relates to genetically modified microorganisms and methods for producing ethylene glycol and ethylene glycol precursors by microbiological fermentation, in particular by microbiological fermentation of a gaseous substrate.

Description of related art

Ethylene glycol, also known as monoethylene glycol (MEG), has a current market value of more than US$33 billion and is an important ingredient in a wide variety of industrial, medical and consumer products. Ethylene glycol is currently produced using chemical catalysis processes that require large amounts of energy and water, produce a number of unwanted by-products, and depend on petrochemical feedstocks. The demand for environmentally friendly materials has led to some technological advances, such as the catalytic production of ethylene glycol from sugarcane-derived ethanol.

Ethylene glycol precursors are also commercially valuable. For example, glycolate is used in leather care, personal care, dyeing, tanning, and as a cleaning agent. Glyoxylate is an intermediate for vanillin, agricultural chemicals, antibiotics, allantoin, and complexing agents.

However, no microorganism is known to be able to biologically produce ethylene glycol, and there is no well-established fully biological pathway for the production of ethylene glycol. Several biological pathways to ethylene glycol from sugars have been described in the literature. For example, Alkim et al., Microb Cell Fact, 14: 127, 2015 demonstrated the production of ethylene glycol from (D)-xylose in E. coli, but noted that aerobic conditions are needed to achieve high yields. Similarly, Pereira et al., Metab Eng, 34: 80-87, 2016, achieved the production of ethylene glycol from pentoses in E. coli. Several studies on the production of ethylene glycol from pentoses have also been performed with S. cerevisiae with conflicting results; see, for example, Uranukul et al., Metab Eng, 51: 2031, 2018.

Gas fermentation provides a way to convert a wide range of readily available low cost C1 feedstocks such as industrial waste gases, synthesis gas, or reformed methane into chemicals or fuels. Since the metabolism of gas fermentation is significantly different from that of sugar fermentation, the use of the aforementioned pathways is impractical, as these pathways would require the production of sugar precursors from gas via gluconeogenesis, an energetically negative process. To date, there is no method for the production of ethylene glycol from gaseous substrates.

In a research paper, Islam et al., Metab Eng, 41: 173-181, 2017, predicted hundreds of hypothetical pathways to produce ethylene glycol from synthesis gases in M. thermoacetia using chemoinformatics tools. However, even for a person skilled in the art, it is not possible to introduce these pathways into a fermenting gas organism, as many of the pathways are not possible due to thermodynamic or other limitations. For example, almost 2000 reactions dependent on oxygen, or oxygen-containing radicals, have been envisaged by Islam et al., which would be impossible in a strictly anaerobic system. The only ones identified by Islam et al. hypothetical pathways, with known reactions, require gluconeogenesis or ethanol as an intermediate. Therefore, there remains a need for proven, energy efficient recombinant production systems that can produce high yields of ethylene glycol and ethylene glycol precursors from gaseous substrates.

The essence of the invention

In contrast to the foregoing rationale, the present invention offers certain advantages and advances over the prior art.

Although the invention disclosed herein is not limited to specific benefits or functionality, the invention provides a genetically modified microorganism capable of producing ethylene glycol or an ethylene glycol precursor from a gaseous substrate.

In some aspects of the microorganism disclosed herein, the microorganism produces ethylene glycol or an ethylene glycol precursor via one or more intermediates selected from the group consisting of 5,10-methylenetetrahydrofolate, oxaloacetate, citrate, malate, and glycine.

In some aspects of the microorganism disclosed herein, the microorganism comprises one or more heterologous enzymes capable of converting oxaloacetate to citrate, heterologous enzymes capable of converting glycine to glyoxylate, heterologous enzymes capable of converting isocitrate to glyoxylate, and heterologous enzymes capable of converting glycolate to glycoaldehyde.

In some aspects of the microorganism disclosed herein, the heterologous enzyme capable of converting oxaloacetate to citrate is citrate [Si] synthase [2.3.3.1], ATP-1 042922 citrate synthase [2.3.3.8]; or citrate (Re)-synthase [2.3.3.3]; heterologous enzyme capable of converting glycine to glyoxylate is alanine glyoxylate transaminase [2.6.1.44], seringlyoxylate transaminase [2.6.1.45], serine pyruvate transaminase [2.6.1.51], glycine oxaloacetate transaminase [2.6.1.35] , glycine transaminase [2.6.1.4], glycine dehydrogenase [1.4.1.10], alanine dehydrogenase [1.4.1.1] or glycine dehydrogenase [1.4.2.1]; the heterologous enzyme capable of converting isocitrate to glyoxylate is isocitrate lyase [4.1.3.1]; and/or a heterologous enzyme capable of converting glycolate to glycoaldehyde is glycolaldehyde dehydrogenase [1.2.1.21], lactaldehyde dehydrogenase [1.2.1.22], succinate semialdehyde dehydrogenase [1.2.1.24], 2,5-dioxovalerate dehydrogenase [1.2 .1.26], aldehyde dehydrogenase [1.2.1.3/4/5], betaine aldehyde dehydrogenase [1.2.1.8] or aldehyde ferredoxin oxidoreductase [1.2.7.5].

In some aspects of the microorganism disclosed herein, the heterologous enzymes are derived from a genus selected from the group consisting of Bacillus, Clostridium, Escherichia, Gluconobacter, Hyphomicrobium, Lysinibacillus, Paenibacillus, Pseudomonas, Sedimenticola, Sporosarcina, Streptomyces, Thermithiobacillus, Thermotoga, and Zea.

In some aspects of the microorganism disclosed herein, one or more heterologous enzymes are codon-optimized for expression in the microorganism.

In some aspects of the microorganism disclosed herein, the microorganism further comprises one or more of: an enzyme capable of converting acetyl-CoA to pyruvate; an enzyme capable of converting pyruvate to oxaloacetate; an enzyme capable of converting pyruvate to malate; an enzyme capable of converting pyruvate to phosphenolpyruvate; an enzyme capable of converting oxaloacetate to citryl-CoA; an enzyme capable of converting citryl-CoA to citrate; an enzyme capable of converting citrate to aconitate and aconitate to isocitrate; an enzyme capable of converting phosphoenolpyruvate to oxaloacetate; an enzyme capable of converting phosphoenolpyruvate to 2-phospho-O-glycerate; an enzyme capable of converting 2-phosphoD-glycerate to 3-phospho-O-glycerate; an enzyme capable of converting 3-phospho-O-glycerate to 3-phosphonooxypyruvate; an enzyme capable of converting 3-phosphonooxypyruvate to 3-phospho-E-serine; an enzyme capable of converting 3-phosphoT-serine to serine; an enzyme capable of converting serine to glycine; an enzyme capable of converting 5,10-methylenetetrahydrofolate to glycine; an enzyme capable of converting serine to hydroxypyruvate; an enzyme capable of converting D-glycerate to hydroxypyruvate; an enzyme capable of converting malate to glyoxylate; an enzyme capable of converting glyoxylate to glycolate; an enzyme capable of converting hydroxypyruvate to glycoaldehyde; and/or an enzyme capable of converting glycoaldehyde to ethylene glycol.

In some aspects of the microorganism disclosed herein, the microorganism overexpresses a heterologous enzyme capable of converting oxaloacetate to citrate, a heterologous enzyme capable of converting glycine to glyoxylate, and/or a heterologous enzyme capable of converting glycolate to glycoaldehyde.

In some aspects of the microorganism disclosed herein, the microorganism overexpresses an enzyme capable of converting pyruvate to oxaloacetate, an enzyme capable of converting citrate to aconitate and aconitate to isocitrate, an enzyme capable of converting phosphoenolpyruvate to oxaloacetate, an enzyme capable of converting serine to glycine, an enzyme capable of converting 5,10methylenetetrahydrofolate to glycine, an enzyme capable of converting glyoxylate to glycolate; and/or an enzyme capable of converting glycoaldehyde to ethylene glycol.

In some aspects of the microorganism disclosed herein, the microorganism contains a disruptive mutation in one or more enzymes selected from the group consisting of isocitrate dehydrogenase, glycerate dehydrogenase, glycolate dehydrogenase, aldehyde ferredoxin oxidoreductase, and aldehyde dehydrogenase.

In some aspects of the microorganism disclosed herein, said microorganism is a member of a genus selected from the group consisting of Acetobacterium, Alkalibaculum, Blautia, Butyribacterium, Clostridium, Eubacterium, Moorella, Oxobacter, Sporomusa, and Thermoanaerobacter.

In some aspects of the microorganism disclosed herein, said microorganism is derived from a parent microorganism selected from the group consisting of Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Butyribacterium methylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium ljungdahlii, Clostridium magnum, Clostridium ragsdalei, Clostridium scatologenes, Eubacterium limosum, Moorella thermautotrophica, Moorella thermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides and Thermoanaerob actor kiuvi.

In some aspects of the microorganism disclosed herein, said microorganism is derived from a parent bacterium selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei.

In some aspects of the microorganism disclosed herein, the microorganism contains a native or heterologous Wood-Lyungdahl pathway.

In some aspects of the microorganism disclosed herein, the microorganism produces glyoxylate or glycolate as an ethylene glycol precursor.

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The invention further provides a method for producing ethylene glycol or an ethylene glycol precursor, comprising cultivating the microorganism disclosed herein in a nutrient medium and in the presence of a substrate, whereby the microorganism produces ethylene glycol or an ethylene glycol precursor.

In some aspects of the method disclosed herein, the substrate contains one or more of CO, CO2 and H ₂ .

In some aspects of the method disclosed herein, at least part of the substrate is a gaseous industrial waste, industrial waste gas, or synthesis gas.

In some aspects of the method disclosed herein, the microorganism produces glyoxylate or glycolate as ethylene glycol precursors.

In some aspects of the method disclosed herein, the method further includes separating the ethylene glycol or ethylene glycol precursor from the growth medium.

In some aspects of the method disclosed herein, the microorganism additionally produces one or more of: ethanol, 2,3-butanediol and succinate.

The invention further provides a composition containing ethylene glycol, obtained by the method described in this document. In some aspects, the composition is an antifreeze, preservative, dehydrating agent, or drilling fluid.

The invention further provides an ethylene glycol-containing polymer obtained by the process described herein. In some aspects, the polymer is a homopolymer or copolymer. In some aspects, the polymer is polyethylene glycol or polyethylene terephthalate.

The invention further provides a composition containing the polymer described herein. In some aspects, the composition is a fiber, resin, film, or plastic.

These and other features and advantages of the present invention will be better understood from the following detailed description, taken in conjunction with the appended claims. It should be noted that the scope of the claims is determined by the wording given therein, and not by a specific consideration of the features and advantages set forth in this description.

Brief description of graphic materials

The following detailed description of embodiments of the present invention may be better understood when read in conjunction with the following drawings, where the same structure is denoted by the same reference numerals and in which:

fig. 1 is a diagram showing the ways of obtaining ethylene glycol, glycolate and glyoxylate from a gaseous substrate containing CO, CO ₂ and/or H ₂ ;

fig. 2A-2E are maps of the plasmids used in Examples 1-4. Fig. 2A is a map of the pIPL12 expression shuttle vector as described in Example 1. FIG. 2B is a map of plasmid pMEG042 which contains B. subtilis citrate synthase, E. coli isocitrate lyase, and G. oxydans glycolaldehyde dehydrogenase as described in Example 1. FIG. 2C is a map of plasmid pMEG058 which contains S. thiotaurini alanine glyoxylate aminotransferase and P. fluorescens aldehyde dehydrogenase as described in Example 2. FIG. 2D is a map of plasmid pMEG059 which contains S. thiotaurini alanine glyoxylate aminotransferase and G. oxydans aldehyde dehydrogenase as described in Example 3. FIG. 2E is a map of plasmid pMEG061 which contains C. acidurici class V aminotransferase and P. fluorescens aldehyde dehydrogenase as described in Example 4;

fig. 3A shows biomass levels (g dry cell weight/L) of C. autoethanogenum expressing pMEG042 (clones 1-3) or C. autoethanogenum containing empty vector (negative control). Fig. 3B shows ethylene glycol produced over time in C. autoethanogenum growing autotrophically and carrying the pMEG042 expression vector compared to a negative control (empty vector). Fig. 3C shows glycolate produced over time in C. autoethanogenum growing autotrophically and harboring the expression vector pMEG042; see example 1;

fig. 4A shows biomass levels (g dry cell weight/L) of C. autoethanogenum expressing pMEG058 (clones 1-2) or C. autoethanogenum containing empty vector (negative control). Fig. 4B shows ethylene glycol produced over time in C. autoethanogenum growing autotrophically and carrying the pMEG058 expression vector compared to a negative control (empty vector); see example 2;

fig. 5A shows biomass levels (g dry cell weight/L) of C. autoethanogenum expressing pMEG059 (clones 1-3) or C. autoethanogenum containing empty vector (negative control). Fig. 5B shows ethylene glycol produced over time in C. autoethanogenum growing autotrophically and carrying the pMEG059 expression vector compared to a negative control (empty vector); see example 3;

fig. 6A shows biomass levels (g dry cell weight/L) of C. autoethanogenum expressing pMEG061 (clone 1) or C. autoethanogenum containing empty vector (negative control). Fig. 6B shows ethylene glycol produced over time in C.

autoethanogenum growing autotrophically and carrying the pMEG061 expression vector compared to a negative control (empty vector); see example 4.

Detailed Description of the Invention

This invention provides microorganisms for the biological production of ethylene glycol. A microorganism is a microscopic organism, such as a bacterium, archaea, virus, or fungus. In a preferred embodiment, the microorganism according to this invention is a bacterium.

The term "non-naturally occurring" when applied to a microorganism is intended to mean that said microorganism has at least one genetic modification not found in a natural strain of the listed species, including wild-type strains of the listed species. Non-naturally occurring microorganisms are usually developed in a laboratory or research facility. The microorganism according to this invention does not occur in nature.

The terms genetic modification, genetic alteration, or genetic engineering broadly refer to the manipulation of the genome or nucleic acids of a microorganism by human hands. Similarly, the terms genetically modified, genetically altered or genetically engineered refer to a microorganism containing such genetic modifications, genetic changes or genetically engineered elements. These terms can be used to differentiate a laboratory-produced microorganism from a naturally occurring microorganism. Methods of genetic modification include, for example, heterologous gene expression, gene or promoter insertion or deletion, nucleic acid mutation, altered gene expression or inactivation, enzyme engineering, directed evolution, knowledge engineering, random mutagenesis methods, gene shuffling, and optimization. codons. The microorganisms according to this invention are genetically engineered.

Recombinant means that the nucleic acid, protein, or microorganism is the product of genetic modification, engineering, or recombination. In general, the term recombinant refers to a nucleic acid, protein, or microorganism that contains or is encoded by genetic material derived from multiple sources, such as two or more different strains or species of microorganisms. The microorganisms according to this invention are generally recombinant.

Wild type refers to the normal form of an organism, strain, gene, or characteristic as it occurs in nature, as opposed to mutated or variant forms.

Endogenous refers to a nucleic acid or protein that is present or expressed in the wild-type microorganism or parental microorganism from which the microorganism of the present invention is derived. For example, an endogenous gene is a gene that is natively present in a wild-type microorganism or a parent microorganism from which the microorganism of the present invention is derived. In one embodiment, expression of an endogenous gene can be controlled by an exogenous regulatory element, such as an exogenous promoter.

Exogenous refers to a nucleic acid or protein that originates outside the microorganism according to the invention. For example, an exogenous gene or enzyme can be created artificially or recombinantly, and introduced into or expressed in the microorganism according to this invention. An exogenous gene or enzyme may also be isolated from a heterologous microorganism and introduced into or expressed in the microorganism according to the present invention. Exogenous nucleic acids can be adapted to be integrated into the genome of the microorganism of the invention, or to be maintained in an extrachromosomal state in the microorganism of the invention, for example, in a plasmid.

Heterologous refers to a nucleic acid or protein that is not present in the wild-type microorganism or parental microorganism from which the microorganism of the present invention is derived. For example, a heterologous gene or enzyme may be obtained from a different strain or species and introduced into or expressed in the microorganism of the present invention. The heterologous gene or enzyme may be introduced or expressed in the microorganism of the present invention in the form in which it occurs in different strains or species. Alternatively, the heterologous gene or enzyme can be modified in some way, such as by optimizing its codons for expression in the microorganism of the invention, or by designing it to change function, such as changing the direction of enzyme activity or changing substrate specificity.

In particular, the heterologous nucleic acid or protein expressed in the microorganism described herein can be obtained from Bacillus, Clostridium, Escherichia, Gluconobacter, Hyphomicrobium, Lysinibacillus, Paenibacillus, Pseudomonas, Sedimenticola, Sporosarcina, Streptomyces, Thermithiobacillus, Thermotoga, Zea, Klebsiella, Mycobacterium, Salmonella, Mycobacteroides, Staphylococcus, Burkholderia, Listeria, Acinetobacter, Shigella, Neisseria, Bordetella, Streptococcus, Enterobacter, Vibrio,

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Legionella, Xanthomonas, Serratia, Cronobacter, Cupriavidus, Helicobacter, Yersinia, Cutibacterium, Francisella, Pectobacterium, Arcobacter, Lactobacillus, Shewanella, Erwinia, Sulfurospirillum, Peptococcaceae, Thermococcus, Saccharomyces, Pyrococcus, Glycine, Homo, Ralstonia, Brevibacter ium, Methylobacterium, Geobacillus, bos gallus usa, Thermoanaerobacter, Schizosaccharomyces, Paenibacillus, Fictibacillus, Lysinibacillus, Ornithinibacillus, Halobacillus, Kurthia, Lentibacillus, Anoxybacillus, Solibacillus, Virgibacillus, Alicyclobacillus, Sporosarcina, Salimicrobium, Sporosarcina, Planococcus, Corynebacterium, Thermaerobacter, Sulfobacillus, or Symbiobacterium.

The terms polynucleotide, nucleotide, nucleotide sequence, nucleic acid, and oligonucleotide are used interchangeably. They refer to the polymeric form of nucleotides of any length, both deoxyribonucleotides and ribonucleotides, or their analogues. Polynucleotides can have any three-dimensional structure, and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), miRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleotide probes and primers. The polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides or nucleotide analogs. Nucleotide structure modifications (if any) can be introduced before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example by conjugation with a labeling moiety.

As used herein, expression refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into an mRNA or other RNA transcript) and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as gene products.

The terms polypeptide, peptide, and protein are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acid compounds. These terms also cover an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling moiety. As used herein, the term amino acid includes naturally occurring and/or non-naturally occurring or synthetic amino acids, including glycine and D- or L-optical isomers, as well as amino acid analogs and peptidomimetics.

Enzymatic activity, or simply activity, broadly refers to enzymatic activity, including, but not limited to, enzyme activity, the amount of enzyme, or the availability of enzyme required to catalyze a reaction. Accordingly, an increase in enzyme activity includes an increase in enzyme activity, an increase in the amount of enzyme, or an increase in the availability of an enzyme necessary to catalyze a reaction. Similarly, a decrease in enzyme activity includes a decrease in enzyme activity, a decrease in the amount of enzyme, or a decrease in the availability of an enzyme necessary to catalyze a reaction.

Mutated refers to a nucleic acid or protein that has been modified in the microorganism of the present invention compared to the wild-type microorganism or parental microorganism from which the microorganism of the present invention is derived. In one embodiment, the mutation may be a deletion, insertion, or substitution in a gene encoding an enzyme. In yet another embodiment, the mutation may be a deletion, insertion, or substitution of one or more amino acids in the enzyme.

The original microorganism is a microorganism used to obtain a microorganism according to this invention. The parent microorganism may be a naturally occurring microorganism (ie, a wild-type microorganism) or a previously modified microorganism (ie, a mutant or recombinant microorganism). The microorganism of the present invention may be modified to express or overexpress one or more enzymes not expressed or overexpressed in the parent microorganism. Similarly, the microorganism according to the invention may be modified to contain one or more genes that were not contained in the original microorganism. The microorganism of the present invention may also be modified not to express or to express lesser amounts of one or more of the enzymes that were expressed in the original microorganism.

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The microorganism according to this invention can be obtained essentially from any original microorganism. In one embodiment, the microorganism of the present invention may be derived from a parent microorganism selected from the group consisting of Clostridium acetobutylicum, Clostridium beijerinckii, Escherichia coli, and Saccharomyces cerevisiae. In other embodiments, the microorganism is derived from a parent microorganism selected from the group consisting of Acetobacterium woodii, Alkalibaculum bacchii, Blautia product, Butyribacterium methylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostrid ium ljungdahlii, Clostridium magnum, Clostridium ragsdalei, Clostridium scatologenes, Eubacterium limosum, Moorella thermautotrophica, Moorella thermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, and Thermoanaerobacter kiuvi. In a preferred embodiment, the parent microorganism is Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment, the parent microorganism is Clostridium autoethanogenum LZ1561 introduced June 7, 2010 to the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)), located at Inhoffenstraβ 7B, D38124 Braunschweig, Germany, June 7, 2010 under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure under accession number DSM23693. This strain is described in International Patent Application No. PCT/NZ2011/000144 published as WO 2012/015317.

The term is derived from meaning that a nucleic acid, protein, or microorganism is modified or adapted from another (eg, parental or wild-type) nucleic acid, protein, or microorganism to produce a new nucleic acid, protein, or microorganism. Such modifications or adaptations typically include the insertion, deletion, mutation, or substitution of nucleic acids or genes. As a rule, the microorganism according to this invention is derived from the original microorganism. In one embodiment, the microorganism according to this invention is derived from Clostridium autoethanogenum, Clostridium ljungdahlii or Clostridium ragsdalei. In a preferred embodiment, the microorganism according to the invention is derived from Clostridium autoethanogenum LZ1561 deposited with the DSMZ under accession number DSM23693.

The microorganism according to this invention can be further classified based on functional characteristics. For example, the microorganism of the present invention may be or may be derived from a C1 fixing microorganism, anaerobe, acetogen, ethanologen, carboxydotroph and/or methanotroph.

In table. 1 shows a typical list of microorganisms and their functional characteristics.

Acetobacterium woodii______________

Alkalibaculum bacchii

Blautia producta_____________________

Butyribacterium methylotrophicum

Clostridium aceticum_________________

Clostridium autoethanogenum

Clostridium carboxidivorans__________

Clostridium coskatii___________________

Clostridium drakei____________________

Clostridium formicoaceticum_________

Clostridium ljungdahlii_________________

Clostridium magnum_______________

Clostridium ragsdalei_________________

Clostridium scatologenes_____________

Eubacterium limosum_______________

Moorella thermautotrophica__________

Moorella thermoacetica (formerly

Clostridium thermoaceticum)

Oxobacter pfennigii

Sporomusa ovata_________________

Sporomusa silvacetica_______________

Sporomusa sphaeroides____________

Thermoanaerobacter kiuvi

Table 1

¹ Acetobacterium woodi can produce ethanol from fructose, but not from gas.

² It has not been investigated whether Clostridium magnum can be grown on CO.

³ One strain from Moorella thermoacetica, Moorella sp. HUC22-1, produces ethanol from gas.

⁴ It has not been investigated whether Sporomusa ovata can be grown on CO.

⁵ It has not been investigated whether Sporomusa silvacetica can be grown on CO.

⁶ It has not been investigated whether Sporomusa sphaeroides can be grown on CO.

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Wood-Ljungdahl refers to the Wood-Ljungdahl carbon fixation pathway described, for example, in Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008. Wood-Ljungdahl microorganisms are expected to refer to microorganisms containing the Wood-Ljungdahl pathway. As a rule, the microorganism according to this invention contains the native Wood-Ljungdahl pathway. As used herein, the Wood-Ljungdahl pathway may be a native, unmodified Wood-Ljungdahl pathway, or may be a Wood-Ljungdahl pathway with some degree of genetic modification (e.g., overexpression, heterologous expression, knockout, etc.) as long as it is used to convert CO , CO ₂ and/or H ₂ in acetyl-CoA.

C1 refers to a molecule containing one carbon atom, such as CO, CO ₂ , CH ₄ or CH3OH. C1-oxygenate refers to a one-carbon molecule that also contains at least one oxygen atom, such as CO, CO ₂ or CH3OH. A C1 carbon source refers to a one-carbon molecule that serves as a partial or sole carbon source for a microorganism according to the present invention. For example, the C1 carbon source may contain one or more of: CO, CO ₂ , CH ₄ , CH 3 OH or CH ₂ O ₂ . The C1 carbon source preferably contains one or more of: CO and CO ₂ . A C1 fixing microorganism is a microorganism capable of producing one or more products from a C1 carbon source. Typically, the microorganism according to this invention is a C1-fixing bacterium. According to a preferred embodiment, the microorganism according to this invention is obtained from the C1-fixing microorganism shown in table. 1.

Anaerobe is a microorganism that does not require oxygen for growth. An anaerobe can react negatively or even die if oxygen is present above a certain threshold. However, some anaerobes are able to tolerate low levels of oxygen (eg 0.000001-5% oxygen), sometimes referred to as microoxide conditions. Often the microorganism according to this invention is an anaerobe. In a preferred embodiment, the microorganism according to this invention is obtained from the anaerobe shown in table. 1.

Acetogens are obligate anaerobic bacteria using the Wood-Ljungdahl pathway as their primary mechanism for energy conservation and the synthesis of acetyl-CoA and acetyl-CoA derived products such as acetate (Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008 ). In particular, acetogens use the Wood-Ljungdahl pathway as (1) a mechanism for the reductive synthesis of acetyl-CoA from CO ₂ , (2) a terminal electron-withdrawing, energy-saving process, (3) a mechanism for fixing (assimilating) CO ₂ during the synthesis of cellular carbon (Drake , Acetogenic Prokaryotes, In: The Prokaryotes, ^3rd edition, p. 354, New York, NY, 2006). All naturally occurring acetogens are C1-fixing, anaerobic, autotrophic, and non-methanotrophic organisms. Typically, the microorganism according to this invention is an acetogen. In a preferred embodiment, the microorganism according to this invention is obtained from the acetogen shown in table. 1.

An ethanologen is a microorganism that produces or is capable of producing ethanol. Typically, the microorganism according to this invention is an ethanologen. In a preferred embodiment, the microorganism according to this invention is obtained from the ethanologen shown in table. 1.

An autotroph is a microorganism that can grow in the absence of organic carbon. Instead, autotrophs use inorganic carbon sources such as CO and/or CO ₂ . As a rule, the microorganism according to this invention is an autotroph. In a preferred embodiment, the microorganism according to this invention is obtained from the autotroph shown in table. 1.

A carboxydotroph is a microorganism capable of using CO2 as its sole source of carbon and energy. As a rule, the microorganism according to this invention is a carboxydotroph. In a preferred embodiment, the microorganism according to this invention is obtained from the carboxydotroph shown in table. 1.

A methanotroph is a microorganism capable of using methane as its sole source of carbon and energy. In certain embodiments, the microorganism according to the invention is a methanotroph, or derived from a methanotroph. In other embodiments, the microorganism of the invention is not a methanotroph or is not derived from a methanotroph.

According to a preferred embodiment, the microorganism according to the present invention is obtained from a Clostridia cluster including Clostridium autoethanogenum, Clostridium ljungdahlii and Clostridium ragsdalei species. Such species were first described and investigated in Abrini, Arch Microbiol, 161: 345-351, 1994 (Clostridium autoethanogenum), Tanner, Int J System Bacteriol, 43: 232-236, 1993 (Clostridium ljungdahlii) and Huhnke, WO 2008/ 028055 (Clostridium ragsdalei).

These three species have many similar properties. In particular, all these species are C1-fixing, anaerobic, acetogenic, ethanologenic, and carboxydotrophic representatives of the genus Clostridium. Such species have similar genotypes and phenotypes, and ways of conserving energy

- 7 042922 and enzymatic metabolism. In addition, these species are united in a cluster of homologous group I clostridial rRNA, and their 16S rRNA DNA is more than 99% identical, the content of G + C in DNA is about 22-30% mol.%, they are gram-positive, characterized by similar morphology and size (the size of logarithmically growing cells is in the range between 0.5-0.7 x 3-5 µm), are mesophilic (optimum growth at a temperature of 30-37°C), are characterized by similar pH ranges of about 4-7.5 (at optimal pH value of about 5.5-6), do not contain cytochromes and store energy through the Rnf complex. In addition, these species have been shown to reduce carboxylic acids to form their respective alcohols (Perez, Biotechnol Bioeng, 110:1066-1077, 2012). It is important to note that all these species also show strong autotrophic growth on CO-containing gases, produce ethanol and acetate (or acetic acid) as the main fermentation products, and under certain conditions form small amounts of 2,3butanediol and lactic acid.

However, these three species also have a number of differences. These species have been isolated from various sources: Clostridium autoethanogenum from rabbit intestines, Clostridium ljungdahlii from chicken coop waste, and Clostridium ragsdalei from freshwater sediments. These species differ in the utilization of various sugars (eg rhamnose, arabinose), acids (eg gluconate, citrate), amino acids (eg arginine, histidine) and other substrates (eg betaine, butanol). In addition, these species differ in auxotrophy for certain vitamins (eg, thiamine, biotin). These species have differences in the nucleic acid and amino acid sequences of the genes and proteins of the Wood-Ljungdahl pathway, although the general organization and number of these genes and proteins have been found to be the same in all species (Kopke, Curr Opin Biotechnol, 22: 320-325, 2011 ).

Thus, in conclusion, many of the characteristics of Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei are not specific to this species, but are rather general characteristics for this cluster of C1-fixing, anaerobic, acetogenic, ethanologenic, and carboxydotrophic members of the genus Clostridium. However, since these species are actually different, genetic modification or manipulation of one of these species may not have the same effect in another of these species. For example, there may be differences in growth, productivity, or product production.

The microorganism according to the present invention can also be obtained from an isolate or mutant of Clostridium autoethanogenum, Clostridium ljungdahlii or Clostridium ragsdalei. Isolates and mutants of Clostridium autoethanogenum include JA1-1 (DSM10061) (Abrini, Arch Microbiol, 161: 345-351, 1994), LBS1560 (DSM19630) (WO 2009/064200) and LZ1561 (DSM23693) (wO 2012/015 317) . Clostridium ljungdahlii isolates and mutants include ATCC 49587 (Tanner, Int J Syst Bacteriol, 43: 232-236, 1993), PETCT (DSM13528, ATCC 55383), ERI-2 (ATCC 55380) (US 5593886), C-01 (ATCC 55988) (US 6368819), 0-52 (ATCC 55989) (US 6368819) and OTA-1 (Tirado-Acevedo, Production of bioethanol from synthesis gas using Clostridium ljungdahlii, PhD thesis, North Carolina State University, 2010). Clostridium ragsdalei isolates and mutants include PI 1 (ATCC BAA-622, ATCC PTA-7826) (WO 2008/028055).

However, in one embodiment, the microorganism of this invention may also be derived from essentially any parent microorganism, such as a parent microorganism selected from the group consisting of Clostridium acetobutylicum, Clostridium beijerinckii, Escherichia coli, and Saccharomyces cerevisiae.

The invention provides microorganisms capable of producing ethylene glycol, glyoxylate and glycolate, as well as methods for producing ethylene glycol, glyoxylate and glycolate, comprising culturing the microorganism according to this invention in the presence of a substrate, whereby the microorganism produces ethylene glycol.

The microorganism according to the invention may contain an enzyme that converts acetyl-CoA, eg acetyl-CoA produced in the Wood-Lungdal pathway, into pyruvate (reactions 1 of FIG. 1). This enzyme can be pyruvate synthase (PFOR) [1.2.7.1] or ATP:pyruvate orthophosphate phosphotransferase [1.2.7.1]. In some embodiments, the enzyme that converts acetyl-CoA to pyruvate is an endogenous enzyme.

The microorganism according to the invention may contain an enzyme that converts pyruvate to oxaloacetate (reaction 2 of FIG. 1). This enzyme may be pyruvate:dioxide ligase [ADP-forming] [6.4.1.1]. In some embodiments, the enzyme that converts pyruvate to oxaloacetate is an endogenous enzyme. In some embodiments, an enzyme is overexpressed that converts pyruvate to oxaloacetate.

The microorganism of the invention may contain an enzyme that converts oxaloacetate to citryl-CoA (reaction 3 of FIG. 1). This enzyme may be citryl-CoAlyase [4.1.3.34]. In some embodiments, the enzyme that converts oxaloacetate to citryl-CoA is an endogenous enzyme.

The microorganism of the invention may contain an enzyme that converts citryl-CoA to citrate (reaction 4 of FIG. 1). This enzyme may be citrate-CoAtransferase [2.8.3.10]. In some embodiments, the enzyme that converts citryl-CoA to citrate is an endogenous enzyme.

- 8 042922

The microorganism according to the invention may contain an enzyme that converts oxaloacetate to citrate (reaction 5 of FIG. 1). This enzyme can be citrate [Si]-synthase [2.3.3.1], ATP-citrate synthase [2.3.3.8], or citrate (Re)-synthase [2.3.3.3]. In some embodiments, the enzyme that converts oxaloacetate to citrate is an endogenous enzyme. In other embodiments, the enzyme that converts oxaloacetate to citrate is a heterologous enzyme. For example, in some embodiments, the microorganism of this invention contains citrate synthase 1 [EC 2.3.3.16] from B. subtilis such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 1, which encodes the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the microorganism of this invention contains citrate (Re)-synthase from C. kluyveri, such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 3, which encodes the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the microorganism of this invention contains citrate (Si)-synthase from Clostridium sp., such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 5, which encodes the amino acid sequence shown in SEQ ID NO : 6. In some embodiments, the microorganism of this invention contains B. subtilis citrate synthase 2 such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 7, which encodes the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, an enzyme is overexpressed that converts oxaloacetate to citrate.

The microorganism according to the invention may contain an enzyme that converts citrate to aconitate and aconitate to isocitrate (reactions 6 of FIG. 1). This enzyme may be aconitate hydratase [4.2.1.3]. In some embodiments, the enzyme that converts citrate to aconitate and aconitate to isocitrate is an endogenous enzyme. In some embodiments, an enzyme is overexpressed that converts citrate to aconitate and aconitate to isocitrate.

The microorganism according to the invention may contain an enzyme that converts isocitrate to glyoxylate (reaction 7 of FIG. 1). This enzyme may be isocitrate lyase [4.1.3.1]. In some embodiments, the microorganism of the invention comprises an isocitrate lyase from Z. mays such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 9, which encodes the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the microorganism according to the invention contains isocitrate lyase from E. coli, such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 11, which encodes the amino acid sequence shown in SEQ ID NO: 12.

The microorganism according to the invention may contain an enzyme that converts glyoxylate to glycolate (reaction 8 of FIG. 1). This enzyme can be glycerate dehydrogenase [1.1.1.29], glyoxylate reductase [1.1.1.26/79] or glycolate dehydrogenase [1.1.99.14]. In some embodiments, the enzyme that converts glyoxylate to glycolate is an endogenous enzyme. In some embodiments, an enzyme is overexpressed that converts glyoxylate to glycolate.

The microorganism according to the invention may contain an enzyme that converts glycolate to glycoaldehyde (reaction 9 of FIG. 1). This enzyme can be glycolaldehyde dehydrogenase [1.2.1.21], lactaldehyde dehydrogenase [1.2.1.22], succinate semialdehyde dehydrogenase [1.2.1.24], 2,5-dioxovalerate dehydrogenase [1.2.1.26], aldehyde dehydrogenase [1.2 .1.3/4/5], betainealdehyde dehydrogenase [1.2.1.8] or aldehyde ferredoxin oxidoreductase [1.2.7.5]. In some embodiments, the enzyme that converts glycolate to glycoaldehyde is an endogenous enzyme. In other embodiments, the enzyme that converts glycolate to glycoaldehyde is a heterologous enzyme. For example, in some embodiments, the microorganism of the present invention comprises gamma-aminobutyraldehyde dehydrogenase from E. coli, such that the microorganism comprises the nucleotide sequence shown in SEQ ID NO: 49, which encodes the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the microorganism of the present invention comprises an aldehyde dehydrogenase from E. coli such that the microorganism comprises the nucleotide sequence shown in SEQ ID NO: 51, which encodes the amino acid sequence shown in SEQ ID NO: 52. In some embodiments, the microorganism according to this invention contains NADP-dependent succinate semialdehyde dehydrogenase I from E. coli, such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 53, which encodes the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, implementation , the microorganism of this invention contains lactaldehyde dehydrogenase/glycolal dehydrogenase from G. oxydans, such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 55, which encodes the amino acid sequence shown in SEQ ID NO: 56. In some embodiments implementation, the microorganism according to this invention

- 9 042922 tenyu contains aldehyde dehydrogenase A from P. fluorescens, so that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 57 or SEQ ID NO: 59, which encodes the amino acid sequence shown in SEQ ID NO: 58 or SEQ ID NO: 60, respectively. Additional non-limiting examples of enzymes that convert glycolate to glycoaldehyde can be found in GenBank accession numbers: WP_003202098, WP_003182567, ACT39044, ACT39074, WP_041112005 and ACT40170. In some embodiments, an enzyme is overexpressed that converts glycolate to glycoaldehyde.

The microorganism according to the invention may contain an enzyme that converts glycoaldehyde to ethylene glycol (reaction 10 of FIG. 1). This enzyme can be lactaldehyde reductase [1.1.1.77], alcohol dehydrogenase [1.1.1.1], alcohol dehydrogenase (NADP+) [1.1.1.2], glycerol dehydrogenase [1.1.1.72], glycerol-3-phosphate dehydrogenase [1.1.1.8] or aldehyde reductase [1.1.1.21]. In some embodiments, the enzyme that converts glycoaldehyde to ethylene glycol is an endogenous enzyme. In some embodiments, an endogenous enzyme that converts glycoaldehyde to ethylene glycol is overexpressed. In other embodiments, the enzyme that converts glycoaldehyde to ethylene glycol is a heterologous enzyme. In some embodiments, the microorganism of the invention comprises lactaldehyde reductase from C. saccharoperbutylacetonicum such that the microorganism comprises the nucleotide sequence shown in SEQ ID NO: 61 that encodes the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the implementation , the microorganism of this invention contains lactaldehyde reductase from C. ljungdahlii such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 63, which encodes the amino acid sequence shown in SEQ ID NO: 64. In some embodiments, the microorganism according to this of the invention contains lactaldehyde reductase from E. coli, such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 65, which encodes the amino acid sequence shown in SEQ ID NO: 66. In some embodiments, the microorganism according to this invention contains lactaldehyde reductase from C. beijerinckii such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 67, which encodes the amino acid sequence shown in SEQ ID NO: 68. In some embodiments, a heterologous enzyme that converts glycoaldehyde to ethylene glycol is overexpressed.

The microorganism according to the invention may contain an enzyme that converts pyruvate to malate (reaction 11 of FIG. 1). This enzyme can be malate dehydrogenase [1.1.1.37], malate dehydrogenase (oxaloacetate decarboxylating) [1.1.1.38], malate dehydrogenase (decarboxylating) [1.1.1.39], malate dehydrogenase (oxaloacetate decarboxylating) (NADP +) [1.1.1.40], malate dehydrogenase (NADP+) [1.1.1.82], D-malate dehydrogenase (decarboxylating) [1.1.1.83], dimethylmalate dehydrogenase [1.1.1.84], 3-isopropyl- malate dehydrogenase [1.1.1.85], malate dehydrogenase [NAD(P)+] [1.1.1.299] or malate dehydrogenase (quinone) [1.1.5.4]. In some embodiments, the enzyme that converts pyruvate to malate is an endogenous enzyme. In other embodiments, the enzyme that converts pyruvate to malate is a heterologous enzyme. For example, in some embodiments, the microorganism of the invention comprises C. autoethanogenum malate dehydrogenase such that the microorganism comprises the nucleotide sequence set forth in SEQ ID NO: 23, which encodes the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the microorganism of the present invention comprises the NAD-dependent malate enzyme from C. autoethanogenum such that the microorganism comprises the nucleotide sequence shown in SEQ ID NO: 25, which encodes the amino acid sequence shown in SEQ ID NO: 26.

The microorganism according to the invention may contain an enzyme that converts malate to glyoxylate (reaction 12 of FIG. 1). This enzyme can be malate synthase [2.3.3.9] or isocitrate lyase [4.1.3.1]. In some embodiments, the enzyme that converts malate to glyoxylate is a heterologous enzyme. For example, in some embodiments, the microorganism of the invention comprises Sporosarcina sp. malate synthase G such that the microorganism comprises the nucleotide sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 33 that encodes the amino acid sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 34, respectively. In some embodiments, the microorganism of the invention comprises Bacillus sp. malate synthase G such that the microorganism comprises the nucleotide sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 35, which encodes the amino acid sequence set forth in SEQ ID NO: : 30 or SEQ ID NO: 36, respectively. In some embodiments, the microorganism of the invention comprises S. coelicolor malate synthase such that the microorganism comprises the nucleotide sequence shown in SEQ ID NO: 31, which encodes the amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the implementation , the microorganism according to the invention contains malate synthase G from B. infantis, so that the microorganism contains a nucleotide sequence

- 10 042922 the value shown in SEQ ID NO: 37, which encodes the amino acid sequence shown in SEQ ID NO: 38. In some embodiments, the microorganism according to this invention contains malate synthase from C. cochlearium, such that the microorganism contains the nucleotide sequence , shown in SEQ ID NO: 39, which encodes the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the microorganism of this invention contains malate synthase G from B. megaterium, such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 41, which encodes the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the microorganism of this invention contains malate synthase from Paenibacillus sp., such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 43, which encodes the amino acid sequence shown in SEQ ID NO: 44. In some embodiments, the microorganism of the invention comprises malate synthase from Lysinibacillus sp., such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 45, which encodes the amino acid sequence shown in SEQ ID NO: 46. In some embodiments, the microorganism of the invention comprises B. cereus malate synthase such that the microorganism comprises the nucleotide sequence shown in SEQ ID NO: 47 that encodes the amino acid sequence shown in SEQ ID NO. :48.

The microorganism according to the invention may contain an enzyme that converts pyruvate to phosphoenolpyruvate (reactions 13 of FIG. 1). This enzyme can be pyruvate kinase [2.7.1.40], pyruvate phosphate dikinase [2.7.9.1], or pyruvate aqueous dikinase [2.7.9.2]. In some embodiments, the enzyme that converts pyruvate to phosphoenolpyruvate is an endogenous enzyme.

The microorganism according to the invention may contain an enzyme that converts phosphoenolpyruvate to 2-phospho-D-glycerate (reaction 14 of FIG. 1). This enzyme may be phosphopyruvate hydratase [4.2.1.11]. In some embodiments, the enzyme that converts phosphoenolpyruvate to 2-phospho-D-glycerate is an endogenous enzyme.

The microorganism according to the invention may contain an enzyme that converts 2phospho-D-glycerate to 3-phospho-D-glycerate (reaction 15 of FIG. 1). This enzyme may be phosphoglycerate mutase [5.4.2.11/12]. In some embodiments, the enzyme that converts 2-phospho-D-glycerate to 3-phospho-D-glycerate is an endogenous enzyme.

The microorganism according to the invention may contain an enzyme that converts 3phospho-D-glycerate to 3-phosphonooxypyruvate (reaction 16 of FIG. 1). This enzyme may be phosphoglycerate dehydrogenase [1.1.1.95]. In some embodiments, the enzyme that converts 3-phospho-D-glycerate to 3-phosphonooxypyruvate is an endogenous enzyme.

The microorganism according to the invention may contain an enzyme that converts 3-phosphonooxypyruvate to 3-phospho-b-serine (reaction 17 of FIG. 1). This enzyme may be a phosphoserine transaminase [2.6.1.52]. In some embodiments, the enzyme that converts 3-phosphonooxypyruvate to 3-phosphoXserine is an endogenous enzyme.

The microorganism according to the invention may contain an enzyme that converts 3phospho-b-serine to serine (reaction 18 of FIG. 1). This enzyme may be a phosphoserine phosphatase [3.1.3.3]. In some embodiments, the enzyme that converts 3-phospho-Lserine to serine is an endogenous enzyme.

The microorganism according to the invention may contain an enzyme that converts serine to glycine (reaction 19 of FIG. 1). This enzyme may be glycine hydroxymethyltransferase [2.1.2.1]. In some embodiments, the enzyme that converts serine to glycine is an endogenous enzyme. In some embodiments, an enzyme is overexpressed that converts serine to glycine.

The microorganism according to the invention may contain an enzyme that converts glycine to glyoxylate (reaction 20 of FIG. 1). This enzyme can be alanine glyoxylate aminotransferase/transaminase [2.6.1.44], serine glyoxylate aminotransferase/transaminase [2.6.1.45], serine pyruvate aminotransferase/transaminase [2.6.1.51], glycine oxaloacetate aminotransferase/transaminase [2.6. 1.35], glycine transaminase [2.6.1.4], glycine dehydrogenase [1.4.1.10], alanine dehydrogenase [1.4.1.1] or glycine dehydrogenase [1.4.2.1]. In some embodiments, the enzyme that converts glycine to glyoxylate is an endogenous enzyme. In other embodiments, the enzyme that converts glycine to glyoxylate is a heterologous enzyme. For example, in some embodiments, the microorganism of this invention comprises a serine glyoxylate aminotransferase from H. methylovorum such that the microorganism comprises the nucleotide sequence shown in SEQ ID NO: 13, which encodes the amino acid sequence shown in SEQ ID NO: 14. B in some embodiments, the microorganism of the invention contains an alanine glyoxylate aminotransferase from S. thiotaurini such that the microorganism contains the nucleotide sequence shown in

- 11 042922

SEQ ID NO: 15, which encodes the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the microorganism of this invention contains an alanine glyoxylate aminotransferase from T. tepidarius, such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: : 17, which encodes the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the microorganism according to this invention contains a class V aminotransferase from C. acidurici, such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 19, which encodes the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the microorganism according to this invention contains a serine pyruvate aminotransferase from T. maritima, such that the microorganism contains the nucleotide sequence shown in SEQ ID NO: 21, which encodes the amino acid sequence shown in SEQ ID NO: 22. In some embodiments, an enzyme that converts glycine to glyoxylate is overexpressed.

The microorganism according to the invention may contain an enzyme that converts serine to hydroxypyruvate (reaction 21 of FIG. 1). This enzyme can be serine pyruvate transaminase [2.6.1.51], serine glyoxylate transaminase [2.6.1.45], alanine dehydrogenase [1.4.1.1], L-amino acid dehydrogenase [1.4.1.5], serine 2-dehydrogenase [1.4. 1.7], alanine transaminase [2.6.1.2], glutamine pyruvate transaminase [2.6.1.15], D-amino acid transaminase [2.6.1.21], alanine glyoxylate transaminase [2.6.1.44], or serine pyruvate transaminase [2.6 .1.51]. In some embodiments, the enzyme that converts serine to hydroxypyruvate is an endogenous enzyme. In other embodiments, the enzyme that converts serine to hydroxypyruvate is a heterologous enzyme. Non-limiting examples of enzymes capable of converting serine to hydroxypyruvate can be found under GenBank accession numbers: WP_009989311 and NP_511062.1. In some embodiments, an enzyme is overexpressed that converts serine to hydroxypyruvate.

The microorganism according to the invention may contain an enzyme that converts hydroxypyruvate to glycoaldehyde (reaction 22 of FIG. 1). This enzyme can be hydroxypyruvate decarboxylase [4.1.1.40] or pyruvate decarboxylase [4.1.1.1]. This enzyme can also be any other decarboxylase [4.1.1.-]. In some embodiments, the enzyme that converts hydroxypyruvate to glycoaldehyde is a heterologous enzyme. Non-limiting examples of enzymes capable of converting hydroxypyruvate to glycoaldehyde can be found under GenBank accession numbers: CCG28866, SVF98953, PA0096, CAA54522, KRU13460, and KLA26356.

The microorganism according to the invention may contain an enzyme that converts D-glycerate to hydroxypyruvate (reaction 23 of FIG. 1). This enzyme can be glyoxylate reductase [EC 1.1.1.26], glycerate dehydrogenase [EC 1.1.1.29], or hydroxypyruvate reductase [EC 1.1.1.81]. In some embodiments, the enzyme that converts D-glycerate to hydroxypyruvate is a heterologous enzyme. Non-limiting examples of enzymes capable of converting D-glycerate to hydroxypyruvate can be found under GenBank accession numbers: SUK16841, RPK22618, KPA02240, AGW90762, CAC11987, Q9CA90, and Q9UBQ7.

The microorganism according to the invention may contain an enzyme complex that converts 5,10-methylenetetrahydrofolate to glycine (reaction 24 of FIG. 1). 5,10-methylenetetrahydrofolate is a cofactor in the reducing branch of the Wood-Ljungdahl pathway and acts as a scaffold in the production of acetyl-CoA. This complex can be a glycine cleavage system, including glycine dehydrogenase [1.4.4.2], dihydro-lipoyl dehydrogenase [1.8.1.4], and aminomethyl transferase (glycine synthase) [2.1.2.10]. In some embodiments, the enzymes of the complex that converts 5,10-methylenetetrahydrofolate to glycine are endogenous enzymes. In some embodiments, the enzymes of the glycine cleavage system are overexpressed.

The microorganism according to the invention may contain an enzyme that converts phosphoenolpyruvate to oxaloacetate (reaction 25 of FIG. 1). This enzyme can be phosphoenol pyruvate carboxykinase (ATP) [4.1.1.49] or (GTP) [4.1.1.32]. In some embodiments, the enzyme that converts phosphoenolpyruvate to oxaloacetate is an endogenous enzyme. In other embodiments, the enzyme that converts phosphoenolpyruvate to oxaloacetate is a heterologous enzyme. In some embodiments, an enzyme is overexpressed that converts phosphoenolpyruvate to oxaloacetate.

In some embodiments, a microorganism containing an enzyme that converts acetyl-CoA to pyruvate (reaction 1 of Fig. 1), an enzyme that converts pyruvate to oxaloacetate (reaction 2 of Fig. 1), an enzyme that converts oxaloacetate to citrate (reaction 5 Fig. 1), an enzyme that converts citrate to aconitate and aconitate to isocitrate (reaction 6 of Fig. 1), an enzyme that converts isocitrate to glyoxylate (reaction 7 of Fig. 1), an enzyme that converts glyoxylate to glycolate (reaction 8 of Fig. 1 1), the enzyme that converts glycolate to glycoaldehyde (reaction 9 of FIG. 1), and the enzyme that converts glycoaldehyde to ethylene glycol (reaction 10 of FIG. 1) produce ethylene glycol. In a non-limiting example, the enzyme that converts oxaloacetate to citrate may be citrate synthase from B. subtilis (SEQ ID NOS: 1-2). In a non-limiting example, the enzyme that converts isocitrate to glyoxylate may be E. coli isocitrate lyase (SEQ ID NO: 1112). In a non-limiting example, the enzyme that converts glycolate to glycoaldehyde can be glycol aldehyde dehydrogenase from G. oxydans (SEQ ID NOs: 55-56) or aldehyde dehydrogenase from P. fluorescens (SEQ ID NOs: 57-58). One or more of the enzymes catalyzing reactions 2, 5, 6, 8, 9 and 10 may be overexpressed as shown in FIG. 1; see, for example, Example 1 and FIG. 3b.

In some embodiments, a microorganism containing an enzyme that converts acetyl-CoA to pyruvate (reaction 1 of FIG. 1), an enzyme that converts pyruvate to phosphoenolpyruvate (reaction 13 of FIG. 1), an enzyme that converts phosphoenolpyruvate to 2-phospho- O-glycerate (reaction 14 of Fig. 1), an enzyme that converts 2-phospho-O-glycerate to 3-phospho-O-glycerate (reaction 15 of Fig. 1), an enzyme that converts 3-phospho-O-glycerate to 3-phosphonooxypyruvate (reaction 16 of Fig. 1), an enzyme that converts 3-phosphonooxypyruvate to 3-phospho-L-serine (reaction 17 of Fig. 1), an enzyme that converts 3-phospho-L-serine to serine (reaction 18 Fig. 1), an enzyme that converts serine to glycine (reaction 19 of Fig. 1), an enzyme that converts glycine to glyoxylate (reaction 20 of Fig. 1), an enzyme that converts glyoxylate to glycolate (reaction 8 of Fig. 1), an enzyme that converts glycolate to glycoaldehyde (reaction 9 of FIG. 1) and an enzyme that converts glycoaldehyde to ethylene glycol (reaction 10 of FIG. 1) produce ethylene glycol. In a non-limiting example, the enzyme that converts glycine to glyoxylate can be an alanine glyoxylate aminotransferase from S. thiotaurini (SEQ ID NOs: 15-16) or a class V aminotransferase from C. acidurici (SEQ ID NOs: 19-20) . In a non-limiting example, the enzyme that converts glycolate to glycoaldehyde can be glycolaldehyde dehydrogenase from G. oxydans (SEQ ID NOs: 55-56) or aldehyde dehydrogenase from P. fluorescens (SEQ ID NOs: 57-58). One or more of the enzymes catalyzing reactions 19, 20, 8, 9 and 10 may be overexpressed as shown in FIG. 1; see, for example, examples 2-4 and FIG. 4V, 5V and 6V.

In some embodiments, a microorganism containing an enzyme that converts acetyl-CoA to pyruvate (reaction 1 of Fig. 1), an enzyme that converts pyruvate to oxaloacetate (reaction 2 of Fig. 1), an enzyme that converts oxaloacetate to citryl-CoA ( reaction 3 of Fig. 1), an enzyme that converts citryl-CoA to citrate (reaction 4 of Fig. 1), an enzyme that converts citrate to aconitate and aconitate to isocitrate (reaction 6 of Fig. 1), an enzyme that converts isocitrate to glyoxylate (reaction 7 of Fig. 1) an enzyme that converts glyoxylate to glycolate (reaction 8 of Fig. 1), an enzyme that converts glycolate to glycoaldehyde (reaction 9 of Fig. 1), and an enzyme that converts glycoaldehyde to ethylene glycol (reaction 10 of Fig. 1) produces ethylene glycol. In a non-limiting example, the enzyme that converts isocitrate to glyoxylate may be E. coli isocitrate lyase (SEQ ID NOS: 11-12). In a non-limiting example, the enzyme that converts isocitrate to glyoxylate may be E. coli isocitrate lyase (SEQ ID NOS: 11-12). In a non-limiting example, the enzyme that converts glycolate to glycoaldehyde can be glycolaldehyde dehydrogenase from G. oxydans (SEQ ID NOs: 55-56) or aldehyde dehydrogenase from P. fluorescens (SEQ ID NOs: 57-58).

One or more of the enzymes catalyzing reactions 2, 6, 8, 9 and 10 may be overexpressed as shown in FIG. 1.

In some embodiments, a microorganism containing an enzyme that converts acetyl-CoA to pyruvate (reaction 1 of Fig. 1), an enzyme that converts pyruvate to malate (reaction 11 of Fig. 1), an enzyme that converts malate to glyoxylate (reaction 12 Fig. 1), an enzyme that converts glyoxylate to glycolate (reaction 8 of Fig. 1), an enzyme that converts glycolate to glycoaldehyde (reaction 9 of Fig. 1), and an enzyme that converts glycoaldehyde to ethylene glycol (reaction 10 of Fig. 1) produces ethylene glycol. In a non-limiting example, the enzyme that converts glycolate to glycoaldehyde can be glycolaldehyde dehydrogenase from G. oxydans (SEQ ID NO: 5556) or aldehyde dehydrogenase from P. fluorescens (SEQ ID NO: 57-58). One or more of the enzymes catalyzing reactions 8, 9 and 10 may be overexpressed as shown in FIG. 1.

In some embodiments, a microorganism containing an enzyme complex that converts 5,10-methylenetetrahydrofolate to glycine (reaction 24 of FIG. 1), an enzyme that converts glycine to glyoxylate (reaction 20 of FIG. 1), an enzyme that converts glyoxylate to glycolate (reaction 8 of Fig. 1), an enzyme that converts glycolate to glycoaldehyde (reaction 9 of Fig. 1), and an enzyme that converts glycoaldehyde to ethylene glycol (reaction 10 of Fig. 1) produce ethylene glycol. In a non-limiting example, the enzyme that converts glycine to glyoxylate can be an alanine glyoxylate aminotransferase from S. thiotaurini (SEQ ID NOs: 15-16) or a class V aminotransferase from C. acidurici (SEQ ID NOs: 19-20) . In a non-limiting example, the enzyme that converts glycolate to glycoaldehyde can be glycolaldehyde dehydrogenase from G. oxydans (SEQ ID NOs: 55-56) or aldehyde dehydrogenase from P. fluorescens (SEQ ID NOs: 57-58). One or more of the enzymes catalyzing reactions 8, 9, 10, 20 and 24 may be overexpressed.

- 13 042922

In some embodiments, a microorganism containing an enzyme that converts acetyl-CoA to pyruvate (reaction 1 of FIG. 1), an enzyme that converts pyruvate to phosphoenolpyruvate (reaction 13 of FIG. 1), an enzyme that converts phosphoenolpyruvate to oxaloacetate (reaction 25 Fig. 1), an enzyme that converts oxaloacetate to citryl-CoA (reaction 3 of Fig. 1), an enzyme that converts citryl-CoA to citrate (reaction 4 of Fig. 1), an enzyme that converts citrate to aconitate and aconitate to isocitrate (reaction 6 of Fig. 1), an enzyme that converts isocitrate to glyoxylate (reaction 7 of Fig. 1), an enzyme that converts glyoxylate to glycolate (reaction 8 of Fig. 1), an enzyme that converts glycolate to glycoaldehyde (reaction 9 of Fig. 1) and the enzyme that converts glycoaldehyde to ethylene glycol (reaction 10 of FIG. 1) produces ethylene glycol. In a non-limiting example, the enzyme that converts isocitrate to glyoxylate may be E. coli isocitrate lyase (SEQ ID NOS: 11-12). In a non-limiting example, the enzyme that converts glycolate to glycoaldehyde can be glycolaldehyde dehydrogenase from G. oxydans (SEQ ID NOs: 55-56) or aldehyde dehydrogenase from P. fluorescens (SEQ ID NOs: 57-58). One or more of the enzymes catalyzing reactions 2, 6, 8, 9, 10 and 25 may be overexpressed as shown in FIG. 1.

In some embodiments, a microorganism containing an enzyme that converts acetyl-CoA to pyruvate (reaction 1 of FIG. 1), an enzyme that converts pyruvate to phosphoenolpyruvate (reaction 13 of FIG. 1), an enzyme that converts phosphoenolpyruvate to oxaloacetate (reaction 25 fig. 1), an enzyme that converts oxaloacetate to citrate (reaction 5 of fig. 1), an enzyme that converts citrate to aconitate and aconitate to isocitrate (reaction 6 of fig. 1), an enzyme that converts isocitrate to glyoxylate (reaction 7 of fig. 1), an enzyme that converts glyoxylate to glycolate (reaction 8 of fig. 1), an enzyme that converts glycolate to glycoaldehyde (reaction 9 of fig. 1), and an enzyme that converts glycoaldehyde to ethylene glycol (reaction 10 of fig. 1), produces ethylene glycol. In a non-limiting example, the enzyme that converts oxaloacetate to citrate may be citrate synthase from B. subtilis (SEQ ID NOS: 1-2). In a non-limiting example, the enzyme that converts isocitrate to glyoxylate may be E. coli isocitrate lyase (SEQ ID NOS: 11-12). In a non-limiting example, the enzyme that converts glycolate to glycoaldehyde can be glycolaldehyde dehydrogenase from G. oxydans (SEQ ID NOs: 55-56) or aldehyde dehydrogenase from P. fluorescens (SEQ ID NOs: 57-58). One or more of the enzymes catalyzing reactions 5, 6, 8, 9, 10 and 25 may be overexpressed as shown in FIG. 1.

In some embodiments, a microorganism containing an enzyme that converts acetyl-CoA to pyruvate (reaction 1 of FIG. 1), an enzyme that converts pyruvate to phosphoenolpyruvate (reaction 13 of FIG. 1), an enzyme that converts phosphoenolpyruvate to 2-phospho- O-glycerate (reaction 14 of Fig. 1), an enzyme that converts 2-phospho-O-glycerate to 3-phospho-O-glycerate (reaction 15 of Fig. 1), an enzyme that converts 3-phospho-O-glycerate to 3 -phosphonooxypyruvate (reaction 16 of Fig. 1), an enzyme that converts 3-phosphonooxypyruvate to 3-phospho-L-serine (reaction 17 of Fig. 1), an enzyme that converts 3-phosphoA-serine to serine (reaction 18 of Fig. 1 ), contains an enzyme that converts serine to hydroxypyruvate (reaction 21 of Fig. 1), an enzyme that converts hydroxypyruvate to glycoaldehyde (reaction 22 of Fig. 1), and an enzyme that converts glycoaldehyde to ethylene glycol (reaction 10 of Fig. 1), produces ethylene glycol. An enzyme catalyzing the conversion of glycoaldehyde to ethylene glycol may be overexpressed.

In some embodiments, a microorganism comprising an enzyme that converts D-glycerate to hydroxypyruvate (reaction 23 of FIG. 1), an enzyme that converts hydroxypyruvate to glycoaldehyde (reaction 22 of FIG. 1), and an enzyme that converts glycoaldehyde to ethylene glycol (reaction 10 Fig. 1) produces ethylene glycol. An enzyme catalyzing the conversion of glycoaldehyde to ethylene glycol may be overexpressed.

The enzymes of the invention may be codon-optimized for expression in the microorganism of the invention. Codon optimization refers to the mutation of a nucleic acid, such as a gene, to optimize or improve the translation of the nucleic acid in a particular strain or species of microorganism. Codon optimization can lead to increased translation rates or higher translation fidelity. In a preferred embodiment, the genes of the invention are codon-optimized for expression in the microorganism of the invention. Although codon optimization refers to the underlying genetic sequence, codon optimization often results in improved translation and thus improved enzyme expression. Accordingly, the enzymes of this invention can also be described as codon-optimized.

One or more of the enzymes of this invention may be overexpressed. Overexpressed refers to an increase in the expression of a nucleic acid or protein in a microorganism of the invention compared to the wild-type microorganism or parental microorganism from which the microorganism of the invention is derived. Overexpression can be achieved by any means known in the art, including altering the copy number of a gene, the rate of gene transcription, the rate of translation of a gene, or the rate of degradation of an enzyme. As described above, one or more enzymes catalyze

- 14 042922 for reactions 2, 5, 6, 8, 9, 10, 19, 20, 24 or 25 in FIG. 1 can be overexpressed.

The enzymes of this invention may contain disruptive mutations. In particular, an offending mutation refers to a mutation that reduces or suppresses (ie, disrupts) the expression or activity of a gene or enzyme. An offending mutation may partially deactivate, completely deactivate, or remove a gene or enzyme. The offending mutation may be a knockout (KO) mutation. An offending mutation can be any mutation that reduces, prevents, or blocks the biosynthesis of a product produced by an enzyme. An offending mutation may include, for example, a mutation in a gene encoding an enzyme, a mutation in a genetic regulatory element involved in the expression of a gene encoding an enzyme, the introduction of a nucleic acid that produces a protein that reduces or inhibits the activity of the enzyme, or the introduction of a nucleic acid ( e.g. antisense RNA, siRNA, CRISPR) or a protein that inhibits the expression of an enzyme. The offending mutation may be introduced using any method known in the art.

In some embodiments, the microorganism of the invention contains a disruptive mutation in isocitrate dehydrogenase [1.1.1.41]. Isocitrate dehydrogenase converts isocitrate to 2-oxoglutarate. Disruption of isocitrate dehydrogenase, for example by deletion of isocitrate dehydrogenase, results in elevated levels of isocitrate.

In some embodiments, the microorganism of the invention contains a disruptive mutation in glycerate dehydrogenase [1.1.1.29]. Glycerate dehydrogenase converts glyoxylate to glycolate. Disruption of glycerate dehydrogenase, for example by deletion of isocitrate dehydrogenase, results in increased levels of glyoxylate.

In some embodiments, the microorganism of the invention contains a disruptive mutation in glycolate dehydrogenase [1.1.99.14]. Glycolate dehydrogenase converts glyoxylate to glycolate. Disruption of glycolate dehydrogenase, for example by removing glycolate dehydrogenase, results in an increase in glyoxylate levels.

In some embodiments, the microorganism of the invention contains a disruptive mutation in aldehyde ferredoxin oxidoreductase [1.2.7.5]. Aldehyde ferredoxin oxidoreductase converts glycolate to glycoaldehyde. Disruption of aldehyde ferredoxin oxidoreductase, for example by deletion of aldehyde ferredoxin oxidoreductase, results in an increase in glycolate levels.

In some embodiments, the microorganism of the invention contains a disruptive mutation in aldehyde dehydrogenase [1.2.1.3/1.2.3.4/1.2.3.5]. Aldehyde dehydrogenase converts glycolate to glycoaldehyde. Disruption of aldehyde dehydrogenase, for example by removing aldehyde dehydrogenase, results in an increase in glycolate levels.

The introduction of an offending mutation results in a microorganism of the invention that produces a non-target product, or a substantially non-target product, or a reduced amount of the desired product, compared to the parent microorganism from which the microorganism of the invention is derived. For example, the microorganism according to this invention may produce an off-target product, or at least about 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% or 95% less of the target product than the original microorganism. For example, the microorganism according to this invention may produce less than about 0.001, 0.01, 0.10, 0.30, 0.50, or 1.0 g/L of the desired product.

While illustrative sequences and sources for enzymes are provided herein, the invention is in no way limited to these sequences and sources, it also encompasses variants. The term variants includes nucleic acids and proteins whose sequence differs from that of the parent nucleic acid and protein, such as the parent nucleic acid and protein sequence disclosed in the prior art or exemplified herein. The invention may be practiced using variants of nucleic acids or proteins that perform essentially the same function as the original nucleic acid or protein. For example, a protein variant may perform essentially the same function or catalyze essentially the same reaction as the original protein. A gene variant may encode the same or substantially the same protein as the original gene. A promoter variant may have substantially the same ability to drive the expression of one or more genes as the original promoter.

Such nucleic acids or proteins may be referred to herein as functionally equivalent variants. For example, functionally equivalent nucleic acid variants may include allelic variants, gene fragments, mutated genes, polymorphisms, and the like. Homologous genes from other microorganisms are also examples of functionally equivalent variants. These include homologous genes in species such as Clostridium acetobutylicum, Clostridium beijerinckii, or Clostridium ljungdahlii, details of which are publicly available on websites such as Genbank or NCBI. Functionally equivalent variants also include nucleic acids whose sequence changes as a result of codon optimization for

- 15 042922 specific microorganism. A functionally equivalent nucleic acid variant will preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or more nucleic acid sequence identity (percent homology) with the parent nucleic acid. A functionally equivalent protein variant will preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or more amino acid sequence identity (percent homology) with the reference protein. The functional equivalence of a nucleic acid or protein variant can be assessed using any method known in the art.

Nucleic acids can be delivered to the microorganism according to this invention using any method known in the art. For example, nucleic acids may be delivered as pure nucleic acids, or may be formulated with one or more agents, such as liposomes. Nucleic acids can be DNA, RNA, cDNA, or combinations thereof, as needed. In certain embodiments, restriction inhibitors may be used. Additional vectors may include plasmids, viruses, bacteriophages, cosmids, and artificial chromosomes. In a preferred embodiment, the nucleic acids are delivered to the microorganism of the invention using a plasmid. For example, transformation (including transduction or transfection) can be achieved by electroporation, ultrasound, polyethylene glycol-mediated transformation, chemical or physical competence, protoplast transformation, prophage induction, or conjugation. In some embodiments with active restriction enzyme systems, it may be necessary to methylate the nucleic acid prior to introducing the nucleic acid into the microorganism.

In addition, nucleic acids can be designed to contain a regulatory element, such as a promoter, to increase or otherwise control the expression of a particular nucleic acid. The promoter may be a constitutive promoter or an inducible promoter. Ideally, the promoter is a Wood-Ljungdahl pathway promoter, a ferredoxin promoter, a pyruvate-ferredoxin oxidoreductase promoter, an Rnf complex operon promoter, an ATP synthase operon promoter, or a phosphotransacetylase/acetate kinase operon promoter.

The substrate refers to a source of carbon and/or energy for the microorganism according to this invention. Typically, the substrate is gaseous and contains a source of C1 carbon, such as CO, CO2 and/or CH ₄ . Preferably, the substrate contains a source of C1-carbon in the form of CO or CO + CO2. The substrate may additionally contain other non-carbon components such as H ₂ , N 2 or electrons. However, in other embodiments, the substrate may be a carbohydrate such as sugar, starch, fiber, lignin, cellulose, or hemicellulose, or a combination thereof. For example, the carbohydrate may be fructose, galactose, glucose, lactose, maltose, sucrose, xylose, or some combination thereof. In some embodiments, the substrate does not contain (D)-xylose (Alkim, Microb Cell Fact, 14: 127, 2015). In some embodiments, the substrate does not contain pentose, such as xylose (Pereira, Metab Eng, 34: 80-87, 2016). In some embodiments, the substrate may contain both gaseous and carbohydrate substrates (mixotrophic fermentation).

In general, the gaseous substrate contains at least some CO, such as about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mol% CO. The gaseous substrate may contain CO in the range of, for example, about 20-80, 30-70, or 40-60 mole % CO. Preferably, the gaseous substrate contains about 40-70 mole % CO (e.g. steel mill gas or blast furnace gas), about 20-30 mole % CO (e.g. converter furnace gas), or about 15-45 mole % CO (e.g. , synthesis gas). In some embodiments, the gaseous substrate may contain a relatively low amount of CO, such as about 1-10 or 1-20 mole % CO. The microorganism according to the invention typically converts at least a portion of the CO in the gaseous substrate to a product. In some embodiments, the gaseous substrate is free or substantially free (<1 mole%) of CO.

The gaseous substrate may contain some H ₂ . For example, the gaseous substrate may contain about 1, 2, 5, 10, 15, 20, or 30 mol % H ₂ . In some embodiments, the gaseous substrate may contain a relatively high amount of H2, such as about 60, 70, 80, or 90 mol% _H2 . In further embodiments, the gaseous substrate is free or substantially free (<1 mole%) of H ₂ .

The gaseous substrate may contain some CO2. For example, the gaseous substrate may contain about 1-80 or 1-30 mol.% CO ₂ . In some embodiments, the implementation, the gaseous substrate may contain less than about 20, 15, 10 or 5 mol.% CO ₂ . In another embodiment, the gaseous substrate is free or substantially free (<1 mole%) of CO ₂ .

The gaseous substrate may also be provided in alternative forms. For example, gas

- 16 042922 shaped substrate can be dissolved in a liquid or adsorbed on a solid carrier.

The gaseous substrate and/or source of C1 carbon may be an exhaust gas obtained as a by-product of an industrial process or from some other source such as vehicle exhaust or biomass gasification. In certain embodiments, the industrial process is selected from the group consisting of ferrous product manufacturing, e.g., steelmaking, nonferrous metal product manufacturing, petroleum refining, coal gasification, power generation, pure carbon production, ammonia production, methanol production, and coke production. . In these embodiments, the gaseous substrate and/or C1 carbon source may be removed from the industrial process prior to release to the atmosphere using any convenient method.

The gaseous substrate and/or source of C1 carbon can be synthesis gas, for example, synthesis gas obtained from gasification of coal or oil refinery residues, gasification of biomass or lignocellulosic material, or natural gas reforming. In another embodiment, synthesis gas may be obtained from the gasification of municipal solid waste or industrial solid waste.

The composition of the gaseous substrate can have a significant impact on the efficiency and/or cost of the reaction. For example, the presence of oxygen (O ₂ ) can reduce the efficiency of an anaerobic fermentation process. Depending on the composition of the substrate, it may be desirable to treat, purify or filter the substrate to remove any undesirable contaminants, such as toxins, undesirable components or dust particles, and/or to increase the concentration of the desired components.

In some embodiments, the fermentation is carried out without carbohydrate substrates such as sugar, starch, lignin, cellulose, or hemicellulose.

In some embodiments, the overall energetics of CO and H ₂ for ethylene glycol (MEG) is preferred over glucose to ethylene glycol, as shown below, where more negative Gibbs free energies, ArG'm, for CO and H ₂ indicate a greater driving force towards ethylene glycol. Calculations of the overall delta G response for comparing glucose with CO as a substrate were performed using an equilibrator (http://equilibrator.weizmann.ac.il/), which is a standard way to evaluate the overall feasibility of a pathway or individual steps in pathways in biological systems ( Flamholz, E. Noor, A. Bar-Even, R. Milo (2012) eQuilibrator - the biochemical thermodynamics calculator Nucleic Acids Res 40:D770-5; Noor, HS Haraldsd0ttir, R. Milo, RMT Fleming (2013) Consistent Estimation of Gibbs Energy Using Component Contributions PLoS Comput Biol 9(7): e1003098 Noor, A. Bar-Even, A. Flamholz, E. Reznik, W. Liebermeister, R. Milo (2014) Pathway Thermodynamics Highlights Kinetic Obstacles in Central Metabolism PLoS Comput Biol 10 (2):e1003483).

Calculations are performed as follows:

glucose (aq) + 3 NADH (aq) 3 MEG (aq) + 3 NAD+ (aq) ArG'm -104 kJ/mol,

CO (aq) + 3 H ₂ (aq) + 6 NADH (aq) 3 MEG (aq) + 6 NAD+ (aq) ArG'm -192 kJ/mol.

Physiological conditions:

glucose (aq) + 3 NADH (aq) 3 MEG (aq) + 3 NAD+ (aq) ArG'm -70 kJ/mol,

CO (aq) + 3 H ₂ (aq) + 6 NADH (aq) 3 MEG (aq) + 6 NAD+ (aq) ArG'm -295 kJ/mol.

In addition to ethylene glycol, glyoxylate and/or glycolate, the microorganism of the invention may be cultured to produce one or more by-products. Thus, for example, the microorganism according to the invention may produce or may be genetically engineered to produce ethanol (WO 2007/117157), acetate (WO 2007/117157), butanol (WO 2008/115080 and WO 2012/053905), butyrate (WO 2008 /115080), 2,3-butanediol (WO 2009/151342 and WO 2016/094334), lactate (WO 2011/112103), butene (WO 2012/024522), butadiene (WO 2012/024522), methyl ethyl ketone (2-butanone ) (WO 2012/024522 and WO 2013/185123), ethylene (WO 2012/026833), acetone (WO 2012/115527), isopropanol (WO 2012/115527), lipids (WO 2013/036147), 3-hydroxypropionate (3 -HP) (WO 2013/180581), isoprene (WO 2013/180584), fatty acids (WO 2013/191567), 2-butanol (WO 2013/185123), 1,2-propanediol (WO 2014/036152), 1 -propanol (WO 2014/0369152), products derived from chorismate (WO 2016/191625), 3-hydroxybutyrate (WO 2017/066498) and 1,3-butanediol (WO 2017/0066498). In some embodiments, in addition to ethylene glycol, the microorganism of this invention also produces ethanol, 2,3-butanediol and/or succinate. In some embodiments, the microbial biomass itself can be considered as a product.

A native product is a product produced by a non-genetically modified microorganism. For example, ethanol, acetate, and 2,3-butanediol are native products of Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei. A non-native product is a product produced by a genetically modified microorganism, however, it is not produced by the non-genetically modified microorganism from which the genetically modified microorganism is derived. What is not known is that ethylene glycol is produced by any natural microorganism, so that it is a non-native product of all microorganisms.

Selectivity refers to the ratio of obtaining the desired product to obtaining all fermentation products produced by the microorganism. The microorganism according to this invention can be designed to produce products with a certain selectivity or with a minimum selectivity. In one embodiment, the target product, such as ethylene glycol, is at least about 5%, 10%, 15%, 20%, 30%, 50%, or 75% of all fermentation products produced by the microorganism according to this invention. In one embodiment, ethylene glycol makes up at least 10% of all fermentations produced by the microorganism of this invention, thus the microorganism of this invention has a selectivity for ethylene glycol of at least 10%. In another embodiment, ethylene glycol makes up at least 30% of all fermentations produced by the microorganism of this invention, thus the microorganism of this invention has a selectivity for ethylene glycol of at least 30%.

As a rule, the cultivation is carried out in a bioreactor. The term bioreactor includes a culture/fermentation device consisting of one or more vessels, columns, or piping, e.g. TBR), bubble column, gas lift fermenter, static mixer, or other vessel or other device suitable for contacting a gas with a liquid. In some embodiments, the bioreactor may comprise a first growth reactor and a second culture/fermentation reactor. One or both of these reactors may be provided with a substrate. As used herein, the terms culture and fermentation are used interchangeably. These terms cover both the growth phase and the biosynthetic phase of the product during cultivation/fermentation.

Cultivation is usually maintained in an aqueous culture medium containing sufficient nutrients, vitamins and/or minerals to ensure growth of the microorganism. The aqueous culture medium is preferably an anaerobic microbial growth medium, such as a minimal anaerobic microbial growth medium. Suitable media are well known in the art.

To obtain ethylene glycol, the cultivation/fermentation is preferably carried out under appropriate conditions. As a rule, cultivation/fermentation is performed under anaerobic conditions. Reaction conditions to be considered include pressure (or partial pressure), temperature, gas flow rate, liquid flow rate, pH of the medium, redox potential of the medium, stirring rate (when using a continuously stirred reactor), inoculum level , maximum gas substrate concentrations to ensure that gas in the liquid phase does not become a limiting factor, and maximum product concentrations to avoid product inhibition. In particular, the rate of introduction of the substrate can be adjusted to ensure that the gas concentration in the liquid phase does not become limiting.

The operation of the bioreactor at elevated pressures makes it possible to increase the rate of gas mass transfer from the gas phase to the liquid phase. Accordingly, it is generally preferred to carry out the culture/fermentation at pressures above atmospheric pressure. In addition, since the desired gas conversion rate depends in part on the retention time of the substrate, with the retention time dictating the required volume of the bioreactor, the use of pressurized systems can significantly reduce the required volume of the bioreactor and hence the capital cost of the culture/fermentation equipment. This, in turn, means that by maintaining the bioreactors at elevated pressure, rather than at atmospheric pressure, it is possible to reduce the retention time, defined as the volume of liquid in the bioreactor divided by the flow rate of the injected gas. The optimum reaction conditions will depend in part on the particular microorganism used. However, it is generally preferred to carry out the fermentation at a pressure above atmospheric pressure. Also, since this gas conversion rate depends in part on the retention time of the substrate, and achieving the required retention time in turn determines the required bioreactor volume, the use of pressurized systems can significantly reduce the required bioreactor volume and hence the capital cost of fermentation equipment. .

In some embodiments, the fermentation is performed in the absence of light or in the presence of some amount of light that is insufficient to meet the energy requirements of the photosynthetic microorganisms. In some embodiments, the microorganism of this invention is a non-photosynthetic microorganism.

The method of the present invention may further include separating the ethylene lycol from the fermentation broth. Ethylene glycol can be separated or purified from the fermentation broth by any method or combination of methods known in the art, including, for example, distillation, fluidized bed processes, membrane purification, evaporation, semi-permeable membrane evaporation, gas stripping, phase separation, ion exchange , or extractive fermentation, including, for example, liquid-liquid extraction. In one embodiment, the ethylene glycol can be concentrated from the fermentation broth using reverse osmosis and/or evaporation through a semi-permeable membrane (US 5,552,023). The water can be removed by distillation and the residues (containing a high proportion of ethylene glycol) can then be recovered using distillation or vacuum distillation to obtain a high purity ethylene glycol fraction. Alternatively, with or without reverse osmosis concentration and/or semi-permeable membrane evaporation, ethylene glycol can be further purified by reactive distillation with aldehyde (Atul, Chem Eng Sci, 59: 2881-2890, 2004) or azeotropic distillation using hydrocarbon (US 2218234). In another approach, ethylene glycol can be captured on activated carbon or polymeric absorbent from an aqueous solution (with or without reverse osmosis and/or evaporation through a semi-permeable membrane) and recovered using a low-boiling organic solvent (Chinn, Recovery of Glycols, Sugars, and Related Multiple -OH Compounds from Dilute-Aqueous Solution by Regenerable Adsorption onto Activated Carbons, University of California Berkeley, 1999). The ethylene glycol can then be recovered from the organic solvent by distillation. In some embodiments, the ethylene glycol is recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating the microbial cells from the broth (typically by filtration), and isolating the ethylene glycol from the broth. By-products such as alcohols or acids can also be separated or refined from the broth. Alcohols and/or acetone can be isolated, for example, by distillation. Acids can be isolated, for example, by adsorption on activated charcoal. The separated microbial cells may be returned to the bioreactor in some embodiments. The cell-free filtrate remaining after removal of the desired products is also preferably returned to the bioreactor, in whole or in part. Additional nutrients (such as B vitamins) can be added to the cell-free filtrate to replenish the medium before it is returned to the bioreactor.

Recovery of diols from aqueous media has been demonstrated in a number of ways. A pseudo-moving bed (SMB) technique has been used to recover 2,3-butanediol from an aqueous mixture of ethanol and related oxygenates (US Pat. No. 8,658.45). Reactive separation has also been demonstrated to efficiently recover the diol. In some embodiments, the recovery of ethylene glycol is carried out by reacting the diol-containing fraction with aldehydes, fractionating and reducing the diol, final fractionating to recover the concentrated diol fraction; see, for example, US Pat. No. 7,951,980.

The invention provides compositions containing ethylene glycol produced by microorganisms and according to the methods described herein. For example, the composition containing ethylene glycol can be an antifreeze, a preservative, a dehydrating agent, or a drilling fluid.

The invention also provides polymers containing ethylene glycol produced by microorganisms and according to the methods described herein. Such polymers can be, for example, homopolymers such as polyethylene glycol or copolymers such as polyethylene terephthalate. Methods for the synthesis of such polymers are well known in the art; see, for example, Herzberger et al., Chem Rev., 116(4): 2170-2243 (2016) and Xiao et al., Ind Eng Chem Res. 54(22): 5862-5869 (2015).

This invention further provides compositions containing polymers containing ethylene glycol produced by microorganisms and according to the methods described herein. For example, the composition may be a fiber, resin, film, or plastic.

Examples

The following examples further illustrate the invention, but of course should not be construed as limiting its scope in any way.

Example 1 Construction of a heterologous expression vector containing B. subtilis citrate synthase, E. coli isocitrate lyase and G. oxydans glycolaldehyde dehydrogenase to produce ethylene glycol from CO and/or CO ₂ and H ₂ in C. autoethanogenum.

Genes encoding citrate synthase from B. subtilis (citZ; SEQ ID NO: 1-2), isocitrate lyase from E. coli (icl; SEQ ID NO: 11-12), and glycolaldehyde dehydrogenase from G. oxydans (aldA1; SEQ ID NO: : 55-56) were codon-adapted and synthesized for expression in C. autoethanogenum. The adapted genes were cloned into a shuttle expression vector, pIPL12, using the Golden Gate Cloning Bsal standard kit (New England Biolabs, Ipswich, MA). pIPL12 contains an origin of replication for both E. coli and C. autoethanogenum, allowing it to replicate and be maintained in both species; pIPL12 also works in most Clostridia. pIPL12 additionally contains 23S rRNA (adenine(2058)-N(6)) methyltransferase Erm (B) conferring resistance to erythromycin/clarithromycin for positive selection, TraJ for conjugative transfer from E. coli, and a promoter for expression heterologous genes; see fig. 2A. The expression vector generated by cloning citZ, icl and aldA1 into pIPL12 is referred to herein as pMEG042 (FIG. 2B).

table 2

Oligonucleotides used to construct the expression vector pMEG042

SEQID NO Name Subsequence 69 plPL12-bb-F CACACCAGGTCTCAAACCATGGAGATCTCGAGGCC TG 70 plPL12-bb-R CACACCAGGTCTCACATATGATAAGAAGACTCTTGG C 71 citZ_Bs1-F CACACCAGGTCTCACATATGACAGCAACAAGGGGC C 72 citZ_Bs1-R CACACCAGGTCTCAATTGTAACACCTCCTTAATTAGT TATGTCTCTTTCTTCTATAGGTACAAATTTTTG 73 lcl_Ec-F CACACCAGGTCTCACAATGAAAACAAGAACTCAACA AATAG 74 lcl_Ec-R CACACCAGGTCTCAGTGTTCCTCCTATGTGTTCTTA AAATTGAGATTCTTCAGTTGAACCTG 75 aldA1_Go-F CACACCAGGTCTCAACACATATGACTGAAAAAAAATA ATTTATTCATAAATGGATC 76 aldA1_Go-R CACACCAGGTCTCAGGTTATGCATTTAGATATATTGT TTTTGTCTGTACG

The construct pMEG042 was transformed into C. autoethanogenum by conjugation. The expression vector was first introduced into the E. coli conjugative donor strain HB101+R702 (CA434) (Williams et al. 1990) (donor) using standard heat shock transformation. Donor cells were reconstituted in SOC medium at 37°C for 1 h, after which they were placed on dishes with LB medium containing 100 μg/ml spectinomycin and 500 μg/ml erythromycin and incubated at 37°C overnight. The next day, 5 ml aliquots of LB containing 100 μg/ml spectinomycin and 500 μg/ml erythromycin were inoculated with several donor colonies and incubated at 37°C with shaking for about 4 h or until the culture became noticeably more concentrated, but has not yet entered the stationary phase. 1.5 ml of donor culture was collected by centrifugation at 4000 rpm and 20-25°C for 2 min and the supernatant was removed. Donor cells were carefully resuspended in 500 μl of sterile PBS buffer and centrifuged at 4000 rpm for 2 min, and the PBS supernatant was removed.

The pellet was introduced into an anaerobic chamber and carefully resuspended in 200 μl during the late exponential phase of the culture of C. autoethanogenum (recipient). C. autoethanogenum DSM10061 and DSM23693 (derived from DSM10061) were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Inhoffenstraβe 7B, 38124 Braunschweig, Germany). The strains were grown at 37° C. in PETC medium (see US Pat. No. 9,738,875) at pH 5.6 using standard anaerobic methods (Hungate 1969; Wolfe 1971).

The conjugation mixture (a mixture of donor and recipient cells) was applied to PETC-MES + fructose agar plates and left to dry. When the spots were no longer visually wet, the cups were placed in a pressure chamber, pressurized with synthesis gas (50% CO, 10% N ₂ , 30% CO ₂ , 10% H ₂ ) to 25-30 psi, and incubated at 37° C. for approximately 24 hours. The conjugation mixture was then removed from the plates by gentle scraping using a 10 μl inoculation loop. The removed mixture was suspended in 200-300 μl of PETC medium. Aliquots of 100 µl of the conjugation mixture were plated on plates with PETS agar medium supplemented with 5 µg/ml clarithromycin to select plasmid-bearing transformants.

Three separate colonies of C. autoethanogenum carrying plasmid pMEG042 were inoculated into 2 ml PETC-MES medium with 5 μg/ml clarithromycin and grown autotrophically at 37°C with 50% CO, 10% N ₂ , 30% CO ₂ , 10% H ₂ and 100 rpm orbital shaking for three days. Cultures were diluted to OD ₆₀₀ 0.05 in 10 ml PETC-MES medium with 5 μg/ml clarithromycin in serum vials and grown autotrophically at 37°C with 50% CO, 10% N ₂ , 30% CO ₂ , 10% H ₂ and 100 rpm orbital shaking for 20 days, sampling daily for biomass and metabolite measurements (FIGS. 3A and 3B). Ethylene glycol production was measured by gas chromatography mass spectrometry (GC-MS) and other metabolites were measured by high performance liquid chromatography (HPLC) as described below.

Ethylene glycol concentrations were measured using a Thermo Scientific ISQ LT GCMS equipped with an Agilent VF-WAXms (15 m x 0.25 µm x 0.25 µm) column and RSH autosampler. Samples were prepared by diluting 200 µl of broth with 200 µl of methanol. Samples were shaken, then centrifuged for 3 min at 14,000 rpm; 200 µl of the supernatant was transferred into a glass vial with an insert. Samples were transferred to an autosampler for analysis using a 1.0 µL injection, a 5:1 split ratio, and an inlet temperature of 240°C. Chromatography was performed with a heating program: 80°C with a hold of 0.5 min to raise 10°C/min to 150°C, to raise 25°C/min to 220°C, with a final hold of 3 min. The column flow rate was 4.0 ml/min with a hold of 0.5 min, then decreased to 1.5 ml/min at a rate of 100 ml/min/min using helium as the carrier gas. The MS ion source was maintained at 260°C and the transition line was set to 240°C. Quantification was performed using linear calibration with an external standard using

- 20 042922

33.0 m/z as quantification peak and 31.0 + 62.0 m/z as confirmation peaks.

Ethanol, acetate, 2,3-butanediol, glyoxylate, and glycolate concentrations were measured by HPLC on an Agilent 1260 Infinity LC with refractive index (RI) determination at 35°C. Samples were prepared by heating for 5 min at 80° C. followed by 3 min centrifugation at 14,000 rpm; the supernatant was transferred into a glass vial for analysis. Separations were performed with a 10 µl Phenomenex Rezex™ ROA-Organic Acid H+ (8%) (300 mm x 7.8 mm x 8 µm) column injection at 0.7 ml/min and 35°C under isocratic conditions, s using a mobile phase in the form of 5 mm sulfuric acid.

After about 3 days of autotrophic growth, the ethylene glycol glycolate precursor was detected, and ethylene glycol production was detected after 10 days (FIG. 3B).

Example 2 Construction of a heterologous expression vector containing S. thiotaurini alanine glyoxylate aminotransferase and P. fluorescens aldehyde dehydrogenase to produce ethylene glycol from CO and/or CO ₂ and H ₂ in C. autoethanogenum.

The genes encoding alanine glyoxylate aminotransferase from S. thiotaurini (pucG; SEQ ID NOs: 15-16) and aldehyde dehydrogenase from P. fluorescens Q8r1-96 (aldA1; SEQ ID NOs: 57-58) were codon-adapted and synthesized for expression in C. autoethanogenum. The codon adapted genes were cloned into pIPL12 (FIG. 2A) and the resulting expression vector, pMEG058, was introduced into C. autoethanogenum as described in Example 1; see fig. 2C.

Table 3

Oligonucleotides used to construct the expression vector pMEG058

SEQID NO Name Subsequence 69 plPL12-bb-F CACACCAGGTCTCAAACCATGGAGATCTCGAGGCC TG 70 plPL12-bb-R CACACCAGGTCTCACATATGATAAGAAGACTCTTGG C 77 PucG_Sthi1-F CACACCAGGTCTCACATATGCAATTTAGGCCTTTTAA TCCACCA 78 PucG_Sthi1-R CACACCAGGTCTCAGTGTTCCTCCTATGTGTTCTTAT GCTTGCGCAAGTGCCT 79 aldA1_Pfq8-F CACACCAGGTCTCAACACATATGTCTTCAGTGCCTG TATTCCAG 80 aldA1_Pfq8-R CACACCAGGTCTCAGGTTAAGACTGGAGATATACTG CATGAG

Two separate colonies of C. autoethanogenum carrying plasmid pMEG058 were inoculated into 2 ml PETC-MES medium with 5 μg/ml clarithromycin and grown autotrophically as described in Example 1; see fig. 4A. After about 3 days of autotrophic growth, glycolate was detected, and after 8 days, ethylene glycol production was detected (FIG. 4B).

Example 3 Construction of a heterologous expression vector containing S. thiotaurini alanine glyoxylate aminotransferase and G. oxydans glycolaldehyde dehydrogenase to produce ethylene glycol from CO and/or CO ₂ and H ₂ in C. autoethanogenum.

The genes encoding alanine glyoxylate aminotransferase from S. thiotaurini (pucG; SEQ ID NO: 15-16) and glycolaldehyde dehydrogenase from G. oxydans (aldA1; SEQ ID NO: 55-56) were codon-adapted and synthesized for expression in C. autoethanogenum. The codon adapted genes were cloned into pIPL12 (FIG. 2A) and the resulting expression vector, pMEG059, was introduced into C. autoethanogenum as described in Example 1; see fig. 2D.

Table 4

Oligonucleotides used to construct the expression vector pMEG059

SEQID NO Name Subsequence 69 plPL12-bb-F CACACCAGGTCTCAAACCATGGAGATCTCGAGGCC TG 70 plPL12-bb-R CACACCAGGTCTCACATATGATAAGAAGACTCTTGG C 77 PucG_Sthi1-F CACACCAGGTCTCACATATGCAATTTAGGCCTTTTAA TCCACCA 78 PucG_Sthi1-R CACACCAGGTCTCAGTGTTCCTCCTATGTGTTCTTAT GCTTGCGCAAGTGCCT 75 aldA1_Go-F CACACCAGGTCTCAACACATATGACTGAAAAAAAATA ATTTATTCATAAATGGATC 76 aldA1_Go-R CACACCAGGTCTCAGGTTATGCATTTAGATATATTGT TTTTGTCTGTACG

Two separate colonies of C. autoethanogenum carrying plasmid pMEG059 were inoculated into 2 ml PETC-MES medium with 5 μg/ml clarithromycin and grown autotrophically as described in Example 1; see fig. 5A. After about 3 days of autotrophic growth, glycolate was detected, and after 10 days, ethylene glycol production was detected (FIG. 5B).

Example 4 Construction of a heterologous expression vector containing alanine glyoxylate aminotransferase and aldehyde dehydrogenase to produce ethylene glycol from CO and/or CO ₂ and H ₂ in C. autoethanogenum.

The genes encoding class V aminotransferase from C. acidurici (SgA; SEQ ID NO: 19, 20) and aldehyde dehydrogenase from P. fluorescens Q8r1-96 (aldA1; SEQ ID NO: 57-58) were codon-adapted and synthesized for expression in C. autoethanogenum. The codon-adapted genes were cloned into pIPL12 (Fig. 2A) and the resulting expression vector, pMEG061, was introduced into C. autoethanogenum as described in

- 21 042922 example 1; see fig. 2E.

Table 5

Oligonucleotides used to construct the expression vector pMEG061

SEQID NO Name Subsequence 69 plPL12-bb-F CACACCAGGTCTCAAACCATGGAGATCTCGAGGCCT G 70 plPL12-bb-R CACACCAGGTCTCACATATGATAAGAAGACTCTTGGC 81 SgaA_Caci1-F CACACCAGGTCTCACATATGAGAACTCCATTTATTAT GAC 82 SgaA_Caci1-R CACACCAGGTCTCAGTGTTCCTCCTATGTGTTCCTAA TCTACAAAGTGCTTG 79 aldA1_Pfq8-F CACACCAGGTCTCAACACATATGTCTTCAGTGCCTGT ATTCCAG 80 aldA1_Pfq8-R CACACCAGGTCTCAGGTTAAGACTGGAGATATACTGC ATGAG

Three separate colonies of C. autoethanogenum carrying plasmid pMEG061 were inoculated into 2 ml PETC-MES medium with 5 μg/ml clarithromycin and grown autotrophically as described in Example 1; see fig. 6A. After about 3 days of autotrophic growth, glycolate was detected, and after 16 days, ethylene glycol production was detected (FIG. 6B).

Example 5 Simulation of Maximum Yields of Various Pathways for Ethylene Glycol.

To predict the maximum yields of different pathways for ethylene glycol, a genome-scale model of Clostridium autoethanogenum metabolism, similar to that described in Marcellin, Green Chem, 18: 3020-3028, 2016, was used. including the production path of a non-native connection. Although the model used for the experimental work described in this document is based on Clostridium autoethanogenum, it can be expected that the results obtained will be applicable to other Wood-Ljungdahl microorganisms, given the similarity of their metabolism.

Ethylene glycol production was modeled using flow balance analysis (FBA) constraint and linear minimization of metabolic adjustment (LMOMA) computational modeling techniques (Maia, Proceedings of the Genetic and Evolutionary Computation Conference Companion on - GECCO '17, New York, New York, ACM Press, 1661-1668, 2017) using cobrapy version 0.8.2 (Ebrahim., COBRApy: COnstraints-Based Reconstruction and Analysis for Python, BMC Syst Biol, 7: 74, 2013), optlang version 1.2.3 (Jensen, Optlang : An Algebraic Modeling Language for Mathematical Optimization, The Journal of Open Source Software, 2, doi:10.21105/joss.00139, 2017) as the solution search interface and Gurobi Optimizer version 7.0.2 as the optimization solution search tool.

The simulation showed a predicted yield of 0.37 mol ethylene glycol/mol CO for the routes described herein in examples 1-4. This is more than double the predicted yield from the hypothetical pathways described in Islam et al. Metab Eng, 41: 173-181, 2017 that require gluconeogenesis; the highest predicted yields were found to be about 0.44 g ethylene glycol/g CO, corresponding to about 0.18 mol ethylene glycol/mol CO.

All references, including publications, patent applications, and patents, cited herein are incorporated herein by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and are set forth herein in their entirety. volume. In this specification, reference to any prior art is not, and should not be construed as an admission that such prior art is part of the public knowledge of the art in any country.

The use of terms in the singular and similar references in the context of the description of this invention (especially in the context of the claims below) should be considered to cover both the singular and the plural, unless otherwise specified in this document or otherwise clearly contradicts the context. The terms containing, having, including, and covering are to be considered as non-limiting terms (i.e., meaning including, but not limited to) unless otherwise noted. The term essentially consisting of limits the scope of the composition, process or method to the specified materials or steps, or to that which does not significantly affect the essential and new characteristics of the composition, process or method. The use of an alternative (eg, or) should be understood to mean one, both, or any combination of the above alternatives. As used herein, the term about means ±20% of the specified range, value, or structure, unless otherwise noted.

The listing in this document of ranges of values is merely intended to serve as a shorthand way of referring individually to each individual value falling within the specified range, unless otherwise noted in this document, with each individual value included in this description as if it were has been provided separately in this document. For example, any concentration range, percentage range, ratio range, integer range, size range, or thickness range should be understood to include the value of any integer within the specified range and, if appropriate, its fractions (e.g., one tenth and one hundredth of an integer ), unless otherwise specified.

All methods described in this document may be performed in any suitable order.

- 22 042922 ke, unless otherwise indicated in this document or otherwise clearly does not contradict the context. The use of any and all examples or introductory words (eg, such as) provided herein is merely intended to better illuminate the present invention and is not intended to limit the scope of the invention unless otherwise stated. None of the expressions given in this description, should not be understood as an indication of any unclaimed element as necessary for the practical implementation of this invention.

This document describes the preferred embodiments of the present invention. Variations of these preferred embodiments will become apparent to those skilled in the art upon reading the above description. The inventors expect those skilled in the art to make use of such variations as needed, and the inventors expect the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter set forth in the appended claims, as provided by applicable law. In addition, any combination of the elements described above in all their possible variations is covered by this invention as long as this document does not indicate otherwise, or otherwise clearly contradicts the context.

Claims (15)

1. A genetically engineered microorganism capable of producing ethylene glycol or an ethylene glycol precursor from a gaseous substrate, wherein the genetically engineered microorganism is Clostridium autoethanogenum transformed with one of the following vectors: (i) an expression vector containing genes for heterologous citrate synthase, isocitrate lyase, and glycolaldehyde dehydrogenase; (ii) an expression vector containing heterologous alanine-glyoxylate aminotransferase and aldehyde dehydrogenase genes; and (iii) an expression vector containing heterologous alanine-glyoxylate aminotransferase and glycolaldehyde dehydrogenase genes. 2. A microorganism according to claim 1, characterized in that said microorganism produces ethylene glycol or an ethylene glycol precursor via one or more intermediates selected from the group consisting of 5,10-methylenetetrahydrofolate, oxaloacetate, citrate, malate and glycine. 3. Microorganism according to claim 1, characterized in that said microorganism contains one or more of: a) a heterologous enzyme capable of converting oxaloacetate to citrate; b) a heterologous enzyme capable of converting glycine to glyoxylate; c) a heterologous enzyme capable of converting isocitrate to glyoxylate; And d) a heterologous enzyme capable of converting glycolate to glycoaldehyde. 4. The microorganism according to claim 3, characterized in that: a) a heterologous enzyme capable of converting oxaloacetate to citrate is citrate [Si]-synthase [2.3.3.1], ATP-citrate synthase [2.3.3.8] or citrate (Re)-synthase [2.3.3.3]; b) a heterologous enzyme capable of converting glycine to glyoxylate is alanine glyoxylate transaminase [2.6.1.44], serine glyoxylate transaminase [2.6.1.45], serine pyruvate transaminase [2.6.1.51], glycine oxaloacetate transaminase [ 2.6.1.35], glycine transaminase [2.6.1.4], glycine dehydrogenase [1.4.1.10], alanine dehydrogenase [1.4.1.1] or glycine dehydrogenase [1.4.2.1]; c) the heterologous enzyme capable of converting isocitrate to glyoxylate is isocitrate lyase [4.1.3.1] and/or d) a heterologous enzyme capable of converting glycolate to glycoaldehyde is glycolaldehyde dehydrogenase [1.2.1.21], lactaldehyde dehydrogenase [1.2.1.22], succinate semialdehyde dehydrogenase [1.2.1.24], 2,5-dioxovalerate dehydrogenase [1.2. 1.26], aldehyde dehydrogenase [1.2.1.3/4/5], betaine aldehyde dehydrogenase [1.2.1.8], or aldehyde ferredoxin oxidoreductase [1.2.7.5]. 5. The microorganism according to claim 3, characterized in that one or more of the heterologous enzymes are codon-optimized for expression in the microorganism. 6. The microorganism according to claim 3, characterized in that said microorganism additionally contains one or more of an enzyme capable of converting acetyl-CoA to pyruvate; an enzyme capable of converting pyruvate to oxaloacetate; an enzyme capable of converting pyruvate to malate; an enzyme capable of converting pyruvate to phosphenolpyruvate; an enzyme capable of converting oxaloacetate to citryl-CoA; an enzyme capable of converting citryl-CoA to citrate; an enzyme capable of converting citrate to aconitate and aconitate to isocitrate; an enzyme capable of converting phosphoenolpyruvate to oxaloacetate; an enzyme capable of converting phosphoenolpyruvate to 2-phospho-D-glycerate; fer - 23 042922 menta capable of converting 2-phospho^-glycerate to 3-phospho^-glycerate; an enzyme capable of converting 3-phospho^-glycerate to 3-phosphonooxypyruvate; an enzyme capable of converting 3-phosphonooxypyruvate to 3-phospho-L-serine; an enzyme capable of converting 3-phospho-L-serine to serine; an enzyme capable of converting serine to glycine; an enzyme capable of converting 5,10-methylenetetrahydrofolate to glycine; an enzyme capable of converting serine to hydroxypyruvate; an enzyme capable of converting D-glycerate to hydroxypyruvate; an enzyme capable of converting malate to glyoxylate; an enzyme capable of converting glyoxylate to glycolate; an enzyme capable of converting hydroxypyruvate to glycoaldehyde; and an enzyme capable of converting glycoaldehyde to ethylene glycol. 7. The microorganism according to claim 3, characterized in that said microorganism overexpresses: a) a heterologous enzyme capable of converting oxaloacetate to citrate; b) a heterologous enzyme capable of converting glycine to glyoxylate; and/or c) a heterologous enzyme capable of converting glycolate to glycoaldehyde. 8. The microorganism according to claim 6, characterized in that said microorganism overexpresses: a) an enzyme capable of converting pyruvate to oxaloacetate; b) an enzyme capable of converting citrate to aconitate and aconitate to isocitrate; c) an enzyme capable of converting phosphoenolpyruvate to oxaloacetate; d) an enzyme capable of converting serine to glycine; e) an enzyme capable of converting 5,10-methylenetetrahydrofolate to glycine; f) an enzyme capable of converting glyoxylate to glycolate; and/or g) an enzyme capable of converting glycoaldehyde to ethylene glycol. 9. A microorganism according to claim 1, characterized in that said microorganism contains a disruptive mutation in one or more of isocitrate dehydrogenase, glycerate dehydrogenase, glycolate dehydrogenase, aldehyde ferredoxin oxidoreductase and aldehyde dehydrogenase. 10. Microorganism according to claim 1, characterized in that the ethylene glycol precursor is glyoxylate or glycolate. 11. A method for producing ethylene glycol or an ethylene glycol precursor, which includes cultivating the microorganism according to claim 1 in a nutrient medium, in the presence of a gaseous substrate, as a result of which the microorganism produces ethylene glycol or an ethylene glycol precursor. 12. The method according to claim 11, characterized in that the gaseous substrate contains one or more of CO and CO ₂ and H ₂ . 13. The method of claim 11, wherein the ethylene glycol precursor is glyoxylate or glycolate. 14. The method of claim 11 further comprising separating the ethylene glycol or ethylene glycol precursor from the growth medium. 15. The method according to claim 11, characterized in that said microorganism additionally produces one or more of ethanol, 2,3-butanediol and succinate.