Construction and use of engineered escherichia coli strain for synthesizing tartaric acid from ethylene glycol

Abstract

The construction and use of an engineered Escherichia coli strain for synthesizing tartaric acid from ethylene glycol. A new way for synthesizing tartaric acid from ethylene glycol is established, and tartaric acid is generated with ethylene glycol as a substrate.

Description

Construction and application of an engineered Escherichia coli strain for synthesizing tartaric acid using ethylene glycol

[0001] This application claims priority to a prior application, patent application number 202311231991X, filed with the State Intellectual Property Office of China on September 22, 2023, entitled “Construction and Application of an Engineered Escherichia coli Strain for Synthesizing Tartaric Acid Using Ethylene Glycol.” The entire text of this prior application is incorporated herein by reference. Technical Field

[0002] The invention belongs to the field of biochemical engineering and relates to the construction and application of an Escherichia coli engineering strain for synthesizing tartaric acid by utilizing ethylene glycol. Background Art

[0003] Ethylene glycol (EG), a widely used chemical, can be used directly as an antifreeze or coolant and is also a substrate for the production of polymers such as polyethylene terephthalate (PET). Over 25 million tons are synthesized annually for use in plastic bottles. The accumulation of PET plastic in nature is a growing concern due to its growing global demand and ongoing negative environmental impacts. If PET waste could be cost-effectively hydrolyzed, the resulting hydrolyzed PET monomer would become a sustainable and abundant source of raw materials for producing useful molecules.

[0004] Tartaric acid, as an important organic acid product, is widely used in chemical, pharmaceutical and food fields. At present, the products of chemical synthesis are all racemic tartaric acid, which has quite different physical and chemical properties from the tartaric acid present in the human body, namely L(+)-tartaric acid. Therefore, the biological synthesis of tartaric acid is particularly important. In 1974, Sato Eiji et al. first reported the biotransformation method of hydrolyzing cis-epoxysuccinic acid to produce L(+)-tartaric acid, and the molar conversion rate can reach 100%. The biotransformation synthesis method is widely used due to its advantages of high conversion rate, high product purity and stable safety. Tartaric acid can form oxaloacetic acid (OAA) in Escherichia coli under the action of tartrate dehydrogenase and enter the tricarboxylic acid cycle, and can also provide a precursor for amino acid synthesis. Tartaric acid dehydrogenase exists in an inactive low molecular weight form and is activated in the presence of Fe 2+ and thiols into active enzymes.

[0005] Escherichia coli is a commonly used host organism in metabolic engineering, but wild-type E. coli cannot utilize ethylene glycol (EG) as a sole carbon source. Early studies found that E. coli strains that utilize EG could be separated from mutants capable of growth on propylene glycol through adaptive evolution. As shown in Figure 1, the promiscuous activity of alcohol oxidoreductase converts ethylene glycol to glycolaldehyde, which is then converted to glycolate by lactaldehyde dehydrogenase. Glycolic acid is finally converted to glyoxylate by endogenous glycolate oxidase for cell growth. Glyoxylate reacts with acetyl-CoA through malate synthase to generate malate, which enters the TCA cycle.

[0006] Summary of the Invention

[0007] The present invention aims to establish a new pathway for synthesizing tartaric acid from ethylene glycol, using ethylene glycol as a substrate to produce tartaric acid. Patent document EP1748076A1 reports that the conversion of ethylene glycol to glyoxylic acid in Escherichia coli requires the participation of three enzymes: lactaldehyde reductase, lactaldehyde dehydrogenase, and glycolate oxidase, encoded by genes fucO, aldA, and glcDEF, respectively. These enzymes sequentially catalyze the conversion of ethylene glycol into glycolaldehyde, glycolic acid, and glyoxylic acid. Furthermore, non-patent literature (JOURNAL OF BACTERIOLOGY, Vol. 153, No. 1, pp. 134-139) reports that propylene glycol oxidoreductase can catalyze the oxidation of ethylene glycol to glycolaldehyde. The inventors of the present application discovered that the NAD-dependent glycerol dehydrogenase encoded by the gldA gene in Escherichia coli and Klebsiella pneumoniae can also react with ethylene glycol as a substrate to produce glycolaldehyde. Based on the metabolic pathway for the production of glyoxylic acid from ethylene glycol, the present invention introduces propionaldehyde dehydrogenase (PduP) to catalyze the synthesis of glycolaldehyde into glycolyl-CoA, and modifies malate synthase (GlcB) to catalyze the reaction of glyoxylic acid with glycolyl-CoA to produce tartaric acid (Figure 1).

[0008] Specific aspects of the present invention are as follows:

[0009] A first aspect of the present invention provides a biological method for producing tartaric acid, comprising the steps of:

[0010] a) Glyoxylic acid reacts with glycolyl-CoA in the presence of an enzyme that catalyzes the Claisen condensation (EC 2.3.3.-) to produce tartaric acid.

[0011] Preferably, the enzyme that can catalyze the Claisen condensation reaction (EC 2.3.3.-) can be malate synthase (EC 2.3.3.9), 2-isopropylmalate synthase (EC 2.3.3.13) or citramalate synthase (EC 2.3.3.21).

[0012] More preferably, malate synthase (GlcB) catalyzes the synthesis of tartaric acid from glyoxylate and glycolyl-CoA.

[0013] In a specific embodiment of the present invention, the method further comprises: b) propionaldehyde dehydrogenase (PduP) catalyzing the synthesis of glycolyl-CoA from glycolaldehyde.

[0014] In a specific embodiment of the present invention, the method further comprises: c) converting ethylene glycol into glycolaldehyde under the catalysis of glycerol dehydrogenase gldA, lactaldehyde reductase fucO or propanediol oxidoreductase.

[0015] In a specific embodiment of the present invention, the method further comprises: d) converting glycolaldehyde into glycolic acid under the catalysis of lactaldehyde dehydrogenase (aldA); and e) converting glycolic acid into glyoxylic acid under the action of glycolate oxidase (glcDEF).

[0016] In a specific embodiment of the present invention, the 2-isopropylmalate synthase (LeuA) is a mutant having enhanced catalytic activity towards glycolyl-CoA;

[0017] Preferably, the parent of the mutant is Escherichia coli 2-isopropylmalate synthase, preferably with protein sequence accession number AAC73185;

[0018] or the mutant has an amino acid sequence derived from Escherichia coli and having at least 98%, or at least 99%, sequence identity to the amino acid sequence represented by protein accession number AAC73185, and has enhanced catalytic activity towards glycolyl-CoA;

[0019] Preferably, the parent of the mutant is Corynebacterium glutamicum 2-isopropylmalate synthase, preferably with protein sequence accession number CAA50295;

[0020] Or the mutant has an amino acid sequence derived from Corynebacterium glutamicum and having at least 98% or at least 99% sequence identity with the amino acid sequence represented by protein accession number CAA50295, and has enhanced catalytic activity for glycolyl-CoA.

[0021] In a specific embodiment of the present invention, the citramalate synthase (CimA) is a mutant having enhanced catalytic activity towards glycolyl-CoA;

[0022] Preferably, the parent of the mutant is Methanocaldococcus jannaschii citramalate synthase, preferably a mutant mj.CimA with a protein sequence accession number of AAB99402 (referred to as wild-type mj.CimA), or a mutant mj.CimA in which six point mutations in brackets [I47V E114V H126Q T204A L238S V373STOP (stop codon)] are simultaneously introduced on the basis of the wild-type mj.CimA.

[0023] In a specific embodiment of the present invention, the malate synthase (GlcB) is a mutant having enhanced catalytic activity towards glycolyl-CoA.

[0024] In a specific embodiment of the present invention, the parent of the mutant is Escherichia coli malate synthase G, preferably protein sequence accession number CAA52639, WP_194154652.1 (L25I), HBP7705285.1 (L64M), WP_130525714.1 (L596M), EFM9907032.1 (L46M), HAI5727248.1 (L514M), EJO9706351.1 (A467S), HCB2230779.1 (A540S), EHC4340872.1 (A322S), WP_1931 30882.1 (A168S), HAW1984968.1 (A701S), WP_222024550.1 (A672S), WP_247168346.1 (A510S) or HBE5017233.1 (V365I); preferably, the mutant comprises 454A and / or 633G; enzymes having the above-mentioned 454A and / or 633G mutation sites and having enhanced catalytic activity for glycolyl-CoA, derived from Escherichia coli and having an amino acid sequence represented by protein accession number CAA52639 with at least 98%, or at least 99% sequence identity are also suitable for use in the present invention.

[0025] In a specific embodiment of the present invention, the glycerol dehydrogenase is a glycerol dehydrogenase or a propylene glycol / glycerol bifunctional dehydrogenase derived from the genus Escherichia, such as AAC43051 derived from Escherichia coli, or a glycerol dehydrogenase derived from Escherichia albertii, such as OSL28309.1; or a glycerol dehydrogenase derived from Klebsiella pneumoniae, such as ABR79620, or a glycerol dehydrogenase derived from Shigella, such as Shigella boydii or Shigella dysenteriae (such as ABB68416.1 or ABB63725.1), or a glycerol dehydrogenase derived from Streptococcal pneumoniae, such as VTQ29411.1, or a glycerol dehydrogenase derived from Bacillus stearothermophilus, such as AAA22477, or a glycerol dehydrogenase derived from Bacillus subtilis. subtilis) such as CAB15083.2, or a glycerol dehydrogenase from Pseudomonas putida such as AAC44426, or a glycerol dehydrogenase from Clostridium saccharobutylicum such as AAA83520.1, or a glycerol dehydrogenase from Clostridium acetobutylicum such as AAK79593.1, or a glycerol dehydrogenase from Salmonella such as CNU06578.1, or an alcohol dehydrogenase (glycerol dehydrogenase) from Corynebacterium glutamicum such as SJM54452.1, or an alcohol dehydrogenase from Gluconobacter oxydans such as AAW60096.1, and can also be a glycerol dehydrogenase from Parasutterella excrementihominis , and natural or artificial mutants of the enzyme.

[0026] Preferably, the glycerol dehydrogenase from Escherichia coli is the protein sequence accession number AAC43051 (ec.gldA); preferably, the glycerol dehydrogenase from Klebsiella pneumoniae is the protein sequence accession number ABR79620 (kp.gldA); or an enzyme that maintains the above enzyme activity and has an amino acid sequence that is at least 96% or 97% or 98%, or at least 99% identical to the amino acid sequence represented by protein accession number AAC43051 or ABR79620.

[0027] In a specific embodiment of the present invention, any step of the method is optionally performed inside or outside the cells of the genetically engineered strain.

[0028] In a particular embodiment of the invention, the method is performed within a cell; optionally within the same cell or within a different cell.

[0029] In a specific embodiment of the present invention, the method is performed outside of a cell; optionally under whole-cell catalysis or cell-free enzyme catalysis conditions.

[0030] In a specific embodiment of the present invention, the pathway for glycolaldehyde to produce ethylene glycol, the pathway for glyoxylic acid to produce glycolic acid, hydroxymalonic acid semialdehyde and malic acid, and the pathway for tartaric acid to produce oxaloacetic acid in the genetically engineered strain are blocked or knocked out; preferably, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, weakened or eliminated:

[0031] i) NADPH-dependent aldehyde reductase yqhD;

[0032] ii) glyoxylate / hydroxypyruvate reductase ycdW;

[0033] iii) glyoxylate aldehyde ligase gcl;

[0034] iv) endogenous malate synthase A, aceB and endogenous malate synthase G, glcB;

[0035] v) tartrate dehydratase α subunit, tartrate dehydratase β subunit ttdAB;

[0036] Preferably, the glyoxylate / hydroxypyruvate reductase ycdW and the glyoxylate-aldehyde ligase gcl are knocked out simultaneously;

[0037] Further preferably, NADPH-dependent aldehyde reductase yqhD, endogenous malate synthase A, aceB and endogenous malate synthase G, glcB, as well as tartrate dehydratase α subunit and tartrate dehydratase β subunit ttdAB are knocked out.

[0038] In a specific embodiment of the present invention, the method is carried out in a genetically engineered strain cell. Preferably, the production pathway of metabolic byproducts such as succinate, lactate, ethanol, acetic acid and formic acid in the genetically engineered strain is blocked or knocked out. In a further preferred embodiment of the present invention, the expression of one, two or more of the following genes in the microorganism is inhibited, weakened or eliminated: phosphate acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvaldehyde synthase mgsA, lactate dehydrogenase ldhAD, and fumarate reductase flavoprotein subunit frdA.

[0039] A second aspect of the present invention provides a biological method for producing glycolaldehyde, the method comprising:

[0040] 1) Ethylene glycol is converted into glycolaldehyde under the catalysis of glycerol dehydrogenase gldA.

[0041] The second aspect of the present invention also provides a method for synthesizing glycolic acid, which comprises first obtaining glycolaldehyde by the above method, and then 2) converting glycolaldehyde into glycolic acid under the catalysis of aldehyde dehydrogenase (aldA).

[0042] The second aspect of the present invention also provides a method for synthesizing glyoxylic acid, comprising: first obtaining glycolic acid by the above method; and then 3) converting the glycolic acid into glyoxylic acid under the catalysis of glycolate oxidase (glcDEF).

[0043] The second aspect of the present invention also provides a method for synthesizing glycolyl-CoA, which comprises first obtaining glycolaldehyde by the above method, and then 4) synthesizing glycolyl-CoA from glycolaldehyde by propionaldehyde dehydrogenase (PduP).

[0044] The glycerol dehydrogenase gldA is as described in the first aspect of the present invention.

[0045] A third aspect of the present invention provides a genetically engineered strain for producing tartaric acid, wherein the strain expresses:

[0046] a) at least one DNA molecule encoding an enzyme that catalyzes the Claisen condensation reaction (EC 2.3.3.-);

[0047] b) at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP).

[0048] In a specific embodiment of the present invention, the genetically engineered strain further expresses: c) at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO.

[0049] In a specific embodiment of the present invention, the genetically engineered strain further expresses: d) at least one DNA molecule encoding lactaldehyde dehydrogenase (aldA) and e) at least one DNA molecule encoding glycolate oxidase (glcDEF).

[0050] The glycerol dehydrogenase gldA is as described in the first aspect of the present invention.

[0051] The enzyme (EC 2.3.3.-) capable of catalyzing the Claisen condensation reaction is as described in the first aspect of the present invention.

[0052] A fourth aspect of the present invention provides a genetically engineered strain capable of utilizing ethylene glycol, wherein the strain expresses any one or a combination of the following DNA molecules:

[0053] At least one DNA molecule encoding an enzyme that catalyzes the Claisen condensation reaction (EC 2.3.3.-);

[0054] at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP);

[0055] at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO;

[0056] At least one DNA molecule encoding lactaldehyde dehydrogenase (aldA)

[0057] at least one DNA molecule encoding glycolate oxidase (glcDEF);

[0058] Preferably, the strain expresses:

[0059] at least one DNA molecule encoding a glycerol dehydrogenase gldA, wherein the strain synthesizes glycolaldehyde;

[0060] or

[0061] at least one DNA molecule encoding a glycerol dehydrogenase gldA, and at least one DNA molecule encoding a lactaldehyde dehydrogenase (aldA), wherein the strain synthesizes glycolate;

[0062] or

[0063] at least one DNA molecule encoding a glycerol dehydrogenase gldA, at least one DNA molecule encoding a lactaldehyde dehydrogenase (aldA), and at least one DNA molecule encoding a glycolate oxidase (glcDEF), wherein the strain synthesizes glyoxylate;

[0064] or

[0065] At least one DNA molecule encoding glycerol dehydrogenase gldA, at least one DNA molecule encoding lactaldehyde dehydrogenase (aldA) and at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP), and the strain synthesizes acetyl-CoA.

[0066] In a specific embodiment of the present invention, the DNA molecule is introduced into the host strain via a plasmid, or integrated into the genome of the host strain by genetic engineering means.

[0067] The host strain is selected from bacteria or fungi; optionally, the host strain is selected from wild or genetically engineered Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces; optionally, the host strain is selected from wild or genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; optionally, the host strain is selected from wild or genetically engineered Escherichia coli.

[0068] Furthermore, the following metabolic pathways of the genetically engineered strain are blocked, weakened or eliminated:

[0069] In a specific embodiment of the present invention, the method is carried out in a genetically engineered strain cell, in which the pathway for glycolaldehyde to produce ethylene glycol, the pathway for glyoxylic acid to produce glycolic acid, hydroxymalonic acid semialdehyde and malic acid, and the pathway for tartaric acid to produce oxaloacetic acid are blocked or knocked out; preferably, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, weakened or eliminated:

[0070] i) NADPH-dependent aldehyde reductase yqhD;

[0071] ii) glyoxylate / hydroxypyruvate reductase ycdW;

[0072] iii) glyoxylate aldehyde ligase gcl;

[0073] iv) endogenous malate synthase A, aceB and endogenous malate synthase G, glcB;

[0074] v) tartrate dehydratase α subunit, tartrate dehydratase β subunit ttdAB;

[0075] Preferably, the glyoxylate / hydroxypyruvate reductase ycdW and the glyoxylate-aldehyde ligase gcl are knocked out simultaneously;

[0076] Further preferably, NADPH-dependent aldehyde reductase yqhD, endogenous malate synthase A, aceB and endogenous malate synthase G, glcB, as well as tartrate dehydratase α subunit and tartrate dehydratase β subunit ttdAB are knocked out;

[0077] In a further preferred embodiment of the present invention: the production pathway of metabolic byproducts such as succinate, lactate, ethanol, acetate and formate in the genetically engineered strain is blocked or knocked out; preferably, the expression of one, two or more of the following genes in the microorganism is inhibited, weakened or eliminated: phosphate acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvaldehyde synthase mgsA, lactate dehydrogenase ldhAD, fumarate reductase flavoprotein subunit frdA.

[0078] The glycerol dehydrogenase gldA is as described in the first aspect of the present invention.

[0079] The malate synthase (GlcB) is as described in the first aspect of the present invention.

[0080] A sixth aspect of the present invention provides a biomaterial or a combination of biomaterials, wherein the biomaterial comprises:

[0081] at least one DNA molecule encoding an enzyme that catalyzes the Claisen condensation reaction (EC 2.3.3.-);

[0082] at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP);

[0083] at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO;

[0084] At least one DNA molecule encoding lactaldehyde dehydrogenase (aldA)

[0085] At least one DNA molecule encoding glycolate oxidase (glcDEF).

[0086] In a specific embodiment of the present invention, the biomaterial combination is a combination of the following biomaterials A and B:

[0087] Biomaterial A, comprising:

[0088] at least one DNA molecule that catalyzes an enzyme (EC 2.3.3.-) that catalyzes the Claisen condensation reaction;

[0089] at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP);

[0090] Biomaterial B, comprising:

[0091] at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO;

[0092] At least one DNA molecule encoding lactaldehyde dehydrogenase (aldA) and at least one DNA molecule encoding glycolate oxidase (glcDEF).

[0093] Preferably, the biological material is an expression cassette, a transposon, a plasmid vector, a cosmid vector, an artificial chromosome (such as a yeast artificial chromosome YAC or a bacterial artificial chromosome BAC), a phage vector or a viral vector.

[0094] The glycerol dehydrogenase gldA is as described in the first aspect of the present invention.

[0095] The enzyme catalyzing the Claisen condensation reaction (EC 2.3.3.-) is as described in the first aspect of the present invention.

[0096] In a seventh aspect, the present invention provides a mutant of malate synthase (GlcB), which has enhanced catalytic activity towards glycolyl-CoA, and the mutant has mutation sites 454A and / or 633G relative to the amino acid sequence encoded by GeneID: 948857, and the mutant has an amino acid sequence that is at least 98%, or at least 99%, identical to the amino acid sequence encoded by GeneID: 948857.

[0097] The seventh aspect of the present invention also provides a gene sequence encoding the above-mentioned malate synthase (GlcB) mutant, or a biological material comprising the above-mentioned gene sequence, or a genetically engineered strain expressing the gene sequence.

[0098] In an eighth aspect, the present invention provides the use of the above-mentioned biological materials or mutants of malate synthase (GlcB) or other encoding genes in constructing genetically engineered strains for producing tartaric acid and / or genetically engineered strains for utilizing ethylene glycol.

[0099] A ninth aspect of the present invention provides a genetically engineered strain, wherein at least one of the pathways for glycolaldehyde to produce ethylene glycol, the pathways for glyoxylic acid to produce glycolic acid, hydroxymalonic acid semialdehyde and malic acid, and the pathways for tartaric acid to produce oxaloacetic acid is blocked or knocked out; preferably, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, weakened or eliminated:

[0100] i) NADPH-dependent aldehyde reductase yqhD;

[0101] ii) glyoxylate / hydroxypyruvate reductase ycdW;

[0102] iii) glyoxylate aldehyde ligase gcl;

[0103] iv) endogenous malate synthase A, aceB and endogenous malate synthase G, glcB;

[0104] v) tartrate dehydratase α subunit, tartrate dehydratase β subunit ttdAB;

[0105] Preferably, the glyoxylate / hydroxypyruvate reductase ycdW and the glyoxylate-aldehyde ligase gcl are knocked out simultaneously;

[0106] Further preferably, NADPH-dependent aldehyde reductase yqhD, endogenous malate synthase A, aceB and endogenous malate synthase G, glcB, as well as tartrate dehydratase α subunit and tartrate dehydratase β subunit ttdAB are knocked out.

[0107] In a further preferred embodiment of the present invention: the production pathway of metabolic byproducts such as succinate, lactate, ethanol, acetate and formate in the genetically engineered strain is blocked or knocked out; preferably, the expression of one, two or more of the following genes in the microorganism is inhibited, weakened or eliminated: phosphate acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvaldehyde synthase mgsA, lactate dehydrogenase ldhAD, fumarate reductase flavoprotein subunit frdA.

[0108] The beneficial technical effects of the present invention are:

[0109] The invention establishes a new way to synthesize tartaric acid from ethylene glycol, and uses ethylene glycol as a substrate to generate tartaric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1: Synthesis pathway from ethylene glycol to tartaric acid: dotted arrows represent gene knockouts, and gray represents the knockout gene name; GlcB mut is a GlcB protein with a point mutation;

[0111] Figure 2: Schematic diagram of the plasmid structure of EG05 and EG08;

[0112] Figure 3: Schematic diagram of the plasmid structures of EG00, EG01 and EG02;

[0113] Figure 4: LC-MS peak spectrum of L-tartaric acid standard;

[0114] Figure 5: LC-MS peak profile of the fermentation broth of the DA7-EG01 strain at 24 h; DETAILED DESCRIPTION

[0115] The synthetic method of the present invention will be described in further detail below in conjunction with specific examples. It should be understood that the following examples are merely illustrative and explain the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies realized based on the foregoing of the present invention are encompassed within the scope that the present invention is intended to protect.

[0116] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the corresponding fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0117] As used herein, the term "amplification" means that the intracellular activity of one or more enzymes encoded by appropriate DNA in a microorganism is increased, for example, by increasing the gene copy number, using a strong promoter, or using a gene encoding an appropriate enzyme with high activity, and optionally combining these methods.

[0118] Unless specifically stated, the terms "first" and "second" do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one object from another.

[0119] As used herein, the term "gene synthesis" refers to the production of DNA or amino acid sequences using recombinant DNA technology or obtained using techniques for synthesizing DNA or amino acid sequences available and known in the art. "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is equivalent to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, which serves as a template for transcription of a gene or cDNA, may be said to encode a protein or other product of that gene or cDNA.

[0120] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter. The terms "overexpression" or "enhanced expression" refer to an increase in the level of mRNA encoding a protein and / or an increase in the level of a protein in a cell compared to a similar, unmodified cell expressing a basal level of mRNA or protein. For example, endogenous or exogenous genes can be introduced into a microorganism to increase the expression level of the corresponding enzyme. Alternatively, endogenous or exogenous gene expression can be enhanced by replacing a strong promoter with a strong promoter or introducing an enhancer.

[0121] As used herein, the term "vector" or "recombinant vector" is a genetic material composition that includes an isolated nucleic acid and that can be used to deliver the isolated nucleic acid to the interior of a cell. The transferred nucleic acid is typically connected to, for example, an inserted vector nucleic acid molecule. The vector may comprise a sequence that directs autonomous replication in the cell or may comprise a sequence sufficient to allow integration into the host cell DNA. Many vectors are known in the art and include, but are not limited to, plasmids, phagemids, artificial chromosomes, bacteriophages, and animal viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The host may be not only a wild strain, but also a mutant strain thereof or an artificial genetic recombinant.

[0122] As used herein and unless otherwise indicated, the term "about" when referring to a measurable value such as an amount, a time period, etc., is meant to include variations of ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the given value, provided that such variations are appropriate to practice the disclosed methods.

[0123] In the present invention, serial numbers are used to describe combinations of reaction steps, DNA molecules, biomaterials, or enzymes. For example, a)-c) includes a combination of the three aspects numbered a), b), and c). In the present invention, the combination of reaction steps indicates that the reaction steps are used to complete the final synthesis, but does not limit the manner in which the reactions of each step are carried out. Each reaction step can be carried out step by step or simultaneously, can be carried out inside or outside the cell, or can be carried out under the catalysis of cell-free enzymes. Unless otherwise stated, the combination of DNA molecules, biomaterials, or enzymes does not mean that the combination of DNA molecules, biomaterials, or enzymes is in the same organism or biomaterial. However, the present invention preferably expresses the combination of DNA molecules and enzymes in the same organism.

[0124] In the present invention, "at least one" refers to at least one of the same or similar references, and is not necessarily present. For example, a combination comprising "at least one DNA molecule encoding malate synthase (GlcB)" refers to comprising one or more DNA molecules encoding malate synthase (GlcB). However, if there is an expression of "optionally comprising" or "optionally in combination", the "DNA molecule encoding malate synthase (GlcB)" may not exist.

[0125] In the present invention, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion or addition of one or more amino acids or nucleotides". In the present invention, the term "mutation" refers to a change in the nucleotide sequence or amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

[0126] In the present invention, the term "natural state" used refers to the activity of the polypeptide in the microorganism in an unmodified state, that is, the activity in the natural state.

[0127] In the present invention, the term "maintaining activity" used has the same or similar meaning as commonly understood by those skilled in the art, and both refer to that the amino acid sequence of a fragment is part of the amino acid sequence of a complete protein or polypeptide and has the same or similar function or activity as the complete protein or polypeptide.

[0128] Therefore, it is readily apparent that further mutations of the enzymes of the present invention can be made to produce further mutants that still possess the corresponding functions and activities. For example, it is well known to those skilled in the art that adding or subtracting a number of amino acid residues, such as preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1, at either end of a polypeptide will not affect the function of the resulting mutant. For example, to facilitate purification, technicians often attach a 6×His tag to either end of the resulting protein, and such a protein has the same function as a protein without the 6×His tag. Therefore, the present invention encompasses conservative mutations obtained based on the present invention.

[0129] In the present invention, the term "conservative mutation" refers to a mutation that can maintain the normal function of a protein. A representative example of a conservative mutation is a conservative substitution.

[0130] As used in the present invention, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, and include residues having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0131] As used herein, a "conservative substitution" generally involves exchanging an amino acid at one or more sites in a protein. Such substitutions may be conservative. Examples of substitutions considered conservative include the substitution of Ala for Ser or Thr, the substitution of Arg for Gln, His or Lys, the substitution of Asn for Glu, Gln, Lys, His or Asp, the substitution of Asp for Asn, Glu or Gln, the substitution of Cys for Ser or Ala, the substitution of Gln for Asn, Glu, Lys, His, Asp or Arg, the substitution of Glu for Gly, Asn, Gln, Lys or Asp, the substitution of Gly for Pro, the substitution of His for Asn, Lys, Gln, Arg or Tyr. Conservative mutations include substitutions of Ile with Leu, Met, Val or Phe, substitutions of Leu with Ile, Met, Val or Phe, substitutions of Lys with Asn, Glu, Gln, His or Arg, substitutions of Met with Ile, Leu, Val or Phe, substitutions of Phe with Trp, Tyr, Met, Ile or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe or Trp, and substitutions of Val with Met, Ile or Leu. Conservative mutations also include naturally occurring mutations resulting from individual differences, strain differences, species differences, etc., from which the gene is derived.

[0132] The term "corresponding to" as used herein has the meaning commonly understood by those skilled in the art. Specifically, "corresponding to" refers to a position in one sequence that corresponds to a specified position in the other sequence after alignment for homology or sequence identity. Thus, for example, with respect to "the amino acid residue corresponding to position 40 of the amino acid sequence set forth in SEQ ID NO: 1," if a 6×His tag is added to one end of the amino acid sequence set forth in SEQ ID NO: 1, then the position corresponding to position 40 in the resulting mutant may be position 46.

[0133] In a specific embodiment, the homology or sequence identity may be greater than 90%, preferably greater than 95%, more preferably 96%, 97%, 98%, or 99%.

[0134] Methods for determining sequence homology or identity that are well known to those of ordinary skill in the art include, but are not limited to, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991, and Carillo, H. and Lipman, D., SIAM J. Applied Biology. Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0135] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0136] The present invention first provides a method or use of NAD-dependent glycerol dehydrogenase encoded by the gldA gene in Escherichia coli and Klebsiella pneumoniae to catalyze the reaction of ethylene glycol to produce ethanolaldehyde. The accession number of the glycerol dehydrogenase protein sequence from Escherichia coli is AAC43051 (ec.gldA), and the accession number of the glycerol dehydrogenase protein from Klebsiella pneumoniae is ABR79620 (kp.gldA). Enzymes that maintain the activity of the above enzymes and have an amino acid sequence that is at least 98% or at least 99% identical to the amino acid sequence represented by the protein accession number are also suitable for use in the present invention.

[0137] The present invention further provides a biological method for producing tartaric acid, comprising the following steps:

[0138] a) Glyoxylate reacts with glycolyl-CoA in the presence of an enzyme that catalyzes the Claisen condensation (EC 2.3.3.-) to produce tartaric acid.

[0139] In a specific embodiment of the present invention, the malate synthase (GlcB) is a mutant having enhanced catalytic activity towards glycolyl-CoA.

[0140] In a specific embodiment of the present invention, the parent of the mutant is Escherichia coli malate synthase G, with protein sequence accession number CAA52639; preferably, the mutant comprises 454A and / or 633G, has enhanced catalytic activity towards glycolyl-CoA, and has an amino acid sequence with at least 98%, or at least 99% sequence identity to the amino acid sequence represented by protein accession number CAA52639. The present invention is also applicable to enzymes.

[0141] It can be expected by those skilled in the art that malate synthase G derived from Escherichia coli and having one mutation site compared to CAA52639 can also be used as a mutant parent, such as WP_194154652.1 (L25I), HBP7705285.1 (L64M), WP_130525714.1 (L596M), EFM9907032.1 (L46M), HAI5727248.1 (L514M), E JO9706351.1(A467S), HCB2230779.1(A540S), EHC4340872.1(A322S), WP_193130882.1(A168S), HAW1984968.1(A701S), WP_222024550.1(A672S), WP_247168346.1(A510S) or HBE5017233.1(V365I).

[0142] In a specific embodiment of the present invention, the method further comprises: b) catalyzing glycolaldehyde to glycolyl-CoA by propionaldehyde dehydrogenase (PduP).

[0143] In a specific embodiment of the present invention, the method further comprises: c) converting ethylene glycol into glycolaldehyde under the catalysis of glycerol dehydrogenase gldA, lactaldehyde reductase fucO or propanediol oxidoreductase.

[0144] In a specific embodiment of the present invention, the glycerol dehydrogenase is a glycerol dehydrogenase protein sequence from Escherichia coli with accession number AAC43051 (ec.gldA) or a glycerol dehydrogenase protein sequence from Klebsiella pneumoniae with accession number ABR79620 (kp.gldA).

[0145] In a specific embodiment of the present invention, the method further comprises: d) converting glycolaldehyde into glycolic acid under the catalysis of lactaldehyde dehydrogenase (aldA); and e) converting glycolic acid into glyoxylic acid under the action of glycolate oxidase (glcDEF).

[0146] The present invention realizes the conversion of ethylene glycol into tartaric acid in recombinant Escherichia coli.

[0147] Those skilled in the art are aware of other microorganisms such as wild or genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris, etc.

[0148] The starting strain of the present invention is Escherichia coli BW25113 (K12 strain). Those skilled in the art will appreciate that other similar Escherichia coli strains, such as other K strains (e.g., MG1665 or W3110), W strains (e.g., ATCC 9637), Crooks strains (e.g., ATCC 8739), B strains (e.g., ATCC 11303, BL21 (DE3) or REL606), C strains (e.g., ATCC 23461), and the Escherichia coli probiotic Nissle 1917 (EcN / DSM 6601) and its derivatives, as well as Escherichia coli of other genetic lineages, can also be used in the present invention.

[0149] The present invention first constructs an Escherichia coli strain with knockout genes encoding metabolic enzymes that are not conducive to tartaric acid accumulation and can divert EG metabolic intermediates. In the genetically engineered strain, the production pathways of metabolic byproducts such as succinate, lactate, ethanol, acetic acid and formic acid are blocked or knocked out to avoid waste of carbon sources.

[0150] The present invention uses gene knockout to inhibit, reduce or eliminate the expression of the corresponding enzyme. Those skilled in the art will know that other methods of inhibiting enzyme expression or reducing / eliminating enzyme functional activity may also be applicable, such as promoter knockout, introduction of enzyme inactivation / deactivation mutations, deletion (or partial deletion) of essential functional elements or regions for gene transcription / translation, introduction of mutations or nucleic acid sequences that accelerate the degradation of mRNA that directs enzyme synthesis, introduction of mutations or protein tags that accelerate enzyme degradation / decomposition / inactivation, inhibition of signal activation, RNA interference and gene silencing.

[0151] In a specific embodiment of the present invention, the expression of one, two or more of the following genes in the microorganism is inhibited, reduced or eliminated:

[0152] i) NADPH-dependent aldehyde reductase yqhD;

[0153] ii) glyoxylate / hydroxypyruvate reductase ycdW;

[0154] iii) glyoxylate aldehyde ligase gcl;

[0155] iv) malate synthase A, aceB and malate synthase G, glcB;

[0156] v) tartrate dehydratase α subunit, tartrate dehydratase β subunit ttdAB.

[0157] In a further preferred embodiment of the present invention, the expression of one, two or more of the following genes in the microorganism is inhibited, weakened or eliminated: phosphate acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvaldehyde synthase mgsA, lactate dehydrogenase ldhAD, fumarate reductase flavoprotein subunit frdA.

[0158] The present invention uses two recombinant plasmids expressing gldA-aldA-glcDEF and glcB mutant-pduP to express the enzyme that uses ethylene glycol to synthesize tartaric acid. Those skilled in the art will know that any other plasmid combination is also applicable to the present invention as long as it expresses the above enzyme.

[0159] Those skilled in the art can also enhance the activity of the corresponding enzyme by replacing a strong promoter, expressing a mutant with higher activity, etc., or enhance the activity of the corresponding enzyme in a spatiotemporally controllable manner by introducing regulatory elements.

[0160] Those skilled in the art also know that other strain construction methods such as genome integration methods are also applicable to the present invention. For example, random integration or site-specific integration technology can be used to construct the corresponding strain.

[0161] Enzymes that catalyze the Claisen condensation reaction (EC 2.3.3.-)

[0162] Enzymes that can catalyze the Claisen condensation reaction are known to those skilled in the art. In the present invention, malate synthase (EC 2.3.3.9), 2-isopropylmalate synthase (EC 2.3.3.13) or citramalate synthase (EC 2.3.3.21) are preferred.

[0163] Biological pathway for the conversion of ethylene glycol to glycolaldehyde

[0164] Glycerol dehydrogenase (GDH) mainly catalyzes the conversion of glycerol to dihydroxyacetone (DHA), but some glycerol dehydrogenases (GDH) also have a certain catalytic activity for the conversion of ethylene glycol. Preferred glycerol dehydrogenases (GDH) of the present invention include: glycerol dehydrogenase or propylene glycol / glycerol bifunctional dehydrogenase from the genus Escherichia, such as AAC43051 from Escherichia coli, or glycerol dehydrogenase from Escherichia albertii such as OSL28309.1; or glycerol dehydrogenase from Klebsiella pneumoniae such as ABR79620, or glycerol dehydrogenase from Shigella such as Shigella boydii or Shigella dysenteriae (such as ABB68416.1 or ABB63725.1), or glycerol dehydrogenase from Streptococcus pneumoniae. pneumoniae, such as VTQ29411.1, or a glycerol dehydrogenase from Bacillus stearothermophilus, such as AAA22477, or a glycerol dehydrogenase (alcohol / choline dehydrogenase) from Bacillus subtilis, such as CAB15083.2, or a glycerol dehydrogenase from Pseudomonas putida, such as AAC44426, or a glycerol dehydrogenase from Clostridium saccharobutylicum, such as AAA83520.1, or a glycerol dehydrogenase from Clostridium acetobutylicum, such as AAK79593.1, or a glycerol dehydrogenase from Salmonella, such as CNU06578.1, or a glycerol dehydrogenase from Corynebacterium glutamicum, such as CNU06578.1. glutamicum), such as SJM54452.1; and may be a propanediol / glycerol bifunctional dehydrogenase MTU07760.1 from Parasutterella excrementihominis, as well as natural or artificial mutants of enzymes from the same species.

[0165] In addition, lactaldehyde reductase fucO or propanediol oxidoreductase can also be used to catalyze the conversion of ethylene glycol to glycolaldehyde.

[0166] Materials and Methods

[0167] The information of the enzyme involved in the present invention is as follows:

[0168] In the context of this application, the enzymes mentioned include mutants that retain enzyme activity, said mutants having an amino acid sequence that is at least 98%, or at least 99% identical to the amino acid sequence represented by the protein accession number in the table.

[0169] The biomaterials constructed by the present invention are shown in the following table:

[0170] Example 1: Construction of DA7 and DT13 strains

[0171] Using the E. coli Keio Knockout Collection 1 Phage was prepared using the corresponding single-gene knockout strain from Horizon Discovery, CO, USA. The target gene deletion DNA fragment, which confers kanamycin (Kan) resistance, was introduced into the starting Escherichia coli BW25113 strain using the P1 phage transfection method. The fragment was plated on LB plates containing 50 mg / L kanamycin and incubated overnight at 37°C to generate a Kan-resistant deletion strain. The plasmid pCP20 was then transformed into the Kan-resistant transformants, plated on LB plates containing 100 mg / L ampicillin, and incubated at 30°C for 24 hours. The correct transformants, with the Kan resistance gene removed from the target gene, were identified by PCR to generate a non-resistant deletion strain. This process was repeated to construct the BW25113-based deletion strain DA7, in which the pathways for the production of metabolic byproducts (succinate, lactate, ethanol, acetate, and formate) were blocked and knocked out to avoid carbon source waste.

[0172] The same method was used to knock out Δgcl, ΔglcB, ΔaceB, ΔycdW and ΔyqhD based on DA7 to obtain the DT12 strain.

[0173] Utilizing λ-Red recombination technology 2 , according to the literature 1 Describe the conditions. Use primers SEQ ID NO: 01 and SEQ ID NO: 02 with plasmid pKD13 as template to amplify a linear DNA product containing the upstream homology arm of ttdA, the kan gene, and the downstream homology arm of ttdB. The purified and recovered DNA fragments were electroporated into the DNA fragments according to the literature. 2Prepared competent cells of the DT12 strain were allowed to recover in LB medium at 37°C for half an hour, then plated onto LB plates containing 50 mg / L kanamycin and cultured overnight at 37°C. Correct transformants, in which the ttdAB gene had been replaced with a kanamycin-resistance gene, were identified, resulting in strain DT13K. PCP20 was then transformed into DT13K and plated onto LB plates containing 100 mg / L ampicillin. After 24 hours of culture at 30°C, PCR was performed to identify correct transformants in which the kanamycin-resistance gene Kan had been deleted from the ttdAB locus, resulting in strain DT13. In strain DT13, genes encoding metabolic enzymes that are detrimental to tartaric acid accumulation and that divert EG metabolic intermediates were deleted.

[0174] Example 2: Recombinant plasmids pZE-kp.gldA-aldA-glcDEF (EG08), pZE-ec.gldA-aldA-glcDEF (EG05), pZA-glcB-pduP (EG00), pZA-glcB 454A -pduP(EG01), pZA-glcB 633G - Construction of pduP(EG02)

[0175] kp.gldA was cloned from the Klebsiella pneumoniae genome, and aldA and glcDEF were cloned from the Escherichia coli BW25113 genome by PCR. The obtained cloned fragments were ligated to the plasmid pZElac (ampicillin resistance, ColE1 replicon, lac promoter) by Gibson Assembly to obtain the recombinant plasmid pZE-kp.gldA-aldA-glcDEF (EG08) encoding the ethylene glycol (EG) utilization pathway.

[0176] As described above, ec.gldA, aldA, and glcDEF were cloned from the Escherichia coli BW25113 genome by PCR. The resulting cloned fragments were ligated into the plasmid pZElac (ampicillin resistance, ColE1 replicon, lac promoter) using Gibson assembly to generate the recombinant plasmid pZE-ec.gldA-aldA-glcDEF (EG05), encoding the ethylene glycol (EG) utilization pathway. The plasmid structures of EG05 and EG08 are shown in Figure 2.

[0177] The malate synthase gene glcB from the E. coli BW25113 genome and the pduP gene from the Salmonella typhimurium LT2 genome were cloned by PCR, and the obtained gene fragments were ligated into the plasmid pZAlac (kanamycin resistance, P15A replicon, lac promoter) by Gibson Assembly to obtain the recombinant plasmid pZA-glcB-pduP (plasmid EG00).

[0178] The optimal substrates for malate synthase (encoded by the glcB gene) are glyoxylate and acetyl-CoA. The present invention achieves the synthesis of tartaric acid shown in Figure 1 by modifying the acetyl-CoA binding region of malate synthase so that it can accept glycolyl-CoA. Using the recombinant plasmid pZA-glcB-pduP (EG00) as a template, circular plasmid PCR site-directed mutagenesis techniques were used to generate PCR products in which the leucine at position 454 of GlcB was mutated to alanine (using primers SEQ ID NO:03 and SEQ ID NO:04), or the alanine at position 633 was mutated to glycine (using primers SEQ ID NO:05 and SEQ ID NO:06). These two PCR products were digested with the restriction endonuclease Dpn I to remove the template, and then transformed into Escherichia coli to obtain the recombinant plasmid pZA-glcB. 454A -pduP (plasmid EG01) and pZA-glcB 633G -pduP (plasmid EG02). The structures of EG00, EG01 and EG02 plasmids are shown in Figure 3.

[0179] Example 3: DA7-EG strain shake flask fermentation

[0180] First, plasmids EG00, EG01, and EG02 were electroporated into the DA7 (BW25113 ΔldhA Δpta ΔpoxB ΔadhE ΔpflB ΔmgsA ΔfrdA) recipient strain containing the EG08 plasmid, generating strains DA7-EG00, DA7-EG01, and DA7-EG02. Shake flask fermentations were performed using the DA7 strain as a control. The fermentation medium formulation was as follows: 30 g / L glucose, 5 g / L yeast extract, 11.3 g / L M9 salts, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 10 g / L ethylene glycol, 5 mg / L VB1, 20 g / L calcium carbonate, and 0.5 mM IPTG. Fermentation was performed at 30°C for 24 hours at 220 rpm. The results of the 24-hour fermentation are shown in Table 1.

[0181] Table 1 Fermentation data of DA7-EG series strains

[0182] Fermentation broths of the recombinant strains were analyzed using LC-MS. Due to the very low tartaric acid production, only qualitative analysis (positive for presence, negative for absence) was performed here. Figures 4 and 5 show the LC-MS peak profiles of the L-tartaric acid standard and fermentation broth (using DA7-EG01 as an example; the results of DA7-EG02 were similar to those of DA7-EG01 and are not shown). A small amount of tartaric acid was detected in the 24-h fermentation broth of the DA7-EG01 strain (qualitative LC-MS analysis), while no tartaric acid was detected in the fermentation broth samples of the control strain DA7 and the wild-type GlcB-overexpressing DA7-EG00 strain. Overall, these analyses indicate that the malate synthase mutants expressed by the EG01 and EG02 plasmids successfully catalyze the synthesis of tartaric acid from glyoxylate and glycolyl-CoA.

[0183] Example 4: DT13-TA strain shake flask fermentation

[0184] Plasmid EG01 was electroporated into the DT13 recipient strain (BW25113 ΔldhA Δpta ΔpoxB ΔadhE ΔpflB ΔmgsA ΔfrdA Δgcl ΔglcB ΔaceB ΔycdW ΔyqhD ΔttdAB) containing plasmids EG05 or EG08, generating strains DT13-TA1 and DT13-TA1. Shake flask fermentations were performed using the following medium: 30 g / L glucose, 5 g / L yeast extract, 11.3 g / L M9 salts, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 10 g / L ethylene glycol, 5 mg / L VB1, 20 g / L calcium carbonate, and 0.5 mM IPTG. Fermentations were performed at 30°C for 24 hours at 220 rpm. 24-hour fermentation results: The EG consumption of the DT13 plasmid strain increased significantly, with DT13-TA1 producing 6.3 mg / L of tartaric acid and DT13-TA2 producing 4.1 mg / L of tartaric acid.

[0185] Table 2 Fermentation data of DT13-EG series strains

[0186] References:

[0187] 1. Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, K. A.; Tomita, M.; Wanner, B. L.; Mori, H., Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology 2006, 2(1), 2006.0008.

[0188] 2. Datsenko, K. A.; Wanner, B. L., One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences 2000, 97(12), 6640-6645.

[0189] Sequence information

Claims

1. A biological method for producing tartaric acid, comprising the steps of: a) Glyoxylic acid reacts with glycolyl-CoA in the presence of an enzyme that catalyzes the Claisen condensation reaction (EC 2.3.3.-) to produce tartaric acid; Preferably, the enzyme that can catalyze the Claisen condensation reaction (EC 2.3.3.-) can be malate synthase (EC 2.3.3.9), 2-isopropylmalate synthase (EC 2.3.3.13) or citramalate synthase (EC 2.3.3.21); Further preferably, malate synthase (GlcB) catalyzes the synthesis of tartaric acid from glyoxylic acid and glycolyl-CoA; Preferably, the method further comprises: b) Propionaldehyde dehydrogenase (PduP) catalyzes the synthesis of glycolyl-CoA from glycolaldehyde; Preferably, the method further comprises: c) catalyzing the conversion of ethylene glycol into glycolaldehyde by glycerol dehydrogenase GldA, lactaldehyde reductase FucO or propanediol oxidoreductase; Further preferably, the method further comprises: d) converting glycolaldehyde into glycolic acid under the catalysis of aldehyde dehydrogenase (AldA) and e) converting glycolic acid into glyoxylic acid under the catalysis of glycolate oxidase (GlcDEF).

2. The method of claim 1, wherein: The 2-isopropylmalate synthase (LeuA) is a mutant having enhanced catalytic activity for ethanolyl-CoA; Preferably, the parent of the mutant is Escherichia coli 2-isopropylmalate synthase, preferably with protein sequence accession number AAC73185; or the mutant has an amino acid sequence derived from Escherichia coli and having at least 98%, or at least 99% sequence identity to the amino acid sequence represented by protein accession number AAC73185, and has enhanced catalytic activity for glycolyl-CoA; Preferably, the parent of the mutant is Corynebacterium glutamicum 2-isopropylmalate synthase, preferably with protein sequence accession number CAA50295; Or the mutant has an amino acid sequence derived from Corynebacterium glutamicum and having at least 98%, or at least 99% sequence identity to the amino acid sequence represented by protein accession number CAA50295, and has enhanced catalytic activity for glycolyl-CoA; The citramalate synthase (CimA) is a mutant having enhanced catalytic activity for ethanolyl-CoA; Preferably, the parent of the mutant is Methanocaldococcus jannaschii citramalate synthase, preferably a mutant mj.CimA with a protein sequence accession number of AAB99402 (referred to as wild-type mj.CimA), or a mutant mj.CimA with six point mutations in brackets [I47V E114V H126Q T204A L238S V373STOP (stop codon)] introduced simultaneously on the basis of the wild-type mj.CimA; The malate synthase (GlcB) is a mutant having enhanced catalytic activity for glycolyl-CoA; Preferably, the parent of the mutant is Escherichia coli malate synthase G, preferably a protein sequence accession number of CAA52639, WP_194154652.1 (L25I), HBP7705285.1 (L64M), WP_130525714.1 (L596M), EFM9907032.1 (L46M), HAI5727248.1 (L514M), EJO970635 1.1(A467S), HCB2230779.1(A540S), EHC4340872.1(A322S), WP_193130882.1(A168S), HAW1984968.1(A701S), WP_222024550.1(A672S), WP_247168346.1(A510S) or HBE5017233.1(V365I); or the mutant has an amino acid sequence derived from Escherichia coli and having at least 98%, or at least 99% sequence identity to the amino acid sequence represented by protein accession number CAA52639, and has enhanced catalytic activity for glycolyl-CoA; Preferably, the mutant comprises 454A and / or 633G mutations.

3. The method of claim 1, wherein: The glycerol dehydrogenase is a glycerol dehydrogenase or a propylene glycol / glycerol bifunctional dehydrogenase derived from the genus Escherichia, such as AAC43051 derived from Escherichia coli, or a glycerol dehydrogenase such as OSL28309.1 derived from Escherichia albertii; or a glycerol dehydrogenase such as ABR79620 derived from Klebsiella pneumoniae, or a glycerol dehydrogenase such as Shigella boydii or Shigella dysenteriae (such as ABB68416.1 or ABB63725.1) derived from Shigella, or a glycerol dehydrogenase such as VTQ29411.1 derived from Streptococcal pneumoniae, or a glycerol dehydrogenase such as AAA22477 derived from Bacillus stearothermophilus, or a glycerol dehydrogenase such as AAA22477 derived from Bacillus subtilis. subtilis) such as CAB15083.2, or a glycerol dehydrogenase from Pseudomonas putida such as AAC44426, or a glycerol dehydrogenase from Clostridium saccharobutylicum such as AAA83520.1, or a glycerol dehydrogenase from Clostridium acetobutylicum such as AAK79593.1, or an alcohol dehydrogenase (glycerol dehydrogenase) from Corynebacterium glutamicum such as SJM54452.1, or an alcohol dehydrogenase from Gluconobacter oxydans such as AAW60096.1, or a glycerol dehydrogenase from Salmonella such as CNU06578.1, and can also be a glycerol dehydrogenase from Parasutterella Propanediol / glycerol bifunctional dehydrogenase MTU07760.1 from the genus excrementihominis, and natural or artificial mutants of the enzyme from the same species; Preferably, the glycerol dehydrogenase from Escherichia coli is a protein sequence accession number AAC43051 (ec.gldA); preferably, the glycerol dehydrogenase from Klebsiella pneumoniae is a protein sequence accession number ABR79620 (kp.gldA); or an enzyme that retains the above enzyme activity and has an amino acid sequence that has at least 96% or 97% or 98%, or at least 99% sequence identity with the amino acid sequence represented by protein accession number AAC43051 or ABR79620.

4. The method of claim 1, wherein: Any step of the method is optionally performed inside or outside the cells of the genetically engineered strain; or The method is performed in a cell; optionally in the same cell or in a different cell; Preferably, the method is performed outside the cell; optionally under whole-cell catalysis or cell-free enzyme catalysis conditions; Preferably, the method is carried out in a genetically engineered strain cell, in which the pathway for glycolaldehyde to generate ethylene glycol, the pathway for glyoxylic acid to generate glycolic acid, hydroxymalonic acid semialdehyde and malic acid, and the pathway for tartaric acid to generate oxaloacetic acid are blocked or knocked out; further preferably, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, weakened or eliminated: i) NADPH-dependent aldehyde reductase yqhD; ii) glyoxylate / hydroxypyruvate reductase ycdW; iii) glyoxylate-aldehyde ligase gcl; iv) endogenous malate synthase A, aceB and endogenous malate synthase G, glcB; v) tartrate dehydratase α subunit, tartrate dehydratase β subunit ttdAB; Preferably, the glyoxylate / hydroxypyruvate reductase ycdW and the glyoxylate-aldehyde ligase gcl are knocked out simultaneously; Further preferably, NADPH-dependent aldehyde reductase yqhD, endogenous malate synthase A, aceB and endogenous malate synthase G, glcB, and tartaric acid dehydratase α subunit, tartaric acid dehydratase β subunit ttdAB are knocked out; Preferably, the production pathway of metabolic byproducts such as succinate, lactate, ethanol, acetate and formate in the genetically engineered strain is blocked or knocked out; further preferably: the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, weakened or eliminated: phosphate acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvaldehyde synthase mgsA, lactate dehydrogenase ldhAD, fumarate reductase flavoprotein subunit frdA.

5. A biological method for synthesizing glycolaldehyde, the method comprising: 1) Ethylene glycol is converted into ethanolaldehyde under the catalysis of glycerol dehydrogenase gldA; Preferably, the glycerol dehydrogenase gldA is as described in claim 3.

6. A method for synthesizing glycolic acid, the method comprising: firstly obtaining glycolaldehyde by the method of claim 5, and then 2) converting glycolaldehyde into glycolic acid under the catalysis of aldehyde dehydrogenase (aldA).

7. A method for synthesizing glyoxylic acid, the method comprising: first obtaining glycolic acid by the method of claim 6, and then 3) converting glycolic acid into glyoxylic acid under the catalysis of glycolate oxidase (glcDEF).

8. A method for synthesizing alcohol acyl-CoA, the method comprising: first obtaining glycolaldehyde by the method of claim 5, and then 4) synthesizing glycolyl-CoA from glycolaldehyde by propionaldehyde dehydrogenase (PduP).

9. A genetically engineered strain for producing tartaric acid, the strain expressing: a) at least one DNA molecule encoding an enzyme that catalyzes the Claisen condensation reaction (EC 2.3.3.-); b) at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP); Preferably, the genetically engineered strain further expresses: c) at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO; Preferably, the genetically engineered strain further expresses: d) at least one DNA molecule encoding lactaldehyde dehydrogenase (aldA) and e) at least one DNA molecule encoding glycolate oxidase (glcDEF).

10. A genetically engineered strain that can utilize ethylene glycol, wherein the strain expresses any one or a combination of the following DNA molecules: at least one DNA molecule encoding an enzyme that catalyzes the Claisen Condensation reaction (EC 2.3.3.-); at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP); at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO; At least one DNA molecule encoding lactaldehyde dehydrogenase (aldA) at least one DNA molecule encoding glycolate oxidase (glcDEF); Preferably, the strain expresses: at least one DNA molecule encoding a glycerol dehydrogenase gldA, wherein the strain synthesizes glycolaldehyde; or at least one DNA molecule encoding a glycerol dehydrogenase gldA, and at least one DNA molecule encoding a lactaldehyde dehydrogenase (aldA), wherein the strain synthesizes glycolic acid; or at least one DNA molecule encoding a glycerol dehydrogenase gldA, at least one DNA molecule encoding a lactaldehyde dehydrogenase (aldA), and at least one DNA molecule encoding a glycolate oxidase (glcDEF), wherein the strain synthesizes glyoxylate; or At least one DNA molecule encoding glycerol dehydrogenase gldA, at least one DNA molecule encoding lactaldehyde dehydrogenase (aldA) and at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP), and the strain synthesizes acetyl-CoA.

11. The genetically engineered strain according to claim 9 or 10, wherein: Preferably, expression of one or two or more of the genes is enhanced; Preferably, the malate synthase (GlcB) is as described in claim 2; Preferably, the glycerol dehydrogenase gldA is as described in claim 3; Preferably, the DNA molecule is introduced into the host strain via a plasmid, or integrated into the genome of the host strain by genetic engineering; Preferably, the host strain is selected from bacteria or fungi; optionally, the host strain is selected from wild or genetically engineered Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces; optionally, the host strain is selected from wild or genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; optionally, the host strain is selected from wild or genetically engineered Escherichia coli; Preferably, the pathway for glycolaldehyde to generate ethylene glycol, the pathway for glyoxylic acid to generate glycolic acid, hydroxymalonic acid semialdehyde and malic acid, and the pathway for tartaric acid to generate oxaloacetic acid in the genetically engineered strain are blocked or knocked out; further preferably, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, weakened or eliminated: i) NADPH-dependent aldehyde reductase yqhD; ii) glyoxylate / hydroxypyruvate reductase ycdW; iii) glyoxylate-aldehyde ligase gcl; iv) endogenous malate synthase A, aceB and endogenous malate synthase G, glcB; v) tartrate dehydratase α subunit, tartrate dehydratase β subunit ttdAB; Preferably, the glyoxylate / hydroxypyruvate reductase ycdW and the glyoxylate-aldehyde ligase gcl are knocked out simultaneously; Further preferably, NADPH-dependent aldehyde reductase yqhD, endogenous malate synthase A, aceB and endogenous malate synthase G, glcB, and tartaric acid dehydratase α subunit, tartaric acid dehydratase β subunit ttdAB are knocked out; Preferably, the production pathways of metabolic byproducts such as succinic acid, lactic acid, ethanol, acetic acid and formic acid in the genetically engineered strain are blocked or knocked out; further preferably, the expression of one, two or more of the following genes in the microorganism is inhibited: Inhibit, weaken or eliminate: phosphate acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvaldehyde synthase mgsA, lactate dehydrogenase ldhAD, fumarate reductase flavoprotein subunit frdA.

12. A combination of biological materials or biological materials, the biological material comprising: at least one DNA molecule encoding an enzyme that catalyzes the Claisen Condensation reaction (EC 2.3.3.-); at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP); at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO; At least one DNA molecule encoding lactaldehyde dehydrogenase (aldA) at least one DNA molecule encoding glycolate oxidase (glcDEF); In a specific embodiment of the present invention, the biomaterial combination is a combination of the following biomaterials A and B: Biomaterial A, comprising: at least one DNA molecule encoding malate synthase (GlcB); at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP); Biomaterial B, comprising: at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO; at least one DNA molecule encoding lactaldehyde dehydrogenase (aldA) and at least one DNA molecule encoding glycolate oxidase (glcDEF); Preferably, the biological material is an expression cassette, a transposon, a plasmid vector, a cosmid vector, an artificial chromosome (such as a yeast artificial chromosome YAC or a bacterial artificial chromosome BAC), a phage vector or a viral vector.

13. A mutant of malate synthase (GlcB), wherein the mutant has enhanced catalytic activity for glycolyl-CoA, wherein the mutant has mutation sites of 454A or / and 633G relative to the amino acid sequence encoded by GeneID: 948857 or the amino acid sequence represented by protein accession number CAA52639, and the mutant has an amino acid sequence with at least 98%, or at least 99% sequence identity with the amino acid sequence encoded by GeneID: 948857 or the amino acid sequence represented by protein accession number CAA52639.

14. The gene sequence of the malate synthase (GlcB) mutant according to claim 13, or a biological material comprising the gene sequence, or a genetically engineered strain expressing the gene sequence.

15. Use of the gene encoding the biological material according to claim 12 or the mutant of malate synthase (GlcB) according to claim 13 or the biological material comprising the gene in constructing a genetically engineered strain producing tartaric acid and / or a genetically engineered strain utilizing ethylene glycol.

16. A genetically engineered strain, wherein at least one of the pathways for glycolaldehyde to generate ethylene glycol, the pathways for glyoxylic acid to generate glycolic acid, hydroxymalonic acid semialdehyde and malic acid, and the pathways for tartaric acid to generate oxaloacetic acid is blocked or knocked out; preferably, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, weakened or eliminated: i) NADPH-dependent aldehyde reductase yqhD; ii) glyoxylate / hydroxypyruvate reductase ycdW; iii) glyoxylate-aldehyde ligase gcl; iv) endogenous malate synthase A, aceB and endogenous malate synthase G, glcB; v) tartrate dehydratase α subunit, tartrate dehydratase β subunit ttdAB; Preferably, the glyoxylate / hydroxypyruvate reductase ycdW and the glyoxylate-aldehyde ligase gcl are knocked out simultaneously; Further preferably, NADPH-dependent aldehyde reductase yqhD, endogenous malate synthase A, aceB and endogenous malate synthase G, glcB, and tartaric acid dehydratase α subunit, tartaric acid dehydratase β subunit ttdAB are knocked out; Also preferably, the production pathways of metabolic byproducts such as succinate, lactate, ethanol, acetate and formate in the genetically engineered strain are blocked or knocked out; preferably, the expression of one, two or more of the following genes in the microorganism is inhibited, weakened or eliminated: phosphate acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvaldehyde synthase mgsA, lactate dehydrogenase ldhAD, fumarate reductase flavoprotein subunit frdA.

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