Microorganisms and methods for the production of oxygenates from hexoses

Abstract

This application relates to recombinant microorganisms that can be used for the biosynthesis of monoethylene glycol (MEG) or optionally MEG and one or more co-products from one or more hexose raw materials. This application also relates to recombinant microorganisms for the biosynthesis of glycolic acid (GA) or optionally GA and one or more co-products from one or more hexose raw materials. This application relates to recombinant microorganisms that can be used for the biosynthesis of xylitol or optionally xylitol and one or more co-products from one or more hexose raw materials. Also provided are methods for producing MEG (or GA or xylitol) or optionally MEG (or GA or xylitol) and one or more co-products from one or more hexose raw materials using the recombinant microorganisms, and compositions comprising the recombinant microorganisms and / or the product MEG (or GA or xylitol) or optionally MEG (or GA or xylitol) and one or more co-products.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 808,247, filed on February 20, 2019, entitled “MICROORGANISMS AND METHODS FOR THE PRODUCTION OF OXYGENATED COMPOUNDS FROM HEXOSES,” the disclosure of which is incorporated herein by reference. Technical Field

[0003] The application relates to recombinant microorganisms that can be used for biosynthesizing monoethylene glycol or monoethylene glycol and one or more co-products (co-product) by one or more hexose raw materials. The application further relates to recombinant microorganisms for biosynthesizing glycolic acid or glycolic acid and one or more co-products by one or more hexose raw materials. The application also relates to methods for producing monoethylene glycol or monoethylene glycol and one or more co-products by one or more hexose raw materials using recombinant microorganisms, and methods for producing glycolic acid or glycolic acid and one or more co-products by one or more hexose raw materials using recombinant microorganisms. The application further relates to compositions comprising one or more of these compounds and / or recombinant microorganisms.

[0004] Statement regarding sequence listing

[0005] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is BRSK-004_02WO_ST25.txt. The text file is approximately 616KB in size, was created on February 18, 2020, and was submitted electronically through EFS-Web. Background Art

[0006] At present, a large amount of chemical compounds are derived from petrochemical products. Chemical compounds such as monoethylene glycol (MEG), glycolic acid, acetone, isopropyl alcohol (IPA), propylene, serine, glycine, monoethanolamine and ethylenediamine are valuable raw materials for producing products such as polyethylene terephthalate (PET) resin (produced by MEG), polypropylene plastic (produced by propylene), polyglycolic acid and other biocompatible copolymers (produced by glycolic acid) and polyurethane fibers (produced by ethylenediamine). Olefins (such as ethylene, propylene, different butenes and pentenes) are used in other fields of plastics industry, fuel and chemical industry. For example, isobutylene is a small and highly reactive molecule, widely used as platform chemical to manufacture various products, including fuel additives, rubber and rubber additives and specialty chemicals.

[0007] However, these compounds are currently produced from precursors derived from fossil fuels, which contributes to climate change. In order to develop more environmentally friendly MEG production processes, researchers have engineered microorganisms with biosynthetic pathways to produce MEG. However, the implementation of these pathways is challenging, and some of the main obstacles to be overcome are loss of product yield, redox balance, and excess biomass formation.

[0008] Therefore, there is a need for improved biosynthetic pathways for the production of MEG and other compounds useful in industrial and pharmaceutical applications. Summary of the invention

[0009] The present application relates to recombinant microorganisms having one or more biosynthetic pathways for producing monoethylene glycol (MEG) or glycolic acid (GA) or optionally MEG (or GA) and one or more co-products from one or more hexose feedstocks.

[0010] The recombinant microorganisms and methods of the present disclosure combine the advantages of fermentative MEG production based on glucose and fermentative MEG production based on xylose. In some embodiments, the recombinant microorganisms and methods of the present disclosure combine the advantages of xylose degradation biochemistry for high yield formation of MEG (or GA) or optional MEG (or GA) and one or more co-products with the advantages of easily available pure hexose feedstocks.

[0011] In some embodiments, the recombinant microorganisms and methods of the present disclosure solve the problem of xylose raw material availability. In some embodiments, the recombinant microorganisms and methods of the present disclosure solve the problem of unaffordable xylose raw material prices. In some embodiments, the recombinant microorganisms and methods of the present disclosure solve the problem of xylose raw material impurities. In some embodiments, the recombinant microorganisms and methods of the present disclosure solve the problem of low efficiency of microbial xylose uptake. In some embodiments, the recombinant microorganisms and methods of the present disclosure solve the problem of suppressed glucose-induced xylose utilization. In some embodiments, the recombinant microorganisms and methods of the present disclosure solve the problem of ATP shortage in the MEG (or GA) production pathway. In some embodiments, the recombinant microorganisms and methods of the present disclosure solve the problem of excessive NADH in the MEG (or GA) production pathway. In some embodiments, the recombinant microorganisms and methods of the present disclosure solve the problem of low overall product yield potential.

[0012] In some embodiments, the recombinant microorganism and method of the present disclosure provide lossless conversion of one or more hexose raw materials to one or more pentose-5-phosphate intermediates. In some embodiments, the one or more pentose-5-phosphate intermediates are used to produce MEG (or GA) or optional MEG (or GA) and one or more co-products by one or more xylose-based fermentation methods. In some embodiments, glucose flow (flux) is transported to the pentose phosphate pathway instead of the glycolytic pathway.

[0013] On the one hand, the present disclosure provides a recombinant microorganism comprising one or more biochemical pathways that produce monoethylene glycol (MEG) (or glycolic acid) from one or more hexose feedstocks through one or more pentose-5-phosphate intermediates. In one embodiment, one or more co-products are co-produced with MEG (or glycolic acid). In another embodiment, the one or more pentose-5-phosphate intermediates are one or more of D-xylulose-5-phosphate, D-ribulose-5-phosphate, or D-ribose-5-phosphate.

[0014] Thus, in one embodiment, the present application relates to a recombinant microorganism comprising one or more biochemical pathways comprising expression of at least one enzyme having activity for lossless conversion of one or more hexose feedstocks into one or more pentose 5-phosphate intermediates.

[0015] In some embodiments, the recombinant microorganism comprises expression of at least one enzyme having transketolase activity. In some embodiments, the enzyme having transketolase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with tktA from Escherichia coli (E. coli). In other embodiments, the enzyme having transketolase activity is tktA from E. coli. In some embodiments, the enzyme having transketolase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with tktB from E. coli. In other embodiments, the enzyme having transketolase activity is tktB from E. coli. In another embodiment, one or more nucleic acid molecules encoding an enzyme having transketolase activity comprise an amino acid sequence selected from SEQ ID NO: 148 and SEQ ID NO: 150. In some embodiments, one or more nucleic acid molecules encoding an enzyme having transketolase activity is tktA or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding an enzyme having transketolase activity is tktB or a homolog thereof. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having transketolase activity is encoded by a nucleic acid sequence selected from SEQ ID NO: 147 and SEQ ID NO: 149.

[0016] In some embodiments, the recombinant microorganism comprises expression of at least one enzyme having transaldolase activity. In some embodiments, the enzyme having transaldolase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to talA or talB from Escherichia coli. In some embodiments, the enzyme having transaldolase activity is talA from Escherichia coli. In other embodiments, the enzyme having transaldolase activity is talB from Escherichia coli. In another embodiment, one or more nucleic acid molecules encoding an enzyme having transaldolase activity comprise an amino acid sequence selected from SEQ ID NO: 152 and SEQ ID NO: 154. In a further embodiment, one or more nucleic acid molecules encoding an enzyme having transaldolase activity are encoded by a nucleic acid sequence selected from SEQ ID NO: 151 and SEQ ID NO: 153.

[0017] In some embodiments, the recombinant microorganism comprises the expression of at least one enzyme having ribulose-5-phosphate 3-epimerase activity. In some embodiments, the enzyme having ribulose-5-phosphate 3-epimerase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with rpe from Escherichia coli. In other embodiments, the enzyme having ribulose-5-phosphate 3-epimerase activity is rpe from Escherichia coli. In another embodiment, one or more nucleic acid molecules encoding an enzyme having ribulose-5-phosphate 3-epimerase activity comprise the amino acid sequence listed in SEQ ID NO: 158. In a further embodiment, one or more nucleic acid molecules encoding an enzyme having ribulose-5-phosphate 3-epimerase activity are encoded by the nucleic acid sequence listed in SEQ ID NO: 157.

[0018] In some embodiments, the recombinant microorganism comprises expression of at least one enzyme having ribose-5-phosphate isomerase activity. In some embodiments, the enzyme having ribose-5-phosphate isomerase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with rpiA from Escherichia coli. In other embodiments, the enzyme having ribose-5-phosphate isomerase activity is rpiA from Escherichia coli. In other embodiments, the enzyme having ribose-5-phosphate isomerase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with rpiB from Escherichia coli. In other embodiments, the enzyme having ribose-5-phosphate isomerase activity is rpiB from Escherichia coli. In another embodiment, one or more nucleic acid molecules encoding an enzyme having ribose-5-phosphate isomerase activity comprise the amino acid sequence listed in SEQ ID NO: 156. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having ribose-5-phosphate isomerase activity is encoded by the nucleic acid sequence set forth in SEQ ID NO:155.

[0019] In some embodiments, the recombinant microorganism comprises expression of at least one enzyme having transketolase activity, transaldolase activity, ribulose-5-phosphate 3-epimerase activity, and ribose-5-phosphate isomerase activity. In other embodiments, the recombinant microorganism further comprises the loss or reduction of activity of one or more endogenous enzymes selected from glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, and phosphoglycerate mutase. In some embodiments, the endogenous glyceraldehyde 3-phosphate dehydrogenase is gapA, the phosphoglycerate kinase is pgk, and the phosphoglycerate mutase is gpmA and / or gpmM.

[0020] In some embodiments, the recombinant microorganism comprises expression of at least one enzyme having fructose 6-phosphate phosphoketolase activity. In some embodiments, the enzyme having fructose-6-phosphate phosphoketolase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with an enzyme having fructose-6-phosphate phosphoketolase activity selected from the group consisting of Bifidobacterium dentium BDP_1006, Bifidobacterium lactis xfp, Lactobacillus paraplantarum xpkA, and Bifidobacterium breve xfp. In a preferred embodiment, the enzyme having fructose-6-phosphate phosphoketolase activity is selected from Bifidobacterium dentium BDP_1006, Bifidobacterium lactis xfp, Lactobacillus paraplantarum xpkA, and Bifidobacterium breve xfp. In another embodiment, the one or more nucleic acid molecules encoding an enzyme having fructose-6-phosphate phosphoketolase activity comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 212, 214, 216 and 218. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having fructose-6-phosphate phosphoketolase activity is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 211, 213, 215 and 217.

[0021] In some embodiments, the recombinant microorganism comprises expression of at least one enzyme having phosphate acetyltransferase activity. In some embodiments, the enzyme having phosphate acetyltransferase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with an enzyme having phosphate acetyltransferase activity selected from the group consisting of Escherichia coli pta and Clostridium acetobutylicum pta. In a preferred embodiment, the enzyme having phosphate acetyltransferase activity is selected from the group consisting of Escherichia coli pta and Clostridium acetobutylicum pta. In another embodiment, one or more nucleic acid molecules encoding an enzyme having phosphate acetyltransferase activity comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 220 and 222. In a further embodiment, one or more nucleic acid molecules encoding an enzyme having phosphate acetyltransferase activity are encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 219 and SEQ ID NOs: 221.

[0022] In some embodiments, the recombinant microorganism comprises expression of at least one enzyme having fructose-6-phosphate phosphoketolase activity, phosphate acetyltransferase activity, transketolase activity, transaldolase activity, ribulose-5-phosphate 3-epimerase activity, and ribose-5-phosphate isomerase activity. In other embodiments, the recombinant microorganism further comprises an endogenous 6-phosphofructokinase activity deletion or reduction. In some embodiments, the endogenous 6-phosphofructokinase is pfkA and / or pfkB.

[0023] In another embodiment, one or more pentose-5-phosphate intermediates produced by lossless conversion of one or more hexose feedstocks can be connected to any known C2 MEG or glycolate production pathway by converting one or more pentose-5-phosphate intermediates into one or more pentoses and / or pentose-1-phosphate intermediates. In some embodiments, the recombinant microorganism comprises expression of at least one enzyme having pentose phosphatase activity, arabitol phosphate dehydrogenase activity, and / or phosphopentose mutase activity. In some embodiments, the phosphopentose mutase is (Pgm3). In some embodiments, the phosphopentose mutase is (Pgm3) from Saccharomyces cerevisiae. In some embodiments, the phosphopentose mutase is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with Pgm3 from Saccharomyces cerevisiae.

[0024] In some embodiments, the recombinant microorganism comprises the expression of at least one enzyme having pentose phosphatase activity. In other embodiments, the at least one enzyme having pentose phosphatase activity is selected from one or more of the following: an enzyme having D-pentose-5-phosphatase activity, an enzyme having D-xylulose-5-phosphatase activity, an enzyme having D-ribose-5-phosphatase activity, and an enzyme having D-ribulose-5-phosphatase activity. In some embodiments, the pentose phosphatase is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with a D-pentose-5-phosphatase selected from the following: Escherichia coli phoA, Escherichia coli yfbT, and Escherichia coli yidA. In some embodiments, the enzyme having D-xylulose-5-phosphatase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with Bacillus subtilis araL. In some embodiments, the enzyme having D-ribose-5-phosphatase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with an enzyme having D-ribose-5-phosphatase activity selected from Arabidopsis thaliana SGPP, Pseudomonas fluorescens PFLU_2693, and Escherichia coli ybiV. In some embodiments, the enzyme having D-ribulose-5-phosphatase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with PF10_0325 of Plasmodium falciparum. In some embodiments, one or more nucleic acid molecules encoding an enzyme having D-pentose-5-phosphatase activity are selected from SEQ ID NOs: 159, 161, 163, 165, 167, 169, 171, and 173. In another embodiment, the one or more nucleic acid molecules encoding an enzyme having D-pentose-5-phosphatase activity comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 160, 162, 164, 166, 168, 170, 172, and 174.

[0025] In some embodiments, the recombinant microorganism comprises expression of at least one enzyme having arabitol phosphate dehydrogenase activity. In some embodiments, the enzyme having arabitol phosphate dehydrogenase activity is selected from one or more of the following: an enzyme having D-arabitol 1-phosphate 4-dehydrogenase activity, an enzyme having D-arabitol 5-phosphate 2-dehydrogenase activity, an enzyme having D-arabitol 1-phosphate 2-dehydrogenase activity, and an enzyme having D-arabitol 5-phosphate 4-dehydrogenase activity.

[0026] In some embodiments, the enzyme having D-arabitol 1-phosphate 4-dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to APDH from Enterococcus avium. In a preferred embodiment, the enzyme having D-arabitol 1-phosphate 4-dehydrogenase activity is APDH from Enterococcus avium. In some embodiments, one or more nucleic acid molecules encoding an enzyme having D-arabitol 1-phosphate 4-dehydrogenase activity are listed in SEQ ID NO: 175. In another embodiment, one or more nucleic acid molecules encoding an enzyme having D-arabitol 1-phosphate 4-dehydrogenase activity comprise the amino acid sequence listed in SEQ ID NO: 176.

[0027] In some embodiments, the enzyme having D-arabitol 5-phosphate 2-dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to APDH from Enterococcus avium. In a preferred embodiment, the enzyme having D-arabitol 5-phosphate 2-dehydrogenase activity is APDH from Enterococcus avium. In some embodiments, one or more nucleic acid molecules encoding an enzyme having D-arabitol 5-phosphate 2-dehydrogenase activity are listed in SEQ ID NO: 175. In another embodiment, one or more nucleic acid molecules encoding an enzyme having D-arabitol 5-phosphate 2-dehydrogenase activity comprise the amino acid sequence listed in SEQ ID NO: 176.

[0028] In some embodiments, the enzyme having D-arabitol 1-phosphate 2-dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with an enzyme having D-arabitol 1-phosphate 2-dehydrogenase activity selected from the group consisting of Candida albicans ARD1, Candida tropicalis ARD1, Scheffersomyces stipitis ARDH, Bacillus subtilis egsA (araM), Aeropyrum pernix egsA, Escherichia coli gpsA, and Saccharomyces cerevisiae GPD1. In a preferred embodiment, the enzyme having D-arabitol 1-phosphate 2-dehydrogenase activity is selected from the group consisting of Candida albicans ARD1, Candida tropicalis ARD1, Scheffersomyces stipitis ARDH, Bacillus subtilis egsA (araM), Aeropyrum pernix egsA, Escherichia coli gpsA, and Saccharomyces cerevisiae GPD1. In some embodiments, the one or more nucleic acid molecules encoding an enzyme having D-arabitol 1-phosphate 2-dehydrogenase activity are selected from SEQ ID NOs: 177, 179, 181, 189, 191, 193, and 195. In another embodiment, the one or more nucleic acid molecules encoding an enzyme having D-arabitol 1-phosphate 2-dehydrogenase activity comprise an amino acid sequence selected from SEQ ID NOs: 178, 180, 182, 190, 192, 194, and 196.

[0029] In some embodiments, the enzyme having D-arabitol 5-phosphate 4-dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to an enzyme having D-arabitol 5-phosphate 4-dehydrogenase activity selected from the group consisting of: Pseudomonas fluorescens mtlD, Klebsiella pneumoniae dalD, Ralstonia solanacearum dalD, Bacillus subtilis egsA (araM), Aeropyrum facilis egsA, Escherichia coli gpsA, and Saccharomyces cerevisiae GPD1. In a preferred embodiment, the enzyme having D-arabitol 5-phosphate 4-dehydrogenase activity is selected from Pseudomonas fluorescens mtlD, Klebsiella pneumoniae dalD, Ralstonia solanacearum dalD, Bacillus subtilis egsA (araM), Aeropyrum facilis egsA, Escherichia coli gpsA and Saccharomyces cerevisiae GPD1. In some embodiments, the one or more nucleic acid molecules encoding the enzyme having D-arabitol 5-phosphate 4-dehydrogenase activity are selected from SEQ ID NOs: 183, 185, 187, 189, 191, 193 and 195. In another embodiment, the one or more nucleic acid molecules encoding the enzyme having D-arabitol 5-phosphate 4-dehydrogenase activity comprise an amino acid sequence selected from SEQ ID NOs: 184, 186, 188, 190, 192, 194 and 196.

[0030] In some embodiments, the recombinant microorganism comprises expression of at least one enzyme having phosphopentose mutase activity. In some embodiments, the enzyme having phosphopentose mutase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with an enzyme having phosphopentose mutase activity selected from the group consisting of: Escherichia coli deoB, Escherichia coli pgm, Bacillus subtilis pgcA, Lactococcus lactis pgmB, Escherichia coli ycjU, Pseudomonas aeruginosa algC, and Escherichia coli cpsG. In a preferred embodiment, the enzyme having pentose phosphate mutase activity is selected from the group consisting of E. coli deoB, E. coli pgm, Bacillus subtilis pgcA, Lactococcus lactis pgmB, E. coli ycjU, Pseudomonas aeruginosa algC and E. coli cpsG. In some embodiments, the one or more nucleic acid molecules encoding the enzyme having pentose phosphate mutase activity are selected from the group consisting of SEQ ID NOs: 197, 199, 201, 203, 205, 207 and 209. In another embodiment, the one or more nucleic acid molecules encoding the enzyme having pentose phosphate mutase activity comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 198, 200, 202, 204, 206, 208 and 210.

[0031] In some embodiments of any of the above-mentioned recombinant microorganisms, the recombinant microorganism further comprises an activity reduced in one or more endogenous enzymes, and the endogenous enzyme is selected from glucose 6-phosphate-1-dehydrogenase, 6-phosphogluconolactonase and 6-phosphogluconate dehydrogenase. In further embodiments, the glucose 6-phosphate-1-dehydrogenase is zwf, the 6-phosphogluconolactonase is pgl, and the 6-phosphogluconate dehydrogenase is gnd.

[0032] In some embodiments, the application provides a recombinant microorganism that co-produces MEG (or glycolic acid) and one or more co-products selected from acetone, isopropanol, propylene, isobutylene and one or more serine pathway compounds. In some preferred embodiments, the one or more serine pathway compounds are selected from serine, glycine, monoethanolamine (MEA) and ethylenediamine (EDA).

[0033] In some embodiments, the one or more hexose raw materials are selected from glucose or oligomers of glucose. In other embodiments, the oligomers of glucose are selected from fructose, sucrose, starch, cellobiose, maltose, lactose and cellulose.

[0034] In some embodiments, in the recombinant microorganism, expression of an enzyme having transketolase activity or an enzyme having fructose-6-phosphate phosphoketolase activity enables lossless conversion of one or more hexose feedstocks into one or more pentose 5-phosphate intermediates.

[0035] In some embodiments, the recombinant microorganism produces MEG or glycolic acid (GA) by conversion of glycolaldehyde in the C2 pathway and conversion of dihydroxyacetone phosphate (DHAP) or pyruvic acid in the C3 pathway. In other embodiments, glycolaldehyde is oxidized to glycolic acid by glycolaldehyde dehydrogenase.

[0036] In some embodiments, the at least one enzyme used to produce MEG or GA by conversion of DHAP or pyruvate in the C3 pathway is selected from at least one enzyme having an activity selected from the group consisting of 3-phosphoglycerate dehydrogenase activity, phosphoserine transaminase activity, 3-phosphohydroxypyruvate phosphatase activity, phosphoserine phosphatase activity, serine transaminase activity, hydroxypyruvate decarboxylase activity, 3-phosphohydroxypyruvate reductase activity, glycolaldehyde reductase activity, glycolaldehyde dehydrogenase activity, serine oxidoreductase (deaminating) or serine-pyruvate transaminase activity, serine decarboxylase activity, ethanolamine transaminase or ethanolamine oxidoreductase (deaminating) activity, glycerate decarboxylase activity, hydroxypyruvate reductase activity, 3-phosphoglycerate phosphatase activity, 2-phosphoglycerate phosphatase activity, glycerate 3-kinase activity, and glycerate 2-kinase activity.

[0037] In some embodiments, the recombinant microorganism produces MEG or glycolic acid (GA) by conversion of glycolaldehyde in the C2 pathway, and produces one or more co-products by conversion of dihydroxyacetone phosphate (DHAP) or pyruvic acid in the C3 pathway. In other embodiments, the one or more co-products are selected from acetone, isopropanol, propylene, isobutylene, and one or more serine pathway compounds. In some preferred embodiments, the one or more serine pathway compounds are selected from serine, glycine, monoethanolamine (MEA), and ethylenediamine (EDA).

[0038] In some embodiments, the at least one enzyme used to produce one or more co-products by conversion of DHAP or pyruvate in the C3 pathway is selected from at least one enzyme having an activity selected from the group consisting of: thiolase or acetyl-CoA acetyltransferase activity, acetyl-CoA:acetoacetate transferase or acetate:acetoacetyl-CoA hydrolase activity, and acetoacetate decarboxylase activity, and the one or more co-products comprise acetone.

[0039] In some embodiments, the at least one enzyme used to produce one or more co-products by conversion of DHAP or pyruvate in the C3 pathway is selected from at least one enzyme having an activity selected from the group consisting of: thiolase or acetyl-CoA acetyltransferase activity, acetyl-CoA:acetoacetate transferase or acetate:acetoacetyl-CoA hydrolase activity, acetoacetate decarboxylase activity, and secondary alcohol dehydrogenase activity, and the one or more co-products comprise isopropanol.

[0040] In some embodiments, the at least one enzyme used to produce one or more co-products by conversion of DHAP or pyruvate in the C3 pathway is selected from at least one enzyme having an activity selected from the group consisting of: thiolase or acetyl-CoA acetyltransferase activity, acetyl-CoA:acetoacetate transferase or acetate:acetoacetyl-CoA hydrolase activity, acetoacetate decarboxylase activity, secondary alcohol dehydrogenase activity, and dehydratase activity, and the one or more co-products comprise propylene.

[0041] In some embodiments, the at least one enzyme used to produce one or more co-products by conversion of DHAP or pyruvate in the C3 pathway is selected from at least one enzyme having an activity selected from the group consisting of thiolase or acetyl-CoA acetyltransferase activity, acetyl-CoA:acetoacetate transferase or acetate:acetoacetyl-CoA hydrolase activity, acetoacetate decarboxylase activity, 3-hydroxyisovalerate (3HIV) synthase activity, hydroxymethylglutaryl-CoA synthase activity, methylglutaconyl-CoA hydratase activity, methylcrotonyl-CoA carboxylase activity, methylcrotonyl-CoA hydratase activity, 3-hydroxyisovaleryl-CoA thioesterase activity, 3HIV kinase activity, 3HIV-3-phosphate decarboxylase activity, and 3HIV decarboxylase activity, and the one or more co-products comprise isobutylene.

[0042] In some embodiments, the at least one enzyme used to produce one or more co-products by conversion of DHAP or pyruvate in the C3 pathway is selected from at least one enzyme having an activity selected from the group consisting of 3-phosphoglycerate dehydrogenase activity, phosphoserine transaminase activity, 3-phosphohydroxypyruvate phosphatase activity, phosphoserine phosphatase activity, serine oxidoreductase (deaminating) or serine-pyruvate transaminase activity, hydroxypyruvate reductase activity, 3-phosphoglycerate phosphatase activity, 2-phosphoglycerate phosphatase activity, glycerate 3-kinase activity, and glycerate 2-kinase activity, and wherein the one or more co-products comprise L-serine.

[0043] In some embodiments, the at least one enzyme used to produce one or more co-products by conversion of DHAP or pyruvate in the C3 pathway is selected from at least one enzyme having an activity selected from the group consisting of serine hydroxymethyltransferase activity, transferase activity, formaldehyde dehydrogenase activity, formate dehydrogenase activity, activity associated with the glycine cleavage system, 3-phosphoglycerate dehydrogenase activity, phosphoserine transaminase activity, 3-phosphohydroxypyruvate phosphatase activity, phosphoserine phosphatase activity, serine transaminase activity, hydroxypyruvate decarboxylase activity. , serine oxidoreductase (deaminating) activity, serine decarboxylase activity, ethanolamine transaminase or ethanolamine oxidoreductase (deaminating) activity, hydroxypyruvate reductase activity, 3-phosphoglycerate phosphatase activity, 2-phosphoglycerate phosphatase activity, glycerate 3-kinase activity, glycerate 2-kinase activity, glycolaldehyde dehydrogenase activity, glycolate dehydrogenase activity, alanine-glyoxylate transaminase activity, alanine transaminase activity and NAD (P) H dependent glutamate dehydrogenase activity, and one or more co-products include glycine. In another embodiment, the activity associated with the glycine cleavage system includes an enzyme or protein selected from the group consisting of glycine decarboxylase (P protein), aminomethyltransferase (T protein), dihydrolipoamide dehydrogenase (L protein) and H protein.

[0044] In some embodiments, the at least one enzyme used to produce one or more co-products by conversion of DHAP or pyruvate in the C3 pathway is selected from at least one enzyme having an activity selected from the group consisting of 3-phosphoglycerate dehydrogenase activity, phosphoserine transaminase activity, 3-phosphohydroxypyruvate phosphatase activity, phosphoserine phosphatase activity, transaminase activity, hydroxypyruvate decarboxylase activity, serine oxidoreductase (deaminating) or serine-pyruvate transaminase activity, serine decarboxylase activity, hydroxypyruvate reductase activity, 3-phosphoglycerate phosphatase activity, 2-phosphoglycerate phosphatase activity, glycerate 3-kinase activity, glycerate 2-kinase activity, acetaldehyde dehydrogenase activity, and ethanolamine ammonia lyase activity, and the one or more co-products comprise monoethanolamine (MEA).

[0045] In some embodiments, the at least one enzyme used to produce one or more co-products by conversion of DHAP or pyruvate in the C3 pathway is selected from at least one enzyme having an activity selected from the group consisting of serine dehydrogenase activity, 2-aminomalonate semialdehyde decarboxylase activity, aminoacetaldehyde transaminase activity, 2-aminomalonate semialdehyde transaminase activity, 2,3-diaminopropionate decarboxylase activity, serine decarboxylase activity, ethanolamine dehydrogenase activity, serine hydroxymethyltransferase activity, aldehyde oxidase activity, N-acetyltransferase or O-acetyltransferase activity, N-acetylserine dehydrogenase activity, transaminase activity, deacetylase activity, serine aminase activity, and 2,3-diaminopropionate ammonia lyase activity, and the one or more co-products comprise ethylenediamine (EDA).

[0046] In some embodiments, at least one enzyme for producing MEG or GA by conversion of ethanolaldehyde in the C2 pathway is selected from at least one enzyme having an activity selected from the following: D-tagatose 3-epimerase activity, D-ribulose kinase activity, D-ribulose-1-phosphate aldolase activity, D-xylulose 1-kinase activity, D-xylulose-1-phosphate aldolase activity, xylose reductase or aldose reductase activity, xylitol dehydrogenase activity, xylose isomerase activity, xylose dehydrogenase activity, xylon lactonase activity, xylonate dehydrogenase activity, 2-keto-3-deoxy-D-pentanoic acid aldolase activity, ethanolaldehyde reductase activity and ethanolaldehyde dehydrogenase activity. In some embodiments, the enzyme having D-xylulose-1-phosphate aldolase activity is aldoB. In some embodiments, the enzyme having D-ribulose-1-phosphate aldolase activity is fucA.

[0047] In some embodiments of any of the above recombinant microorganisms, the recombinant microorganism further comprises one or more modifications to reduce or delete the activity of glycolaldehyde dehydrogenase, lactate dehydrogenase, xylose isomerase, xylulokinase, or a combination thereof.

[0048] In one embodiment, at least a portion of the excess NADH produced in the C3 pathway is used as a source of reducing equivalents in the C2 pathway. In another embodiment, at least a portion of the excess NADH produced in the C3 pathway is used to produce ATP.

[0049] In one embodiment, excess biomass formation is minimized and production of MEG (or glycolic acid) or MEG (or glycolic acid) and one or more co-products is maximized.

[0050] On the other hand, the present application provides a method for producing MEG or glycolic acid (GA) using a recombinant microorganism according to any one of the above embodiments, wherein the method includes culturing a recombinant microorganism in a culture medium containing one or more hexose raw materials providing a carbon source until MEG or GA is produced. In some embodiments, one or more co-products are co-produced with MEG or GA. In further embodiments, the one or more co-products are selected from acetone, isopropanol, propylene, isobutylene and one or more serine pathway compounds. In still further embodiments, the one or more serine pathway compounds are selected from serine, glycine, monoethanolamine (MEA) and ethylenediamine (EDA).

[0051] In another aspect, a method for producing a recombinant microorganism that produces or accumulates MEG or glycolic acid (GA) from one or more exogenous hexose feedstocks via one or more pentose-5-phosphate intermediates, the method comprising: introducing or expressing one or more enzymes in the recombinant microorganism for converting one or more hexose feedstocks into one or more pentose-5-phosphate intermediates; introducing or expressing one or more enzymes in the recombinant microorganism for converting one or more pentose-5-phosphate intermediates into one or more pentoses or pentose-1-phosphate intermediates; introducing or expressing one or more C2 pathways in the recombinant microorganism, comprising one or more enzymes for producing MEG or GA from glycolaldehyde; introducing or expressing one or more C3 pathways in the recombinant microorganism, comprising one or more enzymes for producing MEG or GA derived from DHAP or pyruvate; and culturing the recombinant microorganism in a culture medium containing one or more hexose feedstocks to produce or accumulate MEG or GA, wherein glycolaldehyde and DHAP (or pyruvate) are intermediates produced in the C2 pathway, and wherein MEG or GA is produced in both the C2 and C3 pathways.

[0052] In some embodiments, the present application provides a method for producing a recombinant microorganism, wherein the microorganism produces or accumulates MEG or glycolic acid (GA) and one or more co-products from one or more exogenous hexose feedstocks through one or more pentose-5-phosphate intermediates, the method comprising: introducing or expressing one or more enzymes in the recombinant microorganism for converting one or more hexose feedstocks into one or more pentose-5-phosphate intermediates; introducing or expressing one or more enzymes in the recombinant microorganism for converting one or more pentose-5-phosphate intermediates into one or more pentoses or pentose-1-phosphate intermediates; introducing or expressing one or more C2 pathways in the recombinant microorganism, which contain one or more enzymes for producing MEG or GA from glycolaldehyde; introducing or expressing one or more C3 pathways in the recombinant microorganism, which contain one or more enzymes for producing one or more co-products derived from DHAP or pyruvate; culturing the recombinant microorganism in a culture medium containing one or more hexose feedstocks to produce or accumulate MEG or GA and one or more co-products, wherein glycolaldehyde and DHAP (or pyruvate) are intermediates produced in the C2 pathway, wherein MEG or GA is produced in one or more C2 pathways, and one or more co-products are produced in one or more C3 pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Illustrative embodiments of the present disclosure are illustrated in the accompanying drawings, in which:

[0054] Figure 1 This illustrates the lossless conversion of glucose to pentose phosphate. This means that the enzyme may be down-regulated or inactivated / eliminated, ie the respective gene may be attenuated or deleted.

[0055] Figure 2 MEG and possible co-production pathways via D-xylulose-1-phosphate are illustrated.

[0056] Figure 3 MEG and possible co-production pathways via D-xylonic acid are described.

[0057] Figure 4 MEG and possible co-production pathways via D-ribulose-1-phosphate are illustrated.

[0058] Figure 5 The option of linking the pentose phosphate pathway and the MEG production pathway via pentose phosphatase (PP) is described.

[0059] Figure 6 A scheme for the production of pentose-1-phosphate and derivatives from the pentose phosphate pathway via arabitol phosphate dehydrogenase (APD) is described.

[0060] Figure 7The isomerization of pentose phosphates mediated by arabitol phosphate dehydrogenase (APD) is demonstrated.

[0061] Figure 8 A scheme for the production of pentose-1-phosphate and derivatives from the pentose phosphate pathway by phosphopentose mutase (PPM) is illustrated.

[0062] Fig. 9 Illustrates the lossless conversion of glucose to pentose phosphate and acetyl-CoA. This means that the enzyme may be down-regulated or inactivated / eliminated, ie the respective gene may be attenuated or deleted.

[0063] Fig.10 An overview of the co-production pathways of MEG and Ser, Gly, MEA, and EDA is described.

[0064] Fig.11 Published EDA production pathways are described. From WO 2014 / 049382. Reaction F: Direct amination of L-serine by L-serine aminase. Reaction G: Direct amination of pyruvate by 2,3-diaminopropionate ammonia lyase.

[0065] Fig.12 The biosynthesis pathway of xylitol from glucose using phosphatases is illustrated. Symbols This means that the enzyme will likely be down-regulated or inactivated / eliminated, ie the respective gene may be attenuated or deleted.

[0066] Fig.13 The biosynthesis pathway of xylitol from glucose using arabitol-P dehydrogenase (ADP) is illustrated. This means that the enzyme will likely be down-regulated or inactivated / eliminated, ie the respective gene may be attenuated or deleted.

[0067] Fig.14 The biosynthesis pathway of xylitol from glucose using pentose phosphomutase (PPM) is illustrated. This means that the enzyme may be down-regulated or inactivated / eliminated, ie the respective gene may be attenuated or deleted.

[0068] Fig.15 The present invention is a protocol for the in vitro assay of phosphoglucomutase against its natural substrate glucose-1P.

[0069] Fig.16 The invention is a scheme for converting pentoses into glycolate precursors in vitro by the action of phosphoglucomutase on the key pentose-5P intermediate.

[0070] Fig.17 An in vivo screening protocol for key candidate enzymes that facilitate the conversion of pentose sugars to glycolate.

[0071] Fig.18 The present invention is a scheme for the in vitro conversion of pentoses into glycolate precursors by aldolase acting on the key pentose-1P intermediate. DETAILED DESCRIPTION

[0072] definition

[0073] The following definitions and abbreviations will be used to explain this disclosure.

[0074] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, reference to "the microorganism" includes reference to one or more microorganisms, and so forth.

[0075] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not to an exclusive "or."

[0076] As used herein, the terms "about" and "about" that modify numerical values ​​represent a close range around the explicit value. If "X" is the value, then "about X" or "about X" will indicate a value of 0.9X to 1.1X, or in some embodiments, a value of 0.95X to 1.05X. Any reference to "about X" or "about X" specifically represents at least the following values: X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Therefore, "about X" and "about X" are intended to teach and provide written description support for the limitation (e.g., "0.98X") of the claims.

[0077] As used herein, the terms "microbial" and "microbial organism, microorganism" include any organism that exists as a microscopic cell within the kingdoms Archaea, Bacteria, or Eukaryota, including yeast and filamentous fungi, protozoa, algae, or higher protists. Thus, the term is intended to encompass prokaryotic or eukaryotic cells or organisms of microscopic size, and includes bacteria, archaea, and eubacteria of all species, as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of chemicals.

[0078] As described herein, in some embodiments, the recombinant microorganism is a prokaryotic microorganism. In some embodiments, the prokaryotic microorganism is a bacterium. "Bacteria" or "eubacteria" refers to the kingdom of prokaryotes. Bacteria include at least eleven different groups, as follows: (1) Gram-positive (gram+) bacteria, of which there are two major branches: (1) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, and others); (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, and Mycoplasmas); (2) Proteobacteria, such as purple photosynthetic + non-photosynthetic Gram-negative bacteria (including most of the "common" Gram-negative bacteria); (3) Cyanobacteria, such as oxygenic phototrophs; (4) phototroph); (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides and Flavobacteria; (7) Chlamydia; (8) Green sulfurbacteria; (9) Green non-sulfur bacteria (also anaerobic phototroph); (10) Radioresistant Micrococcus and its relatives; (11) Thermotoga and Thermosipho thermophiles.

[0079] "Gram-negative bacteria" include cocci, nonenteric rods, and enteric rods. Genera of gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, and ibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0080] "Gram-positive bacteria" include cocci, nonsporulating rods, and sporulating rods. The genera of Gram-positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0081] The terms "recombinant microorganism" and "recombinant host cell" are used interchangeably herein and refer to microorganisms that have been genetically modified to express or overexpress endogenous enzymes, express heterologous enzymes (such as those included in vectors, in integration constructs), or the expression of endogenous genes. "Alteration" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more polypeptides or polypeptide subunits, or the activity of one or more polypeptides or polypeptide subunits is upregulated or downregulated so that the expression, level, or activity is greater than or less than the expression, level, or activity observed without the change. For example, the term "alteration" can mean "inhibition", but the use of the term "alteration" is not limited to this definition. It should be understood that the terms "recombinant microorganism" and "recombinant host cell" refer not only to specific recombinant microorganisms, but also to the offspring or potential offspring of such microorganisms. Because some modifications may occur in offspring due to mutations or environmental influences, such offspring may actually be different from the parental cell, but are still included in the scope of the terms used herein.

[0082] The term "expression" associated with a gene sequence refers to the transcription of a gene, and, where appropriate, to the translation of the resulting mRNA transcript into a protein. Therefore, as is clear from the context, the expression of a protein is caused by the transcription and translation of an open reading frame sequence. The expression level of a desired product in a host cell can be determined based on the amount of the corresponding mRNA present in the cell or the amount of the desired product encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantified by qRT-PCR or Northern hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). The protein encoded by the selected sequence can be quantified by various methods, such as by ELISA, by determining the biological activity of the protein, or by using an assay independent of such activity, such as a Western blot or a radioimmunoassay (using antibodies that recognize and bind to the protein). See Sambrook et al., 1989, supra.

[0083] In this article, the term "polynucleotide" is used interchangeably with the term "nucleic acid" and refers to an organic polymer composed of two or more monomers, including nucleotides, nucleosides or their analogs, including but not limited to single-stranded or double-stranded, sense or antisense deoxyribonucleic acid (DNA) of any length, and, where appropriate, single-stranded or double-stranded, sense or antisense ribonucleic acid (RNA) (including siRNA) of any length. The term "nucleotide" refers to any of several compounds consisting of ribose or deoxyribose connected to a purine or pyrimidine base and a phosphate group, and is the basic structural unit of nucleic acid. The term "nucleoside" refers to a compound (such as guanosine or adenosine) composed of a purine or pyrimidine base and a deoxyribose or ribose combination, particularly found in nucleic acids. The term "nucleotide analog" or "nucleoside analog" refers to a nucleotide or nucleoside, respectively, in which one or more individual atoms are replaced by different atoms or different functional groups. Accordingly, the term polynucleotide includes nucleic acids, DNA, RNA, analogs thereof, and fragments thereof of any length. A polynucleotide of three or more nucleotides is also called a nucleotide oligomer or oligonucleotide.

[0084] It should be understood that the polynucleotides described herein include "genes" and the nucleic acid molecules described herein include "vectors" or "plasmids". Accordingly, the term "gene", also referred to as "structural gene", refers to a polynucleotide encoding a specific amino acid sequence, which contains all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine, for example, the conditions for gene expression. The transcribed region of a gene may include untranslated regions, including introns, 5'-untranslated regions (UTRs) and 3'-UTRs, as well as coding sequences.

[0085] As used herein, the term "enzyme" refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, and generally includes enzymes composed entirely or in part of one or more polypeptides, but may also include enzymes composed of different molecules including polynucleotides.

[0086] As used herein, the term "non-naturally occurring", when used in reference to a microorganism or enzyme activity of the present disclosure, is intended to mean that the microorganism or enzyme has at least one genetic alteration that is not normally present in a naturally occurring strain of a reference species (including a wild-type strain of a reference species). Genetic alterations include, for example, modifications that introduce expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions, and / or other functional disruptions of the microorganism's genetic material. Such modifications include, for example, coding regions of heterologous, homologous, or heterologous and homologous polypeptides of the reference species, and functional fragments thereof. Other modifications include, for example, non-coding regulatory regions where the modification alters the expression of a gene or operon. Exemplary non-naturally occurring microorganisms or enzyme activities include the above-mentioned hydroxylation activities.

[0087] As used herein, the term "exogenous" with respect to various molecules (eg, polynucleotides, polypeptides, enzymes, etc.) refers to molecules that are not normally or naturally present in and / or produced by a given yeast, bacteria, organism, microorganism or cell in nature.

[0088] On the other hand, as used herein, the term "endogenous" or "native" with respect to various molecules (e.g., polynucleotides, polypeptides, enzymes, etc.) refers to molecules that normally or naturally occur in and / or are produced by a given yeast, bacteria, organism, microorganism or cell in nature.

[0089] As used in the context of this article, the term "heterologous" of a modified host cell refers to various molecules (e.g., polynucleotides, polypeptides, enzymes, etc.) wherein at least one of the following is true: (a) the molecule is foreign ("exogenous") to the host cell (i.e., not naturally occurring in the host cell); (b) the molecule naturally occurs (e.g., is "endogenous") in a given host microorganism or host cell, but is produced in a non-natural location or in a non-natural amount in the cell; and / or (c) the molecule differs in nucleotide or amino acid sequence from an endogenous nucleotide or amino acid sequence, such that the molecule that differs in nucleotide or amino acid sequence from an endogenously occurring endogenous nucleotide or amino acid is produced in a non-natural amount (e.g., greater than naturally occurring) in the cell.

[0090] As used herein, the term "homologue" of an original enzyme or gene of a first family or species refers to a different enzyme or gene of a second family or species that is determined by function, structure or genome analysis to correspond to an enzyme or gene of a second family or species of the original enzyme or gene of the first family or species. Homologues typically have functional, structural or genomic similarities. Known techniques can easily use genetic probes and PCR to clone homologues of enzymes or genes. Functional assays and / or genome mapping of genes can be used to confirm the characteristics of cloned sequences as homologues.

[0091] If the amino acid sequence encoded by a gene is similar to the amino acid sequence of a second gene, then the protein has "homology" or is "homologous" to a second protein. Alternatively, if two proteins have "similar" amino acid sequences, then the protein has homology to the second protein. Therefore, the term "homologous proteins" is intended to mean that two proteins have similar amino acid sequences. In some cases, the homology between two proteins indicates that they have a common ancestor that is related in evolution. The terms "homologous sequences" or "homologs" are considered, believed or known to be functionally related. The functional relationship can be expressed in any of a variety of ways, including but not limited to: (a) the degree of sequence identity and / or (b) the same or similar biological function. Preferably, (a) and (b) are indicated simultaneously. The degree of sequence identity can vary, but in one embodiment, is at least 50% (when using standard sequence alignment programs known in the art), at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 98.5%, or at least about 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987) Appendix 30, Section 7.718, Table 7.71. MacVector (Oxford Molecular Ltd, Oxford, UK) and ALIGN Plus (Scientific and Educational Software, Pennsylvania) are some alignment programs. Other non-limiting alignment programs include Sequencher (Gene Codes, Ann Arbor, Michigan), AlignX, and Vector NTI (Invitrogen, Carlsbad, CA). Similar biological functions may include, but are not limited to: catalyzing the same or similar enzymatic reactions; having the same or similar selectivity for substrates or cofactors; having the same or similar stability; having the same or similar tolerance to various fermentation conditions (temperature, pH, etc.); and / or having the same or similar tolerance to various metabolic substrates, products, by-products, intermediates, etc.The degree of similarity of biological functions can vary, but in one embodiment, is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 98.5%, or at least about 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%, for a given biological function as determined by one or more assays known to those of skill in the art.

[0092] The term "variant" refers to any polypeptide or enzyme as described herein. Variant also encompasses one or more components of a multimer, a multimer comprising a single component, a multimer comprising a plurality of single components (e.g., a multimer of a reference molecule), chemical decomposition products, and biological decomposition products. In particular, in non-limiting embodiments, due to the change of any part of the polypeptide sequence encoding the reference enzyme, the enzyme can be a "variant" relative to the reference enzyme. In the standard assay for measuring the enzymatic activity of a reference enzyme preparation, the variant of the reference enzyme can have at least 10%, at least 30%, at least 50%, at least 80%, at least 90%, at least 100%, at least 105%, at least 110%, at least 120%, at least 130% or higher enzymatic activity. In some embodiments, variants may also refer to polypeptides having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the full-length or unprocessed enzyme of the disclosure. In some embodiments, variants may also refer to polypeptides having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature or processed enzyme of the disclosure.

[0093] As used herein, the term "signal sequence" refers to an amino acid sequence that targets peptides and polypeptides to a cellular location or the extracellular environment. The signal sequence is typically located at the N-terminal portion of the polypeptide and is typically removed enzymatically. A polypeptide having its signal sequence is referred to as full-length and / or unprocessed. A polypeptide from which its signal sequence has been removed is referred to as mature and / or processed.

[0094] As used herein, the term "yield potential" refers to the yield of a product from a biosynthetic pathway. In one embodiment, the yield potential can be expressed as the weight percentage of the final product per weight of the starting compound.

[0095] As used herein, the term "thermodynamic maximum yield" refers to the maximum yield of a product obtained by fermentation of a given raw material (such as glucose) based on the energy value of the product compared to the raw material. For example, in normal fermentation, if no additional energy (such as light, hydrogen or methane or electricity) is used, the product cannot contain more energy than the raw material. Thermodynamic maximum yield represents the product yield at which all energy and mass from the raw material are converted into the product. The yield can be calculated and is independent of a specific pathway. If the yield of a specific pathway leading to a product is lower than the thermodynamic maximum yield, it loses quality and is likely to be improved or replaced with a more efficient pathway leading to the product.

[0096] The term "redox balanced" refers to a set of reactions that overall produce as many redox cofactors as they consume. Designing metabolic pathways and engineering organisms so that redox cofactors are balanced or close to balanced generally results in more efficient production of desired compounds and higher yields. Redox reactions always occur simultaneously because two half reactions, one oxidation reaction and the other reduction reaction, occur simultaneously. In a redox process, a reductant transfers electrons to an oxidant. Thus, in this reaction, a reductant (reductant, reducing agent) loses electrons and is oxidized, and an oxidant (oxidant, oxidizing agent) gains electrons and is reduced. In one embodiment, redox reactions occur in biological systems. Biological energy is often stored and released through redox reactions. Photosynthesis involves the reduction of carbon dioxide to sugars and the oxidation of water to molecular oxygen. The reverse reaction, respiration, oxidizes sugars to produce carbon dioxide and water. As an intermediate step, the reduced carbon compound is used to reduce nicotinamide adenine dinucleotide (NAD+), which then promotes the generation of a proton gradient that drives the synthesis of adenosine triphosphate (ATP) and is maintained by the reduction of oxygen. The term redox state is often used to describe the balance of GSH / GSSG, NAD+ / NADH, and NADP+ / NADPH in a biological system (such as a cell or organ). The redox state is reflected in the balance of several groups of metabolites (e.g., lactate and pyruvate, β-hydroxybutyrate and acetoacetate), whose interconversion depends on these ratios. Abnormal redox states can develop in various harmful situations (such as hypoxia, shock, and sepsis).

[0097] As used herein, the term "C2 pathway," "C2 branch pathway," "C2 biochemical pathway," or "C2 stream" refers to a biochemical pathway in which MEG can be produced via glycolaldehyde.

[0098] As used herein, the terms "C3 pathway," "C3 branch pathway," "C3 biochemical pathway," or "C3 stream" refer to a biochemical pathway in which MEG and / or one or more co-products (e.g., acetone, isopropanol, propylene, isobutylene, and / or serine pathway compounds) can be produced via pyruvate, acetyl-CoA, or dihydroxyacetone phosphate (DHAP).

[0099] introduction

[0100] The present disclosure combines the advantages of xylose degradation biochemistry for high yield formation of MEG (or glycolic acid) or MEG (or glycolic acid) and co-products with the advantages of easily available pure hexose raw materials. It is achieved as follows: by any of the aforementioned D-xylose-based methods, the hexose glucose is provided to be converted into an intermediate D-xylulose-5-phosphate without loss, and further converted into a pentose D-xylulose or D-ribulose, to be used as an intermediate for producing MEG (or glycolic acid) or MEG (or glycolic acid) and co-products. In addition to glucose, other hexoses (such as fructose) or hexose oligosaccharides (such as starch or sucrose or cellobiose) can also be used. In some embodiments, hexose can be selected from D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose, D-tagatose, D-sorbose, D-fructose, D-psicose and other hexoses known in the art. In some embodiments, pentose can be selected from D-xylose, D-ribose, D-arabinose, D-lyxose, D-xylulose, D-ribulose and other pentoses known in the art. In some embodiments, hexose and pentose can be selected from the left-handed or right-handed enantiomers of any hexose and pentose disclosed herein.

[0101] Compared to other glucose-based MEG or glycolic acid production methods, the present method solves the following problems: ATP shortage (if co-production is utilized); large excess of NADH; and low overall product yield potential.

[0102] Compared to other xylose-based MEG or glycolic acid production methods, the present method addresses the following challenges and issues: xylose-dependent process (availability / market restrictions, price, purity); glucose-induced inhibition of xylose utilization.

[0103] Fermentative MEG production is described in WO2010 / 076324 (or US2011 / 0294178; Metabolic Explorer), which is incorporated herein in its entirety. The application suggests the production of diols by decarboxylation and reduction of 2-ketoacids, including the production of intermediate hydroxypyruvic acid and further production of ethylene glycol based on the pathway of serine biosynthesis. However, the disclosed pathway has a reduced overall yield potential of 0.69 g_MEG / g_glucose, while the thermodynamic maximum yield of glucose→MEG conversion is 0.82 g / g. The pathway is also not redox balanced and has a high excess of NADH, with 2 mol of excess NADH corresponding to each mol of glucose consumed, requiring reoxidation of all NADH to allow cells to survive. In aerobic fermentation, this NADH can be used to generate ATP, but ATP will be in large excess (2NADH→6ATP), resulting in the formation of excessive biomass in the production stage, thereby reducing product formation and yield.

[0104] Therefore, the fermentative MEG production pathway disclosed in WO2010 / 076324 has an ATP shortage (-1 ATP per MEG), excess NADH (+1 NADH per MEG), low yield potential (ymax = 0.69 g_MEG / g_glucose), and is a challenging pathway that has not yet been demonstrated to have high efficiency / productivity.

[0105] The disclosure of WO2011 / 130378A1 (or US2011 / 0312049; Genomatica) proposes a process similar to WO2010 / 076324 for producing MEG from glucose via hydroxypyruvate, but also mentions pathway variations with alternative but related key intermediates glycerate or ethanolamine.

[0106] The disclosure of WO2011 / 130378A1 has the same disadvantages as WO2010 / 076324, except for the shortage of ATP. Each MEG can correspond to +0 ATP or +1 ATP, depending on the enzyme utilized.

[0107] In the case of glycolate, the pathway described from glucose also passes through reactions of the 3-phosphoglycerate and serine pathways, or through the glyoxylate bypass. In both cases, one CO is lost per glycolate. 2 , resulting in a maximum yield (0.84 g / g) that is well below the thermodynamic maximum yield potential (1.7 g / g).

[0108] It has been confirmed that the production of MEG (WO2013 / 126721) by xylose fermentation through ribulose-1-phosphate has a high yield potential (0.82g_MEG / g_xylose), which is equal to the thermodynamic maximum yield. It produces MEG through the two different parallel active pathways of 2-carbon stream (through glycolaldehyde) and 3-carbon stream (through dihydroxyacetone phosphate). C2 stream is easy to implement, but C3 stream is difficult to implement efficiently through metabolic engineering. C3 stream utilizes the approach shown in WO2010 / 076324 or WO2011 / 130378.

[0109] Assuming that xylose import is typically driven by ATP, the entire process is at least ATP neutral. Therefore, in order to obtain some excess ATP required for cell growth and maintenance, some xylose and therefore yield will be lost.

[0110] However, glucose is the preferred carbon source for most microorganisms, and xylose uptake is not as efficient and rapid as glucose uptake. In addition, the presence of glucose in the culture medium often inhibits the utilization of other sugars such as xylose. For a more efficient process, it is necessary to disrupt the organism regulation that leads to this preferential consumption and adapt the strain to xylose priority or sugar co-consumption.

[0111] However, the key challenge is to obtain xylose as an affordable and clean raw material. Xylose as a pure chemical is expensive and not available in large quantities. Xylose in hemicellulose hydrolysate is more abundant and may cost less than glucose, but is accompanied by many impurities and substances that inhibit fermentation.

[0112] Therefore, the production of MEG (or glycolic acid) by xylose fermentation (WO2013 / 126721) presents challenges to the use of xylose as a feedstock (availability, price, purity, inhibition of xylose utilization by glucose) and the use of the C3 pathway, which has not yet been demonstrated at high efficiency / productivity. In addition, there is an ATP shortage, +0 ATP (-1 ATP if glycerate kinase is not used), which is insufficient to sustain the cells.

[0113] Further confirmed production of MEG from xylose fermentation via xylulose-1-phosphate (Alkim et al., MicrobCell Fact (2015) 14: 127), is very similar to the pathway described in WO2013 / 126721. It has the same high yield potential (0.82 g / g), the difficulty in implementing C3 flow of MEG production via DHAP, ATP shortage and raw material challenges.

[0114] The other confirmed production of MEG (WO2013 / 119020) by xylose fermentation through xylonic acid has similarities with the approach described in WO2013 / 126721. It produces glycolaldehyde and pyruvic acid as key intermediates, to allow the production of MEG by glycolaldehyde with a yield potential of 0.41g / g. This represents a high relative yield, because its realization uses only half of the stream. However, WO2013 / 119020 or elsewhere does not introduce the approach of converting the remaining pyruvic acid into MEG. At present, there is no known realistic and effective approach to convert pyruvic acid into MEG. Although pyruvic acid itself makes the whole process redox neutral (+0NADH) as a co-product, it is not an economically interesting product, and the process will lack 1ATP (for pyruvic acid output, there may be more than ~2ATP). Therefore, ideally, a pyruvic acid-derived, economically interesting high-yield co-product is needed to deliver excess ATP. Therefore, the production of MEG from xylose via xylonic acid fermentation (WO2013 / 119020) presents challenges in terms of the use of xylose as a feedstock (availability, price, purity, inhibition of xylose utilization by glucose), low absolute yield of MEG, ATP shortage (which can range from -1 to -3 ATP with pyruvate, depending on the co-product), and the need for a pyruvate-derived co-product with high yield potential and excess ATP.

[0115] U.S. Application Nos. 62 / 305,814, 62 / 430,742, and 62 / 406,684, each of which is incorporated herein in its entirety, describe high yield and easily implemented routes for co-producing MEG and compounds such as acetone, isopropyl alcohol (IPA), propylene, or isobutylene, and provide solutions to most of the challenges described above for the MEG production process. In addition, they provide solutions to the challenges encountered when producing IPA or isobutylene from glucose as described above.

[0116] All currently known methods for producing MEG (or glycolic acid) using glucose as a feedstock have low yield potential. This is an inherent disadvantage of the biochemistry by which glucose is degraded to MEG, and for all proposed and known pathways, one decarboxylation occurs per MEG (or glycolic acid) molecule produced. However, one decarboxylation per MEG is too much to achieve redox neutrality and therefore an optimal yield.

[0117] All the MEG (or glycolic acid) production methods using xylose as a raw material have high yield potential or high relative yield potential due to the use of specific and favorable xylose degradation biochemistry, resulting in or approaching the ideal 0.5 decarboxylation reaction per MEG molecule. However, the challenge common to all these methods is the use of xylose as a raw material, such as its market limitations and technical challenges caused by raw material impurities.

[0118] The present disclosure combines the advantages of xylose degradation biochemistry for high yield formation of MEG (or glycolic acid) or optional MEG (or glycolic acid) and one or more co-products with the advantages of easily available pure hexose raw materials. It is achieved by: by any of the aforementioned D-xylose-based methods, providing lossless conversion of the hexose glucose into an intermediate D-xylulose-5-phosphate, and further converted into pentose D-xylulose or D-ribulose, to be used as an intermediate for producing MEG (or glycolic acid) or optional MEG (or glycolic acid) and one or more co-products. By oxidizing glycolaldehyde, glycolic acid (GA) can be produced instead of MEG. In addition to glucose, other hexoses (such as fructose) or hexose oligosaccharides (such as starch or sucrose) can also be used.

[0119] Compared to other glucose-based MEG (or glycolic acid) production methods, the recombinant microorganisms and methods of the present invention solve: the problem of ATP shortage (if co-production is utilized); the problem of large excess of NADH; and the problem of low overall product yield potential.

[0120] Compared to other xylose-based MEG (or glycolic acid) production methods, the recombinant microorganisms and methods of the present invention address the challenges of xylose-dependent processes (availability / market limitations, price, purity), and the problem of glucose-induced inhibition of xylose utilization.

[0121] The present disclosure relates to the production of MEG, or optionally MEG and one or more co-products, from hexoses, preferably in E. coli. Alternatively, glycolic acid (GA) may be produced instead of MEG by oxidation of glycolaldehyde.

[0122] If glucose is utilized via glycolysis (the standard degradation pathway for E. coli and most other organisms), it will be degraded via 3-phosphoglycerate (a common key intermediate for all glucose-to-MEG processes described so far). However, it is true for all described pathway variations that this 3-carbon compound is degraded to a 2-carbon compound (MEG) with a loss of one CO per MEG. 2 , where NADH is in excess and implies a significant loss in yield potential (only 0.69 g_MEG per gram of sugar, versus the thermodynamic maximum yield potential of 0.82 g_MEG).

[0123] Utilizes non-oxidative entry into the pentose phosphate pathway

[0124] In the present disclosure, glucose flux is routed to the pentose phosphate pathway rather than the glycolytic pathway ( Figure 1). Transketolase (such as that encoded by tktA or tktB from Escherichia coli) is used as a non-oxidative entry into the pentose phosphate pathway, converting the glycolytic intermediates fructose-6-phosphate and glyceraldehyde-3-phosphate to D-xylulose-5-phosphate and D-erythrose-4-phosphate ( Figure 1 ). This produces the key intermediate D-xylulose-5-phosphate.

[0125] Alternatively, fructose 6-phosphate phosphoketolase (Fpk) and phosphate acetyltransferase (PTA) can be used as an entry into the pentose phosphate pathway to produce one erythrose 4-phosphate and one acetyl-CoA ( Fig. 9 ).

[0126] Complete conversion to pentose intermediates using the pentose phosphate pathway

[0127] In order to convert all hexose carbons into pentose intermediates without loss, D-erythrose-4-phosphate needs to be further processed. Transaldolase (such as encoded by talA or talB from Escherichia coli) catalyzes the conversion of D-erythrose-4-phosphate and D-fructose-6-phosphate to generate D-sedoheptulose-7-phosphate and D-glyceraldehyde-3-phosphate. These intermediates are further processed by transketolase (such as encoded by tktA or tktB from Escherichia coli) to generate D-ribose-5-phosphate and D-xylulose-5-phosphate. D-ribose-5-phosphate can be easily converted into D-ribulose-5-phosphate by ribose-5-phosphate isomerase (such as encoded by rpiA or rpiB in Escherichia coli), respectively, and further converted into D-xylulose-5-phosphate by ribulose-5-phosphate 3-epimerase (such as encoded by rpe in Escherichia coli). Therefore, all glucose or fructose can be completely converted to D-xylulose-5-phosphate. The total stoichiometry is:

[0128] 2.5 glucose + 2.5 ATP + 0.5 phosphate → 2D-xylulose-5-phosphate + D-ribose-5-phosphate

[0129] Of course, the bi-isomerase and epimerase reactions can also convert the two D-xylulose-5-phosphate and one D-ribose-5-phosphate molecules produced into three D-ribulose-5-phosphate. The overall net conversion ultimately depends on whether the intermediate actually consumed by the subsequent pathway is D-xylulose-5-phosphate or D-ribulose-5-phosphate.

[0130] If alternatively entering the pentose phosphate pathway via Fpk ( Fig. 9 ), the stoichiometry is:

[0131] 2 glucose + 2 ATP + Coenzyme A -> 2 D-xylulose-5-phosphate + 1 acetyl Coenzyme A

[0132] Optimization of non-oxidative flux into the pentose phosphate pathway

[0133] Inactivation of the oxidative branch of the pentose phosphate pathway

[0134] The common pathway in E. coli (i.e., oxidation into the pentose phosphate pathway via 6-phosphoglucono-1,5-lactone and oxidative decarboxylation to D-ribulose-5-phosphate) should not be used because this pathway loses one carbon when converting the hexose glucose to the pentose ( Figure 1 It is advantageous to inhibit at least one or more enzymes catalyzing one or more appropriate reactions in the oxidative branch of the pentose phosphate pathway (i.e. glucose-6-phosphate-1-dehydrogenase, 6-phosphogluconolactonase and 6-phosphogluconate dehydrogenase); said inhibition is achieved by deleting one or more of the responsible genes, for example in E. coli, said genes are zwf (glucose 6-phosphate-1-dehydrogenase), pgl (6-phosphogluconolactonase) and gnd (6-phosphogluconate dehydrogenase).

[0135] Down-regulation of downstream reactions of glycolysis

[0136] The upper part of glycolysis is required to convert 2.5 glucose or fructose into the key intermediates 2x fructose-6-phosphate and 1x glyceraldehyde-3-phosphate. To reduce or eliminate further flow to the lower part of glycolysis, namely the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphospho-D-glycerate and its subsequent conversion to 3-phospho-D-glycerate and 2-phospho-D-glycerate, the activities of glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, and phosphoglycerate mutase can be reduced in E. coli by gapA, pgk, and gpmA / gpmM, respectively.

[0137] However, if alternative access to the pentose phosphate pathway via fructose 6-phosphate phosphoketolase (Fpk) is utilized, glyceraldehyde 3-phosphate is not required and the appropriate 6-phosphofructokinase activity can be reduced or deleted (pfkA and / or pfkB genes in E. coli).

[0138] Connecting pentose-phosphate intermediates to the MEG production pathway

[0139] In order to connect the pentose phosphate pathway intermediate D-xylulose-5-phosphate to any of the known MEG or glycolate production pathways, the D-xylulose-5-phosphate intermediate needs to be dephosphorylated by pentose 5-phosphatase to produce D-xylulose ( Figure 2 and Figure 5 Similarly, the D-ribulose-5-phosphate intermediate can be linked to any of the known MEG or glycolate production pathways by dephosphorylation by pentose 5-phosphatase to produce D-ribulose ( Figure 4 and Figure 5 ).

[0140] In the case of the xylonic acid-based MEG pathway, a xylose isomerase function (such as XylA from E. coli) is further required to convert the generated D-xylulose into D-xylose ( Figure 3 ).

[0141] Alternatives and variations for linking the pentose phosphate pathway to the MEG production pathway

[0142] In the case of a D-ribulose-1-phosphate based pathway, as described, a D-xylulose-5-phosphate intermediate can be linked to the pentose phosphate pathway via the formation of D-xylulose, followed by epimerization and phosphorylation (mediated by dte and fucK) ( Figure 4 Alternatively, D-ribulose-5-phosphate can be used as an entry point and degraded to D-ribulose by utilizing D-ribulose 5-phosphatase. In this way, the necessary isomerization reaction is performed at the level of the RPE, rather than DTE ( Figure 4 ).

[0143] In some embodiments, two consecutive arabitol dehydrogenase reactions can be used to convert pentose 5-phosphate to pentose 1-phosphate ( Figure 6 Here, the 2-keto position of D-xylulose-5-phosphate or D-ribulose-5-phosphate is reduced, and then the 4-hydroxyl position is oxidized to the keto group, converting pentose-5-phosphate into pentose-1-phosphate ( Figure 7 ).

[0144] In a further embodiment, pentose-5-phosphate can be directly converted to pentose-1-phosphate by the action of pentose phosphate mutase (also known as phosphopentose mutase or PPM). Similar to phosphoglucomutase or phosphomannose mutase, it transfers the phosphate residue from the last position to the 1-position of the phosphate sugar.

[0145] Utilization of hexose

[0146] The molecules that enter the pathway in the present disclosure are fructose-6-phosphate and glyceraldehyde-3-phosphate, both of which are obtained in most organisms by normal glycolytic degradation of glucose or fructose. If the organism has the ability to consume starch or sucrose or cellulose (e.g., through the expression of a sucrose invertase), it will still generate glucose (and fructose), allowing it to produce the same benefits using the methods of the present disclosure in the same manner and to the same extent.

[0147] Monoethylene glycol (MEG)

[0148] Monoethylene glycol (MEG) is an important raw material for industrial applications. The main use of MEG is in the manufacture of polyethylene terephthalate (PET) resins, films, and fibers. In addition, MEG is also important in the production of antifreeze, coolants, aircraft anti-icing agents, de-icing agents, and solvents. MEG is also known as ethylene-1,2-diol.

[0149] Ethylene glycol is also used as a medium for convective heat transfer, such as in automobiles and liquid-cooled computers.

[0150] Ethylene glycol is a useful desiccant due to its high boiling point and affinity for water. Ethylene glycol is widely used to inhibit the formation of natural gas clathrates (hydrates) in long multiphase pipelines that transport natural gas from remote gas fields to natural gas processing facilities. Ethylene glycol can be recovered from natural gas and reused as an inhibitor after purification treatment to remove water and inorganic salts.

[0151] Minor uses of ethylene glycol include: use in capacitor manufacturing, as a chemical intermediate in the manufacture of 1,4-dioxane, and as an additive to prevent corrosion in personal computer liquid cooling systems. Ethylene glycol is also used in the manufacture of some vaccines; as a minor component in shoe polish, inks, and dyes; for treating wood decay and fungi; and as a preservative for biological specimens.

[0152] Glycolic acid

[0153] Glycolic acid is used as a dye and tanning agent in the textile industry, as a flavoring agent and preservative in food processing, and as a skin care agent in the pharmaceutical industry. It is also used in adhesives and plastics. Glycolic acid is often included in emulsion polymers, solvents, and additives for inks and coatings to improve flow properties and impart gloss. It is used in surface treatment products that increase the coefficient of friction of tile floors.

[0154] Due to its excellent ability to penetrate the skin, glycolic acid is used in skin care products to improve the appearance and texture of the skin. It can be used as a chemical peel performed by a dermatologist at a concentration of 20% to 70%, or in a lower concentration between 10% and 20% for at-home kits. In addition to concentration, pH also plays a large role in determining the effectiveness of glycolic acid in solution.

[0155] Glycolic acid can be synthesized in a variety of ways. The primary method uses the catalytic reaction of formaldehyde with synthesis gas (carbonylation of formaldehyde) because of its low cost. Glycolic acid can also be made by reacting chloroacetic acid with sodium hydroxide followed by re-acidification. Other methods not apparently in use include the hydrogenation of oxalic acid and the hydrolysis of cyanohydrins derived from formaldehyde. Some glycolic acid today does not contain formic acid. Glycolic acid can be isolated from natural sources such as sugar cane, sugar beets, pineapple, cantaloupe, and unripe grapes.

[0156] Glycolic acid is an organic synthesis intermediate that can be used in a range of reactions, including redox, esterification, and long-chain polymerization. It is used as a monomer for the preparation of polyglycolic acid and other biocompatible copolymers (e.g., PLGA). Commercially, important derivatives include the methyl (CAS#96-35-5) and ethyl (CAS#623-50-7) esters that are easily distilled. The butyl ester is a component of some paints and is popular because it is nonvolatile and has good solubility properties.

[0157] acetone

[0158] Acetone (also called propanone) is a chemical with the molecular formula (CH3) 2 An organic compound of CO. It is a colorless, volatile, flammable liquid and the simplest ketone.

[0159] Acetone is miscible with water and is an important solvent commonly used in laboratories for cleaning purposes. Global production exceeds 6.7 million tons, primarily used as a solvent and in the production of methyl methacrylate and bisphenol A. It is a common building block in organic chemistry. Common household uses of acetone are as an active ingredient in nail polish remover and as a paint thinner.

[0160] Isopropyl alcohol

[0161] Isopropyl alcohol (IUPAC name is 2-propanol, also known as isopropanol) is a 3 H 8 O or C 3 H 7 OH or CH 3 CHOHCH 3 It is a colorless, flammable compound with a strong odor. It is the simplest example of a secondary alcohol, in which the alcohol carbon atom is attached to two other carbon atoms, sometimes shown as (CH3) 2 CHOH. It is a structural isomer of propanol. It has a wide range of industrial and household uses.

[0162] Propylene (propene, propylene), also known as methylethylene, is a 3 H 6 An unsaturated organic compound. It has one double bond and is the second simplest member of the olefin class of hydrocarbons.

[0163] Propylene is produced from fossil fuels (petroleum, natural gas, and to a much lesser extent coal). Propylene is a by-product of oil refining and natural gas processing.

[0164] Isobutylene

[0165] Isobutene (isobutylene, also known as 2-methylpropylene) is an industrially significant hydrocarbon. It is a four-carbon branched alkene (alkene, olefin) and one of the four isomers of butene (butylene, butene). It is a colorless, flammable gas at standard temperature and pressure.

[0166] Isobutylene is used as an intermediate in the production of various products. It reacts with methanol and ethanol in the manufacture of the gasoline oxygenates methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE), respectively. Alkylation with butane produces isooctane, another fuel additive. Isobutylene is also used in the production of methacrolein. Polymerization of isobutylene produces butyl rubber (polyisobutylene). Antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are produced by Friedel-Crafts alkylation of phenol with isobutylene.

[0167] Polymer and chemical grade isobutylene is usually obtained by dehydration of tert-butyl alcohol or catalytic dehydrogenation of isobutane. Gasoline oxygenates MTBE and ETBE are usually produced by reacting methanol or ethanol with isobutylene contained in a butene stream from an olefin steam cracker or refinery. Isobutylene is not separated before the reaction because it is simpler to separate the ether from the remaining butenes.

[0168] Serine pathway compounds

[0169] Compounds that can be co-produced with MEG (or glycolic acid) include serine pathway compounds such as serine, glycine, monoethanolamine (MEA), and ethylenediamine (EDA).

[0170] Serine is a non-essential amino acid that can be synthesized in the human body. Due to its high water solubility, serine is used as a moisturizer in emulsions in the pharmaceutical and cosmetic industries. In addition, serine has a huge market in the chemical industry because it can be converted into other chemicals such as plastics, detergents, dietary supplements, and various other products. In fact, serine has been mentioned as one of the 30 most promising biosubstances to replace chemicals in the petroleum industry.

[0171] α-Decarboxylation of serine produces ethanolamine, which is an industrial product used as an intermediate in the herbicide, textile, metal, detergent, plastic and personal care product industries, with a production volume of up to hundreds of thousands of tons per year (Scott, E. et al. (2007) Biomass in the manufacture of industrial products—the use of proteins and amino acids. Appl Microbiol Biotechnol. 75(4): 751-762).

[0172] Glycine is the simplest amino acid and is valuable for pharmaceutical and industrial applications. It is included in pet food and animal feed as an additive. For humans, glycine is sold as a sweetener / flavor enhancer. Some food supplements and protein drinks contain glycine. Some pharmaceutical preparations contain glycine to improve gastric absorption of drugs. Glycine is used as a buffering agent in antacids, analgesics, antiperspirants, cosmetics, and toiletries. Many miscellaneous products use glycine or its derivatives, such as the production of rubber sponge products, fertilizers, and metal complexing agents. Glycine is also valuable as an intermediate in the synthesis of various chemical products. It is used to make the herbicide glyphosate. Glycine can be converted to oxalic acid, which is used as a bleaching agent in the textile and pulp industries and in wastewater treatment. Glycine is also widely used in laboratory research, such as in gel electrophoresis.

[0173] Ethylenediamine (EDA) (1,2-diaminoethane, C 2 H 4 (NH 2 ) 2) is used in large quantities to produce many industrial chemicals. It forms derivatives with carboxylic acids (including fatty acids), nitriles, alcohols (at elevated temperatures), alkylating agents, carbon disulfide, and aldehydes and ketones. Due to its bifunctional nature with two amines, it readily forms heterocycles such as imidazolidinones. The most prominent derivative of ethylenediamine is the chelating agent EDTA, which is derived from ethylenediamine by the Strecker synthesis involving cyanide and formaldehyde. Hydroxyethylethylenediamine is another commercially important chelating agent. Many biologically active compounds and drugs contain N-CH2-CH2-N bonds, including some antihistamines. Ethylenebisdithiocarbamates are commercially important fungicides with the trade names maneb, mancozeb, zineb, and metiram. Some imidazoline-containing fungicides are derived from ethylenediamine. Ethylenediamine is a component of the common bronchodilator aminophylline, which is used to dissolve the active ingredient theophylline. Ethylenediamine has also been used in dermatological preparations. When used as a pharmaceutical excipient, its bioavailability after oral administration is about 0.34 due to a significant first-pass effect. Less than 20% is eliminated through urinary excretion. Since ethylenediamine contains two amine groups, it is a precursor of various widely used polymers. Condensates derived from formaldehyde are plasticizers. It is widely used in the production of polyurethane fibers. PAMAM-type dendrimers are derived from ethylenediamine. The bleach activator tetraacetylethylenediamine is generated from ethylenediamine. The derivative N,N-ethylenebis(stearamide) (EBS) is a commercially significant release agent, as well as a surfactant in gasoline and motor oil.

[0174] Ethylenediamine is also used as a solvent for proteins such as albumin and casein; in certain electroplating baths; as a corrosion inhibitor in paints and coolants; as a chemical for color photographic development, binders, adhesives, fabric softeners, epoxy resin hardeners, and dyes. Ethylenediamine dihydroiodide (EDDI) is added to animal feed as a source of iodide.

[0175] Xylitol

[0176] Xylitol is a chemical compound, a sugar alcohol, which has considerable value as a sweetener. The human palate detects its sweetness as being similar to that of sucrose, and it is non-toxic and non-cariogenic.

[0177] One method of producing xylitol is to use xylan (a hemicellulose) extracted from hardwood and corn cobs. Xylan can be hydrolyzed to xylose, which is then catalytically hydrogenated to xylitol. The catalytic pathway for xylitol is cost and energy intensive because a large number of separation and purification steps are used in the process, and the overall yield is low. Another method of producing xylitol includes using fermentation and biocatalytic processes in bacteria, fungi and / or yeast cells.

[0178] Enzymes

[0179] Exemplary enzymes are listed in Table 1 that can be used in the biosynthetic pathways of MEG (or glycolic acid) or optionally MEG (or glycolic acid) and one or more co-products disclosed herein.

[0180] Table 1

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] D-Tagatose 3-epimerase (EC 5.1.3.31)

[0195] The present disclosure describes enzymes that can catalyze the following reactions: epimerization of various ketoses at the C-3 position, interconversion of D-fructose and D-psicose, D-tagatose and D-sorbose, D-ribulose and D-xylulose, and L-ribulose and L-xylulose. The specificity depends on the species. The enzymes from Pseudomonas cichorii and Rhodobacter sphaeroides require Mn 2+ In one embodiment, the enzyme is D-tagatose 3-epimerase (DTE). In another embodiment, D-tagatose 3-epimerase catalyzes the conversion of D-xylulose to D-ribulose.

[0196]

[0197] In one embodiment, the D-tagatose 3-epimerase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from Pseudomonas sp., Mesorhizobium sp., and Rhodobacter sp. In some embodiments, the D-tagatose 3-epimerase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from Pseudomonas cichorii, Pseudomonas sp. ST-24, Mesorhizobium loti, and Rhodobacter sphaeroides. In some embodiments, the one or more nucleic acid molecules are dte and / or FJ851309.1, or homologs thereof. In a further embodiment, the D-tagatose 3-epimerase comprises an amino acid sequence selected from SEQ ID NOs: 3 and 5. In a still further embodiment, the D-tagatose 3-epimerase is encoded by a nucleic acid sequence selected from SEQ ID NOs: 1, 2, and 4.

[0198] D-tagatose 3-epimerase may also be referred to as L-ribulose 3-epimerase or ketose 3-epimerase.

[0199] D-Ribulokinase (EC 2.7.1.16)

[0200] The present disclosure describes enzymes that can catalyze the following reactions:

[0201] L-fucose + ATP → L-fucose 1-phosphate + ADP + H+

[0202] D-ribulose + ATP → D-ribulose 1-phosphate + ADP + H+

[0203] D-Ribulose kinase may also be referred to as L-fuculokinase, fucokinase, ATP:L-fucose 1-phosphotransferase or L-fuculose kinase.

[0204] Thus, in some embodiments, the present disclosure provides enzymes that function in the fucose degradation pathway, the super pathway of fucose and rhamnose degradation, and / or the D-arabinose degradation I pathway.

[0205] In some embodiments, the enzyme can function as both an L-fucokinase and a D-ribulosekinase, the second enzyme of the L-fucose and D-arabinose degradation pathways, respectively.

[0206] In a specific embodiment, the enzyme converts D-ribulose to D-ribulose-1-phosphate. In one embodiment, the D-ribulose kinase is encoded by one or more nucleic acid molecules obtained from Escherichia coli. In some embodiments, the one or more nucleic acid molecules are fucK or a homologue thereof. In a further embodiment, the D-ribulose kinase comprises the amino acid sequence set forth in SEQ ID NO: 8. In a still further embodiment, the D-ribulose kinase is encoded by a nucleic acid sequence selected from SEQ ID NO: 6 and 7.

[0207] D-Ribulose-1-phosphate aldolase (EC 4.1.2.17)

[0208] The present disclosure describes enzymes that can catalyze the following reversible reaction:

[0209]

[0210]

[0211] D-ribulose-1-phosphate aldolase may also be referred to as L-fucose-phosphate aldolase, L-fucose 1-phosphate aldolase, or L-fucose-1-phosphate (S)-lactaldehyde-lyase.

[0212] Thus, in some embodiments, the present disclosure provides enzymes that function in the fucose degradation pathway, the super pathway of fucose and rhamnose degradation, and / or the D-arabinose degradation pathway I. In one embodiment, the enzyme can be used with Zn 2+ As a cofactor. In another embodiment, the inhibitor of the enzyme may be phosphoglycohydroxamic acid.

[0213] In some embodiments, the enzyme can function as both an L-fucose-phosphate aldolase and a D-ribulose-phosphate aldolase, the third enzyme of the L-fucose and D-arabinose degradation pathways, respectively.

[0214] The substrate specificity of the enzyme has been tested using partially purified preparations from E. coli strains.

[0215] The crystal structure of the enzyme and a number of point mutants have been solved. The combination of structural data and enzyme activity of the mutants allowed modeling and refinement of the catalytic mechanism of the enzyme. The enantioselectivity of the enzyme has been studied.

[0216] In a specific embodiment, the enzyme converts D-ribulose-1-phosphate into glycolaldehyde and DHAP. In one embodiment, the D-ribulose-1-phosphate aldolase is encoded by one or more nucleic acid molecules obtained from Escherichia coli. In some embodiments, the one or more nucleic acid molecules are fucA or a homolog thereof. In a further embodiment, the D-ribulose-1-phosphate aldolase comprises the amino acid sequence set forth in SEQ ID NO: 11. In a still further embodiment, the D-ribulose-1-phosphate aldolase is encoded by a nucleic acid sequence selected from SEQ ID NO: 9 and 10.

[0217] Glycolaldehyde reductase (EC 1.1.1.77)

[0218] The present disclosure describes enzymes that can catalyze the following reversible reaction:

[0219]

[0220]

[0221] Glycolaldehyde reductase may also be referred to as lactaldehyde reductase, propanediol oxidoreductase, (R)[or (S)]-propane-1,2-diol:NAD+ oxidoreductase or L-1,2-propanediol oxidoreductase.

[0222] Thus, in some embodiments, the disclosure provides enzymes that function in an ethylene glycol degradation pathway, a superpathway for ethylene glycol metabolism and degradation, an anaerobic L-lactaldehyde degradation pathway, and / or a superpathway for fucose and rhamnose degradation. In one embodiment, the enzyme may be used with Fe 2+ As a cofactor.

[0223] L-1,2-propanediol oxidoreductase is an iron-dependent group III dehydrogenase that anaerobically reduces L-lactaldehyde (a product of the L-fucose and L-rhamnose catabolic pathway) to L-1,2-propanediol, which is then excreted from the cell.

[0224] The crystal structure of the enzyme has been solved, showing a domain-swapped dimer in which the metal, cofactor, and substrate binding sites may be located. An aspartate and three conserved histidine residues are required to realize Fe 2+ binding and enzymatic activity.

[0225] In vitro, high concentrations of NAD+ can reactivate the enzyme, Fe 3+ and ascorbic acid or Fe 2+ and H 2 O 2 The enzyme was effectively inactivated by a mixture of 2,4-dihydro-1,4-dihydro-2-nitropropene and 1,4-dihydro-1,4-dihydro-2-nitropropene. Metal-catalyzed oxidation of the conserved His277 residue was thought to be responsible for the inactivation.

[0226] Expression of FucO enabled one-turn reversal of the engineered β-oxidation cycle. FucO activity facilitated the conversion of isobutyraldehyde to isobutanol in the engineered strain.

[0227] In specific embodiments, the enzyme converts glycolaldehyde to MEG. In some embodiments, the glycolaldehyde reductase is from Escherichia coli. In some embodiments, the glycolaldehyde reductase is encoded by the fucO gene.

[0228] In one embodiment, the glycolaldehyde reductase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from Escherichia coli and Saccharomyces cerevisiae. In another embodiment, the one or more nucleic acid molecules are selected from gldA, GRE2, GRE3, yqhD, ydjG, fucO, yafB (dkgB) and / or yqhE (dkgA), or homologs thereof. In another embodiment, the one or more nucleic acid molecules are yqhD. In some embodiments, yqhD comprises a G149E mutation. In a further embodiment, the glycolaldehyde reductase comprises an amino acid sequence selected from SEQ ID NO: 13, 15, 17, 20, 23, 25, 28, 30 and 32. In a still further embodiment, the glycolaldehyde reductase is encoded by a nucleic acid sequence selected from SEQ ID NO: 12, 14, 16, 18, 19, 21, 22, 24, 26, 27, 29 and 31.

[0229] Aldehyde reductase

[0230] A number of aldehyde reductases can be used to convert glycolaldehyde to MEG.

[0231] NADPH-dependent aldehyde reductase (YqhD) can catalyze the following reactions:

[0232]

[0233]

[0234] Aldehyde+NADP++H 2 O→Carboxylate+NADPH+2H+(EC 1.2.1.4)

[0235]

[0236]

[0237] YqhD is a NADPH-dependent aldehyde reductase that may be involved in glyoxal detoxification and / or be part of a glutathione-independent response to lipid peroxidation.

[0238] It has been reported that various alcohols, aldehydes, amino acids, sugars and α-hydroxy acids have been tested as substrates for YqhD. The purified protein showed only NADP-dependent alcohol dehydrogenase activity, with a preference for alcohols longer than C(3), but with a Km value in the millimolar range, indicating that it is not a physiological substrate. In contrast, YqhD does exhibit short-chain aldehyde reductase activity on substrates such as propionaldehyde, acetaldehyde and butyraldehyde, as well as acrolein and malondialdehyde. In metabolically engineered strains, endogenous aldehyde reductases YqhD, YjgB and YahK reduce phenylacetaldehyde and 4-hydroxyphenylacetaldehyde to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol.

[0239] Overexpression of YqhD increases the cellular 1,3-propanediol oxidoreductase activity. E. coli has been engineered to express YqhD for industrial production of 1,3-propanediol. YqhD activity also contributes to the production of isobutanol, 1,2-propanediol, 1,2,4-butanetriol, and acetol. Mutation of yqhD enables the production of butanol via a single-turn reversal of the engineered β-oxidation cycle.

[0240] YqhD possesses furfural reductase activity, which was shown to cause growth inhibition due to NADPH depletion in metabolically engineered strains producing ethanol from lignocellulosic biomass.

[0241] Has been The crystal structure of YqhD was solved at a resolution of 1.3 Å. YqhD is an asymmetric dimer of dimers with Zn 2+ ions. The NADPH cofactor is modified with hydroxyl groups at positions 5 and 6 in the nicotinamide ring.

[0242] Overexpression of yqhD resulted in increased resistance to reactive oxygen-generating compounds such as hydrogen peroxide, paraquat, chromate, and potassium tellurite. yqhD deletion mutants showed increased sensitivity to these compounds and glyoxal and contained increased levels of reactive aldehydes generated during lipid peroxidation. Conversely, yqhD deletion resulted in increased tolerance to furfural.

[0243] In specific embodiments, the NADPH-dependent aldehyde reductase converts glycolaldehyde to MEG. In some embodiments, the NADPH-dependent aldehyde reductase is from Escherichia coli. In some embodiments, the NADPH-dependent aldehyde reductase is encoded by the yqhD gene.

[0244] The multifunctional methylglyoxal reductase (DkgA) can catalyze the following reactions:

[0245]

[0246]

[0247]

[0248]

[0249] 2-Keto-L-gulonic acid + NADP + ← 2,5-didehydro-D-gluconic acid + NADPH + H + (this reaction favors the opposite direction, EC 1.1.1.346)

[0250] DkgA (YqhE) belongs to the aldehyde-keto reductase (AKR) family and has been shown to possess methylglyoxal reductase and β-ketoester reductase activities.

[0251] dkgA is reported to encode 2,5-diketo-D-gluconate reductase (25DKGR)A, one of the two 25DKG reductases in Escherichia coli. The enzyme uses NADPH as a preferred electron donor and is thought to be involved in ketogluconate metabolism. The specific activity of the enzyme toward 2,5-diketo-D-gluconate is reported to be nearly 1000-fold lower than its activity toward methylglyoxal.

[0252] Due to its low Km for NADPH, furan reduction by DkgA may deplete the NADPH pool, thereby limiting cellular biosynthesis. An extensive survey of aldehyde reductases revealed that DkgA is one of several endogenous aldehyde reductases that contribute to the degradation of aldehyde end products required for metabolic engineering.

[0253] Has been The crystal structure of DkgA was solved at a resolution of 1.

[0254] In specific embodiments, the multifunctional methylglyoxal reductase converts glycolaldehyde to MEG. In some embodiments, the multifunctional methylglyoxal reductase is from Escherichia coli. In some embodiments, the multifunctional methylglyoxal reductase is encoded by the dkgA gene.

[0255] The multifunctional methylglyoxal reductase (DkgB) can catalyze the following reactions:

[0256]

[0257]

[0258] 2-Keto-L-gulonic acid + NADP + ← 2,5-didehydro-D-gluconic acid + NADPH + H + (this reaction favors the opposite direction, EC 1.1.1.346)

[0259] DkgB (YafB) is a member of the aldehyde-keto reductase (AKR) subfamily 3 F. DkgB has been shown to have 2,5-diketo-D-gluconate reductase, methylglyoxal reductase, and 4-nitrobenzaldehyde reductase activities.

[0260] dkgB is reported to encode 2,5-diketo-D-gluconate reductase (25DKGR) B, one of the two 25DKG reductases in Escherichia coli. The enzyme uses NADPH as a preferred electron donor and is thought to be involved in ketogluconate metabolism. However, the specific activity of the enzyme toward 2,5-diketo-D-gluconate was reported to be nearly 1000-fold lower than its activity toward methylglyoxal.

[0261] In specific embodiments, the multifunctional methylglyoxal reductase converts glycolaldehyde to MEG. In some embodiments, the multifunctional methylglyoxal reductase is from Escherichia coli. In some embodiments, the multifunctional methylglyoxal reductase is encoded by the dkgB gene.

[0262] Methylglyoxal reductase (YeaE) can catalyze the following reaction:

[0263]

[0264] YeaE has been shown to have methylglyoxal reductase activity.

[0265] The subunit structure of YeaE has not been determined, but the similarity of its amino acid sequence to the aldehyde-keto reductases DkgA (YqhE) and DkgB (YafB) suggests that it may be monomeric.

[0266] In specific embodiments, the methylglyoxal reductase converts glycolaldehyde to MEG. In some embodiments, the methylglyoxal reductase is from Escherichia coli. In some embodiments, the methylglyoxal reductase is encoded by the yeaE gene.

[0267] L-Glyceraldehyde 3-phosphate reductase (yghZ) can catalyze the following reactions:

[0268] L-glyceraldehyde 3-phosphate + NADPH + H+ → sn-glycerol 3-phosphate + NADP+ (EC 1.1.1.-)

[0269]

[0270] YghZ is an L-glyceraldehyde 3-phosphate (L-GAP) reductase. The enzyme is also able to detoxify methylglyoxal at a low rate. YghZ defines the AKR14 (aldoketone reductase 14) protein family.

[0271] L-GAP is not a natural metabolite and is toxic to E. coli. L-GAP is a substrate for the glycerol-3-phosphate and hexose phosphate transport systems of E. coli K-12. It is speculated that the physiological role of YghZ is the detoxification of L-GAP, which may be formed by non-enzymatic racemization of GAP or by unknown cellular processes.

[0272] The crystal structure of the E. coli enzyme has been determined and is thought to be a tetramer. However, others have found, based on gel filtration and electron microscopy studies, that the protein forms an octamer.

[0273] In specific embodiments, L-glyceraldehyde 3-phosphate reductase converts glycolaldehyde into MEG. In some embodiments, L-glyceraldehyde 3-phosphate reductase is from Escherichia coli. In some embodiments, L-glyceraldehyde 3-phosphate reductase is encoded by yghZ gene.

[0274] L-1,2-propanediol dehydrogenase / glycerol dehydrogenase (GldA) can catalyze the following reactions:

[0275]

[0276] Aminoacetone + NADH + H + → (R)-1-aminopropan-2-ol + NAD + (EC 1.1.1.75)

[0277]

[0278] The physiological function of the GldA enzyme has long been unclear. The enzyme was isolated independently as a glycerol dehydrogenase and a D-1-amino-2-propanol:NAD+ oxidoreductase. At the time, D-1-amino-2-propanol was considered to be an intermediate in the biosynthesis of vitamin B12, and although E. coli is not able to synthesize vitamin B12 de novo, enzymes that catalyze the synthesis of this compound have been sought. Later, it was discovered that GldA is responsible for both activities.

[0279] It has recently been proposed that the primary in vivo role of GldA is to remove dihydroxyacetone by converting it to glycerol. However, a dual role in glycerol fermentation has also recently been established. Glycerol dissimilatory degradation in E. coli can be accomplished via two distinct pathways. The glycerol and glycerophosphodiester degradation pathways require the presence of a terminal electron acceptor and utilize the ATP-dependent kinase of the Glp system, which phosphorylates glycerol to glycerol-3-phosphate. However, after inactivating the kinase and selecting for growth on glycerol, the NAD+-linked dehydrogenase GldA was found to be able to support glycerol fermentation. GldA has recently been shown to participate in glycerol fermentation both as a glycerol dehydrogenase, producing dihydroxyacetone, and as a 1,2-propanediol dehydrogenase, regenerating NAD+ by producing 1,2-propanediol from acetone.

[0280] The enzyme exists in two catalytically active forms, a large form of eight subunits and a small form of two subunits. The large form represents the major species.

[0281] In specific embodiments, the L-1,2-propanediol dehydrogenase / glycerol dehydrogenase converts glycolaldehyde to MEG. In some embodiments, the L-1,2-propanediol dehydrogenase / glycerol dehydrogenase is from E. coli. In some embodiments, the L-1,2-propanediol dehydrogenase / glycerol dehydrogenase is encoded by the gldA gene.

[0282] NADPH-dependent methylglyoxal reductase (GRE2) from Saccharomyces cerevisiae catalyzes the following reaction:

[0283]

[0284]

[0285] Gre2 is a multifunctional enzyme that catalyzes the stereoselective reduction of a wide range of substrates including aliphatic and aromatic ketones, diketones, and aldehydes using NADPH as a cofactor.

[0286] Has been The crystal structure of Gre2 in its apo form from Saccharomyces cerevisiae was solved at a resolution of The crystal structure of Gre2 from Saccharomyces cerevisiae in complex with NADPH was solved at a resolution of 1.3 Å. Gre2 forms a homodimer, each subunit of which contains an N-terminal Rossmann-fold domain involved in substrate recognition and a variable C-terminal domain. Binding to the cofactor NADPH induces a fit that moves the two domains toward each other, creating an interdomain cleft that is more suitable for the substrate. Computational simulations combined with site-directed mutagenesis and enzyme activity analysis allowed the characterization of a potential substrate binding pocket that determines the strict substrate stereoselectivity of the catalysis.

[0287] Gre2 catalyzes the irreversible reduction of the cytotoxic compound methylglyoxal (MG) to (S)-lactaldehyde as an alternative to the detoxification of MG by the glyoxalase I GLO1. MG is synthesized from dihydroxyacetone phosphate via a glycolytic bypass and is believed to play a role in cell cycle regulation and stress adaptation. GRE2 also catalyzes the reduction of isovaleraldehyde to isopentanol. The enzyme inhibits isopentanol-induced filamentation by regulating the levels of isovaleraldehyde, and cells respond to the isovaleraldehyde signal by filamentation. GRE2 is also involved in ergosterol metabolism.

[0288] In specific embodiments, the NADPH-dependent methylglyoxal reductase converts glycolaldehyde to MEG. In some embodiments, the NADPH-dependent methylglyoxal reductase is from Saccharomyces cerevisiae. In some embodiments, the NADPH-dependent methylglyoxal reductase is encoded by the GRE2 gene.

[0289] Thiolase / Acetyl-CoA Acetyltransferase (EC 2.3.1.9)

[0290] The present disclosure describes enzymes that can catalyze the following reactions:

[0291]

[0292] Thiolase / acetyl-CoA acetyltransferase may also be referred to as acetyl-CoA AC-acetyltransferase, acetoacetyl-CoA thiolase, acetyl-CoA:acetyl-CoA C-acetyltransferase, or thiolase II.

[0293] Thus, in some embodiments, the present disclosure provides enzymes that play a role in the degradation of acetoacetate (to acetyl-CoA). In one embodiment, the inhibitor of the enzyme may be acetoacetyl-CoA.

[0294] In specific embodiments, the enzyme converts acetyl-CoA to acetoacetyl-CoA. In one embodiment, the thiolase or acetyl-CoA acetyltransferase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from Clostridium species, Bacillus species, Escherichia coli, Saccharomyces species, and Marinobacter species (Marinobacter sp.). In some embodiments, the thiolase or acetyl-CoA acetyltransferase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from Clostridium acetobutylicum, Clostridium thermosaccharolyticum, Bacillus cereus, Escherichia coli, Saccharomyces cerevisiae, and Marinobacter hydrocarbonoclasticus. In some embodiments, the one or more nucleic acid molecules are thlA, atoB, and / or ERG10, or homologs thereof. In further embodiments, the thiolase or acetyl-CoA acetyltransferase comprises an amino acid sequence selected from SEQ ID NOs: 35, 37, and 40. In a still further embodiment, the thiolase or acetyl-CoA acetyltransferase is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 33, 34, 36, 38 and 39.

[0295] Acetyl-CoA:acetoacetyl-CoA transferase (EC 2.8.3.-)

[0296] The present disclosure describes enzymes that can catalyze the following reactions:

[0297]

[0298] Acetyl-CoA:acetoacetyl-CoA transferase may also be referred to as acetate:acetoacetyl-CoA transferase or acetoacetyl-CoA transferase.

[0299] Thus, in some embodiments, the present disclosure provides enzymes that play a role in the degradation of acetoacetate (to acetyl-CoA). In one embodiment, inhibitors of the enzyme may include acetyl-CoA and CoA.

[0300] E. coli grows on short-chain fatty acids (C3-C6) and needs to activate these acids into their respective thioesters. Acetoacetyl-CoA transferase catalyzes this activation. The reaction occurs in two half-reactions involving a covalent enzyme-CoA. The enzyme undergoes two detectable conformational changes during the reaction. It is believed that the reaction may proceed via a ping-pong mechanism. The enzyme can utilize a variety of short-chain acyl-CoA and carboxylic acid substrates, but exhibits maximum activity on normal and 3-keto substrates.

[0301] In specific embodiments, the enzyme converts acetoacetyl-CoA to acetoacetate. In some embodiments, the acetyl-CoA:acetoacetate-CoA transferase is from Clostridium. In some embodiments, the acetyl-CoA:acetoacetate-CoA transferase is from Clostridium acetobutylicum. In some embodiments, the acetyl-CoA:acetoacetate-CoA transferase is from Escherichia coli. In some embodiments, the acetyl-CoA:acetoacetate-CoA transferase is encoded by the atoA and atoD genes. In another embodiment, the subunit composition of the acetoacetyl-CoA transferase is [(AtoA) 2 ][(AtoD) 2 ], where (AtoA) 2 It is a β complex, (AtoD) 2 is an alpha complex. In one embodiment, the acetyl-CoA:acetoacetate-CoA transferase is a fused acetyl-CoA:acetoacetate-CoA transferase: alpha subunit / beta subunit. In another embodiment, the acetyl-CoA:acetoacetate-CoA transferase is encoded by the ydiF gene.

[0302] Acetate: acetoacetyl-CoA hydrolase (EC 3.1.2.11)

[0303] The present disclosure describes enzymes that can catalyze the following reactions:

[0304]

[0305] Acetoacetyl-CoA hydrolase may also be referred to as acetoacetyl coenzyme A hydrolase or acetoacetyl CoAdeacylase or acetoacetyl coenzyme A deacylase.

[0306] This enzyme belongs to the family of hydrolases, specifically those acting on thioester bonds.

[0307] In specific embodiments, the enzyme converts acetoacetyl-CoA to acetoacetate. In some embodiments, the acetate:acetoacetyl-CoA hydrolase is from Clostridium. In some embodiments, the acetate:acetoacetyl-CoA hydrolase is from Clostridium acetobutylicum. In another embodiment, the acetoacetyl-CoA hydrolase is encoded by the ctfA (subunit A) and / or ctfB (subunit B) genes.

[0308] In a further embodiment, the acetyl-CoA:acetoacetate-CoA transferase or acetate:acetoacetyl-CoA hydrolase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 46, 97, 99, 101, and 103. In a still further embodiment, the acetyl-CoA:acetoacetate-CoA transferase or acetate:acetoacetyl-CoA hydrolase is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 41, 42, 44, 45, 96, 98, 100, and 102.

[0309] Acetoacetate decarboxylase (EC 4.1.1.4)

[0310] The present disclosure describes enzymes that can catalyze the following reactions:

[0311] Acetoacetate + H+ → Acetone + CO 2

[0312] Acetoacetate decarboxylase may also be referred to as ADC, AADC or acetoacetate carboxyl-lyase.

[0313] Thus, in some embodiments, the present disclosure provides enzymes that function in isopropanol biosynthesis, pyruvate fermentation to acetone, the C. acetobutylicum acidogenic and solventogenic fermentation superpathway, and / or the C. acetobutylicum solventogenic fermentation superpathway.

[0314] Acetoacetate decarboxylase (ADC) plays a key role in solvent production in Clostridium acetobutylicum. During the acidogenic phase of growth, acid accumulation leads to a metabolic shift toward solvent production. In this phase, acid is reabsorbed and metabolized to produce acetone, butanol, and ethanol.

[0315] Initial purification and crystallization of the enzyme revealed the involvement of a lysine residue in the active site. The enzyme is a large complex composed of 12 copies of a single type of subunit.

[0316] The enzyme has been purified from Clostridium acetobutylicum ATCC 824 and the adc gene encoding it has been cloned. The enzyme has also been purified from the related strain Clostridium acetobutylicum DSM 792 and the gene has been cloned and sequenced. The decarboxylation reaction proceeds via the formation of a Schiff base intermediate.

[0317] ADC is the key enzyme for acid uptake, which effectively pulls the CoA transferase reaction in the direction of acetoacetate formation.

[0318] In a specific embodiment, the enzyme converts acetoacetate into acetone. In one embodiment, the acetoacetate decarboxylase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from Clostridium species, Bacillus species, Chromobacterium sp. and Pseudomonas species. In another embodiment, the acetoacetate decarboxylase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from Clostridium beijerinckii, Clostridium cellulolyticum, Bacillus polymyxa, Chromobacterium violaceum and Pseudomonas putida. In some embodiments, the one or more nucleic acid molecules encoding the acetoacetate decarboxylase are adc or homologs thereof. In a further embodiment, the acetoacetate decarboxylase comprises an amino acid sequence selected from SEQ ID NO: 49 and 52. In a still further embodiment, the acetoacetate decarboxylase is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 47, 48, 50 and 51.

[0319] Alcohol dehydrogenase (EC 1.1.1.-)

[0320] The present disclosure describes enzymes that can catalyze the reversible oxidation of primary or secondary alcohols to aldehydes or ketones, respectively. In one embodiment, the enzyme is a secondary alcohol dehydrogenase (S-ADH) and catalyzes the reduction of ketones (such as acetone) to secondary alcohols (such as 2-propanol (isopropanol)).

[0321] In some embodiments, S-ADH is from Burkholderia sp. In some embodiments, S-ADH is from Burkholderia sp. AIU 652. In some embodiments, S-ADH is from Alcaligenes sp. In some embodiments, S-ADH is from Alcaligenes eutrophus. In some embodiments, S-ADH is from Clostridium. In some embodiments, S-ADH is from Clostridium ragsdalei. In some embodiments, S-ADH is from Clostridium beijer. In some embodiments, S-ADH is from Thermoanaerobacter sp. In some embodiments, S-ADH is from Thermoanaerobacter brockii. In some embodiments, S-ADH is from Thermoanaerobacter ethanolicus (Clostridium thermohydrosulfuricum). In some embodiments, S-ADH is encoded by the adhB gene. In some embodiments, S-ADH is from a species of the genus Phytomonas sp. In some embodiments, S-ADH is from a species of the genus Rhodococcus sp. In some embodiments, S-ADH is from Rhodococcus ruber. In some embodiments, S-ADH is from Methanobacterium palustre. In some embodiments, S-ADH is from a methanogenic archaeon, Methanogenium liminatans. In some embodiments, S-ADH is from the parasitic protist Entamoeba histolytica (EhAdh1). In some embodiments, the S-ADH is from the parasitic protozoan Tritrichomonas foetus. In some embodiments, the S-ADH is from the human parasite Trichomonas vaginalis.

[0322] In some embodiments, S-ADH is predicted based on homology, and the S-ADH can be from Thermoanaerobacter mathranii, Micrococcus luteus, Nocardiopsis alba, Mycobacterium hassiacum, Helicobacter suis, Candida albicans, Candida parapsilosis, Candida orthopsilosis, Candida metapsilosis, Grosmannia clavigera, and Scheffersomyces stipitis.

[0323] In some embodiments, the alcohol dehydrogenase has at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with an alcohol dehydrogenase from Clostridium. In other embodiments, the alcohol dehydrogenase is an alcohol dehydrogenase selected from Clostridium beijerinckii adh and Clostridium carboxidivorans adh. In further embodiments, the alcohol dehydrogenase comprises an amino acid sequence selected from SEQ ID NOs: 138 and 140. In a still further embodiment, the alcohol dehydrogenase is encoded by a nucleic acid sequence selected from SEQ ID NOs: 136, 137, and 139.

[0324] Dehydratase (EC 4.2.1.-)

[0325] The present disclosure describes enzymes that can catalyze the following reactions:

[0326]

[0327] D-Xylulose 1-kinase (EC 2.7.1.-)

[0328] The present disclosure describes enzymes that can catalyze the conversion of D-xylulose to D-xylulose-1-phosphate. In some embodiments, human ketohexokinase C (khk-C), also known as fructokinase, can catalyze the conversion.

[0329] Ketohexokinase or fructokinase phosphorylates fructose to fructose-1-phosphate. The enzyme is involved in fructose metabolism, which is part of carbohydrate metabolism. It is present in the liver, intestines and kidney cortex.

[0330] In human liver, purified fructokinase in combination with aldolase has been found to contribute to an alternative mechanism for the production of oxalate from xylitol. In a combined sequence, fructokinase and aldolase produce glycolaldehyde (a precursor of oxalate) from D-xylulose via D-xylulose 1-phosphate.

[0331] In a specific embodiment, the enzyme converts D-xylulose to D-xylulose-1-phosphate. In one embodiment, the D-xylulose 1-kinase is encoded by one or more nucleic acid molecules obtained from humans. In some embodiments, the one or more nucleic acid molecules encoding D-xylulose 1-kinase are ketohexokinase C (khk-C) or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding D-xylulose 1-kinase comprise the amino acid sequence set forth in SEQ ID NO: 55 or 256. In a further embodiment, the one or more nucleic acid molecules encoding D-xylulose 1-kinase are encoded by a nucleic acid sequence selected from SEQ ID NO: 53 and 54.

[0332] D-Xylulose-1-phosphate aldolase (EC 4.1.2.-)

[0333] The present disclosure describes enzymes that can catalyze the conversion of D-xylulose-1-phosphate to glycolaldehyde and DHAP. In some embodiments, human aldolase B can catalyze the conversion, which is also known as fructose-bisphosphate aldolase B or liver-type aldolase.

[0334] Aldolase B is one of three isozymes (A, B, and C) of the class I fructose 1,6-bisphosphate aldolase (EC 4.1.2.13), which plays a key role in both glycolysis and gluconeogenesis. The general fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate (FBP) to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP), and the reversible cleavage of fructose 1-phosphate (F1P) to glyceraldehyde and dihydroxyacetone phosphate. In mammals, aldolase B is preferentially expressed in the liver, whereas aldolase A is expressed in muscle and erythrocytes, and aldolase C is expressed in the brain. Subtle differences in the isozyme structure result in different activities towards the two substrate molecules, FBP and fructose 1-phosphate. Aldolase B shows no preference and thus catalyzes both reactions, whereas aldolases A and C prefer FBP.

[0335] Aldolase B is a homotetrameric enzyme composed of four subunits. Each subunit has a molecular weight of 36 kDa and contains an eight-chain α / β barrel containing lysine 229 (an amino acid that forms a Schiff base and is critical for catalysis).

[0336] In a specific embodiment, the enzyme converts D-xylulose-1-phosphate into glycolaldehyde and DHAP. In one embodiment, the D-xylulose-1-phosphate aldolase is encoded by one or more nucleic acid molecules obtained from humans. In another embodiment, the one or more nucleic acid molecules encoding the D-xylulose-1-phosphate aldolase are aldolase B (aldoB) or homologs thereof. In some embodiments, the one or more nucleic acid molecules encoding the D-xylulose-1-phosphate aldolase comprise the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the one or more nucleic acid molecules encoding the D-xylulose-1-phosphate aldolase are encoded by a nucleic acid sequence selected from SEQ ID NO: 56 and 57.

[0337] D-Xylose Isomerase (EC 5.3.1.5)

[0338] The present disclosure describes enzymes that can catalyze the following reversible reaction:

[0339]

[0340] D-xylose isomerase may also be referred to as xylose isomerase or D-xylose ketol isomerase.

[0341] Thus, in some embodiments, the present disclosure provides enzymes that function in xylose degradation.

[0342] Xylose isomerase catalyzes the first reaction in the catabolism of D-xylose.

[0343] Two conserved histidine residues, H101 and H271, have been shown to be required for catalytic activity. During xylose binding, the fluorescence of two conserved tryptophan residues, W49 and W188, is quenched, and W49 has been shown to be required for catalytic activity. 2+ , Mn 2+ or Co 2+ The presence of protects the enzyme from heat denaturation.

[0344] The subunit composition has not been determined experimentally.

[0345] In a specific embodiment, the enzyme converts D-xylose into D-xylulose. In one embodiment, the recombinant microorganism further comprises an endogenous or exogenous xylose isomerase that catalyzes the conversion of D-xylose into D-xylulose. In one embodiment, the xylose isomerase is exogenous. In another embodiment, the xylose isomerase is encoded by one or more nucleic acid molecules obtained from Pyromyces sp. or Escherichia coli. In another embodiment, the one or more nucleic acid molecules encoding the xylose isomerase are xylA or homologues thereof. In yet another embodiment, the one or more nucleic acid molecules encoding the xylose isomerase comprise an amino acid sequence selected from SEQ ID NOs: 95 and 144. In a further embodiment, the one or more nucleic acid molecules encoding the xylose isomerase are encoded by a nucleic acid sequence selected from SEQ ID NOs: 93, 94 and 143.

[0346] In some embodiments, the recombinant microorganism producing MEG or GA, or optionally MEG or GA and one or more co-products, comprises a deletion, insertion, or loss-of-function mutation in the gene encoding D-xylose isomerase to prevent the conversion of D-xylose to D-xylulose and to shunt the reaction to the conversion of D-xylose to D-xylonic acid.

[0347] D-Xylulose 5-kinase / Xylulose kinase

[0348] The present disclosure describes enzymes that can catalyze the following reactions:

[0349] D-xylulose + ATP → D-xylulose 5-phosphate + ADP + H+ (EC 2.7.1.17)

[0350] ATP + 1-deoxy-D-xylulose → 1-deoxy-D-xylulose 5-phosphate + ADP + H+ (EC 2.7.1.-)

[0351] D-xylulose 5-kinase may also be referred to as xylulose kinase or xylulokinase.

[0352] Xylulokinase catalyzes the phosphorylation of D-xylulose, the second step in the xylose degradation pathway, to produce D-xylulose-5-phosphate, an intermediate of the pentose phosphate pathway.

[0353] In the absence of substrate, the ATPase activity of xylulokinase is very weak. Xylulokinase can also catalyze the phosphorylation of 1-deoxy-D-xylulose. This will allow potential salvage pathways for the generation of 1-deoxy-D-xylulose 5-phosphate, which is used in the biosynthesis of terpenoids, thiamine and pyridoxal. The phosphorylation rate of 1-deoxy-D-xylulose is 32 times lower than that of D-xylulose.

[0354] The kinetics of this bacterial enzyme have been studied, indicating a predominantly ordered reaction mechanism. The enzyme undergoes significant conformational changes upon binding of substrate and ATP. Two conserved aspartate residues, D6 and D233, have been shown to be essential for catalytic activity, and a catalytic mechanism has been proposed.

[0355] Has been and The crystal structures of bacterial xylulokinase in its apo form and bound to D-xylulose were determined at 1.3 Å resolution.

[0356] In specific embodiments, the enzyme converts D-xylulose into D-xylulose-5-phosphate. In some embodiments, D-xylulose 5-kinase is from Escherichia coli. In some embodiments, D-xylulose 5-kinase is encoded by the xylB gene. In some embodiments, D-xylulose 5-kinase is from Saccharomyces cerevisiae. In some embodiments, D-xylulose 5-kinase is encoded by the XKS1 gene. In some embodiments, D-xylulose 5-kinase is from Pichia stipitis. In some embodiments, D-xylulose 5-kinase is encoded by the XYL3 gene.

[0357] In some embodiments, the D-xylulose 5-kinase is encoded by an amino acid sequence having at least 70% sequence identity with xylB from E. coli. In a further embodiment, the D-xylulose 5-kinase is encoded by an amino acid sequence having at least 80% sequence identity with xylB from E. coli. In a still further embodiment, the D-xylulose 5-kinase is encoded by an amino acid sequence having at least 90% sequence identity with xylB from E. coli. In other embodiments, the D-xylulose 5-kinase is xylB from E. coli.

[0358] In one embodiment, the D-xylulose 5-kinase is encoded by one or more nucleic acid molecules obtained from Escherichia coli. In some embodiments, the one or more nucleic acid molecules encoding the D-xylulose 5-kinase are xylB or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding the D-xylulose 5-kinase comprise the amino acid sequence set forth in SEQ ID NO: 146. In a further embodiment, the one or more nucleic acid molecules encoding the D-xylulose 5-kinase are encoded by the nucleic acid sequence set forth in SEQ ID NO: 145.

[0359] Xylose dehydrogenase (EC 1.1.1.175 or EC 1.1.1.179)

[0360] The present disclosure describes enzymes that can catalyze the following reactions:

[0361] Aldehyde-D-xylose + NAD++H 2O→D-xylonic acid+NADH+2H+

[0362]

[0363] Xylose dehydrogenase may also be referred to as D-xylose dehydrogenase, D-xylose 1-dehydrogenase, (NAD+)-linked D-xylose dehydrogenase, NAD+-D-xylose dehydrogenase, or D-xylose:NAD+1-oxidoreductase.

[0364] D-xylose dehydrogenase catalyzes the NAD+-dependent oxidation of D-xylose to D-xylonolactone. This is the first reaction in the oxidative, non-phosphorylation pathway for D-xylose degradation in Caulobacter crescentus. This pathway is similar to the pathway for L-arabinose degradation in Azospirillum brasilense. The amino acid sequence of the Caulobacter crescentus enzyme is unrelated to the amino acid sequence of the xylose dehydrogenase from the archaeon Haloarcula marismortui or the L-arabinose 1-dehydrogenase from Azospirillum brasilense.

[0365] D-xylose is the preferred substrate for the recombinant D-xylose dehydrogenase from Caulobacter crescentus. The enzyme can use L-arabinose, but it is a poor substrate. The Km for L-arabinose is 166 mM. As measured by NADH production, other substrates such as D-arabinose, L-xylose, D-ribose, D-galactose, D-glucose and D-glucose-6-phosphate show little or no activity in the assay. Caulobacter crescentus D-xylose dehydrogenase can directly convert D-xylose into D-xylonic acid.

[0366] The partially purified native D-xylose dehydrogenase from Caulobacter crescentus had a Km for D-xylose of 70 μM. This value is lower than the Km of the recombinant His-tagged enzyme (760 μM).

[0367] In some embodiments, the D-xylose dehydrogenase is from the halophilic archaeon Haloferax volcanii. Haloferax volcanii D-xylose dehydrogenase catalyzes the first reaction in the oxidative xylose degradation pathway of the halophilic archaeon Haloferax volcanii. Haloferax volcanii D-xylose dehydrogenase has 59% amino acid sequence identity with the functionally characterized xylose dehydrogenase from Haloarcula marismortui, 56% identity with the ortholog in Halorubrum lacusprofundi, but only 11% identity with the bacterial NAD+-dependent xylose dehydrogenase from Caulobacter crescentus CB15.

[0368] In a specific embodiment, the enzyme converts D-xylose into D-xylonic acid lactone. In one embodiment, the xylose dehydrogenase is encoded by one or more nucleic acid molecules obtained from microorganisms, and the microorganism is selected from Caulobacter sp., Haloarcula sp., Haloferax sp., Halorubrum sp. and Trichoderma sp. In another embodiment, the xylose dehydrogenase is encoded by one or more nucleic acid molecules obtained from microorganisms, and the microorganism is selected from Caulobacter sp., Haloarcula marismortui, Haloferax sp., Halorubrum lacusprofundi and Trichoderma reesei. In some embodiments, one or more nucleic acid molecules encoding xylB, xdh1 (HVO_B0028) and / or xyd1, or homologues thereof. In a further embodiment, the one or more nucleic acid molecules encoding xylose dehydrogenase comprise an amino acid sequence selected from SEQ ID NOs: 61, 63 and 65. In yet another embodiment, the one or more nucleic acid molecules encoding xylose dehydrogenase are encoded by a nucleic acid sequence selected from SEQ ID NOs: 59, 60, 62 and 64.

[0369] Xylonase (3.1.1.68)

[0370] The present disclosure describes enzymes that can catalyze the following reactions:

[0371]

[0372] This enzyme belongs to the family of hydrolases, specifically those acting on carboxylic acid ester bonds. This enzyme is involved in the interconversion of pentoses and glucuronic acid.

[0373] Xylonolactonase may also be referred to as D-xylonolactonase, xylono-1,4-lactonase, xylono-γ-lactonase or D-xylono-1,4-lactone lactone hydrolase.

[0374] In a specific embodiment, the enzyme converts D-xylonolactone into D-xylonic acid. In one embodiment, the xylonolactonase is encoded by one or more nucleic acid molecules obtained from a microorganism, and the microorganism is selected from Caulobacter sp. and Haloferax sp. In another embodiment, the xylonolactonase is encoded by one or more nucleic acid molecules obtained from a microorganism, and the microorganism is selected from Caulobacter sp., Haloferax sp. and Haloferax gibbonsii. In some embodiments, the one or more nucleic acid molecules encoding xylonolactonase are xylC or homologues thereof. In a further embodiment, the one or more nucleic acid molecules encoding xylonolactonase comprise the amino acid sequence listed in SEQ ID NO: 67. In yet another embodiment, the one or more nucleic acid molecules encoding xylonolactonase are encoded by the nucleic acid sequence listed in SEQ ID NO: 66.

[0375] Xylonate dehydratase (EC 4.2.1.82)

[0376] The present disclosure describes enzymes that can catalyze the following reactions:

[0377]

[0378] This enzyme belongs to the lyase family, specifically the water lyase, which cleaves carbon-oxygen bonds. This enzyme is involved in the interconversion of pentoses and glucuronic acid.

[0379] Xylonic acid dehydratase may also be referred to as D-xylonic acid hydrolyase, D-xyluronate dehydratase or D-xylonic acid dehydratase.

[0380] In a specific embodiment, the enzyme converts D-xylonic acid into 2-keto-3-deoxy-D-xylonic acid. In one embodiment, the xylonate dehydratase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from a species of the genus Caulobacter, a species of the genus Sulfolobus, and Escherichia coli. In another embodiment, the xylonate dehydratase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from Caulobacter crescentus, Sulfolobus solfataricus, and Escherichia coli. In some embodiments, one or more nucleic acid molecules encoding xylonate dehydratase are selected from xylD, yjhG and / or yagF, or homologs thereof. In a further embodiment, one or more nucleic acid molecules encoding xylonate dehydratase comprise an amino acid sequence selected from SEQ ID NOs: 69, 72, and 75. In yet another embodiment, one or more nucleic acid molecules encoding xylonate dehydratase are encoded by a nucleic acid sequence selected from SEQ ID NOs: 68, 70, 71, 73, and 74.

[0381] 2-Keto-3-deoxy-D-pentosonic acid aldolase (4.1.2.28)

[0382] The present disclosure describes enzymes that can catalyze the following reactions:

[0383]

[0384] This enzyme belongs to the lyase family, specifically aldehyde lyase, which cleaves carbon-carbon bonds. This enzyme is involved in the interconversion of pentoses and glucuronic acid.

[0385] 2-keto-3-deoxy-D-pentosonic acid aldolase may also be referred to as 2-dehydro-3-deoxy-D-pentosonic acid glycolaldehyde lyase (forming pyruvate), 2-dehydro-3-deoxy-D-pentosonic acid aldolase, 3-deoxy-D-pentosonic acid aldolase, and 2-dehydro-3-deoxy-D-pentosonic acid glycolaldehyde lyase.

[0386] YjhH behaves as a 2-dehydro-3-deoxy-D-pentosonic acid aldolase. Genetic evidence suggests that YagE can also function as a 2-dehydro-3-deoxy-D-pentosonic acid aldolase. yagE is part of the prophage CP4-6.

[0387] The yjhH yagE double mutant is unable to use D-xyloglucanate as a sole carbon source and crude cell extracts contain no 2-dehydro-3-deoxy-D-pentosonic acid aldolase activity. Both phenotypes were complemented by providing yjhH on a plasmid.

[0388] ArcA was shown to activate yjhH gene expression under anaerobic conditions. Two putative ArcA binding sites were identified at 211 bp and 597 bp upstream of the gene, but the promoter upstream of them has not been identified.

[0389] The crystal structure of YagE shows that the protein is a homotetramer. The cocrystal structure of YagE in the presence of pyruvate and 2-keto-3-deoxygalactonic acid has been solved.

[0390] In a specific embodiment, the enzyme converts 2-keto-3-deoxy-xylonic acid into glycolaldehyde and pyruvic acid. In one embodiment, the 2-keto-3-deoxy-D-pentose acid aldolase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from Pseudomonas species and Escherichia coli. In another embodiment, the 2-keto-3-deoxy-D-pentose acid aldolase is encoded by one or more nucleic acid molecules obtained from Escherichia coli. In some embodiments, one or more nucleic acid molecules encoding 2-keto-3-deoxy-D-pentose acid aldolase are selected from yjhH and / or yagE, or homologs thereof. In a further embodiment, one or more nucleic acid molecules encoding 2-keto-3-deoxy-D-pentose acid aldolase comprise an amino acid sequence selected from SEQ ID NOs: 78 and 81. In yet another embodiment, one or more nucleic acid molecules encoding 2-keto-3-deoxy-D-pentose acid aldolase are encoded by a nucleic acid sequence selected from SEQ ID NOs: 76, 77, 79 and 80.

[0391] Glycolaldehyde dehydrogenase (1.2.1.21)

[0392] The present disclosure describes enzymes that can catalyze the following reactions:

[0393]

[0394] This enzyme belongs to the family of oxidoreductases, specifically those enzymes that act on aldehyde or oxo groups from donors using NAD+ or NADP+ as acceptors. This enzyme is involved in glyoxylate and dicarboxylic acid metabolism.

[0395] Glycolaldehyde dehydrogenase can also be called glycolaldehyde:NAD+ oxidoreductase or glycolaldehyde dehydrogenase.

[0396] In Escherichia coli, aldehyde dehydrogenase A (AldA) is an enzyme with relatively broad substrate specificity for small α-hydroxyaldehyde substrates. It is therefore utilized in a variety of metabolic pathways.

[0397] L-fucose and L-rhamnose are metabolized by parallel pathways that converge after their corresponding aldolase reactions to produce the same products: dihydroxyacetone phosphate and L-lactaldehyde. Aerobically, aldehyde dehydrogenase A oxidizes L-lactaldehyde to L-lactic acid.

[0398] In a parallel pathway utilizing the same enzymes, D-arabinose and L-xylose can be metabolized to dihydroxyacetone phosphate and glycolaldehyde, which is oxidized to glycolic acid by aldehyde dehydrogenase A.

[0399] The crystal structures of the individual enzymes as well as ternary and binary complexes have been solved.

[0400] Aldehyde dehydrogenase A is present only under aerobic conditions and is most highly induced in the presence of fucose, rhamnose or glutamate. The enzyme is inhibited by NADH, which acts as a switch from oxidizing lactaldehyde to reducing it by propanediol oxidoreductase. AldA is upregulated during short-term adaptation to glucose limitation.

[0401] Based on sequence similarity, AldA was predicted to be a succinate semialdehyde dehydrogenase.

[0402] The regulation of aldA expression has been studied. This gene is regulated by repression by catabolites, repression by ArcA under anaerobic conditions and induction by carbon sources.

[0403] In specific embodiments, the enzyme converts glycolaldehyde to glycolic acid. In some embodiments, the glycolaldehyde dehydrogenase is from Escherichia coli. In some embodiments, the glycolaldehyde dehydrogenase is encoded by the aldA gene.

[0404] In some embodiments, the recombinant microorganism producing MEG, or optionally MEG and one or more co-products, comprises a deletion, insertion or loss-of-function mutation in the gene encoding glycolaldehyde dehydrogenase to prevent the production of glycolic acid from glycolaldehyde and to divert the reaction to the conversion of glycolaldehyde to MEG. In some embodiments, the glycolaldehyde dehydrogenase activity that catalyzes the conversion of glycolaldehyde to glycolic acid is partially deleted, destroyed, mutated and / or reduced, wherein a certain amount of glycolic acid is still produced.

[0405] In other embodiments, the glycolic acid-producing recombinant microorganism comprises or expresses at least one nucleic acid molecule encoding a glycolaldehyde dehydrogenase.

[0406] Lactate dehydrogenase (1.1.1.28)

[0407] The present disclosure describes enzymes that can catalyze the following reactions:

[0408] (R)-Lactate + NAD + ← Pyruvate + NADH + H +

[0409] Lactate dehydrogenase (LDH) is an enzyme present in almost all living cells, such as animals, plants and prokaryotes. LDH catalyzes the conversion of lactate to pyruvate and vice versa, and catalyzes the conversion of NADH to NAD+ and vice versa. Dehydrogenases are enzymes that transfer hydride from one molecule to another.

[0410] There are four different enzyme classes of LDH. The most common one is the NAD(P)-dependent L-lactate dehydrogenase. Other LDHs act on D-lactate and / or are dependent on cytochrome c: D-lactate dehydrogenase (cytochrome) and L-lactate dehydrogenase (cytochrome).

[0411] LDH is of medical interest because it is widely present in body tissues, such as blood cells and heart muscle. Because it is released during tissue damage, it is a marker for common injuries and diseases, such as heart failure.

[0412] Lactate dehydrogenase may also be called lactate dehydrogenase, (R)-lactate:NAD+ oxidoreductase or fermentative D-lactate dehydrogenase.

[0413] In Escherichia coli, lactate dehydrogenase (LdhA) is a soluble NAD-linked lactate dehydrogenase (LDH) that specifically produces D-lactate. LdhA is a homotetramer that shows positive cooperativity at higher pH conditions.

[0414] E. coli contains two other lactate dehydrogenases: D-lactate dehydrogenase and L-lactate dehydrogenase. Both are membrane-associated flavoproteins required for aerobic growth on lactate.

[0415] LdhA is present under aerobic conditions but is induced when E. coli is grown under acidic pH and anaerobic conditions with various sugars. Unlike most genes involved in anaerobic respiration, ldhA is not activated by Fnr; instead, the ArcAB system and several genes involved in controlling carbohydrate metabolism (csrAB and mlc) show regulated expression. The expression of ldhA is negatively influenced by the transcriptional regulator ArcA. ldhA belongs to the σ32 regulon.

[0416] The ldhA gene is a common target for mutation in metabolic engineering, most often to eliminate the production of unwanted fermentation byproducts, but also to specifically produce D-lactic acid.

[0417] In specific embodiments, the enzyme converts pyruvate into lactate. In some embodiments, the lactate dehydrogenase is from Escherichia coli. In some embodiments, the lactate dehydrogenase is encoded by the ldhA gene.

[0418] In some embodiments, a recombinant microorganism that produces MEG or GA, or optionally MEG or GA and one or more co-products, comprises a deletion, insertion, or loss-of-function mutation in a gene encoding lactate dehydrogenase to prevent the production of lactate from pyruvate and to divert the reaction to the production of one or more co-products.

[0419] Xylose reductase or aldose reductase (EC 1.1.1.21)

[0420] The present disclosure describes enzymes that can catalyze the following reactions:

[0421]

[0422]

[0423] Aldose reductase may also be called sugar alcohol: NAD(P)+1-oxidoreductase, polyol dehydrogenase or aldehyde reductase.

[0424] Aldose reductase is a cytosolic oxidoreductase that catalyzes the reduction of a variety of aldehydes and carbonyl groups, including monosaccharides.

[0425] Aldose reductase can be considered the prototype enzyme of the aldo-keto reductase superfamily. The enzyme contains 315 amino acid residues and folds into a β / α-barrel structural motif consisting of eight parallel β strands. Adjacent strands are connected by eight peripheral α-helical segments that are antiparallel to the β fold. The catalytic active site is located in the core of the barrel. The NADPH cofactor is located at the top of the β / α barrel, with its nicotinamide ring protruding downward in the center of the barrel, and the pyrophosphate straddling the edge of the barrel.

[0426] The reaction mechanism of aldose reductase in the aldehyde reduction direction follows a sequentially ordered pathway in which NADPH binds, followed by substrate binding. Binding of NADPH induces a conformational change (enzyme·NADPH–>enzyme*·NADPH) that involves a hinge-like movement of a surface loop (residues 213-217) that caps a portion of NADPH in a manner similar to a seatbelt. The alcohol product is formed by transferring the pro-R hydride of NADPH to the opposite side of the carbonyl carbon of the substrate. After release of the alcohol product, another conformational change occurs (E*·NAD(P)+–>E·NAD(P)+) to release NADP+. Kinetic studies indicate that reorientation of this loop to allow release of NADP+ appears to represent the rate-limiting step in the aldehyde reduction direction. Since the rate of coenzyme release limits the catalytic rate, it can be seen that perturbations of the interactions that stabilize coenzyme binding can have a significant effect on the maximum velocity (Vmax).

[0427] It has been shown that the D-xylose fermenting yeasts Pichia stipitis and Candida shehatae produce a single aldose reductase (ALR) that is active on both NADPH and NADH. Other yeasts such as Pachysolen tannophilus and C. tropicalis synthesize multiple forms of ALRs with different coenzyme specificities. The pronounced dual coenzyme specificity distinguishes the enzymes of Pichia stipitis and C. shehatae from most other ALRs isolated to date from mammalian or microbial sources. The yeast Candida tenuis CBS 4435 produces considerable aldehyde reducing activity linked to both NADH and NADPH during growth on D-xylose.

[0428] In a specific embodiment, the enzyme converts D-xylose to xylitol. In some embodiments, the xylose reductase or aldose reductase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from the group consisting of Hypocrea sp., Scheffersomyces sp., Saccharomyces sp., Pachysolen sp., Pichia sp., Candida sp., Aspergillus sp., Neurospora sp., and Cryptococcus sp. In some embodiments, the xylose reductase or aldose reductase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from the group consisting of Hypocrea jecorina, Scheffersomyces stipitis, Saccharomyces cerevisiae, Pachysolen tannophilus, Pichia stipitis, Pichia quercuum, Candida shehatae, Candida gracilis, Candida tropicalis, Aspergillus niger, Neurospora crassa, and Cryptococcus lactativorus. In another embodiment, the one or more nucleic acid molecules encoding the xylose reductase or aldose reductase are xyl1 and / or GRE3 or homologs thereof. In some embodiments, the one or more nucleic acid molecules encoding the xylose reductase or aldose reductase comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 84 and 87. In some embodiments, the one or more nucleic acid molecules encoding xylose reductase or aldose reductase are encoded by a nucleic acid sequence selected from SEQ ID NOs: 82, 83, 85, and 86.

[0429] Xylitol dehydrogenase (1.1.1.9)

[0430] The present disclosure describes enzymes that can catalyze the following reactions:

[0431]

[0432] Xylitol dehydrogenase may also be referred to as D-xylulose reductase, NAD+-dependent xylitol dehydrogenase, erythritol dehydrogenase, 2,3-cis-polyol (DPN) dehydrogenase (C3-5), pentitol-DPN dehydrogenase, xylitol-2-dehydrogenase, or xylitol:NAD+2-oxidoreductase (forming D-xylulose).

[0433] Xylitol dehydrogenase (XDH) is one of several enzymes responsible for the uptake of xylose into eukaryotic metabolism and can be used to ferment xylose contained in agricultural byproducts to produce ethanol. In order to efficiently utilize xylose at high flow rates, the co-substrate should be cycled between NAD+-specific XDH and NADPH-preferring xylose reductase, another enzyme in the pathway.

[0434] In a specific embodiment, the enzyme converts xylitol into D-xylulose. In one embodiment of any aspect disclosed above, the xylitol dehydrogenase is encoded by one or more nucleic acid molecules obtained from a microorganism, and the microorganism is selected from Scheffersomyces sp., Trichoderma species, Pichia species, Saccharomyces species, Gluconobacter species (Gluconobacter sp.), Galactoocandida sp., Neurospora species (Neurospora sp.) and Serratia species (Serratia sp.). In another embodiment, the xylitol dehydrogenase is encoded by one or more nucleic acid molecules obtained from a microorganism, and the microorganism is selected from Scheffersomyces stipitis, Trichoderma reesei, Pichia stipitis, Saccharomyces cerevisiae, Gluconobacter oxydans (Gluconobacter oxydans), Galactoocandida mastotermitis, Neurospora crassa (Neurospora crassa) and Serratia marcescens (Serratia marcescens). In another embodiment, the one or more nucleic acid molecules encoding xylitol dehydrogenase are xyl2 and / or xdh1, or homologs thereof. In some embodiments, the one or more nucleic acid molecules encoding xylose dehydrogenase comprise an amino acid sequence selected from SEQ ID NOs: 90 and 92. In some embodiments, the one or more nucleic acid molecules encoding xylose dehydrogenase are encoded by a nucleic acid sequence selected from SEQ ID NOs: 88, 89 and 91. In another embodiment, the enzyme converts D-xylulose into xylitol. In another embodiment, the enzyme has high activity for converting D-xylulose into xylitol, and preferably has low activity or no activity for the reverse reaction (i.e., for converting xylitol into D-xylulose). In another embodiment, it is achieved by enzyme engineering.

[0435] Soluble pyridine nucleotide transhydrogenase (EC 1.6.1.1.)

[0436] The present disclosure describes enzymes that can catalyze the following reactions:

[0437]

[0438] Soluble pyridine nucleotide transhydrogenase may also be referred to as NAD(P)+ transhydrogenase (B-specific), STH, pyridine nucleotide transhydrogenase or transhydrogenase.

[0439] Escherichia coli contains soluble and membrane-bound pyridine nucleotide transhydrogenases. Soluble pyridine nucleotide transhydrogenases are the products of sthA or udhA genes; their main physiological role is the reoxidation of NADPH. Membrane-bound proton transhydrogenases are the products of pntAB genes; PntAB is the main source of NADPH.

[0440] UdhA contains non-covalently bound FAD and exists as a seven- or eight-monomer complex.

[0441] Moderate overexpression of UdhA (SthA) allows an increase in the maximal growth rate of phosphoglucose isomerase mutants, whereas pgi sthA double mutants are inviable. These phenotypes are probably due to the ability of UdhA to restore cellular redox balance under conditions of excessive NADPH formation. Mutations in sthA arose during the adaptation of the pgi mutant to growth in glucose-minimal medium.

[0442] Growth on glycerol down-regulated the transcription of sthA.

[0443] In some embodiments, the expression of the transhydrogenase can increase the activity of NADPH-dependent alcohol dehydrogenase, resulting in an increase in the conversion of acetone to 2-propanol. In one embodiment, the soluble pyridine nucleotide transhydrogenase is encoded by one or more nucleic acid molecules obtained from Escherichia coli. In another embodiment, the one or more nucleic acid molecules encoding the soluble pyridine nucleotide transhydrogenase are udhA, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding the soluble pyridine nucleotide transhydrogenase comprise the amino acid sequence set forth in SEQ ID NO: 142. In some embodiments, the one or more nucleic acid molecules encoding the soluble pyridine nucleotide transhydrogenase are encoded by the nucleic acid sequence set forth in SEQ ID NO: 141.

[0444] Hydroxymethylglutaryl-CoA synthase (EC 2.3.3.-)

[0445] The present disclosure describes enzymes that can catalyze the following reactions:

[0446]

[0447] Hydroxymethylglutaryl-CoA synthase may also be referred to as (S)-3-hydroxy-3-methylglutaryl-CoA acetoacetyl-CoA-lyase (CoA-acetylation), 3-hydroxy-3-methylglutaryl-CoA synthetase (3-hydroxy-3-methylglutaryl CoA synthetase, 3-hydroxy-3-methylglutaryl coenzyme Asynthetase), 3-hydroxy-3-methylglutaryl-CoA synthase (3-hydroxy-3-methylglutaryl coenzymeA synthase, 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-coenzyme A synthase), β-hydroxy-β-methylglutaryl-CoA synthase, HMG-CoA synthase, acetoacetyl-CoA transacetylase, hydroxymethylglutaryl-CoA synthase and hydroxymethylglutaryl-CoA condensing enzyme.

[0448] Hydroxymethylglutaryl-CoA synthase catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA to form (S)-3-hydroxy-3-methylglutaryl-CoA, which is an early step in the synthesis of the cholesterol precursor (R)-mevalonate.

[0449] The enzyme catalyzes a complex reaction that can be divided into four steps. The first step involves the formation of the enzyme-acetyl-CoA binary complex, followed by the transfer of the acetyl group from the CoA thioester to a cysteine ​​residue on the enzyme, forming a thioester acyl-enzyme intermediate. In the next step, the now reduced CoA dissociates and the second substrate, acetoacetyl-CoA, binds to the enzyme. The third step involves the formation of a carbanion by removing a proton from the methyl group of acetylcysteine. The activated acetylcysteine ​​then undergoes a Claisen-like condensation with the γ-carbon of the acetoacetyl-CoA ligand to form HMG-CoA while retaining the thioester bond to the enzyme. The final step involves the hydrolysis of this bond, producing free HMG-CoA.

[0450] The HMGCS1 gene from humans has been cloned and sequenced (Russ AP et al. (1992) Amplification and direct sequencing of a cDNA encoding human cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase. Biochim Biophys Acta 1132 (3): 329-31). The gene was expressed in E. coli, and the recombinant protein has been purified and characterized (Rokosz LL et al. (1994) Humancytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase: expression, purification, and characterization of recombinant wild-type and Cys129 mutant enzymes. Arch Biochem Biophys 312 (1): 1-13). The enzyme is a 120 kDa homodimer. Catalysis is carried out by forming a covalent acetyl-enzyme intermediate. Kinetic data indicate that the two substrates (acetyl-CoA and acetoacetyl-CoA) compete for binding to the same site.

[0451] In one embodiment, the hydroxymethylglutaryl-CoA synthase may have 3-hydroxyisovalerate (3HIV) synthase activity and may catalyze the following reaction:

[0452]

[0453] In one embodiment, 3HIV synthase is encoded by one or more nucleic acid molecules obtained from microorganisms, and the microorganisms are selected from Mus sp., Saccharomyces sp., Lactobacillus sp. and Polaromonas sp. In another embodiment, 3HIV synthase is encoded by one or more nucleic acid molecules obtained from microorganisms, and the microorganisms are selected from mice, Saccharomyces cerevisiae, Lactobacillus crispatus and Polaromonas naphthalenivorans. In some embodiments, one or more nucleic acid molecules encoding 3HIV synthase are selected from Hmgcs1, ERG13, PksG and / or Pnap_0477, or homologs thereof. In a further embodiment, one or more nucleic acid molecules encoding 3HIV synthase comprise an amino acid sequence selected from SEQ ID NO: 105, 107, 109 and 111. In yet another embodiment, one or more nucleic acid molecules encoding 3HIV synthase are encoded by a nucleic acid sequence selected from SEQ ID NO: 104, 106, 108 and 110. In some embodiments, the one or more nucleic acid molecules encoding hydroxymethylglutaryl-CoA synthase are hmgS, or a homolog thereof. In a further embodiment, the one or more nucleic acid molecules encoding hydroxymethylglutaryl-CoA synthase comprise the amino acid sequence set forth in SEQ ID NO: 123. In yet another embodiment, the one or more nucleic acid molecules encoding hydroxymethylglutaryl-CoA synthase are encoded by the nucleic acid sequence set forth in SEQ ID NO: 122.

[0454] Methylglutaconyl-CoA hydratase (EC 4.2.1.18)

[0455] The present disclosure describes enzymes that can catalyze the following reactions:

[0456]

[0457] The enzyme catalyzes the syn-hydration of 3-methylglutaconyl-CoA to (S)-3-hydroxy-3-methylglutarate-CoA in the leucine degradation pathway. The bacterial enzyme has been characterized in Pseudomonas putida. It differs from the mammalian enzyme in that its active site has only one glutamyl residue instead of two, resulting in a different reaction mechanism. These enzymes are members of the crotonase superfamily (Wong BJ and Gerlt JA (2004) Evolution of function in the crotonase superfamily: (3S)-methylglutaconyl-CoA hydratase from Pseudomonas putida. Biochemistry 43(16): 4646-4654) and are reviewed in (Hamed RB et al. (2008) Mechanisms and structures of crotonase superfamily enzymes--how nature controls enolate and oxyanion reactivity. Cell Mol Life Sci 65(16): 2507-2527).

[0458] The recombinant enzyme was expressed, purified and characterized in E. coli. The apparent molecular weight of the 10-His-tagged polypeptide was determined to be 32.251 kDa by ESI-MS. The 10-His tag was subsequently removed before characterizing the enzyme (Wong and Gerlt, 2004).

[0459] In one embodiment, the methylglutaconyl-CoA hydratase is encoded by one or more nucleic acid molecules obtained from Pseudomonas species. In another embodiment, the methylglutaconyl-CoA hydratase is encoded by one or more nucleic acid molecules obtained from Pseudomonas putida. In some embodiments, the one or more nucleic acid molecules encoding the methylglutaconyl-CoA hydratase is liuC, or a homolog thereof. In a further embodiment, the one or more nucleic acid molecules encoding the methylglutaconyl-CoA hydratase comprises the amino acid sequence set forth in SEQ ID NO: 125. In yet another embodiment, the one or more nucleic acid molecules encoding the methylglutaconyl-CoA hydratase is encoded by the nucleic acid sequence set forth in SEQ ID NO: 124.

[0460] Methylcrotonyl-CoA carboxylase (EC 6.4.1.4)

[0461] The present disclosure describes enzymes that can catalyze the following reactions:

[0462]

[0463] The enzyme activity is associated with the 3-methylcrotonyl-CoA carboxylase complex. The enzyme is a biotin-dependent carboxylase containing biotin that is involved in the L-leucine (and isovaleric acid) degradation pathway of Pseudomonas aeruginosa PAO1. This pathway is also the final stage of the acyclic terpene utilization pathway (citronellol degradation and cis-geranyl-CoA degradation pathway). The enzyme is not expressed in cells grown with citronellol or citronellol acid, but is expressed in cells grown with isovaleric acid. The genes liuB and liuD encode the two subunits of 3-methylcrotonyl-CoA carboxylase. These subunits are encoded in the liuRABCDE gene cluster of this organism (Hoschle B et al. (2005) Methylcrotonyl-CoA and geranyl-CoAcarboxylases are involved in leucine / isovalerate utilization (Liu) and acyclicterpene utilization (Atu), and are encoded by liuB / liuD and atuC / atuF, in Pseudomonas aeruginosa. Microbiology 151(Pt11): 3649-3656; Forster-Fromme K and Jendrossek D (2010). Catabolism of citronellol and related acyclic terpenoids in pseudomonads. Appl Microbiol Biotechnol 87(3): 859-869).

[0464] The enzyme was purified from cell extracts by avidin affinity chromatography and trypsin fingerprinting and ESI-MS of the subunits separated by SDS gels allowed the identification of their corresponding genes (Hoschle et al., 2005).

[0465] Earlier work characterized the 3-methylcrotonyl-CoA carboxylase from Pseudomonas citronellolis (Hector ML and Fall RR (1976) Multiple acyl-coenzyme A carboxylases in Pseudomonas citronellolis. Biochemistry 15(16): 3465-3472; Fall RR and Hector ML (1977) Acyl-coenzyme A carboxylases. Homologous 3-methylcrotonyl-CoA and geranyl-CoA carboxylases from Pseudomonas citronellolis. Biochemistry 16(18): 4000-4005; Fall RR (1981) 3-Methylcrotonyl-CoA and geranyl-CoA carboxylases from Pseudomonas citronellolis. Methods Enzymol 71 Pt C: 791-799).

[0466] In one embodiment, the methyl crotonyl-CoA carboxylase is encoded by one or more nucleic acid molecules obtained from Pseudomonas species. In another embodiment, the methyl crotonyl-CoA carboxylase is encoded by one or more nucleic acid molecules obtained from Pseudomonas aeruginosa. In some embodiments, the one or more nucleic acid molecules encoding the methyl crotonyl-CoA carboxylase are selected from liuB and / or liuD, or homologs thereof. In a further embodiment, the one or more nucleic acid molecules encoding the methyl crotonyl-CoA carboxylase comprise an amino acid sequence selected from SEQ ID NOs: 127 and 129. In yet another embodiment, the one or more nucleic acid molecules encoding the methyl crotonyl-CoA carboxylase are encoded by the nucleic acid sequences of SEQ ID NOs: 126 and 128.

[0467] Methylcrotonyl-CoA hydratase (EC 4.2.1.17)

[0468] The present disclosure describes enzymes that can catalyze the following reactions:

[0469]

[0470] An exemplary enzyme is 3-ketoacyl-CoA thiolase. It participates in the degradation of fatty acids through the β-oxidation cycle. It has a wide range of chain length specificity for substrates, although it exhibits the highest activity for medium-chain substrates. It is part of a multienzyme complex and is encoded by the fadA gene (Yang SY et al. (1990) Nucleotide sequence of the fadA gene. Primary structure of 3-ketoacyl-coenzyme A thiolase from Escherichia coli and the structural organization of the fadAB operon. J Biol Chem 265 (18): 10424-10429).

[0471] 3-Ketoacyl-CoA thiolase may also be referred to as acetyl-CoA C-acyltransferase, β-ketothiolase, acetyl-CoA acyltransferase, and acyl-CoA:acetyl-CoA C-acyltransferase.

[0472] Another exemplary enzyme is enoyl-CoA hydratase. The alpha subunit has four enzyme activities associated with it. It is part of a multienzyme complex. Two of the activities, enoyl-CoA hydratase (EC 4.2.1.17) and 3-OH acyl-CoA epimerase (EC 5.1.2.3), are carried out by the same N-terminal active site (Yang SY and Elzinga M (1993) Association of both enoyl coenzyme A hydratase and 3-hydroxyacyl coenzyme A epimerase with an active site in the amino-terminal domain of the multifunctional fatty acid oxidation protein from Escherichia coli. J Biol Chem 268 (9): 6588-6592).

[0473] In one embodiment, the methylcrotonyl-CoA hydratase is a 3-ketoacyl-CoA thiolase. In another embodiment, the methylcrotonyl-CoA hydratase is encoded by one or more nucleic acid molecules obtained from Escherichia coli. In some embodiments, the one or more nucleic acid molecules encoding the methylcrotonyl-CoA hydratase are fadA, or a homolog thereof. In a further embodiment, the one or more nucleic acid molecules encoding the methylcrotonyl-CoA hydratase comprise the amino acid sequence set forth in SEQ ID NO: 131. In yet another embodiment, the one or more nucleic acid molecules encoding the methylcrotonyl-CoA hydratase are encoded by the nucleic acid sequence set forth in SEQ ID NO: 130.

[0474] In one embodiment, the methylcrotonyl-CoA hydratase is an enoyl-CoA hydratase. In another embodiment, the methylcrotonyl-CoA hydratase is encoded by one or more nucleic acid molecules obtained from Escherichia coli. In some embodiments, the one or more nucleic acid molecules encoding the methylcrotonyl-CoA hydratase are fadB, or a homolog thereof. In a further embodiment, the one or more nucleic acid molecules encoding the methylcrotonyl-CoA hydratase comprise the amino acid sequence set forth in SEQ ID NO: 133. In yet another embodiment, the one or more nucleic acid molecules encoding the methylcrotonyl-CoA hydratase are encoded by the nucleic acid sequence set forth in SEQ ID NO: 132.

[0475] 3-Hydroxy-isovaleryl-CoA thioesterase (EC 3.1.2.-)

[0476] The present disclosure describes enzymes that can catalyze the following reactions:

[0477]

[0478] Acyl-CoA+H 2 O→carboxylic acid + coenzyme A + H +

[0479] An exemplary acyl-CoA thioesterase is TesB. Thioesterase II (TesB) is one of the many thioesterases present in Escherichia coli. The enzyme has a relatively broad substrate specificity, cleaving both medium-chain and long-chain acyl-CoA substrates; the best substrate tested was 3,5-tetradecadienoyl-CoA (Nie L et al. (2008) A novel paradigm of fatty acid beta-oxidation exemplified by the thioesterase-dependent partial degradation of conjugated linoleic acid that fully supports growth of Escherichia coli. Biochemistry 47(36): 9618-9626). Thioesterase II is one of the thioesterases that supports growth on oleic acid or conjugated linoleic acid as the sole carbon source (Nie et al., 2008).

[0480] Has been The crystal structure of the enzyme was solved at a resolution of 1.5 Å. The D204 residue was predicted to be located in the active site; kinetic analysis of mutants confirmed its importance (Li J et al. (2000) Crystal structure of the Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme. Nat Struct Biol 7(7): 555-559).

[0481] Strains lacking or overproducing tesB have no obvious defects (Narasimhan ML et al. (1986) Genetic and biochemical characterization of an Escherichia coli K-12 mutant deficientin acyl-coenzyme A thioesterase II. J Bacteriol 165(3):911-917; Naggert J et al. (1991) Cloning, sequencing, and characterization of Escherichia colithioesterase II. J Biol Chem 266(17):11044-11050). Overproduction of TesB alleviates the inhibition of fatty acid synthesis by long-chain acyl-ACP molecules accumulated during glycerol starvation (Jiang P and Cronan JE (1994) Inhibition of fatty acid synthesis in Escherichia coli in the absence of phospholipid synthesis and release of inhibition by thioesterase action. J Bacteriol 176(10):2814-2821).

[0482] In one embodiment, the 3-hydroxy-isovaleryl-CoA thioesterase is encoded by one or more nucleic acid molecules obtained from Escherichia coli. In some embodiments, the one or more nucleic acid molecules encoding the 3-hydroxy-isovaleryl-CoA thioesterase is tesB, or a homolog thereof. In a further embodiment, the one or more nucleic acid molecules encoding the 3-hydroxy-isovaleryl-CoA thioesterase comprises the amino acid sequence set forth in SEQ ID NO: 135. In yet another embodiment, the one or more nucleic acid molecules encoding the 3-hydroxy-isovaleryl-CoA thioesterase is encoded by the nucleic acid sequence set forth in SEQ ID NO: 134.

[0483] Mevalonate-3-kinase (EC 2.7.1.-)

[0484] The present disclosure describes enzymes that can catalyze the following reactions:

[0485]

[0486] (R)-Mevalonate + ATP → (R)-Mevalonate 3-phosphate + ADP + H+

[0487] Mevalonate-3-kinase may also be referred to as (R)-MVA 3-phosphotransferase or 3-hydroxyisovalerate (3HIV) kinase.

[0488] The subunit structure of this enzyme from Thermoplasma acidophilum has not been reported.

[0489] Mevalonate-3-kinase from the thermophilic archaeon Thermoplasma acidophilum is thought to be involved in a variant of the mevalonate pathway found in archaea (Azami Y et al. (2014) (R)-Mevalonate 3-Phosphate Is an Intermediate of the Mevalonate Pathway in Thermoplasma acidophilum. J Biol Chem 289(23):15957-15967; Vinokur JM et al. (2014) Evidence of a Novel Mevalonate Pathway in Archaea. Biochemistry 53(25):4161-4168).

[0490] The recombinant His-tagged enzyme was expressed, purified and characterized in E. coli. Despite its homology to mevalonate diphosphomevalonate decarboxylase, it exhibits no decarboxylase activity (Azami et al., 2014; Vinokur et al., 2014). The enzyme exhibits weak phosphomevalonate kinase activity, producing small amounts of (R)-mevalonate diphosphate (Azami et al., 2014). It does not have mevalonate-5-kinase activity (Vinokur et al., 2014).

[0491] In one embodiment, 3HIV kinase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from Thermoplasma sp. and Picrophilus sp. In another embodiment, 3HIV kinase is encoded by one or more nucleic acid molecules obtained from a microorganism selected from Thermoplasma sp. and Picrophilus sp. In some embodiments, the one or more nucleic acid molecules encoding 3HIV kinase are TA1305 and / or PTO1356, or homologs thereof. In some embodiments, TA1305 comprises an L200E mutation. In a further embodiment, the one or more nucleic acid molecules encoding 3HIV kinase comprise an amino acid sequence selected from SEQ ID NOs: 113, 115, and 117. In yet another embodiment, the one or more nucleic acid molecules encoding 3HIV kinase are encoded by a nucleic acid sequence selected from SEQ ID NOs: 112, 114, and 116.

[0492] Mevalonate diphosphate decarboxylase (EC 4.1.1.-)

[0493] The present disclosure describes enzymes that can catalyze the following reactions:

[0494] (R)-Mevalonate diphosphate + ATP → isopentenyl diphosphate + CO 2 +ADP+Phosphate

[0495] 3-Phosphinoylisovaleric acid → CO 2 +Isobutylene

[0496] 3-Hydroxyisovaleric acid → CO 2 +Isobutylene

[0497] Mevalonate diphosphate decarboxylase may also be referred to as pyrophosphomevalonate decarboxylase, mevalonate-5-pyrophosphate decarboxylase, pyrophosphomevalonic acid decarboxylase, 5-pyrophosphomevalonate decarboxylase, mevalonate 5-diphosphate decarboxylase and ATP:(R)-5-diphosphomevalonate carboxyl-lyase (dehydratase), 3-phosphinoisovalerate decarboxylase, 3-hydroxyisovalerate-3-phosphate decarboxylase, 3HIV-3-phosphate decarboxylase, 3-hydroxyisovalerate decarboxylase and 3HIV decarboxylase.

[0498] The enzyme converts mevalonate 5-diphosphate (MVAPP) to isopentenyl diphosphate (IPP) via ATP-dependent decarboxylation. The two substrates of the enzyme are ATP and mevalonate 5-diphosphate, while its four products are ADP, phosphate, isopentenyl diphosphate, and CO. 2 .

[0499] Mevalonate diphosphate decarboxylase catalyzes the last step of the mevalonate pathway. The mevalonate pathway is responsible for the biosynthesis of isoprenoids from acetate. This pathway plays a key role in multiple cellular processes through the synthesis of sterol isoprenoids (such as cholesterol) and non-sterol isoprenoids (such as dolichol, heme A, tRNA prenyltransferase, and ubiquinone). The enzyme belongs to the lyase family, specifically the carboxyl lyase, which cleaves carbon-carbon bonds.

[0500] Mevalonate diphosphate decarboxylase recognizes and binds two substrates: ATP and mevalonate 5-diphosphate. After binding, the enzyme performs three types of reactions, which can be divided into two main stages. First, phosphorylation occurs. This produces a reactive intermediate, which undergoes concerted dephosphorylation and decarboxylation in the second stage.

[0501] In one embodiment, catalysis of 3-phosphinoylisovaleric acid → CO 2 + isobutylene-reactive enzyme is 3HIV-3-phosphate decarboxylase. In another embodiment, 3HIV-3-phosphate decarboxylase is encoded by one or more nucleic acid molecules obtained from Streptococcus species. In some embodiments, the microorganism is selected from Streptococcus mitis and / or Streptococcus gordonii. In some embodiments, one or more nucleic acid molecules encoding 3HIV-3-phosphate decarboxylase comprise an amino acid sequence selected from SEQ ID NOs: 119 and 121. In a further embodiment, one or more nucleic acid molecules encoding 3HIV-3-phosphate decarboxylase are encoded by a nucleic acid sequence selected from SEQ ID NOs: 118 and 120.

[0502] In one embodiment, the catalytic reaction 3-hydroxyisovalerate → CO 2+ isobutylene enzyme is 3HIV decarboxylase. In another embodiment, 3HIV decarboxylase is encoded by one or more nucleic acid molecules obtained from microorganisms selected from Streptococcus species, Thermoplasma species and Acidophilus Archaea species. In another embodiment, 3HIV decarboxylase is encoded by one or more nucleic acid molecules obtained from microorganisms selected from Streptococcus gordonii, Thermoplasma acidophilus and Acidophilus Archaea toseri. In some embodiments, one or more nucleic acid molecules encoding 3HIV decarboxylase include mvaD, TA1305 and / or PTO1356, or homologs thereof. In a further embodiment, one or more nucleic acid molecules encoding 3HIV decarboxylase comprise an amino acid sequence selected from SEQ ID NOs: 113, 117 and 121. In yet another embodiment, one or more nucleic acid molecules encoding 3HIV decarboxylase are encoded by a nucleic acid sequence selected from SEQ ID NOs: 112, 116 and 120.

[0503] Transketolase (EC 2.2.1.1)

[0504] The present disclosure describes enzymes that can catalyze the following reactions:

[0505]

[0506]

[0507] Transketolase catalyzes the reversible transfer of a ketol group between several donor and acceptor substrates. This key enzyme is a reversible link between glycolysis and the pentose phosphate pathway. The enzyme is involved in the catabolism of pentoses, the formation of D-ribose 5-phosphate, and the provision of D-erythrose 4-phosphate, a precursor for aromatic amino acids and PLP. Escherichia coli contains two transketolase isozymes, TktA and TktB. TktA is responsible for the main transketolase activity.

[0508] In addition to its function in central carbon metabolism, transketolase has also been shown to have an unexpected role in chromosome structure; the tktA mutant affects chromosome topology.

[0509] The crystal structure of TktA in complex with donor and acceptor substrates has been solved, elucidating the reaction mechanism and mode of action of transketolase. A computational model of transketolase activity using quantum mechanical / molecular mechanics methods has been proposed, defining a new pathway for thiamine diphosphate activation. Transketolase I (TktA) is a homodimer. The urea denaturation pathway of TktA wild-type and active site mutants has been studied, and the effects of temperature and pH on the structure, stability, aggregation and activity of transketolase have been determined. The receptor specificity of TktA has been studied.

[0510] The abundance of TktA was affected by the SOS inducer and mutagen 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000). tktA was negatively regulated during entry into stationary phase. The effect of RpoS may be indirect and may be mediated by intermediate regulators that are themselves directly regulated by RpoS.

[0511] The subunit structure of transketolase II (TktB) has not been clearly determined. Overproduction of TktB suppresses the tktA mutant phenotype. Expression of tktB is increased in the tyrR mutant in the presence of phenylalanine. tktB expression increases during stationary phase and is positively regulated by RpoS and ppGpp. Levels of TktB protein increase during osmotic stress under aerobic but not anaerobic growth conditions. TktB appears to associate with the degradosome and may link carbon metabolism to replication.

[0512] The expression of tktA and tktB is complementary, resulting in approximately constant levels of transketolase expression throughout growth.

[0513] In some embodiments, the transketolase is encoded by an amino acid sequence having at least 70% sequence identity with tktA from E. coli. In a further embodiment, the transketolase is encoded by an amino acid sequence having at least 80% sequence identity with tktA from E. coli. In a still further embodiment, the transketolase is encoded by an amino acid sequence having at least 90% sequence identity with tktA from E. coli. In other embodiments, the transketolase is tktA from E. coli. In some embodiments, the transketolase is encoded by an amino acid sequence having at least 70% sequence identity with tktB from E. coli. In a further embodiment, the transketolase is encoded by an amino acid sequence having at least 80% sequence identity with tktB from E. coli. In a still further embodiment, the transketolase is encoded by an amino acid sequence having at least 90% sequence identity with tktB from E. coli. In other embodiments, the transketolase is tktB from E. coli.

[0514] In some embodiments, the one or more nucleic acid molecules encoding a transketolase are tktA, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding a transketolase are tktB, or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding a transketolase comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 148 and 150. In a further embodiment, the one or more nucleic acid molecules encoding a transketolase are encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 147 and 149.

[0515] Transaldolase (EC 2.2.1.2)

[0516] The present disclosure describes enzymes that can catalyze the following reactions:

[0517]

[0518] Transaldolase may also be called dihydroxyacetone transferase; dihydroxyacetone synthase; formaldehyde transketolase.

[0519] Transaldolase B is an enzyme of the nonoxidative branch of the pentose phosphate pathway. Together with transketolase, transaldolase establishes a reversible link between the pentose phosphate pathway and glycolysis. It catalyzes the interconversion of glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate into fructose-6-phosphate and erythrose-4-phosphate. The reversibility of this reaction and the carbon flow through the pentose phosphate pathway have been solved experimentally and theoretically.

[0520] There are two closely related transaldolases in Escherichia coli, encoded by talA and talB. Only transaldolase B has been characterized biochemically. TalB is a dimer in solution and in the crystal structure. Mutation of the R300 residue results in the formation of a catalytically active monomer. The active site residues important for catalysis were identified by site-directed mutagenesis.

[0521] The crystal structure of transaldolase B has been determined, confirming the presence of a Schiff base intermediate in the active site and leading to a proposed reaction mechanism.

[0522] The talB null mutant has no growth defect on minimal medium with glucose as the carbon source.

[0523] In some embodiments, the transaldolase is encoded by an amino acid sequence having at least 70% sequence identity with talA or talB from Escherichia coli. In a further embodiment, the transaldolase is encoded by an amino acid sequence having at least 80% sequence identity with talA or talB from Escherichia coli. In a still further embodiment, the transaldolase is encoded by an amino acid sequence having at least 90% sequence identity with talA or talB from Escherichia coli. In other embodiments, the transaldolase is talA from Escherichia coli. In still further embodiments, the transaldolase is talB from Escherichia coli.

[0524] In some embodiments, the one or more nucleic acid molecules encoding transaldolase are talA, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding transaldolase are talB, or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding transaldolase comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 152 and 154. In a further embodiment, the one or more nucleic acid molecules encoding transaldolase are encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 151 and 153.

[0525] Ribose-5-phosphate isomerase (EC 5.3.1.6)

[0526] The present disclosure describes enzymes that can catalyze the following reactions:

[0527]

[0528] Ribose-5-phosphate isomerase may also be referred to as pentose phosphate isomerase; phosphoribose isomerase; ribose phosphate isomerase; 5-phosphoribose isomerase; D-ribose 5-phosphate isomerase; D-ribose-5-phosphate ketol isomerase.

[0529] There are two physically and genetically distinct ribose-5-phosphate isomerases in Escherichia coli. Constitutive ribose-5-phosphate isomerase A (rpiA) normally accounts for more than 99% of the ribose-5-phosphate isomerase activity in the cell and functions in the pentose phosphate pathway (non-oxidative branch). Inducible ribose-5-phosphate isomerase B (rpiB) can replace the function of rpiA if its expression is induced. There is no sequence similarity between the two enzymes.

[0530] The crystal structure of rpiA has been solved, and the active site residues and acid-base catalytic mechanism have been predicted. The rpiA mutant requires ribose for growth.

[0531] In some embodiments, the ribose-5-phosphate isomerase is encoded by an amino acid sequence having at least 70% sequence identity with rpiA or rpiB from E. coli. In a further embodiment, the ribose-5-phosphate isomerase is encoded by an amino acid sequence having at least 80% sequence identity with rpiA or rpiB from E. coli. In a still further embodiment, the ribose-5-phosphate isomerase is encoded by an amino acid sequence having at least 90% sequence identity with rpiA or rpiB from E. coli. In other embodiments, the ribose-5-phosphate isomerase is rpiA or rpiB from E. coli.

[0532] In some embodiments, the one or more nucleic acid molecules encoding ribose-5-phosphate isomerase are rpiA, or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding ribose-5-phosphate isomerase comprise the amino acid sequence set forth in SEQ ID NO: 156. In a further embodiment, the one or more nucleic acid molecules encoding ribose-5-phosphate isomerase are encoded by the nucleic acid sequence set forth in SEQ ID NO: 155. In some embodiments, the one or more nucleic acid molecules encoding ribose-5-phosphate isomerase are rpiB, or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding ribose-5-phosphate comprise the amino acid sequence set forth in SEQ ID NO: 253. In a further embodiment, the one or more nucleic acid molecules encoding ribose-5-phosphate isomerase are encoded by the nucleic acid sequence set forth in SEQ ID NO: 254.

[0533] Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1)

[0534] The present disclosure describes enzymes that can catalyze the following reactions:

[0535]

[0536] Ribulose-5-phosphate 3-epimerase may also be referred to as ribulose-phosphate 3-epimerase; ribulose phosphate epimerase; erythrose-4-phosphate isomerase; phosphotopentose 3-epimerase; xylulose phosphate 3-epimerase; phosphotopentose epimerase; D-ribulose phosphate-3-epimerase; D-ribulose 5-phosphate epimerase; D-ribulose-5-P 3-epimerase; D-xylulose-5-phosphate 3-epimerase; pentose-5-phosphate 3-epimerase.

[0537] Ribulose-5-phosphate 3-epimerase (Rpe) is an enzyme of the nonoxidative branch of the pentose phosphate pathway.

[0538] Rpe requires ferrous iron for activity and is susceptible to H2 due to Fenton chemistry. 2 O 2 Mn 2+ 、Co 2+ and Zn 2+ Can replace Fe to varying degrees 2+ , and Rpe containing these alternative cations are not susceptible to H 2 O 2 Induction of manganese transporters can protect Rpe from H 2 O 2 damage.

[0539] In some embodiments, the ribulose-5-phosphate 3-epimerase is encoded by an amino acid sequence having at least 70% sequence identity with RPE from Escherichia coli. In a further embodiment, the ribulose-5-phosphate 3-epimerase is encoded by an amino acid sequence having at least 80% sequence identity with RPE from Escherichia coli. In a still further embodiment, the ribulose-5-phosphate 3-epimerase is encoded by an amino acid sequence having at least 90% sequence identity with RPE from Escherichia coli. In other embodiments, the ribulose-5-phosphate 3-epimerase is RPE from Escherichia coli.

[0540] In some embodiments, the one or more nucleic acid molecules encoding ribulose-5-phosphate 3-epimerase are rpe, or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding ribulose-5-phosphate 3-epimerase comprise the amino acid sequence set forth in SEQ ID NO: 158. In a further embodiment, the one or more nucleic acid molecules encoding ribulose-5-phosphate 3-epimerase are encoded by the nucleic acid sequence set forth in SEQ ID NO: 157.

[0541] Fructose 6-phosphate phosphoketolase (Fpk, EC 4.1.2.22)

[0542] The present disclosure describes enzymes that can catalyze the following reactions:

[0543]

[0544] The phosphoketolase reaction of converting β-D-fructofuranosyl 6-phosphate into D-erythrose 4-phosphate and acetyl phosphate is one of the key reactions in the Bifidobacterium bypass. There is evidence that there are two different F6P-phosphoketolase enzymes in Bifidobacterium. One is specific only to F6P, while the other is able to utilize F6P and D-xylulose 5-phosphate (EC: 4.1.2.9), which appears later in the Bifidobacterium bypass. The enzyme encoded by the xfp gene was originally found in Bifidobacterium animalis lactis, which is a dual-specific enzyme. Phosphoketolase has also been purified from Leuconostoc mesenteroides (LEUM_1961).

[0545] In some embodiments, the enzyme having fructose-6-phosphate phosphoketolase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with an enzyme having fructose-6-phosphate phosphoketolase activity selected from the group consisting of Bifidobacterium dentium BDP_1006, Bifidobacterium lactis xfp, Lactobacillus paraplantarum xpkA, and Bifidobacterium breve xfp. In a preferred embodiment, the enzyme having fructose-6-phosphate phosphoketolase activity is selected from the group consisting of Bifidobacterium dentium BDP_1006, Bifidobacterium lactis xfp, Lactobacillus paraplantarum xpkA, and Bifidobacterium breve xfp. In another embodiment, the one or more nucleic acid molecules encoding the fructose-6-phosphate phosphoketolase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 212, 214, 216 and 218. In a further embodiment, the one or more nucleic acid molecules encoding the fructose-6-phosphate phosphoketolase is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 211, 213, 215 and 217.

[0546] Phosphate acetyltransferase (EC 2.3.1.8)

[0547] The present disclosure describes enzymes that can catalyze the following reactions:

[0548]

[0549] Phosphate acetyltransferase (Pta) catalyzes the reversible conversion between acetyl-CoA and acetyl phosphate, a step in acetate metabolism. Both pyruvate and phosphoenolpyruvate activate the enzyme in the direction of acetyl phosphate synthesis and inhibit the enzyme in the direction of acetyl-CoA synthesis. The formation of acetate from the acetyl-CoA I pathway has been a target of metabolic engineering to reduce the flow to acetate and increase the yield of commercially desirable end products. It has also been studied using systems biology approaches such as metabolic modeling and flow balance analysis.

[0550] Pta consists of three domains; only the C-terminal domain is required for phosphate acetyltransferase activity. The N-terminal domain is involved in the stabilization of the native quaternary structure and in metabolic regulation.

[0551] Pta may be able to utilize both acetyl-CoA and propionyl-CoA. The ack pta double mutant has reduced levels of propionate from L-threonine, suggesting that the enzyme is part of an anaerobic pathway that metabolizes L-threonine to propionate. The pta mutant cannot grow on acetate as the sole carbon source. Both the pta and pta ackA mutants are impaired in their ability to survive glucose starvation. The growth defect of the pta mutant appears to be due to a perturbation in the flow of acetyl-CoA. The pta mutant produces large amounts of lactic acid when grown on glucose as a carbon source under microaerobic conditions. The effects of the pta mutation on metabolism, enzyme activity, and gene expression have recently been thoroughly studied. The pta and recBC mutants are synthetically growth inhibited.

[0552] Growth on acetic acid and under low pH conditions reduced the levels of Pta. pta belongs to the CreBC regulon. FNR had a slight positive effect on pta expression. The growth rate-dependent expression pattern of pta-ackA was measured.

[0553] The pta gene encoding the enzyme has been cloned from Clostridium acetobutylicum, sequenced and expressed in E. coli. The gene is adjacent to the ackA gene, which encodes the enzyme acetate kinase that catalyzes the second step. Enzyme activity assays performed on cell extracts from E. coli and Clostridium acetobutylicum containing the subclone showed an increase in activity. The enzyme showed a decrease in specific activity when the organism reached the solvent formation stage.

[0554] Enzymes with phosphate acetyltransferase activity or phosphate acetyltransferase genes have also been identified or measured from Escherichia coli (eutD, pta), Roseovarius nubinhibens ISM, Clostridium kluyveri, Chlamydomonas reinhardtii (PAT2), Dasytricharuminantium, Pelobacter acetylenicus, Gottschalkia acidurici, Lactobacillus sanfranciscensis, Paracoccus denitrificans NKNIS, Eubacterium oxidoreducens G41, Mycoplasma pneumoniae M129, Thermotoga maritima, Moorella thermoacetica, Methanosarcinathermophile, Clostridium propionicum propionicum and Fusobacteriumnucleatum.

[0555] In some embodiments, the enzyme having phosphate acetyltransferase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with an enzyme having phosphate acetyltransferase activity selected from the group consisting of Escherichia coli pta and Clostridium acetobutylicum pta. In a preferred embodiment, the enzyme having phosphate acetyltransferase activity is selected from the group consisting of Escherichia coli pta and Clostridium acetobutylicum pta. In another embodiment, the one or more nucleic acid molecules encoding phosphate acetyltransferase comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 220 and 222. In a further embodiment, the one or more nucleic acid molecules encoding phosphate acetyltransferase are encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 219 and 221.

[0556] Pentose phosphatase (EC 3.1.3.23 and EC 3.1.3.1)

[0557] The present disclosure describes enzymes that can catalyze the following reactions:

[0558] D-pentose 5-phosphate + H 2 O→D-pentose + phosphate

[0559] D-ribose 5-phosphate + H2 O→D-ribose+phosphate

[0560] D-Xylulose 5-phosphate + H 2 O→D-xylulose+phosphate

[0561] D-Ribulose 5-phosphate + H 2 O→D-ribulose+phosphate

[0562] Sugar phosphate + H 2 O→Sugar+Phosphate

[0563] Escherichia coli YbiV is a sugar phosphatase that belongs to the type II haloacid dehalogenase (HAD)-like hydrolase family. It shows low-level discrimination between its preferred substrates. In addition, YbiV uses monophosphate as a phosphate donor and exhibits low-level phosphotransferase activity. The phosphatase activity of YbiV has also been found in high-throughput screening of purified proteins. The crystal structure of YbiV has been resolved and a catalytic mechanism has been proposed. YbiV may exist in solution as a homodimer.

[0564] Escherichia coli YidA is a promiscuous sugar phosphatase that belongs to the haloacid dehalogenase (HAD)-like hydrolase superfamily. Its preferred substrate is erythrose-4-phosphate. YidA selectively hydrolyzes α-D-glucose-1-phosphate, while having no activity on the β form. The reaction proceeds via a classical phosphomonoester hydrolase mechanism involving the cleavage of the PO bond, rather than the cleavage of the C1-O bond. The phosphatase activity of YidA was initially discovered in a high-throughput screen of purified proteins. Mutagenesis of the predicted catalytic Asp residue in YidA resulted in a loss of phosphatase activity. YidA does not catalyze phosphoryl transfer to sugar acceptors.

[0565] Escherichia coli alkaline phosphatase (phoA) is a periplasmic homodimeric enzyme that catalyzes the hydrolysis and transphosphorylation of a wide variety of phosphomonoesters. The reaction proceeds through a phosphoseryl intermediate, followed by the release of inorganic phosphate and alcohol. The transphosphorylation reaction results in the transfer of the phosphoryl group to the alcohol of an acceptor such as Tris or ethanolamine. Alkaline phosphatase is a metalloenzyme, with each monomer binding two zinc atoms and one magnesium ion. Alkaline phosphatase exists in three major forms, called isozymes 1, 2, and 3, the relative proportions of which depend on growth conditions. 2The isozymes are distinguished by the presence or absence of a -terminal arginine residue: present in both subunits of isozyme 1, absent in both subunits of isozyme 3, and heterozygous in isozyme 2. The membrane-bound proteolytic enzyme Iap catalyzes the removal of the N-terminal arginine. The precursor polypeptide is secreted across the inner membrane into the periplasmic space with simultaneous removal of the signal sequence. The periplasmic protein DsbA catalyzes the folding of PhoA in vivo and is thought to occur as the polypeptide elongates from the ribosome. PhoA is part of the phosphoregulon; its expression is positively regulated by the PhoB transcriptional regulator.

[0566] In some embodiments, the pentose phosphatase is an alkaline phosphatase. In some preferred embodiments, the alkaline phosphatase can evolve to preferably act on the pentose 5-phosphate of targeting. In some embodiments, the pentose phosphatase is a sugar phosphatase. In other embodiments, the sugar phosphatase can evolve to preferably act on the pentose 5-phosphate of targeting. In some embodiments, the pentose phosphatase is a halogen acid dehalogenase sample hydrolase. In another embodiment, the halogen acid dehalogenase sample hydrolase can evolve to preferably act on the pentose 5-phosphate of targeting.

[0567] In some embodiments, the pentose phosphatase is selected from one or more of the following: D-pentose-5-phosphatase, D-xylulose-5-phosphatase, D-ribose-5-phosphatase and D-ribulose-5-phosphatase. In some embodiments, the pentose phosphatase is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity or at least 90% sequence identity with a D-pentose-5-phosphatase selected from the following: Escherichia coli phoA, Escherichia coli yfbT and Escherichia coli yidA. In some embodiments, the D-xylulose-5-phosphatase is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity or at least 90% sequence identity with Bacillus subtilis araL. In some embodiments, the D-ribose-5-phosphatase is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with a D-ribose-5-phosphatase selected from Arabidopsis thaliana SGPP, Pseudomonas fluorescens PFLU_2693, and Escherichia coli ybiV. In some embodiments, the D-ribulose-5-phosphate isomerase is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with Plasmodium falciparum PF10_0325.

[0568] In some embodiments, the one or more nucleic acid molecules encoding a D-pentose-5-phosphatase are selected from the group consisting of SEQ ID NOs: 159, 161, 163, 165, 167, 169, 171, and 173. In another embodiment, the one or more nucleic acid molecules encoding a D-pentose-5-phosphatase comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 160, 162, 164, 166, 168, 170, 172, and 174.

[0569] Arabitol phosphate dehydrogenase (EC 1.1.1.-)

[0570] The present disclosure describes enzymes that can catalyze the following reactions:

[0571]

[0572]

[0573]

[0574]

[0575] D-arabitol phosphate dehydrogenase APDH from Enterococcus avium has been purified to homogeneity. The protein forms a homotetramer and catalyzes the dehydrogenation of D-arabitol 1-phosphate and D-arabitol 5-phosphate to produce D-xylulose 5-phosphate and D-ribulose 5-phosphate, respectively. The maximum rate with D-arabitol 1-phosphate is 10 times that with D-arabitol 5-phosphate. The purified protein was partially sequenced and the APDH gene encoding it was cloned. The enzyme requires Mn 2+ , and cannot utilize Zn for activity 2+ . Accepts both NAD(+) and NADP(+) as cofactors, but the reaction rate with NAD+ / NADH is about 14 times higher than with NADP+ / NADPH. The enzyme catalyzes a reversible reaction, but the rate of the reduction reaction is much higher than the oxidation reaction. Kinetic data indicate that the enzyme forms a ternary complex with its substrate and NADH. Biochemical evidence and protein sequence homology comparisons indicate that similar enzymes are widely present in Gram-positive bacteria and are involved in arabitol catabolism.

[0576] In some embodiments, the pentanol dehydrogenase activity can be evolved to be applicable to pentanol phosphate.

[0577] In some embodiments, the arabitol phosphate dehydrogenase is selected from one or more of the following: an enzyme having D-arabitol 1-phosphate 4-dehydrogenase activity, an enzyme having D-arabitol 5-phosphate 2-dehydrogenase activity, an enzyme having D-arabitol 1-phosphate 2-dehydrogenase activity, and an enzyme having D-arabitol 5-phosphate 4-dehydrogenase activity.

[0578] In some embodiments, the enzyme having D-arabitol 1-phosphate 4-dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to APDH from Enterococcus avium. In a preferred embodiment, the enzyme having D-arabitol 1-phosphate 4-dehydrogenase activity is APDH from Enterococcus avium. In some embodiments, one or more nucleic acid molecules encoding D-arabitol 1-phosphate 4-dehydrogenase are listed in SEQ ID NO: 175. In another embodiment, one or more nucleic acid molecules encoding D-arabitol 1-phosphate 4-dehydrogenase comprise the amino acid sequence listed in SEQ ID NO: 176.

[0579] In some embodiments, the enzyme having D-arabitol 5-phosphate 2-dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to APDH from Enterococcus avium. In a preferred embodiment, the enzyme having D-arabitol 5-phosphate 2-dehydrogenase activity is APDH from Enterococcus avium. In some embodiments, one or more nucleic acid molecules encoding D-arabitol 5-phosphate 2-dehydrogenase are listed in SEQ ID NO: 175. In another embodiment, one or more nucleic acid molecules encoding D-arabitol 5-phosphate 2-dehydrogenase comprise the amino acid sequence listed in SEQ ID NO: 176.

[0580] In some embodiments, the enzyme having D-arabitol 1-phosphate 2-dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with an enzyme having D-arabitol 1-phosphate 2-dehydrogenase activity selected from the group consisting of Candida albicans ARD1, Candida tropicalis ARD1, Scheffersomyces stipitis ARDH, Bacillus subtilis egsA (araM), Aeropyrum swiftii egsA, Escherichia coli gpsA, and Saccharomyces cerevisiae GPD1. In some embodiments, the enzyme having D-arabitol 1-phosphate 2-dehydrogenase activity is selected from the group consisting of Candida albicans ARD1, Candida tropicalis ARD1, Scheffersomyces stipitis ARDH, Bacillus subtilis egsA (araM), Aeropyrum swiftii egsA, Escherichia coli gpsA, and Saccharomyces cerevisiae GPD1. In some embodiments, the one or more nucleic acid molecules encoding D-arabitol 1-phosphate 2-dehydrogenase are selected from SEQ ID NOs: 177, 179, 181, 189, 191, 193, and 195. In another embodiment, the one or more nucleic acid molecules encoding D-arabitol 1-phosphate 2-dehydrogenase comprise an amino acid sequence selected from SEQ ID NOs: 178, 180, 182, 190, 192, 194, and 196.

[0581] In some embodiments, the enzyme having D-arabitol 5-phosphate 4-dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to an enzyme having D-arabitol 5-phosphate 4-dehydrogenase activity selected from the group consisting of: Pseudomonas fluorescens mtlD, Klebsiella pneumoniae dalD, Ralstonia solanacearum dalD, Bacillus subtilis egsA (araM), Aeropyrum facilis egsA, Escherichia coli gpsA, and Saccharomyces cerevisiae GPD1. In a preferred embodiment, the enzyme having D-arabitol 5-phosphate 4-dehydrogenase activity is selected from Pseudomonas fluorescens mtlD, Klebsiella pneumoniae dalD, Ralstonia solanacearum dalD, Bacillus subtilis egsA (araM), Aeropyrum facilis egsA, Escherichia coli gpsA and Saccharomyces cerevisiae GPD1. In some embodiments, the one or more nucleic acid molecules encoding D-arabitol 5-phosphate 4-dehydrogenase are selected from SEQ ID NOs: 183, 185, 187, 189, 191, 193 and 195. In another embodiment, the one or more nucleic acid molecules encoding D-arabitol 5-phosphate 4-dehydrogenase comprise an amino acid sequence selected from SEQ ID NOs: 184, 186, 188, 190, 192, 194 and 196.

[0582] Pentose phosphate mutase (EC 5.4.2.-)

[0583] The present disclosure describes enzymes that can catalyze the following reactions:

[0584]

[0585]

[0586]

[0587] 2-Deoxy-α-D-ribose 1-phosphate → 2-deoxy-D-ribose 5-phosphate

[0588] Escherichia coli pentosephosphomutase deoB is a catabolic enzyme that catalyzes the transfer of phosphate groups between the C1 and C5 carbon atoms of ribose and deoxyribose, respectively.

[0589] Mutations in deoB suppress the high thymine requirement for growth of the thy mutant and improve the survival of the thyA mutant in stationary phase. Transposon insertion mutations in deoB suppress the growth defect of the tktA tktB mutant. Deletion of deoB increases glycerol consumption and hydrogen and ethanol production and lycopene production in the engineered strain compared with the wild type. The deo operon has a complex regulatory pattern. Nitrogen starvation downregulates the expression of deoB. Escherichia coli pentosephosphomutase behaves biochemically and structurally differently from mammalian pentosephosphomutases, making it a potential target for antibiotic development.

[0590] In some embodiments, the phosphopentose mutase (PPM), also referred to herein as a phosphosugar mutase (PSM), also has phosphoglucomutase activity and is Saccharomyces cerevisiae phosphoribosylmutase PRM15 (also referred to as PGM3) (SEQ ID NO: 255 or SEQ ID NO: 258). In some embodiments, the phosphopentose mutase is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to Pgm3 from Saccharomyces cerevisiae.

[0591] In some embodiments, the phosphopentose mutase is human phosphoglucomutase-2 (PGM2).

[0592] In some embodiments, the pentose phosphate mutase activity can be evolved to be applicable to D-xylulose 5-phosphate. In other embodiments, the pentose phosphate mutase activity can be evolved to be applicable to D-ribulose 5-phosphate. In some embodiments, the α-phosphoglucomutase activity can be evolved to be applicable to D-ribulose 5-phosphate or D-xylulose 5-phosphate. It is reported that the enzyme from the EC 5.4.2.2 class requires α-glucose 1,6-bisphosphate as a cofactor. In further embodiments, the β-phosphoglucomutase activity can be evolved to be applicable to D-ribulose 5-phosphate or D-xylulose 5-phosphate. It is reported that the enzyme from the EC 5.4.2.6 class can be phosphorylated by itself, so external glucose 1,6-bisphosphate is not required as a cofactor. In still further embodiments, the phosphomannose mutase activity can be evolved to be applicable to D-ribulose 5-phosphate or D-xylulose 5-phosphate. This enzyme from the EC 5.4.2.8 class is reported to require either α-glucose 1,6-bisphosphate or α-D-mannose 1,6-bisphosphate as a cofactor.

[0593] In some embodiments, the enzyme having pentose phosphate mutase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with an enzyme having pentose phosphate mutase activity selected from the group consisting of Escherichia coli deoB, Escherichia coli pgm, Bacillus subtilis pgcA, Lactococcus lactis pgmB, Escherichia coli ycjU, Pseudomonas aeruginosa algC, and Escherichia coli cpsG. In a preferred embodiment, the enzyme having pentose phosphate mutase activity is selected from the group consisting of Escherichia coli deoB, Escherichia coli pgm, Bacillus subtilis pgcA, Lactococcus lactis pgmB, Escherichia coli ycjU, Pseudomonas aeruginosa algC, Saccharomyces cerevisiae PGM3, and Escherichia coli cpsG. In some embodiments, the one or more nucleic acid molecules encoding the pentose phosphate mutase are selected from SEQ ID NOs: 197, 199, 201, 203, 205, 207, and 209. In another embodiment, the one or more nucleic acid molecules encoding the pentose phosphate mutase comprise an amino acid sequence selected from SEQ ID NOs: 198, 200, 202, 204, 206, 208, and 210.

[0594] Glucose-6-phosphate 1-dehydrogenase (EC 1.1.1.49)

[0595] The present disclosure describes enzymes that can catalyze the following reactions:

[0596]

[0597] Glucose-6-phosphate 1-dehydrogenase may also be referred to as glucose-6-phosphate dehydrogenase (NADP+); NADP-glucose-6-phosphate dehydrogenase; intermediate fermentation (Zwischenferment); D-glucose 6-phosphate dehydrogenase; glucose 6-phosphate dehydrogenase (NADP); NADP-dependent glucose 6-phosphate dehydrogenase; 6-phosphoglucose dehydrogenase; Entner-Doudoroff enzyme; G6PDH; GPD; glucose-6-phosphate dehydrogenase.

[0598] Glucose-6-phosphate dehydrogenase (G6PDH) is the first enzyme in the pentose phosphate pathway and provides most of the NADPH required for anabolism.

[0599] Escherichia coli G6PDH displays a strong preference for NADP+ over NAD+. The structural basis of this preference was investigated using molecular modeling, kinetic characterization of site-directed mutants, and phylogenetic analysis.

[0600] Using GC-MS and 13Metabolic flux through central carbon metabolic pathways was measured using C labeling and 2D NMR spectroscopy, and regulation of these pathways was measured at the level of enzyme expression and activity under different growth conditions.

[0601] Replacing the native NADPH-producing enzyme with NADH-producing glucose-6-phosphate dehydrogenase reduced the growth rate of wild-type cells while increasing the growth rate of the Δpgi mutant, suggesting that whether NADH production by G6PDH is beneficial or detrimental in vivo depends on the operation of the upstream Embden-Meyerhof pathway.

[0602] In addition to its role in central carbon metabolism, G6PDH was found to be the source of a linear peptide with the amino acid sequence Asn-Asn-Trp-Asn-Asn (NNWNN), which acts as an "extracellular death factor" (EDF) for MazEF-mediated cell death. The peptide exerts its effects by increasing the endoribonuclease activity of the toxins MazF and ChpBK. EDF production under stress conditions is due to MazF cleaving zwf mRNA at a specific ACA site, generating a leaderless truncated mRNA. The position of the EDF coding region relative to the MazF cleavage site is important and requires a trans-translation system.

[0603] zwf is one of the most consistently flux-coupled genes, genes whose expression switching patterns upon perturbation correlate with their corresponding flux values. The expression of zwf is regulated at the transcriptional level by growth rate. G6PDH activity is higher in rapidly growing cells and is higher under nitrogen-limited compared to carbon-limited growth conditions. zwf is part of the SoxRS regulon that responds to superoxide stress. Other regulators have been shown to activate the transcription of zwf. Exposure to tellurite activates the transcription of zwf, thereby increasing the synthesis of NADPH.

[0604] The zwf null mutation does not significantly affect the growth rate. However, central carbon metabolism and metabolic flux are altered. The pgizwf double mutant cannot grow on glucose as the sole carbon source. In the presence of glucose, it accumulates high levels of glucose-6-phosphate and inhibits the activity of fructose-1,6-bisphosphatase I. The loss of zwf reduces the organic solvent tolerance of Escherichia coli JM109.

[0605] In some embodiments, a recombinant microorganism that produces MEG (or glycolic acid), or optionally MEG (or glycolic acid) and one or more co-products, comprises a deletion, insertion, or loss-of-function mutation in a gene encoding glucose-6-phosphate dehydrogenase to prevent the flow of glucose-6-phosphate through the oxidative branch of the pentose phosphate pathway and instead shunts glucose-6-phosphate through the non-oxidative branch of the pentose phosphate pathway to produce one or more pentose 5-phosphate intermediates.

[0606] 6-Phosphogluconolactonase (EC 3.1.1.31)

[0607] The present disclosure describes enzymes that can catalyze the following reactions:

[0608] 6-phospho-D-glucono-1,5-lactone+H 2 O→D-glucose 6-phosphate + H+

[0609] 6-Phosphogluconolactonase may also be referred to as phosphogluconolactonase; 6-PGL.

[0610] 6-Phosphogluconolactonase is an enzyme of the oxidative pentose phosphate pathway.

[0611] The pgl mutant grows only slightly slower than the wild type on glucose as the sole carbon source. Growth on glucose is probably due to nonenzymatic hydrolysis of 6-phospho-D-glucono-1,5-lactone or an alternative pathway involving dephosphorylation and export of glucono-lactone, hydrolysis to gluconic acid, followed by re-import and phosphorylation of gluconic acid. When grown on maltose medium, strains lacking Pgl activity turn blue upon iodine treatment. The phenotype of the pgl deletion strain can be compensated by expression of the pgl gene from Pseudomonas putida, although there is no detectable similarity between the two genes.

[0612] The metabolic engineering strategy for Escherichia coli to produce riboflavin included overexpression of pgl, which resulted in increased riboflavin titers.

[0613] pgl is part of a genomic region that is deleted in E. coli B strain BL21 but is present in K-12 strain MG1655.

[0614] In some embodiments, a recombinant microorganism that produces MEG (or glycolic acid), or optionally MEG (or glycolic acid) and one or more co-products, comprises a deletion, insertion, or loss-of-function mutation in a gene encoding 6-phosphogluconolactonase to prevent the flow of glucose-6-phosphate through the oxidative branch of the pentose phosphate pathway and instead shunts glucose-6-phosphate through the non-oxidative branch of the pentose phosphate pathway to produce one or more pentose 5-phosphate intermediates.

[0615] 6-Phosphogluconate dehydrogenase, decarboxylating (EC 1.1.1.44)

[0616] The present disclosure describes enzymes that can catalyze the following reactions:

[0617] D-glucose 6-phosphate + NADP + → D-ribulose 5-phosphate + CO 2 +NADPH

[0618] 6-phosphogluconate dehydrogenase may also be referred to as phosphogluconate dehydrogenase (NADP+ dependent, decarboxylating); phosphogluconate dehydrogenase; 6-phosphogluconate dehydrogenase; 6-phosphogluconate carboxylase; 6-phospho-D-gluconate dehydrogenase; glyceraldehyde 3-phosphate dehydrogenase.

[0619] 6-Phosphogluconate dehydrogenase is an enzyme of the oxidative branch of the pentose phosphate pathway.

[0620] Three crystal structures of the enzyme in complex with substrate and co-substrate compounds have been solved. Binding of NADP+ can induce conformational changes in the enzyme. A catalytic mechanism has been proposed.

[0621] gnd is a highly polymorphic gene in E. coli populations, probably due to inter-strain transfer and recombination. This may be because it is close to the rfb region that determines the structure of the O antigen.

[0622] Expression of 6-phosphogluconate dehydrogenase is regulated by growth rate. Most of the growth rate-dependent increase in Gnd levels is due to increased transcription, resulting in higher mRNA levels. Posttranscriptional regulation involves secondary structural elements between codons 67 and 78 of the gnd mRNA. This region may act by sequestering the translation initiation region into mRNA secondary structure, thereby reducing the efficiency of translation initiation. However, the effectors of this regulatory mechanism have apparently not yet been identified. The truA(hisT) mutant reduces the growth rate-dependent increase in Gnd expression via posttranscriptional regulation. Growth under acidic conditions upregulates the expression of gnd. gnd is one of the most consistently flow-coupled genes (FCGs), genes whose expression switching patterns upon perturbation correlate with their corresponding flow values.

[0623] Certain growth conditions selected for deletion mutations in the promoter region resulted in increased transcription of gnd and increased enzyme activity. The edd gnd double mutant was unable to grow on gluconic acid. Null mutations in gnd did not significantly alter growth rate. However, cellular metabolism and metabolic flux were altered; succinate production was increased during growth on glucose or glycerol. The gnd deletion mutant exhibited enhanced ethanol and H production during anaerobic growth on glycerol compared to wild type. 2In different highly engineered strains, overexpression of gnd increased ethanol and H 2 of production.

[0624] In some embodiments, a recombinant microorganism that produces MEG (or glycolic acid), or optionally MEG (or glycolic acid) and one or more co-products, comprises a deletion, insertion, or loss-of-function mutation in a gene encoding 6-phosphogluconate dehydrogenase to prevent the flow of glucose-6-phosphate through the oxidative branch of the pentose phosphate pathway and instead shunts glucose-6-phosphate through the non-oxidative branch of the pentose phosphate pathway to produce one or more pentose 5-phosphate intermediates.

[0625] Glyceraldehyde 3-phosphate dehydrogenase, phosphorylating (EC 1.2.1.12)

[0626] The present disclosure describes enzymes that can catalyze the following reactions:

[0627]

[0628] Glyceraldehyde 3-phosphate dehydrogenase may also be referred to as glyceraldehyde-3-phosphate dehydrogenase (phosphorylating); triose phosphate dehydrogenase; dehydrogenase, glyceraldehyde phosphate; glyceraldehyde phosphate dehydrogenase; 3-phosphate glyceraldehyde dehydrogenase; NAD+-dependent glyceraldehyde phosphate dehydrogenase; glyceraldehyde phosphate dehydrogenase (NAD+); glyceraldehyde-3-phosphate dehydrogenase (NAD+); NADH-glyceraldehyde phosphate dehydrogenase; glyceraldehyde-3-P-dehydrogenase.

[0629] Glyceraldehyde 3-phosphate dehydrogenase A catalyzes the reversible oxidative phosphorylation of D-glyceraldehyde-3-phosphate to 1,3-bisphospho-D-glycerate in the presence of NAD+ and phosphate during glycolysis and gluconeogenesis in Escherichia coli. This enzyme is also present in many other organisms and its properties have been extensively studied.

[0630] E. coli is unusual in having two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activities, encoded by gapA and epd (gapB). However, the enzyme encoded by gapA has efficient phosphorylated glyceraldehyde-3-phosphate dehydrogenase activity and low phosphorylated erythrose 4-phosphate dehydrogenase activity, while the enzyme encoded by epd has efficient non-phosphorylated erythrose-4-phosphate dehydrogenase activity and very low phosphorylated glyceraldehyde-3-phosphate dehydrogenase activity.

[0631] The GapA protein has a sequence that is more similar to eukaryotic sequences than to thermophilic bacterial enzymes and generally more similar to prokaryotic enzymes. Glycolysis requires the gapA product, while the epd product is not. Both enzymes can participate in the production of pyridoxal 5'-phosphate (PLP).

[0632] Early studies on a gapA mutant from Escherichia coli K-10 suggested its role in glycolysis and demonstrated some of its catalytic properties. The gapA mutant exhibited a growth defect and also displayed increased aggregation and lysis phenotypes that could be rescued by high salt media.

[0633] Regulation of expression of the gapA gene has been studied. Regulation of the fkpA, gapA and hslT genes has been affected by evolution under conditions of chronic heat stress.

[0634] The E. coli sequence contains several amino acids that are conserved among all GAPDHs and are postulated to be involved in the NAD+ binding or catalytic mechanism.

[0635] Has been The crystal structure of the wild-type enzyme in the presence of NAD+ was determined at a resolution of 1.3 Å, similar to other GAPDH crystal structures. The crystal structure of the N313T mutant was determined at a resolution of 1.3 Å. Several other crystal structures of E. coli GAPDH with and without bound NAD+, as well as in a hemiacetal intermediate state, have been reported.

[0636] The molecular factors responsible for the stereospecificity of the NAD+ cofactor have been studied using site-directed mutagenesis. The enzyme is a B-specific dehydrogenase that catalyzes the transfer of the pro-S hydrogen and binds NAD(H) in the cis-nicotinamide orientation. The triggering factors for the refolding of denatured E. coli GAPDH in the presence of the chaperone protein Tig have been studied.

[0637] ADP-ribosylated GAPDH is a secreted virulence factor in some fungi and Gram-positive pathogens, as well as in pathogenic strains of Escherichia coli. Nonpathogenic E. coli does not secrete GAPDH. Evidence suggests that E. coli GAPDH is also involved in DNA repair.

[0638] A series of vectors that can inducibly express paired-end antisense RNA were constructed to silence central carbon metabolism in the host Escherichia coli K-12MG1655. Vectors that silenced gapA with 93% efficiency resulted in severe growth inhibition. It has been demonstrated that regulating the expression of gapA in engineered E. coli by temperature changes can control glycolysis.

[0639] In some embodiments, the recombinant microorganism producing MEG (or glycolic acid) or optionally MEG (or glycolic acid) and one or more co-products comprises a deletion, insertion, or loss-of-function mutation in the gene encoding glyceraldehyde 3-phosphate dehydrogenase to prevent the conversion of glyceraldehyde 3-phosphate to 1,3-bisphospho-D-glycerate, but instead allows D-glyceraldehyde 3-phosphate to be converted to D-xylulose-5-phosphate (and simultaneously converts fructose-6-phosphate to D-erythrose-4-phosphate) by transketolase, thereby producing the pentose 5-phosphate intermediate required for the production of MEG (or glycolic acid) or optionally MEG (or glycolic acid) and one or more co-products, and providing more D-erythrose-4-phosphate for the non-oxidative branch of the pentose phosphate pathway to further produce one or more pentose 5-phosphate intermediates.

[0640] 6-Phosphofructokinase (EC 2.7.1.11)

[0641] Phosphofructokinase (Pfk) catalyzes the phosphorylation of fructose-6-phosphate at the C1 carbon during glycolysis. E. coli contains two Pfk isozymes, Pfk-1 (pfkA) and Pfk-2 (pfkB), which have no sequence similarity. More than 90% of the phosphofructokinase activity in wild-type E. coli can be attributed to Pfk-1.

[0642] PfkA catalyzes the phosphorylation of fructose-6-phosphate and is a key enzyme regulating the glycolytic pathway. The enzyme cannot catalyze the reverse reaction in vivo. The enzyme shows cooperative kinetics with the substrate fructose-6-phosphate, but not with another substrate ATP. It has recently been shown that PfkA also catalyzes the phosphorylation of sedoheptulose-7-phosphate as part of the sedoheptulose diphosphate bypass. The crystal structure of PfkA has been solved in the presence and absence of activators and inhibitors. Based on sequence similarity, PfkA is predicted to be an NAD+ kinase.

[0643] PfkB is a member of the ribokinase family of sugar kinases. Unlike PfkA, PfkB does not show cooperative interaction with fructose-6-phosphate, inhibition of PEP, or activation of ADP. MgATP 2- is the true substrate of the enzyme. PfkB can also use tagatose-6-phosphate as a substrate. This reaction is part of the galactitol catabolic pathway. The crystal structure of the tetrameric form of PfkB inhibited by MgATP was solved at a resolution of 1.3 Å. Comparison of this structure with the crystal structure of PfkB in complex with fructose-6-phosphate revealed a negative interaction between fructose-6-phosphate binding and MgATP binding.

[0644] In some embodiments, the recombinant microorganism producing MEG (or glycolic acid) or optionally MEG (or glycolic acid) and one or more co-products comprises a deletion, insertion or functional loss mutation in the gene encoding 6-phosphofructokinase to prevent the conversion of fructose-6-phosphate to 1,6-bisphosphate, but to allow the conversion of fructose-6-phosphate to erythrose-4-phosphate and acetyl phosphate by 6-phosphofructophosphoketolase, and to provide more erythrose-4-phosphate for the non-oxidative branch of the pentose phosphate pathway to further produce one or more pentose-5-phosphate intermediates required for the production of MEG (or GA) or optionally MEG (or GA) and one or more co-products. In some embodiments, the 6-phosphofructokinase is pfkA and / or pfkB.

[0645] Hydroxypyruvate decarboxylase, 2-oxoglutarate decarboxylase, 2-ketoacid decarboxylase (EC 4.1.1.-)

[0646] The present disclosure describes enzymes that can catalyze the following reactions:

[0647]

[0648] 2-Oxoglutarate + Coenzyme A + NAD + → Succinyl Coenzyme A + CO 2 +NADH

[0649] 4-Methyl-2-oxopentanoic acid + H+ → 3-methylbutyraldehyde + CO 2

[0650] 3-Methyl-2-oxobutyric acid + H+ → isobutyraldehyde + CO 2

[0651] Hydroxypyruvate decarboxylase may also be referred to as hydroxypyruvate carboxyl-lyase.

[0652] 2-Oxoglutarate decarboxylase may also be referred to as oxoglutarate decarboxylase; alpha-ketoglutarate decarboxylase (alpha-ketoglutarate decarboxylase, alpha-ketoglutaric decarboxylase); pre-2-oxoglutarate decarboxylase; 2-oxoglutarate carboxyl lyase.

[0653] Escherichia coli SucA is responsible for the 2-oxoglutarate decarboxylase activity of the 2-oxoglutarate dehydrogenase multienzyme complex (OGDHC), which catalyzes the conversion of 2-oxoglutarate (2-ketoglutarate) to succinyl-CoA and CO 2 , and produce NADH.

[0654] OGDHC is a member of the 2-oxoacid dehydrogenase family. Members of this family contain multiple copies of three enzyme components: 2-oxoglutarate decarboxylase (E1), lipoamide acyltransferase (E2), and lipoamide dehydrogenase (E3). In most Gram-positive bacteria and mitochondria, the E1 component is a heterodimer composed of two subunits, while in most (but not all) Gram-negative bacteria, it consists of a single type of subunit. In both cases, multiple copies of the E1 component and multiple copies of the E3 component are assembled around an E2 core of 24 subunits with octahedral symmetry or an E2 core of 60 subunits with icosahedral symmetry (depending on the complex and species). In Escherichia coli, the pyruvate dehydrogenase and glycine cleavage multienzyme complexes share the E3 component. The E1 and E2 of the 2-oxoglutarate and pyruvate dehydrogenase complexes are slightly different and are designated as (o) and (p) to distinguish them.

[0655] E. coli OGDHC contains 12 units of the E1(o) component 2-oxoglutarate decarboxylase, which requires thiamine and is encoded by sucA, 24 units of the E2(o) component dihydrolipoyl transsuccinylase, which is encoded by sucB, and 2 units of the E3 component thiamine dehydrogenase, which is encoded by lpd. The 24 E2(o) units form the octahedral core of the complex. It contains the binding sites for lipoyl lysine and the E1(o) and E3 subunit dimers. Electron cryo-tomography shows that it is flexibly attached to the E2 core.

[0656] In the OGDHC reaction cycle, SucA binds 2-oxoglutarate and decarboxylates it. SucA is an enzyme that contains thiamine diphosphate as a cofactor. The crystal structure of a truncated apo form of SucA lacking the N-terminal 77 residues was determined at 1.30 Å resolution. The resolution of thiamine diphosphate and Mg 2+ The truncated form retains decarboxylase activity but does not assemble into an OGDH complex with E2(o). The data also indicate the presence of an AMP binding site. 2 The side reaction generates oxygen-dependent thiamine free radicals.

[0657] Studies of engineered SucA prepared by saturation mutagenesis of His260 and His298 showed that His260 is required for substrate recognition, but His298 can be replaced by a hydrophobic residue of similar size. The data also suggest that E2(o) plays a role in specificity.

[0658] In early work, the sucA gene was cloned and sequenced, and the regulation of sucABCD was studied. It was shown that the sucAB and sucCD genes are mutually essential, and either pair is sufficient to produce succinyl-CoA, but the lack of both sucAB and sucCD will not survive.

[0659] α-Ketoisovalerate decarboxylase catalyzes the decarboxylation of 3-methyl-2-oxobutyrate to isobutyraldehyde. The enzyme is highly specific for 3-methyl-2-oxobutyrate, but also shows activity on other branched 2-keto acids (4-methyl-2-oxopentanoate, relative activity 22.7%; (S)-3-methyl-2-oxopentanoate, 16.7%, 2-oxo-3-phenylpropionic acid, 7.1%, and 4-(methylthio)-2-oxobutyric acid, 5.8%).

[0660] The enzyme is a homotetramer encoded by the sequenced and cloned kivd gene. The deduced protein sequence is 98.6% identical (in its first 438 amino acids) to the protein encoded by the ipd gene of Lactococcus lactis strain IL1403 (which is interrupted at position L439 by the insertion of the IS983 element). The kivd gene does not have any homology with any gene in the sequenced genomes of Lactococcus lactis strains MG1363 and SK11.

[0661] A study on the Kivd activity detection of 156 strains of lactic acid bacteria (Lactococcus, Lactobacillus, Leuconostoc) showed that only strains of Lactococcus lactis had this activity, and even among the Lactococcus strains, only 7 out of 45 strains had this activity.

[0662] A homologous protein from Lactococcus lactis strain B1157 was described as a branched-chain alpha-ketoacid decarboxylase. This protein has 89.8% identity to Kivd and also has a preference for 2-ketoisovalerate.

[0663] In some embodiments, an enzyme having 2-ketoacid decarboxylase activity, an enzyme having hydroxypyruvate decarboxylase activity, or an enzyme having 2-oxoglutarate decarboxylase activity converts hydroxypyruvate into ethanolaldehyde. In some embodiments, the enzyme that converts hydroxypyruvate into ethanolaldehyde is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to Kivd or SucA. In some embodiments, the enzyme having 2-ketoacid decarboxylase activity is Kivd. In some embodiments, the enzyme having 2-oxoglutarate decarboxylase activity is SucA.

[0664] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having 2-oxoglutarate decarboxylase activity are sucA, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding an enzyme having 2-ketoacid decarboxylase activity are Kivd, or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding an enzyme having 2-ketoacid decarboxylase activity, an enzyme having hydroxypyruvate decarboxylase activity, or an enzyme having 2-oxoglutarate decarboxylase activity comprise an amino acid sequence selected from SEQ ID NOs: 224 and 226. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having 2-ketoacid decarboxylase activity, an enzyme having hydroxypyruvate decarboxylase activity, or an enzyme having 2-oxoglutarate decarboxylase activity are encoded by a nucleic acid sequence selected from SEQ ID NOs: 223 and 225.

[0665] 2-oxoglutarate reductase, 3-phospho-hydroxypyruvate reductase, 3-phosphoglycerate dehydrogenase (EC 1.1.1.-)

[0666] The present disclosure describes enzymes that can catalyze the following reactions:

[0667]

[0668]

[0669]

[0670] 3-Phosphoglycerate dehydrogenase can also be called phosphoglycerate dehydrogenase (phosphoglyceratedehydrogenase); PHGDH (gene name); D-3-phosphoglycerate: NAD+ oxidoreductase; α-phosphoglycerate dehydrogenase; 3-phosphoglycerate dehydrogenase; D-3-phosphoglycerate dehydrogenase; glycerate 3-phosphate dehydrogenase; glycerate-1,3-phosphate dehydrogenase; phosphoglycerate oxidoreductase; phosphoglyceric acid dehydrogenase (phosphoglyceric aciddehydrogenase); SerA; 3-phosphoglycerate: NAD+2-oxidoreductase; SerA 3PG dehydrogenase; 3PHP reductase.

[0671] 3-Phosphoglycerate dehydrogenase catalyzes the first critical step in the biosynthesis of L-serine. The enzyme is regulated by allosteric end-product inhibition that exhibits synergistic effects. Inhibition by serine acts primarily by reducing the catalytic rate, with little effect on the Km of the substrate; therefore, SerA is classified as a type V allosteric enzyme.

[0672] The basis for allosteric and cooperative inhibition by serine has been extensively studied. Occupation of two of the four serine binding sites in the homotetramer resulted in 85% inhibition of activity. Further binding of serine showed negative cooperativity. Phosphate was able to reduce the site cooperative effect on serine binding; this effect was mainly due to the presence of intrinsically bound NADH. The Trp139Gly mutation caused the homodimeric enzyme to lose cooperativity in serine binding and allosteric inhibition. Site-directed mutagenesis of residues within the effector binding site, the regulatory interface between subunits, and the flexible hinge region supported a model in which movement of adjacent domains is involved in inhibiting enzyme activity. Transient kinetic analysis showed that the cooperative effect in inhibiting catalytic activity is due to conformational changes caused by serine binding. Enzymes lacking the regulatory domains are no longer inhibited by serine, but other kinetic parameters remain unchanged. The heterotetramer provides further insight into the mechanism of allosteric inhibition.

[0673] Site-directed mutagenesis has allowed the identification of residues within the active site that contribute to substrate binding and catalysis. Mutations in the hinge region between the substrate and nucleotide binding domains affect the enzyme's Kcat; certain mutations uncouple serine binding and catalytic inhibition.

[0674] Extensive site-directed mutagenesis and structural studies have helped to provide a detailed understanding of the interplay between allosteric regulation, cooperativity, and catalytic activity. Stopped-flow kinetic analysis provided further insight into the catalytic pathway, indicating that the rate-limiting step in both catalytic directions is a conformational change in the enzyme. Serine binding was shown to result in the formation of an inactive-end quaternary complex between the enzyme, coenzyme, substrate, and effector, eliminating the conformational change upon substrate binding.

[0675] The enzyme also has α-ketoglutarate reductase activity, generating 2-hydroxyglutarate. Although the metabolic role of this reaction is unclear, it is thought to play a role in regulating serine biosynthesis and recycling NADH back to NAD+, especially during anaerobic processes.

[0676] The crystal structures of the wild-type enzyme and a number of point mutants have been solved. The structures show that each subunit of the homotetramer is composed of three distinct domains, a nucleotide-binding domain, a substrate-binding domain, and a regulatory / serine-binding domain.

[0677] serA is required for growth in glycerol minimal medium; addition of serine can rescue the growth defect.

[0678] In some embodiments, the enzyme with 3-phosphoglycerate dehydrogenase activity can be an enzyme with 3-phospho-hydroxypyruvate reductase activity or an enzyme with 2-oxoglutarate reductase activity. In some embodiments, an enzyme with 3-phosphoglycerate dehydrogenase activity, an enzyme with 3-phospho-hydroxypyruvate reductase activity or an enzyme with 2-oxoglutarate reductase activity catalyzes the conversion of glycerate 3-phosphate into 3-phosphohydroxypyruvate. In some embodiments, an enzyme with 3-phosphoglycerate dehydrogenase activity, an enzyme with 3-phospho-hydroxypyruvate reductase activity or an enzyme with 2-oxoglutarate reductase activity is encoded by an amino acid sequence with at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with serA. In some embodiments, an enzyme with 3-phosphoglycerate dehydrogenase activity, an enzyme with 3-phospho-hydroxypyruvate reductase activity or an enzyme with 2-oxoglutarate reductase activity is serA.

[0679] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having 3-phosphoglycerate dehydrogenase activity is serA, or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding an enzyme having 3-phosphoglycerate dehydrogenase activity, an enzyme having 3-phospho-hydroxypyruvate reductase activity, or an enzyme having 2-oxoglutarate reductase activity comprise the amino acid sequence set forth in SEQ ID NO: 228. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having 3-phosphoglycerate dehydrogenase activity, an enzyme having 3-phospho-hydroxypyruvate reductase activity, or an enzyme having 2-oxoglutarate reductase activity are encoded by the nucleic acid sequence set forth in SEQ ID NO: 227.

[0680] Phosphoserine aminotransferase, Serine aminotransferase, L-serine aminotransferase (EC 2.6.1.52)

[0681] The present disclosure describes enzymes that can catalyze the following reactions:

[0682]

[0683]

[0684]

[0685] Phosphoserine aminotransferase may also be called phosphoserine transaminase; PSAT; 3-phosphoserine aminotransferase; hydroxypyruvate phospho-glutamate aminotransferase; L-phosphoserine aminotransferase; phosphohydroxypyruvate aminotransferase; phosphohydroxypyruvate-glutamate aminotransferase; 3-O-phospho-L-serine:2-oxoglutarate aminotransferase; SerC; PdxC; 3PHP aminotransferase.

[0686] The enzyme encoded by serC is a phosphoserine / phosphohydroxythreonine aminotransferase that functions in the biosynthesis of serine and pyridoxine by using different substrates. Pyridoxal 5'-phosphate is a cofactor for both enzyme activities, suggesting that it may function in an autocatalytic manner, stimulating its own biosynthesis.

[0687] Redundancy and promiscuity between transaminases have been investigated. No activity was observed on non-phosphorylated substrates; however, 3-hydroxypyruvate was able to be used as a substrate for SerC enzyme activity assays. In addition, genetic experiments have shown that SerC is a minor alanine transaminase.

[0688] Normal activities of two enzymes, ArgD and SerC, are sufficient for the biosynthesis of succinyldiaminopimelate (SDAP) and lysine; a third enzyme, AstC, is sufficient for the biosynthesis of SDAP but alone cannot meet the cell's demand for lysine. Other enzymes, including GabT and PuuE, may contribute to the biosynthesis of SDAP. Expression of argD, astC, serC, aspC, gabT, hisC, ilvE, patA, puuE, or tyrB from plasmids allowed the triple ΔargD serC astC mutant to grow in minimal medium.

[0689] The crystal structures of the enzyme in its unligated form and in complex with the substrate analog α-methyl-L-glutamate have been solved, and a molecular reaction mechanism has been proposed.

[0690] serC is required for growth in glycerol minimal medium; addition of serine and pyridoxol / pyridoxine can rescue the growth defect.

[0691] In some embodiments, the enzyme having phosphoserine aminotransferase activity can be an enzyme having L-serine aminotransferase activity or an enzyme having serine aminotransferase activity. In some embodiments, the enzyme having phosphoserine aminotransferase activity, the enzyme having L-serine aminotransferase activity or the enzyme having serine aminotransferase activity catalyzes the conversion of L-serine into hydroxypyruvate. In some embodiments, the enzyme having phosphoserine aminotransferase activity, the enzyme having L-serine aminotransferase activity or the enzyme having serine aminotransferase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with serC. In some embodiments, the enzyme having phosphoserine aminotransferase activity, the enzyme having L-serine aminotransferase activity or the enzyme having serine aminotransferase activity is serC.

[0692] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having phosphoserine aminotransferase activity is serC, or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding an enzyme having phosphoserine aminotransferase activity, an enzyme having L-serine aminotransferase activity, or an enzyme having serine aminotransferase activity comprise the amino acid sequence set forth in SEQ ID NO: 230. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having phosphoserine aminotransferase activity, an enzyme having L-serine aminotransferase activity, or an enzyme having serine aminotransferase activity are encoded by the nucleic acid sequence set forth in SEQ ID NO: 229.

[0693] 3-Phospho-Hydroxypyruvate Phosphatase

[0694] The present disclosure describes enzymes that can catalyze the following reactions:

[0695] 3-Phospho-hydroxypyruvate + H 2 O→Hydroxypyruvate+Phosphate

[0696] YeaB (NudL) belongs to the Nudix hydrolase family and is predicted to have CoA pyrophosphohydrolase activity.

[0697] yeaB (nudL) was isolated as a multicopy suppressor of flhDC transcriptional repression in the pgsA mutant. This repression may be due to the σ S Reduction in expression.

[0698] yeaB (nudL) was also isolated as a multicopy suppressor of PLP auxotrophy in a pdxB deletion strain. NudL was found to be part of an unexpectedly discovered metabolic pathway that produces 4-phospho-hydroxy-L-threonine, an intermediate in the pyridoxal 5'-phosphate biosynthetic pathway I, downstream of PdxB. This pathway diverts 3-phosphohydroxypyruvate from serine biosynthesis. NudL is an inefficient catalyst (K) for the conversion of 3-phosphohydroxypyruvate to hydroxypyruvate. cat 5.7×10 -5 ), but its activity was shown to be sufficient for the production of PLP.

[0699] In some embodiments, the enzyme having 3-phospho-hydroxypyruvate phosphatase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to yeaB. In some embodiments, the enzyme having 3-phospho-hydroxypyruvate phosphatase activity is yeaB.

[0700] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having 3-phospho-hydroxypyruvate phosphatase activity is yeaB, or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding an enzyme having 3-phospho-hydroxypyruvate phosphatase activity comprises the amino acid sequence set forth in SEQ ID NO: 232. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having 3-phospho-hydroxypyruvate phosphatase activity is encoded by the nucleic acid sequence set forth in SEQ ID NO: 231.

[0701] Phosphoserine phosphatase (EC 3.1.3.3)

[0702] The present disclosure describes enzymes that can catalyze the following reactions:

[0703] 3-phospho-L-serine+H 2 O→L-serine+phosphate

[0704] Phosphoserine phosphatases catalyze the final step in serine biosynthesis. The enzyme belongs to the haloacid dehalogenase (HAD)-like hydrolase superfamily. Enzymatic studies were initially performed using a partially purified enzyme from Escherichia coli strain W; this purified enzyme was analyzed as part of studies of the HAD enzyme superfamily.

[0705] SerB is required for growth in glycerol minimal medium; addition of serine rescues the growth defect. Gph, HisB, and YtjC were identified as multicopy suppressors of the conditional ΔserB phenotype. Directed evolution experiments identified mutations that increased the fitness and enzymatic activity of these suppressors.

[0706] In some embodiments, the enzyme having phosphoserine phosphatase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to serB. In some embodiments, the enzyme having phosphoserine phosphatase activity is serB.

[0707] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having phosphoserine phosphatase activity is serB, or a homolog thereof. In another embodiment, the one or more nucleic acid molecules encoding an enzyme having phosphoserine phosphatase activity comprises the amino acid sequence set forth in SEQ ID NO: 234. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having phosphoserine phosphatase activity is encoded by the nucleic acid sequence set forth in SEQ ID NO: 233.

[0708] Serine-pyruvate aminotransferase (EC 2.6.1.51)

[0709] The present disclosure describes enzymes that can catalyze the following reactions:

[0710]

[0711]

[0712] Serine-pyruvate aminotransferase may also be referred to as alanine-glyoxylate aminotransferase.

[0713] Both peroxisomal serine-pyruvate aminotransferase (AGXT1) and mitochondrial-localized alanine-glyoxylate aminotransferase 2 (AGXT2) catalyze the conversion of glyoxylate to glycine using alanine as an amino donor. Unlike AGXT2, AGXT1 cannot utilize asymmetric dimethylarginine (ADMA) as an amino donor.

[0714] Peroxisomal serine-pyruvate aminotransferase is a pyridoxal phosphate-dependent, liver-specific enzyme that consists of a homodimer. Its location in the peroxisome is crucial for proper enzyme activity. The C-terminal peroxisome targeting sequence (PTS1) is required for translocation into the peroxisome.

[0715] Dysfunction or mistargeting of serine-pyruvate aminotransferase results in a loss of hepatic peroxisomes, causing glyoxylate to escape into the cytoplasm where it is further metabolized to oxalate and glycolate. Oxalate cannot be further metabolized in the body, leading to the formation of insoluble calcium oxalate in the kidneys and urinary tract. Mutations in the AGXT1 gene result in improper peroxisome targeting and lead to an autosomal recessive metabolic disorder (primary hyperoxaluria type 1) that causes irreversible renal damage. One-third of patients with primary hyperoxaluria type 1 have a unique protein sorting defect in which enzymes from the hepatic peroxisomes are mislocalized to the mitochondria.

[0716] In some embodiments, the enzyme having serine-pyruvate aminotransferase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to human AGXT1. In some embodiments, the enzyme having serine-pyruvate aminotransferase activity is human AGXT1.

[0717] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having serine-pyruvate aminotransferase activity is AGXT1, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding an enzyme having serine-pyruvate aminotransferase activity comprises the amino acid sequence set forth in SEQ ID NO: 244. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having serine-pyruvate aminotransferase activity is encoded by the nucleic acid sequence set forth in SEQ ID NO: 243.

[0718] Serine decarboxylase (EC 4.1.1.65)

[0719] The present disclosure describes enzymes that can catalyze the following reactions:

[0720] 3-O-sn-phosphatidyl-L-serine + H+ → L-1-phosphatidylethanolamine + CO 2

[0721] Serine decarboxylase may also be referred to as phosphatidylserine decarboxylase; PS decarboxylase; phosphatidyl-L-serine carboxyl lyase.

[0722] Phosphatidylserine decarboxylase is one of a small class of enzymes that use a covalently bound pyruvyl prosthetic group. The pyruvyl group is thought to act in a manner similar to the pyridoxal phosphate cofactor by forming a Schiff base with the amino group of the substrate and then acting as an electron sink to facilitate decarboxylation.

[0723] Four of these enzymes, histidine decarboxylase (EC 4.1.1.22), phosphatidylserine decarboxylase, aspartate 1-decarboxylase, and S-adenosylmethionine decarboxylase are decarboxylases that form important biogenic amines. All of these enzymes are known to have a pyruvyl prosthetic group attached to the amino terminus of the α subunit via an amide. The other two enzymes in this group are D-proline reductase and glycine reductase (EC 1.21.4.2).

[0724] Enzymes containing pyruvyl groups are expressed as zymogens, which are post-translationally processed by a self-maturation cleavage called serinolysis. In this process, the pyruvyl group is formed from the serine residue, splitting the precursor protein into two parts, α and β subunits. In some cases, additional subunits may be involved.

[0725] The enzyme differs from other pyruvyl-dependent decarboxylases composed of different subunits in that the pyruvate prosthetic group is associated with the smaller subunit. The enzyme is a multimer of an unknown number of heterodimers.

[0726] In some embodiments, the enzyme having serine decarboxylase activity catalyzes the conversion of L-serine to ethanolamine.

[0727] In some embodiments, the enzyme having serine decarboxylase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to Arabidopsis thaliana SDC. In some embodiments, the enzyme having serine decarboxylase activity is Arabidopsis thaliana SDC.

[0728] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having serine decarboxylase activity are SDC, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding an enzyme having serine decarboxylase activity comprise the amino acid sequence set forth in SEQ ID NO: 236. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having serine decarboxylase activity are encoded by the nucleic acid sequence set forth in SEQ ID NO: 235.

[0729] Ethanolamine oxidoreductase (deaminating) (EC 1.4.3.8), ethanolamine transaminase (EC 2.6.1.-)

[0730] The present disclosure describes enzymes that can catalyze the following reactions:

[0731] Ethanolamine + oxygen + H 2 O→ammonium + hydrogen peroxide + glycolaldehyde

[0732] Ethanolamine + 2-oxoglutaric acid → glycolaldehyde + L-glutamic acid

[0733] Ethanolamine oxidoreductase (deaminating) can also be called ethanolamine oxidase. This enzyme belongs to the oxidoreductase family, specifically those that use oxygen as an acceptor to react with the donor CH-NH 2 Group of enzymes.

[0734] In some embodiments, the ethanolamine oxidase or ethanolamine transaminase catalyzes the conversion of ethanolamine to ethanolaldehyde.

[0735] In some embodiments, the enzyme having ethanolamine oxidase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to E. coli tynA. In some embodiments, the enzyme having ethanolamine oxidase activity is E. coli tynA.

[0736] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having ethanolamine oxidase activity is tynA, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding an enzyme having ethanolamine oxidase activity comprises the amino acid sequence set forth in SEQ ID NO: 238. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having ethanolamine oxidase activity is encoded by the nucleic acid sequence set forth in SEQ ID NO: 237.

[0737] In some embodiments, the enzyme having ethanolamine aminotransferase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to E. coli alaA. In some embodiments, the enzyme having ethanolamine aminotransferase activity is E. coli alaA.

[0738] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having ethanolamine aminotransferase activity is alaA, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding an enzyme having ethanolamine aminotransferase activity comprises the amino acid sequence set forth in SEQ ID NO: 240. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having ethanolamine aminotransferase activity is encoded by the nucleic acid sequence set forth in SEQ ID NO: 239.

[0739] Hydroxypyruvate reductase (EC 1.1.1.-)

[0740] The present disclosure describes enzymes that can catalyze the following reactions:

[0741]

[0742] Hydroxypyruvate reductase may also be referred to as β-hydroxypyruvate reductase; NADH: hydroxypyruvate reductase; D-glycerate dehydrogenase.

[0743] Hydroxypyruvate reductase is an enzyme present in higher plants, algae, mammalian tissues and bacteria. In most cases, it has been assumed to convert hydroxypyruvate to glycerate. However, most enzymes also reduce glyoxylate to glycolate.

[0744] In serine-cycling methylotrophs, hydroxypyruvate reductase plays a key role in carbon assimilation. It catalyzes the conversion of hydroxypyruvate to glycerate, a key step in the serine cycle, but the enzyme also plays an important role in the metabolism of C2 compounds through the interconversion of glyoxylate and glycolate.

[0745] In some embodiments, the hydroxypyruvate reductase catalyzes the conversion of glycerate to hydroxypyruvate.

[0746] In some embodiments, the enzyme having hydroxypyruvate reductase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to E. coli ghrB. In some embodiments, the enzyme having hydroxypyruvate reductase activity is E. coli ghrB.

[0747] In some embodiments, the one or more nucleic acid molecules encoding the hydroxypyruvate reductase are ghrB, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding the enzyme having hydroxypyruvate reductase activity comprise the amino acid sequence set forth in SEQ ID NO: 242. In a further embodiment, the one or more nucleic acid molecules encoding the enzyme having hydroxypyruvate reductase activity are encoded by a nucleic acid sequence selected from SEQ ID NO: 241.

[0748] Glycerate decarboxylase

[0749] The present disclosure describes enzymes that can catalyze the following reactions:

[0750] D-Glyceric acid + H+ → Ethylene glycol + CO 2

[0751] In some embodiments, the enzyme having glycerate decarboxylase activity catalyzes the conversion of glycerate to ethylene glycol.

[0752] 3-phosphoglycerate phosphatase (EC 3.1.3.38) or 2-phosphoglycerate phosphatase (EC 3.1.3.20)

[0753] The present disclosure describes enzymes that can catalyze the following reactions:

[0754] 3-phospho-D-glyceric acid + H 2 O→D-glyceric acid + phosphoric acid

[0755] 2-phospho-D-glyceric acid + H 2 O→D-glyceric acid+phosphoric acid

[0756] 3-phosphoglycerate phosphatase may also be referred to as D-3-phosphoglycerate phosphatase; 3-PGA phosphatase. 2-phosphoglycerate phosphatase may also be referred to as D-2-phosphoglycerate phosphatase; 2-PGA phosphatase. These enzymes belong to the family of hydrolases, specifically those that act on phosphomonoester bonds.

[0757] In some embodiments, the enzyme having 3-phosphoglycerate phosphatase activity or the enzyme having 2-phosphoglycerate phosphatase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to E. coli phoA. In some embodiments, the enzyme having 3-phosphoglycerate phosphatase activity or the enzyme having 2-phosphoglycerate phosphatase activity is E. coli phoA.

[0758] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having 3-phosphoglycerate phosphatase activity or an enzyme having 2-phosphoglycerate phosphatase activity are phoA, or homologs thereof. In some embodiments, the one or more nucleic acid molecules encoding an enzyme having 3-phosphoglycerate phosphatase activity or an enzyme having 2-phosphoglycerate phosphatase activity comprise the amino acid sequence set forth in SEQ ID NO: 246. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having 3-phosphoglycerate phosphatase activity or an enzyme having 2-phosphoglycerate phosphatase activity are encoded by the nucleic acid sequence set forth in SEQ ID NO: 245.

[0759] Glycerate kinase (EC 2.7.1.31)

[0760] The present disclosure describes enzymes that can catalyze the following reactions:

[0761]

[0762]

[0763] Glycerate kinase may also be referred to as glycerate 3-kinase; glycerate kinase (phosphorylating) (ambiguous); D-glycerate 3-kinase; D-glycerate kinase (ambiguous); glycerate kinase (ambiguous); GK (ambiguous); D-glycerate kinase (ambiguous); ATP:(R)-glycerate 3-phosphotransferase.

[0764] This enzyme belongs to the family of transferases, specifically those enzymes that transfer phosphorus-containing groups using alcohol groups as acceptors (phosphotransferases). This enzyme is involved in three metabolic pathways: serine / glycine / threonine metabolism, glycerolipid metabolism, and glyoxylate-dicarboxylic acid metabolism.

[0765] In some embodiments, the enzyme having glycerate kinase activity catalyzes the conversion of 3-phosphoglycerate to glycerate. In other embodiments, the enzyme having glycerate kinase activity catalyzes the conversion of 2-phosphoglycerate to glycerate.

[0766] In some embodiments, the enzyme with glycerate kinase activity is glycerate 3-kinase. In some embodiments, the enzyme with glycerate 3-kinase activity is encoded by an amino acid sequence with at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to Arabidopsis thaliana GLYK. In some embodiments, glycerate 3-kinase is Arabidopsis thaliana GLYK.

[0767] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having glycerate 3-kinase activity is GLYK, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding an enzyme having glycerate 3-kinase activity comprises the amino acid sequence set forth in SEQ ID NO: 248. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having glycerate 3-kinase activity is encoded by the nucleic acid sequence set forth in SEQ ID NO: 247.

[0768] In some embodiments, the enzyme having glycerate kinase activity is an enzyme having glycerate 2-kinase activity. In some embodiments, the enzyme having glycerate 2-kinase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to E. coli glxK. In some embodiments, the enzyme having glycerate 2-kinase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to E. coli garK. In other embodiments, the enzyme having glycerate 2-kinase activity is E. coli glxK. In some embodiments, glycerate 2-kinase is E. coli garK.

[0769] In some embodiments, the one or more nucleic acid molecules encoding an enzyme having glycerate 2-kinase activity is glxK, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding an enzyme having glycerate 2-kinase activity is garK, or a homolog thereof. In some embodiments, the one or more nucleic acid molecules encoding an enzyme having glycerate 2-kinase activity comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 250 and 252. In a further embodiment, the one or more nucleic acid molecules encoding an enzyme having glycerate 2-kinase activity is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 249 and 251.

[0770] Transferases that transfer one carbon group (EC 2.1.2.-)

[0771] The present disclosure describes enzymes that can catalyze the following reactions:

[0772]

[0773] Transferases such as hydroxymethyl-, formyl- and related transferases can be used. Examples of hydroxymethyl-, formyl- and related transferases include glycine hydroxymethyltransferase, phosphoribosylglycinamide formyltransferase, phosphoribosylaminoimidazole formyltransferase, glycine imidomethyltransferase, glutamate imidomethyltransferase, D-alanine 2-hydroxymethyltransferase, deoxycytidylic acid 5-hydroxymethyltransferase, methionyl-tRNA formyltransferase, aminomethyltransferase, 3-methyl-2-oxobutyrate hydroxymethyltransferase and UDP-4-amino-4-deoxy-L-arabinose formyltransferase.

[0774] Serine hydroxymethyltransferase (EC 2.1.2.1)

[0775] The present disclosure describes enzymes that can catalyze the following reactions:

[0776]

[0777] Serine hydroxymethyltransferase (GlyA) converts serine to glycine while simultaneously transferring a methyl group to tetrahydrofolate, thereby forming 5,10-methylenetetrahydrofolate (M-THF). M-THF is the major source of C1 units in cells, making GlyA a key enzyme in the biosynthesis of purines, thymidine, methionine, choline, and lipids.

[0778] The enzyme also catalyzes a variety of side reactions, including the hydrolysis of 5,10-methyleneTHF to 5-formylTHF and the reversible cleavage of 3-hydroxyamino acids (L-threonine, allo-threonine, 3-phenylserine) to glycine and aldehydes. D-alanine inactivates the enzyme by reacting with the pyridoxal phosphate prosthetic group to form pyridoxamine phosphate.

[0779] The Thr226 residue in the conserved region of the enzyme appears to be involved in substrate discrimination. The His228 residue plays a role in determining the specificity of the reaction. Lys229 appears to not play a catalytic role. Arg363 appears to be a binding site for the carboxyl group of the amino acid substrate. The hydroxyl group of Tyr65 may be involved in the conversion of the active site from a closed conformation to an open conformation. Both Tyr55 and Arg235 are required for the transaldehyde reaction.

[0780] Studies on enzyme refolding have shown that pyridoxal 5'-phosphate (PLP) binds to the dimeric apoenzyme only at the end of the folding pathway. The mechanism of PLP incorporation has been further investigated. At high concentrations of PLP, a second PLP molecule can bind at Lys346. The stability of the PLP binding site involves a conserved hydrophobic contact region. Tyr55 is required for the correct positioning of the PLP cofactor.

[0781] The crystal structures of wild-type and mutant serine hydroxymethyltransferases have been solved.

[0782] The glyA mutant is unable to use glycine as a sole nitrogen source. The glyA mutant is auxotrophic for glycine; it was later shown that glyA is required for growth in glycerol minimal medium.

[0783] The 3' sequence of the structural gene in the glyA mRNA is essential for mRNA stability.

[0784] In some embodiments, the enzyme having serine hydroxymethyltransferase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to E. coli glyA. In some embodiments, one or more nucleic acid molecules encoding an enzyme having serine hydroxymethyltransferase activity comprise the amino acid sequence listed in UniProt ID P0A825. In a further embodiment, one or more nucleic acid molecules encoding an enzyme having serine hydroxymethyltransferase activity are encoded by a nucleic acid sequence listed in Gene ID 947022.

[0785] Formaldehyde dehydrogenase (EC 1.2.1.46 and EC 1.2.1.-)

[0786] The present disclosure describes enzymes that can catalyze the following reactions:

[0787] Formaldehyde + NAD + +H 2 O→formate+NADH+2H +

[0788] Formaldehyde dehydrogenase may also be referred to as NAD-linked formaldehyde dehydrogenase, NAD-dependent formaldehyde dehydrogenase, or formaldehyde:NAD+ oxidoreductase.

[0789] Most formaldehyde dehydrogenases found in animals, plants, and bacteria belong to the group of class III alcohol dehydrogenases and require the addition of glutathione for activity. In fact, it was shown that the real substrate of these enzymes is not formaldehyde, but S-hydroxymethylglutathione formed non-enzymatically from formaldehyde and glutathione.

[0790] Unlike those enzymes, the enzyme isolated from Pseudomonas putida catalyzes the irreversible oxidation of formaldehyde to formic acid without the addition of glutathione. Since its substrate is formaldehyde, this is essentially a "true" formaldehyde dehydrogenase. Like other formaldehyde dehydrogenases, Pseudomonas putida FDH is a metalloenzyme containing zinc. It also requires NAD+ as an electron acceptor. However, unlike the enzymes belonging to the Class III alcohol dehydrogenase group, it is sensitive to 4-methylpyrazole. In one embodiment, the formaldehyde dehydrogenase is from Pseudomonas putida. In some embodiments, the formaldehyde dehydrogenase is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with Pseudomonas putida fdhA. In some embodiments, one or more nucleic acid molecules encoding the formaldehyde dehydrogenase comprise the amino acid sequence listed in GenBank Accession No. BAA04743.1. In a further embodiment, the one or more nucleic acid molecules encoding formaldehyde dehydrogenase are encoded by the nucleic acid sequence set forth in GenBank Accession No. D21201.1.

[0791] In the industrially important actinomycete Corynebacterium glutamicum ATCC13032, there is evidence that two enzymes contribute to the degradation of toxic formaldehyde, the mycothiol-dependent formaldehyde dehydrogenase encoded by the fadH gene and, to a lesser extent, the acetaldehyde dehydrogenase encoded by the ald (acetaldehyde dehydrogenase) gene. A mutant lacking both enzymes is unable to grow in medium containing formaldehyde. The mutant also does not grow in medium containing vanillic acid, since oxidation of vanillic acid produces intracellular formaldehyde. Detoxification of formaldehyde is necessary when this soil bacterium encounters formaldehyde in its habitat or when formaldehyde is produced during metabolism of environmental compounds such as vanillic acid. The formate produced by fadH can be further oxidized to CO by formate dehydrogenase, encoded by the fdhF gene. 2 .

[0792] Compared with wild type, the formaldehyde degradation of ald mutant has been reduced by about 30%. The inactivation of chromosome ald gene leads to the loss of acetaldehyde dehydrogenase activity and the loss of the ability of the organism to grow with ethanol or utilize ethanol, indicating that ethanol is oxidized to acetic acid through two steps. The expression of ald gene depends on the transcriptional regulator RamA, and RamB has a slight negative effect on expression. In one embodiment, formaldehyde dehydrogenase is from Corynebacterium glutamicum ATCC 13032. In some embodiments, formaldehyde dehydrogenase is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity or at least 90% sequence identity with the ald of Corynebacterium glutamicum ATCC 13032. In some embodiments, one or more nucleic acid molecules encoding formaldehyde dehydrogenase comprise the amino acid sequence listed in UniProt ID Q8NLZ0. In a further embodiment, one or more nucleic acid molecules encoding formaldehyde dehydrogenase are encoded by the nucleic acid sequence listed in Gene ID 1020739.

[0793] In some embodiments, the enzyme having formaldehyde dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, having at least 80% sequence identity, or having at least 90% sequence identity to Pseudomonas oleovorans alkH.

[0794] In Saccharomyces cerevisiae, two tandemly duplicated genes, ALD2 and ALD3, encode two cytoplasmic stress-inducible isoforms of aldehyde dehydrogenase. Expression of these isoforms is dependent on the general stress transcription factors Msn2 and Msn4 but independent of the HOG MAP kinase pathway. Both forms can use the cofactor NAD+ more efficiently than NADP+ and are not activated by any cation. While ALD3 is induced by a variety of stresses, including osmotic shock, heat shock, glucose depletion, oxidative stress, and drugs, ALD2 is only induced by osmotic stress and glucose depletion.

[0795] In some embodiments, the enzyme having formaldehyde dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to Saccharomyces cerevisiae ALD2. In other embodiments, the enzyme having formaldehyde dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to Saccharomyces cerevisiae ALD3. In some embodiments, one or more nucleic acid molecules encoding an enzyme having formaldehyde dehydrogenase activity comprise an amino acid sequence selected from UniProt ID P47771 and UniProt ID P54114. In a further embodiment, one or more nucleic acid molecules encoding an enzyme having formaldehyde dehydrogenase activity are encoded by a nucleic acid sequence selected from Gene ID 855206 and Gene ID 855205.

[0796] In some embodiments, the enzyme having formaldehyde dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to human ALDH3A2. In other embodiments, the enzyme having formaldehyde dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to human ALDH9A1. In some embodiments, one or more nucleic acid molecules encoding an enzyme having formaldehyde dehydrogenase activity comprise an amino acid sequence selected from UniProt ID P51648 and UniProtID P49189. In a further embodiment, one or more nucleic acid molecules encoding an enzyme having formaldehyde dehydrogenase activity are encoded by a nucleic acid sequence selected from Gene ID 224 and Gene ID 223.

[0797] Formate dehydrogenase (EC 1.2.1.-)

[0798] The present disclosure describes enzymes that can catalyze the following reactions:

[0799] Formate + oxidized electron acceptor + H+ → CO 2 + Reduced electron acceptor

[0800] Formic acid + H+ → CO 2 +H 2 (Catalyzed by complex)

[0801] Formate + Hydrogenase 3 oxidized → CO 2 + Reduced Hydrogenase 3

[0802] Formate dehydrogenase-H is one of three membrane-associated formate dehydrogenase isozymes in Escherichia coli. All isozymes function in the anaerobic metabolism of the organism.

[0803] Formate dehydrogenase-H (FDH-H) is located in the cytoplasm and forms a formate-hydrogen lyase complex with hydrogenase-3. The enzyme is oxygen-sensitive and contains selenium due to the co-translational incorporation of selenocysteine ​​at the position of the in-frame UGA stop codon in the FdhF open reading frame. The crystal structure of FDH-H was solved at 1.3 Å resolution, confirming the presence of the [4Fe-4S] cluster, the coordination of selenocysteine ​​to the Mo cofactor, and the location of the inhibitor nitrate binding site. Formate and the absence of an external electron acceptor induced fdhF expression, while nitric acid, nitrous acid, trimethylamine N-oxide, and oxygen inhibited fdhF expression. Formate could overcome the inhibition of nitric acid, but not oxygen. Inhibition of DNA gyrase enhanced fdhF expression.

[0804] fdnGHI encodes a membrane-bound formate dehydrogenase N (FDH-N), which is a respiratory enzyme that catalyzes the oxidation of formate to carbon dioxide, donating electrons to the quinone pool to reduce anaerobic respiratory substrates (such as nitrate and trimethylamine N-oxide). FDH-N is a member of the complex iron-sulfur-molybdenum enzyme (CISM) family. The oxidation of formate by FDH-N is electrogenic (H+ / e-=1); the oxidation of formate in the periplasm is accompanied by the reduction of menaquinone on the cytoplasmic side of the inner membrane. Nitric acid and anaerobic bacteria induce the expression of formate dehydrogenase-N, which is mediated by NarL and Fnr, respectively. The purified FDH-N contains three subunits, namely α (FdnG), β (FdnH), and γ (FdnI). The solved crystal structure shows that this subcomplex is further organized into a physiologically relevant trimer, with the α and β subunits positioned toward the periplasmic face of the inner membrane, and the γ subunit positioned toward the cytoplasm. Electrons are transferred across the membrane from the formate oxidation site in the α subunit to the menaquinone reduction site in the γ subunit. At the menaquinone reduction site, protons are taken up from the cytoplasm.

[0805] fdoGHI encodes formate dehydrogenase O (FDH-O), a respiratory molybdenum enzyme that catalyzes the oxidation of formate to carbon dioxide, donating electrons to the membrane-soluble quinone pool for the reduction of nitrate. FDH-O and nitrate reductase Z participate in the electron transfer pathway from formate to nitrate that is active when cells switch from aerobic to anaerobic conditions. The pathway operates via menaquinone or ubiquinone. FDH-O appears constitutively expressed; unlike formate dehydrogenase N (FDH-N), it is not regulated by Fnr or NarL. FDH-O expression increases under aerobic conditions; under anaerobic conditions, nitrate slightly stimulates expression; the global regulators H-NS and CRP may play a role in the regulation of FDH-O expression. FDH-O may contribute to the ability of cells to rapidly adapt to anaerobic conditions while FDH-N levels remain insufficient. FDH-O is a heterotrimeric complex composed of α (FdoG), β (FdoH), and γ (FdoI) subunits that shares extensive sequence similarity and common immunological properties with the anaerobically expressed FDH-N.

[0806] Candida boidinii formate dehydrogenase FDH1 is an NAD-dependent enzyme that mediates the detoxification of formate and is regulated by Cu 2+ , Hg, p-chloromercuric benzoate, cyanide, azide, thiocyanate, and cyanic acid. The inhibition by cyanide is reversible and competes with formate. Methanol induces protein expression, while glucose inhibits protein expression. Since the enzymatic reaction catalyzed by this formate dehydrogenase can regenerate NADH, the enzyme has been cloned into E. coli to optimize engineered pathways that require NADH.

[0807] In the industrially important actinomycete Corynebacterium glutamicum ATCC 13032, there is evidence that formate dehydrogenase catalyzes the oxidation of formate to CO 2 Both formic acid and toxic formaldehyde are present in the environment and can be dissimilated by this soil bacterium through the oxidation of formaldehyde to formic acid. This can be achieved by FadH and Ald. Formic acid is then converted to CO by FdhF 2 . FdhF is a molybdenum cofactor-dependent formate dehydrogenase that is active under aerobic conditions and is presumed to be involved in stress responses. The exact electron acceptor used by FdhF has not been defined. The fdhF gene is part of a gene cluster containing the related genes fdhD and cg0617, and mutant analysis showed that these genes are required for formate dehydrogenase activity. In the presence of formate, the growth of Corynebacterium glutamicum ATCC 13032 was somewhat inhibited, while strains lacking formate dehydrogenase activity showed a stronger inhibition. Radiotracer experiments showed that when Corynebacterium glutamicum ATCC 13032 was fed glucose and 13 During growth, formate is metabolized to 13C-CO2. The fdhF deletion mutant cannot metabolize formate. Growth studies also showed that Mo is required 2+ Protein sequence analysis suggests that FdhF is not an integral membrane protein but is likely to be cytosolic or membrane-associated. Putative orthologs have been identified in a variety of other soil bacteria.

[0808] When Cupriavidus oxalaticus grows with formate as the main carbon and energy source, NAD+-dependent formate dehydrogenase is the enzyme that generates NADH and CO 2 The latter enters the ribulose bisphosphate carboxylase reaction. The enzyme has been purified to homogeneity from cells grown on formate. The enzyme is a complex flavoprotein containing 2 FMN (flavin mononucleotides), 18-25 non-heme iron atoms and 15-20 acid-labile sulfides. The specific activity is 42 units / mg. The enzyme is specific for its natural substrate, formate, but can accept a variety of non-physiological electron acceptors, including methyl viologen, phenazine methylsulfate, methylene blue, nitro blue tetrazolium salts, FMN, FAD, riboflavin and oxygen. It has been shown that the enzyme can also catalyze the reaction in the opposite direction. However, under the conditions used, the enzyme catalyzes the oxidation of formate to CO 2 Restore is about 30 times faster.

[0809] NAD+-dependent formate dehydrogenase from Gottschalkia acidurici catalyzes the reversible reaction of formate oxidation to CO 2 At the same time, it also catalyzes the reduction of the latter to formate, which is then converted into acetic acid. The enzyme has been partially purified and found to be a large enzyme complex (molecular weight of at least 200 kDa) that is very sensitive to oxygen and light. The enzyme contains L-selenocysteine. The crude preparation of the enzyme can be coupled to the reduction of NAD during formate oxidation by ferredoxin. When the artificial electron acceptor methyl viologen is used instead of NAD, ferredoxin is not required. Cyanide inhibits the enzyme by 90%. The basal formate oxidation activity in cell extracts is 0.85 μmol / min / mg protein, but it increases 12-fold after the addition of tungstic acid and selenous acid. Interestingly, the enzyme from the related organism Clostridium cylindrosporum, although it has a similar requirement for selenous acid, requires molybdic acid rather than tungstic acid, which antagonizes it.

[0810] The tungsten-containing NAD+-dependent formate dehydrogenase from Methylobacterium extorquens is a heterodimer containing an iron-sulfur cluster, FMN, and tungsten. Although several other examples have been found, it is somewhat unusual to find a tungsten-containing enzyme in an aerobic bacterium. The smaller β subunit appears as a fusion protein with its N-terminal domain associated with a NueE-like subunit and its C-terminal domain associated with a NuoF-like subunit of a known NADH-ubiquinone oxidoreductase.

[0811] Two groups independently purified two different forms of formate dehydrogenase FDH from Methylosinus trichosporium OB3b and found that the two proteins had different properties. The protein is composed of two types of subunits, which have distinct α 2 β 2 The protein contains non-heme iron and sulfide, no other metals, and appears to require FMN.

[0812] The NAD+-dependent formate dehydrogenase fdh of Moraxella sp. is a relatively simple dimeric protein without a prosthetic group.

[0813] In some embodiments, the enzyme having formate dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to a formate dehydrogenase selected from the group consisting of: Escherichia coli fdhF (chlF, FDH-H), Escherichia coli FDH-N, Escherichia coli FDH-O, Candida boidinii FDH1, Corynebacterium glutamicum fdhF, NAD+-dependent formate dehydrogenase of Cupriavidus oxalaticus, NAD+-dependent formate dehydrogenase of Gottschalkia acidurici, Methylobacterium extorquens Fdh1, formate dehydrogenase of Methylosinus trichosporium, and NAD+-dependent formate dehydrogenase fdh of Moraxella species. In some embodiments, one or more nucleic acid molecules encoding a formate dehydrogenase or a formate dehydrogenase subunit comprise an amino acid sequence selected from the group consisting of: UniProt ID P07658, UniProt ID P0AEK7, UniProt ID P0AAJ3, UniProt ID P24183, UniProt ID P32176, UniProt ID P0AAJ5, UniProt ID P0AEL0, UniProt ID O13437, UniProt ID Q8NSY6, UniProt ID Q8KTI7, UniProt ID Q8KTI8, and UniProt ID O08375. In a further embodiment, the one or more nucleic acid molecules encoding formate dehydrogenase or formate dehydrogenase subunits are encoded by a nucleic acid sequence selected from the group consisting of Gene ID 948584, Gene ID 946038, Gene ID 948794, Gene ID 946035, Gene ID 948394, Gene ID 948395, Gene ID 948383, GenBank Accession No. AJ011046.2, Gene ID 1021531, GenBank Accession No. AF489516, and GenBank Accession No. Y13245.1.

[0814] Formate hydrogenolyase complex

[0815] The present disclosure describes enzymes that can catalyze the following reactions:

[0816] Formic acid + H+ → CO 2 +H 2 (Catalyzed by complex)

[0817] The component enzymes of the formate hydrogenase complex are: 1) formate dehydrogenase H (also known as ChlF, FdhF, FDH-H), 2) hydrogenase 3, which has multiple subunits (hycBCDEFG genes: hycB, hycC, hycD, hycE, hycF, hycG).

[0818] Formate dehydrogenase-H (FDH-H) is as described above.

[0819] Microbial hydrogenases catalyze the reversible reduction of protons to molecular hydrogen. Escherichia coli hydrogenase 3 is a multisubunit enzyme encoded by the hyc genes (hycD, hycC, hycF, hycG, hycB, and hycE) and is part of the formate hydrogenolyase (FHL) complex responsible for the fermentation or anaerobic oxidation of formate to carbon dioxide and molecular hydrogen.

[0820] Hydrogenase 3 is mainly involved in the production of H 2 and for the production of H at acidic pH 2 In strains lacking hydrogenases 1 and 2, hydrogen uptake was further reduced by introducing the hycE mutation, suggesting that hydrogenase 3 may also play a role in hydrogen uptake. Hydrogenase 3 showed activity for the product (H 2 ) is highly resistant to inhibition.

[0821] Hydrogenase 3 is a membrane-associated H 2 Evolved respiratory [NiFe] hydrogenase. It contains large (HycE) and small (HycG) subunits characteristic of "standard" NiFe hydrogenases, plus two additional hydrophilic subunits (HycB and HycF) and two inner membrane subunits (HycC and HycD). The Fe-S prosthetic group located in the hydrophilic part of the complex allows for the formation of an electron transfer pathway. Isolation of FHL using affinity chromatography revealed the presence of a core complex containing HycE, HycB, HycF, HycG and FdhF, which possesses formate hydrogenase activity in vitro; a larger complex containing the membrane-associated subunits HyC and HycD was isolated in the presence of detergent.

[0822] Oxidation of formate generates membrane potential in an anaerobically grown fermenting Escherichia coli strain lacking hydrogenase 1 and hydrogenase 2 enzymes.

[0823] Sequence similarities between the genes encoding hydrogenase 3 and those encoding subunits forming the core of the energy-storing NADH:quinone oxidoreductase (complex I) have been reported, and an evolutionary relationship between the two has been proposed.

[0824] Strains with gene insertion mutations within the hyc operon are defective in hydrogenase activity.

[0825] Hydrogenase 3 is a Fe-S protein containing nickel.

[0826] The hyc operon is co-regulated with the structural genes of formate dehydrogenase H. Under fermentative growth conditions, oxygen and nitric acid repress expression, while formate induces expression. Formate is an obligate inducer of formate hydrogenase complex genes.

[0827] E. coli K-12 contains three other hydrogenases: hydrogenase 1 and hydrogenase 2 (in H 2 uptake) and hydrogenase 4 (less well characterized; possibly silent).

[0828] In some embodiments, the hydrogenase 3 is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to a hydrogenase 3 selected from the group consisting of: E. coli hycB, E. coli hycC, E. coli hycD, E. coli hycE, E. coli hycF, and E. coli hycG. In some embodiments, one or more nucleic acid molecules encoding the hydrogenase 3 or formate hydrogenase complex comprise an amino acid sequence selected from the group consisting of: UniProt ID P0AAK1, UniProt ID P16429, UniProt ID P16430, UniProt ID P16431, UniProt ID P16432, and UniProt ID 16433. In a further embodiment, the one or more nucleic acid molecules encoding the formate hydrogenase complex or formate hydrogenase subunits are encoded by a nucleic acid sequence selected from the group consisting of Gene ID 948002, Gene ID 945327, Gene ID 948994, Gene ID 947396, Gene ID 947048 and Gene ID 947191.

[0829] Glycine cleavage system

[0830] The glycine cleavage system consists of four proteins: three enzymes and a carrier protein. In animals, this system is loosely associated with the inner mitochondrial membrane. These enzymes are i) P-protein (pyridoxal phosphate-containing protein) or glycine dehydrogenase (decarboxylation) (EC 1.4.4.2), ii) T-protein or aminomethyltransferase (EC 2.1.2.10), and iii) L-protein or dihydrolipoamide dehydrogenase (EC 1.8.1.4). The carrier protein is called H-protein (lipoic acid-containing protein).

[0831] The glycine cleavage reaction catalyzes the following reversible reaction:

[0832]

[0833] The system is divided into three partial reactions. The reaction is completely reversible, and the aminomethyl moiety bound to the lipoic acid of the H-protein represents an intermediate in the glycine cleavage and glycine synthesis, which is subsequently degraded to methylenetetrahydrofolate (M-THF) and ammonia by the action of the T-protein, or can be formed from M-THF and ammonia. Possibly, the reaction may involve a ternary complex of the P-protein, the aminomethyl moiety of glycine, and the H-protein as a key intermediate state.

[0834] P-protein catalyzed reactions

[0835] The first partial reaction of glycine degradation is a decarboxylation reaction catalyzed by the P-protein (glycine decarboxylase). The H-protein acts as a co-substrate. One of the most characteristic features of the glycine cleavage reaction is that, although the P-protein should belong to a class of pyridoxal phosphate-dependent amino acid decarboxylases, the P-protein requires the H-protein to significantly catalyze the decarboxylation of glycine. The reaction proceeds by a sequential stochastic mechanism, in which the carboxyl carbon of glycine is converted into carbon dioxide. The remaining part of the glycine molecule is transferred to one of the sulfhydryl groups, which is formed by reductive cleavage of a disulfide bond in lipoic acid attached to the H-protein.

[0836] The P-protein is a protein of about 200 kDa containing pyridoxal phosphate, which is a dimer of homodimers or heterodimers. The former has one molecule of pyridoxal phosphate per subunit, and the latter has one molecule of this cofactor per dimer on the β subunit. The pyridoxal cofactor is attached to a specific lysine residue. The pyridoxal cofactor interacts non-covalently with the active site pocket. The active site of the T.thermophilus P-protein is connected to the molecular surface through a channel with a wide entrance facing the solvent. The molecular surface around the channel consists of several positively charged amino acid residues, which may be involved in forming a complex with the H protein.

[0837] In some embodiments, the enzyme having glycine decarboxylase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to E. coli gcvP. In some embodiments, the one or more nucleic acid molecules encoding the enzyme having glycine decarboxylase activity comprise the amino acid sequence listed in UniProt IDP33195. In a further embodiment, the one or more nucleic acid molecules encoding the enzyme having glycine decarboxylase activity are encoded by the nucleic acid sequence listed in Gene ID 947394.

[0838] T-protein catalyzed reactions

[0839] The glycine decarboxylation part that is connected with H-protein is further degraded under the catalysis of T-protein (aminomethyltransferase).This reaction requires THF and produces ammonia, M-THF and the H-protein of the lipoic acid with reduction.In the absence of THF, formaldehyde is produced instead of M-THF, but the reaction rate is less than 0.05% of the reaction rate measured in the presence of THF.In the reverse reaction, T-protein forms the aminomethyl lipoic acid intermediate of H-protein binding by M-THF, ammonia and the H-protein of the lipoic acid with reduction through orderly Ter Bi mechanism catalysis, wherein H-protein is the first substrate combined, followed by M-THF and ammonia.The order of product release is THF and the H-protein loaded with methylamine.

[0840] The T-protein is a monomer of about 40 kDa and forms a 1:1 complex with the H-protein. A cross-linking study using E. coli proteins showed that the interaction of the H-protein with the T-protein leads to conformational changes in the T-protein. Intermolecular contacts between Lys-288 of the T-protein and Asp-43 of the H-protein were found. The N-terminal region of the T-protein is required for the interaction with the H-protein and for keeping the T-protein in a compact form. The crystal structure of human T-protein in free form and bound to N5-methyl-tetrahydrofolate (an analog of M-THF) has been analyzed. The overall structure consists of three clover-like structures with the THF cofactor bound to the pteridine ring buried deep in a hydrophobic pocket in the central cavity and the glutamyl group pointing to the C-terminal side surface. The structure is similar to the structures of bacterial T-proteins from Termotoga naritima, E. coli, and Pyrococcus horikoshii OT3. Structural and mutational analyses of the human T-protein suggest that the invariant Asp-101 may play a key role in the initiation of catalysis by increasing the nucleophilic properties of the N10 atom of the folate substrate.

[0841] The residues involved in folate binding have been identified by cross-linking and site-directed mutagenesis.The N-terminal region of GvcT is important for the correct conformation of GvcT to enable interaction with the H-protein.

[0842] In some embodiments, the enzyme having aminomethyltransferase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to E. coli gcvT. In some embodiments, the one or more nucleic acid molecules encoding the enzyme having aminomethyltransferase activity comprise the amino acid sequence listed in UniProt IDP27248. In a further embodiment, the one or more nucleic acid molecules encoding the enzyme having aminomethyltransferase activity are encoded by the nucleic acid sequence listed in Gene ID 947390.

[0843] L-protein catalyzed reactions

[0844] The final step of the glycine cleavage reaction is to be connected to the reoxidation of the reduced lipoic acid of H-protein under the catalysis of L-protein.L-protein is the E3 protein component of known dihydrolipoamide dehydrogenase, lipoamide dehydrogenase, dihydrolipoyl dehydrogenase or 2-oxyacid (pyruvic acid, 2-oxoglutaric acid and branched 2-oxyacid) dehydrogenase multienzyme complex.Its catalysis electron is to the transfer of final acceptor NAD.

[0845] Experiments using pea L-protein and H-protein showed that oxidation of dihydrolipoyl H-protein was not affected by the presence of structurally related analogs such as apoH-protein or octanoylated H-protein. Structural interactions between L-protein and H-protein may not be necessary for the oxidation reaction.

[0846] The kinetics of this reaction have been studied and a modified ping-pong mechanism has been proposed. Site-directed mutagenesis was used to identify and characterize redox-active disulfides and charged residues that affect the redox potential of the FAD cofactor. Insertion of the FAD cofactor is essential for dimerization and full activity.

[0847] The lpd null mutant produced more pyruvate and L-glutamate under aerobic conditions. Metabolic flux analysis showed that the Entner-Doudoroff pathway I and the glyoxylate shunt were activated. Another dihydrolipoate dehydrogenase activity was detected in the E. coli lpd mutant; therefore, isozymes may exist.

[0848] Mutation of the lpd gene in Escherichia coli results in a pyruvate dehydrogenase complex that is less sensitive to NADH inhibition and is active during anaerobic growth. Amino acid substitution at Glu354 reduces the sensitivity of the enzyme to NADH inhibition and is proposed to function by limiting the mobility of NADH.

[0849] It has been shown that suppressor mutations in lpd restore growth of redox-deficient mutants lacking thioredoxin and the glutathione / glutaredoxin reduction pathway. The suppressor mutation reduces Lpd activity, leading to accumulation of dihydrolipoamide, which can then act as an electron donor via the reduction of glutaredoxin. Reoxidation of Lpd restores the function of the TCA cycle.

[0850] lpd shows different codon adaptation in aerotolerant microorganisms compared to obligate anaerobes, resulting in different translation efficiency profiles. lpd is therefore predicted to play a role in the oxidative stress response. lpd deletion mutants have been shown to be more sensitive than the wild type specifically to hydrogen peroxide exposure (but not to other stresses).

[0851] In some embodiments, the enzyme with dihydrolipoamide dehydrogenase activity is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with Escherichia coli lpd (lpdA, E3 subunit). In some embodiments, one or more nucleic acid molecules encoding the enzyme with dihydrolipoamide dehydrogenase activity comprise the amino acid sequence listed in UniProt ID P0A9P0. In a further embodiment, one or more nucleic acid molecules encoding the enzyme with dihydrolipoamide dehydrogenase activity are encoded by the nucleic acid sequence listed in Gene ID 944854.

[0852] H-protein

[0853] H-protein is a monomeric heat-stable protein of about 14kDa. Vertebrate H-protein is composed of 125 amino acid residues, and lipoic acid is covalently linked to Lys-59. The X-ray crystal structure of lipoylated pea leaf H-protein (131 residues) has revealed that lipoyl-lysine is located at the protein surface. As mentioned above, the lipoyl-lysine arm on the H-protein transports reaction intermediates and reducing equivalents between the active sites of the glycine cleavage system components. This mechanism is similar to the mechanism found in the 2-oxoacid dehydrogenase complex.

[0854] Lipoic acid protein ligase A (LplA) in Escherichia coli catalyzes the lipoylation of the H-protein and the acyltransferase (E2) component of the 2-oxoacid dehydrogenase complex. The enzyme catalyzes the formation of lipoyl-AMP from lipoic acid and ATP and the transfer of the lipoyl moiety of lipoyl-AMP to the H-protein and the E2 component. X-ray crystallographic studies have shown that LplA consists of a large N-terminal domain and a small C-terminal domain, with a substrate binding pocket at the interface between the two domains.

[0855] In mammals, lipoylation is an intramitochondrial event. Lipoic acid is first activated to lipoyl-GMP by lipoyl activating enzyme, using GTP as the high-energy compound. Lipoic acid activating enzyme is identical to the protein known as xenobiotic metabolizing medium-chain fatty acid: CoA ligase III. Lipoic acid is then transferred from lipoyl-GMP to the apoprotein by the action of lipoyltransferase.

[0856] The H-protein is a lipoyl protein encoded by the gcvH gene in E. coli that is reduced when the methylamine group of glycine is transported from the P-protein to the T-protein and is reoxidized by dihydrolipoamide dehydrogenase. GcvH functions as a substrate for the three enzymes of the gcv complex.

[0857] Based on the conservation of residues 61-65 and their correspondence to the lipoic acid attachment site of pea (Pisum sativum) proteins, these residues were predicted to contain lipoyl modification (on lysine).

[0858] The interaction between GcvH and GcvT has been examined. The interaction between these two proteins is likely required for the formation of the folate binding site, where the polyglutamyl region of folate binds to expose the pteridine ring. The GcvT N-terminus is important for interaction with GcvH, probably by mediating conformational changes, and the D43 residue of GcvH comes into proximity with GcvT in the GcvH-GcvT complex.

[0859] In some embodiments, the H-protein is encoded by an amino acid sequence having at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to E. coli gcvH. In some embodiments, the one or more nucleic acid molecules encoding the H-protein comprise the amino acid sequence listed in UniProt ID P0A6T9. In a further embodiment, the one or more nucleic acid molecules encoding the H-protein are encoded by the nucleic acid sequence listed in Gene ID 947393.

[0860] Glycolate dehydrogenase or glycolate oxidase (EC 1.1.99.14)

[0861] The present disclosure describes enzymes that can catalyze the following reactions:

[0862] Glycolate + oxidized electron acceptor → glyoxylate + reduced electron acceptor

[0863] Glycolate dehydrogenase may also be referred to as glycolate oxidase, glycolate oxidoreductase, and glycolate:(acceptor)2-oxidoreductase.

[0864] Glycolate oxidase catalyzes the first step in the utilization of glycolate as a sole carbon source. The enzyme may be membrane associated. Cytoplasmic membrane-associated glycolate oxidoreductase activity has been isolated from Escherichia coli ATCC11775 (serotype O1:K1:H7), and the GlcF subunit itself could only be det...

Claims

1. A recombinant microorganism for producing monoethylene glycol (MEG) or glycolic acid (GA) from one or more hexose feedstocks via D-xylulose-5-phosphate and / or D-ribulose-5-phosphate; the recombinant microorganism comprising: (i) an exogenous and / or overexpressed enzyme for converting D-xylulose-5-phosphate and / or D-ribulose-5-phosphate into D-xylulose-1-phosphate or D-ribulose-1-phosphate, wherein the exogenous and / or overexpressed enzyme is S. cerevisiae Pgm3; and (ii) one or more exogenous and / or overexpressed enzymes having transketolase activity or fructose-6-phosphate phosphoketolase activity.

2. The recombinant microorganism according to claim 1, wherein the recombinant microorganism further produces one or more co-products selected from acetone, isopropanol, propylene or isobutene.

3. The recombinant microorganism according to claim 1, wherein the recombinant microorganism comprises at least one exogenous and / or overexpressed enzyme having transketolase activity.

4. The recombinant microorganism according to claim 3, wherein the at least one exogenous and / or overexpressed enzyme having transketolase activity comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 148 or SEQ ID NO:

150.

5. The recombinant microorganism according to claim 1, wherein the recombinant microorganism further comprises at least one exogenous and / or overexpressed enzyme having transaldolase activity.

6. The recombinant microorganism according to claim 5, wherein the at least one exogenous and / or overexpressed enzyme having transaldolase activity comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 152 or SEQ ID NO:

154.

7. The recombinant microorganism according to claim 1, wherein the recombinant microorganism further comprises at least one exogenous and / or overexpressed enzyme having ribulose-5-phosphate 3-epimerase activity.

8. The recombinant microorganism according to claim 7, wherein the at least one exogenous and / or overexpressed enzyme having ribulose-5-phosphate 3-epimerase activity comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

158.

9. The recombinant microorganism according to claim 1, wherein the recombinant microorganism further comprises at least one exogenous and / or overexpressed enzyme having ribose-5-phosphate isomerase activity.

10. The recombinant microorganism according to claim 9, wherein the at least one exogenous and / or overexpressed enzyme having ribose-5-phosphate isomerase activity comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 156 or SEQ ID NO:

253.

11. The recombinant microorganism according to claim 1, wherein the recombinant microorganism further comprises a deletion or reduced activity of at least one endogenous enzyme selected from glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase and / or phosphoglycerate mutase.

12. The recombinant microorganism according to claim 1, wherein the recombinant microorganism comprises at least one exogenous and / or overexpressed enzyme having fructose-6-phosphate phosphoketolase activity.

13. The recombinant microorganism according to claim 12, wherein the at least one exogenous and / or overexpressed enzyme having fructose-6-phosphate phosphoketolase activity comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216 or SEQ ID NO:

218.

14. The recombinant microorganism according to claim 12, wherein the recombinant microorganism further comprises at least one exogenous and / or overexpressed enzyme having phosphoacetyltransferase activity.

15. The recombinant microorganism according to claim 14, wherein the at least one exogenous and / or overexpressed enzyme having phosphoacetyltransferase activity comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 220 or SEQ ID NO:

222.

16. The recombinant microorganism according to claim 1, wherein the recombinant microorganism further comprises a deletion or reduced activity of the endogenous enzyme 6-phosphofructokinase.

17. The recombinant microorganism according to claim 1, wherein the recombinant microorganism further comprises a deletion or reduced activity of one or more endogenous enzymes selected from glucose 6-phosphate-1-dehydrogenase, 6-phosphogluconolactonase or 6-phosphogluconate dehydrogenase.

18. The recombinant microorganism according to claim 1, wherein the one or more hexose raw materials are selected from glucose or oligomers of glucose, and the oligomers of glucose are selected from fructose, sucrose, starch, cellobiose, maltose, lactose and cellulose.

19. The recombinant microorganism according to claim 1, wherein the recombinant microorganism further comprises one or more modifications to reduce or eliminate the activity of glyoxylate dehydrogenase, lactate dehydrogenase, xylose isomerase, xylulokinase or a combination thereof.

20. A method for producing MEG or glycolic acid (GA) using the recombinant microorganism according to claim 1, wherein the method comprises culturing the recombinant microorganism in a culture medium containing one or more hexose raw materials providing a carbon source until MEG or GA is produced.

21. The method according to claim 20, wherein the recombinant microorganism further produces one or more co-products selected from acetone, isopropanol, propylene or isobutene.

22. A method for producing a recombinant microorganism that produces or accumulates monoethylene glycol (MEG) or glycolic acid (GA) from one or more exogenous hexose raw materials through one or more pentose-5-phosphate intermediates, the method comprises: expressing in the recombinant microorganism one or more enzymes for converting one or more hexose raw materials into one or more pentose-5-phosphate intermediates, wherein the one or more pentose-5-phosphate intermediates are D-xylulose-5-phosphate and / or D-ribulose-5-phosphate; Overexpressing an enzyme in a recombinant microorganism, the enzyme being used to convert D-xylulose-5-phosphate and / or D-ribulose-5-phosphate into D-xylulose-1-phosphate or D-ribulose-1-phosphate, wherein the enzyme is Saccharomyces cerevisiae Pgm3; Overexpressing at least one enzyme having transketolase activity or at least one enzyme having fructose-6-phosphate phosphoketolase activity in a recombinant microorganism; Expressing a C2 pathway in a recombinant microorganism, the pathway comprising one or more enzymes for producing MEG or GA from glycolaldehyde; and Expressing a C3 pathway in a recombinant microorganism, the pathway comprising one or more enzymes for producing MEG or GA from DHAP or pyruvate; and Culturing the recombinant microorganism in a culture medium containing one or more hexose feedstocks to produce or accumulate MEG or GA, wherein glycolaldehyde and DHAP or pyruvate are intermediates produced in the C2 pathway, and wherein MEG or GA is produced in both the C2 and C3 pathways.

23. A method for producing a recombinant microorganism, the recombinant microorganism producing or accumulating monoethylene glycol (MEG) or glycolic acid (GA) and one or more co-products from one or more exogenous hexose feedstocks through one or more pentose-5-phosphate intermediates, the method comprising: Expressing in a recombinant microorganism one or more enzymes for converting one or more hexose feedstocks into one or more pentose-5-phosphate intermediates, wherein the one or more pentose-5-phosphate intermediates are D-xylulose-5-phosphate and / or D-ribulose-5-phosphate; Overexpressing an enzyme in a recombinant microorganism, the enzyme being used to convert D-xylulose-5-phosphate and / or D-ribulose-5-phosphate into D-xylulose-1-phosphate or D-ribulose-1-phosphate, wherein the enzyme is Saccharomyces cerevisiae Pgm3; Overexpressing at least one enzyme having transketolase activity or at least one enzyme having fructose-6-phosphate phosphoketolase activity in a recombinant microorganism; Expressing a C2 pathway in a recombinant microorganism, the pathway comprising one or more enzymes for producing MEG or GA from glycolaldehyde; and Expressing a C3 pathway in a recombinant microorganism, the pathway comprising one or more enzymes for producing one or more co-products from DHAP or pyruvate; and Culturing the recombinant microorganism in a culture medium containing one or more hexose feedstocks to produce or accumulate MEG or GA and one or more co-products, wherein glycolaldehyde and DHAP or pyruvate are intermediates produced in the C2 pathway, wherein MEG or GA is produced in the C2 pathway and one or more co-products are produced in the C3 pathway; wherein the one or more co-products are selected from acetone, isopropanol, propylene, and isobutene.

24. The method according to claim 22 or 23, wherein the method further comprises inactivating or reducing the activity of one or more endogenous enzymes in the recombinant microorganism, the endogenous enzymes being selected from glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, or phosphoglycerate mutase.

25. The method according to claim 22 or 23, wherein the method further comprises inactivating or reducing the activity of endogenous 6-phosphofructokinase in the recombinant microorganism.

26. The method according to claim 22 or 23, wherein the method further comprises: inactivating or reducing the activity of one or more endogenous enzymes in the recombinant microorganism, wherein the endogenous enzyme is selected from glucose 6-phosphate-1-dehydrogenase, 6-phosphogluconolactonase, or 6-phosphogluconate dehydrogenase.

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