Construction and application of escherichia coli engineering strain for synthesizing tartaric acid by using ethylene glycol

Abstract

The invention discloses construction and application of an Escherichia coli engineering strain capable of synthesizing tartaric acid by using ethylene glycol, establishes a new way for synthesizing tartaric acid by using ethylene glycol, and generates tartaric acid by using ethylene glycol as a substrate.

Description

Technical Field

[0001] 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 using ethylene glycol. Background Art

[0002] Ethylene glycol (EG) is a widely used chemical that can be used directly as an antifreeze or coolant, and is also a substrate for the production of polymers such as polyethylene terephthalate (PET). More than 25 million tons are synthesized each year for the manufacture of plastic bottles. The accumulation of PET plastic in nature is becoming increasingly concerning due to its growing global demand and continued negative impact on the environment. If PET waste can be hydrolyzed cost-effectively, the resulting hydrolyzed PET monomers will become a sustainable and abundant source of raw materials for the production of useful molecules.

[0003] Tartaric acid, as an important organic acid product, is widely used in the fields of chemical industry, medicine and food. 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, Eiji Sato 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. It can also provide precursors for amino acid synthesis. Tartaric acid dehydrogenase exists in an inactive low molecular weight form and is activated in Fe 2+ and thiols to be converted into active enzymes.

[0004] E. coli is a commonly used host organism in metabolic engineering, but wild-type E. coli cannot utilize EG as the sole carbon source. Early studies have found that E. coli strains that utilize EG can be separated from mutants that can grow on propanediol through adaptive evolution. Figure 1 As shown, the promiscuous activity of alcohol oxidoreductase can convert ethylene glycol into glycolaldehyde, which is then converted into glycolic acid by lactaldehyde dehydrogenase, and glycolic acid is finally converted into glyoxylate by endogenous glycolate oxidase for cell growth. Glyoxylate and acetyl-CoA can generate malate to enter the TCA cycle under the action of malate synthase. Summary of the invention

[0005] The present invention aims to establish a new way to synthesize tartaric acid from ethylene glycol, and to generate tartaric acid using ethylene glycol as a substrate. Patent document EP1748076A1 reports that the conversion of ethylene glycol to glyoxylic acid in Escherichia coli requires three enzymes to participate in the catalytic reaction, namely lactaldehyde reductase, lactaldehyde dehydrogenase and glycolate oxidase, and the encoding genes are fucO, aldA and glcDEF, respectively, which catalyze the conversion of ethylene glycol into glycolaldehyde, glycolic acid and glyoxylic acid in turn. In addition, non-patent document (JOURNAL OF BACTERIOLOGY, Vol. 153, No. 1, p. 134-139) also reports that propylene glycol oxidoreductase can catalyze the oxidation of ethylene glycol to glycolaldehyde. The inventors of the present application have found 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. The present invention is based on the metabolic pathway of ethylene glycol to generate glyoxylic acid. On the one hand, propionaldehyde dehydrogenase (PduP) is introduced to catalyze the synthesis of glycolyl-CoA from glycolaldehyde, and on the other hand, malate synthase (GlcB) is modified to catalyze the reaction of glyoxylic acid with glycolyl-CoA to generate tartaric acid ( Figure 1 ).

[0006] The specific aspects of the present invention are as follows:

[0007] The first aspect of the present invention provides a biological method for producing tartaric acid, comprising the following steps:

[0008] 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.

[0009] 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).

[0010] More preferably, malate synthase (GlcB) catalyzes the synthesis of tartaric acid from glyoxylate and glycolyl-CoA.

[0011] In a specific embodiment of the present invention, the method further comprises: b) propionaldehyde dehydrogenase (PduP) catalyzes the synthesis of glycolyl-CoA from glycolaldehyde.

[0012] 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.

[0013] 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).

[0014] In a specific embodiment of the present invention, the 2-isopropylmalate synthase (LeuA) is a mutant having enhanced catalytic activity towards glycolyl-CoA;

[0015] Preferably, the parent of the mutant is Escherichia coli 2-isopropylmalate synthase, preferably with protein sequence accession number AAC73185;

[0016] 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;

[0017] Preferably, the parent of the mutant is Corynebacterium glutamicum 2-isopropylmalate synthase, preferably with protein sequence accession number CAA50295;

[0018] 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.

[0019] In a specific embodiment of the present invention, the citramalate synthase (CimA) is a mutant having enhanced catalytic activity for glycolyl-CoA;

[0020] Preferably, the parent of the mutant is Methanocaldococcus jannaschii citramalate synthase, preferably a mutant mj.CimA with a protein sequence accession number of AAB99402 (called 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.

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

[0022] In a specific embodiment of the present invention, 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), EJ09706351.1 (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; an enzyme having the above-mentioned 454A and / or 633G mutation sites and having enhanced catalytic activity for ethanolyl-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 is also suitable for use in the present invention.

[0023] In a specific embodiment of the present invention, the glycerol dehydrogenase is a glycerol dehydrogenase or a propanediol / 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 glycerol dehydrogenase from Pseudomonas putida, such as AAC44426, or glycerol dehydrogenase from Clostridium saccharobutyl saccharobutylicum) such as AAA83520.1, or a glycerol dehydrogenase derived from Clostridium acetobutylicum such as AAK79593.1, or a glycerol dehydrogenase derived from Salmonella such as CNU06578.1, or an alcohol dehydrogenase (glycerol dehydrogenase) derived from Corynebacterium glutamicum such as SJM54452.1, and can also be a propanediol / glycerol bifunctional dehydrogenase MTU07760.1 derived from Parasutterella excrementihominis, as well as natural or artificial mutants of enzymes from the same species.

[0024] 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.

[0025] 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.

[0026] In a specific embodiment of the invention, the method is performed within a cell; optionally within the same cell or within a different cell.

[0027] In a specific embodiment of the present invention, the method is carried out outside a cell; optionally under whole-cell catalysis or cell-free enzyme catalysis conditions.

[0028] 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:

[0029] i) NADPH-dependent aldehyde reductase yqhD;

[0030] ii) glyoxylate / hydroxypyruvate reductase ycdW;

[0031] iii) glyoxylate-aldehyde ligase gcl;

[0032] iv) endogenous malate synthase A, aceB and endogenous malate synthase G, glcB;

[0033] v) tartrate dehydratase α subunit, tartrate dehydratase β subunit ttdAB.

[0034] In a specific embodiment of the present invention, the method is carried out in a genetically engineered strain cell, and 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; 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.

[0035] A second aspect of the present invention provides a biological method for producing glycolaldehyde, the method comprising:

[0036] 1) Ethylene glycol is converted into ethanolaldehyde under the catalysis of glycerol dehydrogenase gldA.

[0037] The second aspect of the present invention also provides a method for synthesizing glycolic acid, the method comprising: firstly obtaining glycolaldehyde by the above method, and then 2) converting glycolaldehyde into glycolic acid under the catalysis of aldehyde dehydrogenase (aldA).

[0038] The second aspect of the present invention also provides a method for synthesizing glyoxylic acid, the method comprising: firstly obtaining glycolic acid by the above method, and then 3) converting glycolic acid into glyoxylic acid under the catalysis of glycolate oxidase (glcDEF).

[0039] The second aspect of the present invention also provides a method for synthesizing alcohol acyl-CoA, the method comprising first obtaining glycolaldehyde by the above method, and then 4) synthesizing glycolyl-CoA from glycolaldehyde by propionaldehyde dehydrogenase (PduP).

[0040] The glycerol dehydrogenase gldA is as described in the first aspect of the present invention.

[0041] The third aspect of the present invention provides a genetically engineered strain for producing tartaric acid, wherein the strain expresses:

[0042] a) at least one DNA molecule encoding an enzyme catalyzing the Claisen condensation reaction (EC 2.3.3.-);

[0043] b) at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP).

[0044] 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.

[0045] 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).

[0046] The glycerol dehydrogenase gldA is as described in the first aspect of the present invention.

[0047] The enzyme that can catalyze Claisen Condensation reaction (EC 2.3.3.-) is as described in the first aspect of the present invention.

[0048] The fourth aspect of the present invention provides a genetically engineered strain that can utilize ethylene glycol, wherein the strain expresses any one or a combination of the following DNA molecules:

[0049] At least one DNA molecule encoding an enzyme that catalyzes the Claisen Condensation reaction (EC 2.3.3.-);

[0050] at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP);

[0051] at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO;

[0052] At least one DNA molecule encoding lactaldehyde dehydrogenase (aldA)

[0053] at least one DNA molecule encoding glycolate oxidase (glcDEF);

[0054] Preferably, the strain expresses:

[0055] at least one DNA molecule encoding a glycerol dehydrogenase gldA, wherein the strain synthesizes glycolaldehyde;

[0056] or

[0057] 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;

[0058] or

[0059] 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;

[0060] or

[0061] 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.

[0062] 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.

[0063] 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.

[0064] Furthermore, the following metabolic pathways of the genetically engineered strain are blocked, weakened or eliminated:

[0065] 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 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; preferably, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, weakened or eliminated:

[0066] i) NADPH-dependent aldehyde reductase yqhD;

[0067] ii) glyoxylate / hydroxypyruvate reductase ycdW;

[0068] iii) glyoxylate-aldehyde ligase gcl;

[0069] iv) endogenous malate synthase A, aceB and endogenous malate synthase G, glcB;

[0070] v) tartrate dehydratase α subunit, tartrate dehydratase β subunit ttdAB.

[0071] 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.

[0072] The glycerol dehydrogenase gldA is as described in the first aspect of the present invention.

[0073] Wherein malate synthase (GlcB) is as described in the first aspect of the present invention.

[0074] A sixth aspect of the present invention provides a biomaterial or a combination of biomaterials, wherein the biomaterial comprises:

[0075] at least one DNA molecule encoding an enzyme catalyzing the Claisen Condensation reaction (EC 2.3.3.-);

[0076] at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP);

[0077] at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO;

[0078] At least one DNA molecule encoding lactaldehyde dehydrogenase (aldA)

[0079] At least one DNA molecule encoding glycolate oxidase (glcDEF).

[0080] In a specific embodiment of the present invention, the biomaterial combination is a combination of the following biomaterials A and B:

[0081] Biomaterial A, comprising:

[0082] at least one DNA molecule that catalyzes an enzyme (EC 2.3.3.-) that catalyzes the Claisen Condensation reaction;

[0083] at least one DNA molecule encoding propionaldehyde dehydrogenase (PduP);

[0084] Biomaterial B, comprising:

[0085] at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO;

[0086] At least one DNA molecule encoding lactaldehyde dehydrogenase (aldA) and at least one DNA molecule encoding glycolate oxidase (glcDEF).

[0087] 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.

[0088] The glycerol dehydrogenase gldA is as described in the first aspect of the present invention.

[0089] The enzyme catalyzing Claisen Condensation reaction (EC 2.3.3.-) is as described in the first aspect of the present invention.

[0090] The seventh aspect of the present invention provides a mutant of malate synthase (GlcB), which has enhanced catalytic activity for ethanolyl-CoA, and the mutant has mutation sites of 454A and / or 633G relative to the amino acid sequence encoded by GeneID: 948857, 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.

[0091] 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.

[0092] The eighth aspect of the present invention provides the use of the above-mentioned biological material 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.

[0093] The beneficial technical effects of the present invention are:

[0094] 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

[0095] Figure 1 : Synthesis pathway from ethylene glycol to tartaric acid: dotted arrows represent gene knockouts, and gray is the name of the knockout gene; GlcB mut is a GlcB protein with a point mutation;

[0096] Figure 2 : Schematic diagram of the plasmid structure of EG05 and EG08;

[0097] Figure 3 : Schematic diagram of plasmid structures of EG00, EG01 and EG02;

[0098] Figure 4 : LC-MS peak diagram of L-tartaric acid standard;

[0099] Figure 5 : LC-MS peak diagram of the fermentation broth of DA7-EG01 strain at 24h; DETAILED DESCRIPTION

[0100] The following will be further described in detail with reference to specific examples. It should be understood that the following examples are only exemplary descriptions and explanations of 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.

[0101] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0102] As used herein, the term "amplification" means that the intracellular activity of one or more enzymes encoded by appropriate DNA in a microorganism is improved, for example, by increasing the gene copy number, using a strong promoter or using a gene encoding an appropriate enzyme with high activity, and selectively combining these methods.

[0103] 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.

[0104] As used herein, the term "gene synthesis" refers to the production using recombinant DNA technology or the acquisition using synthetic DNA or amino acid sequence technology available and well 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 be used as a template to synthesize other polymers and macromolecules in biological processes, which have any of a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties produced thereby. Therefore, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is equivalent to the mRNA sequence and is usually provided in the sequence listing, and the non-coding strand used as a template for transcribing a gene or cDNA, can be referred to as encoding a protein or other product of that gene or cDNA.

[0105] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter, and the term "overexpression" or "enhanced expression" refers to an increase in the level of mRNAs encoding a protein, and / or refers to an increase in the level of a protein in a cell compared to a similar corresponding unmodified cell expressing a basal level of mRNAs or having a basal level of protein. For example, an endogenous or exogenous gene of a microorganism is introduced to increase the expression level of the corresponding enzyme, and endogenous or exogenous gene expression can also be increased by replacing a strong promoter or introducing an enhancer.

[0106] 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, inserted into a carrier nucleic acid molecule. The vector may contain a sequence that directs autonomous replication in the cell or may contain a sequence sufficient to allow integration into the host cell DNA. Many vectors are known in the art, including but 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.

[0107] As used herein and unless otherwise specified, 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, as long as such variations are suitable for practicing the disclosed methods.

[0108] In the present invention, serial numbers are used to express the combination of reaction steps, DNA molecules, biomaterials or enzymes. For example, a)-c) includes the combination of three aspects with serial numbers a), b) and c). In the present invention, the combination of reaction steps means that the final synthesis is completed by using the reaction steps therein, but it does not limit the way in which the reactions of each step are carried out. Each reaction step can be carried out step by step or simultaneously, and can be carried out in cells or outside cells, or under the catalysis of cell-free enzymes. Unless otherwise stated, the combination of DNA molecules, the combination of biomaterials or the combination of enzymes does not mean that the combination of DNA molecules, the combination of biomaterials or the combination of enzymes are in the same organism or biomaterial, but the present invention preferably expresses the combination of DNA molecules and enzymes in the same organism.

[0109] In the present invention, "at least one" refers to at least one of the same or similar references, but 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.

[0110] In the present invention, the term "amino acid mutation" or "nucleotide mutation" used 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 a nucleotide sequence or an amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

[0111] In the present invention, the term "native state" used refers to the activity of a polypeptide in a microorganism in an unmodified state, that is, the activity in the natural state.

[0112] 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 refers 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.

[0113] Therefore, it is obvious that the enzyme of the present invention can be further mutated to obtain further mutants that still have the corresponding functions and activities. For example, it is well known to those skilled in the art that adding or reducing 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 amino acid residue at either end of the polypeptide will not affect the function of the obtained mutant. For example, for the convenience of purification, technicians often carry a 6×His tag at either end of the obtained protein, and this protein has the same function as the protein without the 6×His tag. Therefore, the present invention should include conservative mutations obtained on the basis of the present invention.

[0114] 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.

[0115] As used in the present invention, the term "conservative substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, and include amino acids 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), nonpolar 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).

[0116] As used in the present invention, "conservative substitutions" generally exchange an amino acid at one or more sites in a protein. Such substitutions may be conservative. Exemplary substitutions that are considered conservative substitutions include substitutions of Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr. Conservative mutations include substitutions of Ile to Leu, Met, Val or Phe, substitutions of Leu to Ile, Met, Val or Phe, substitutions of Lys to Asn, Glu, Gln, His or Arg, substitutions of Met to Ile, Leu, Val or Phe, substitutions of Phe to Trp, Tyr, Met, Ile or Leu, substitutions of Ser to Thr or Ala, substitutions of Thr to Ser or Ala, substitutions of Trp to Phe or Tyr, substitutions of Tyr to His, Phe or Trp, and substitutions of Val to Met, Ile or Leu. In addition, conservative mutations also include naturally occurring mutations caused by individual differences, strains, species differences, etc. from which genes are derived.

[0117] The term "corresponding to" used in the present invention has the meaning generally understood by those of ordinary skill in the art. Specifically, "corresponding to" means that after two sequences are aligned for homology or sequence identity, a position in one sequence corresponds to a specified position in another sequence. Thus, for example, with respect to "the amino acid residue corresponding to position 40 of the amino acid sequence shown in SEQ ID NO. 1", if a 6×His tag is added to one end of the amino acid sequence shown in SEQ ID NO. 1, then the position corresponding to position 40 in the resulting mutant may be position 46 of the amino acid sequence shown in SEQ ID NO. 1.

[0118] 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%.

[0119] Methods for determining sequence homology or identity 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, ed., 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., ed., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied 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 the 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.

[0120] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0121] 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, wherein the glycerol dehydrogenase protein sequence accession number from Escherichia coli is AAC43051 (ec.gldA), and the glycerol dehydrogenase protein sequence accession number from Klebsiella pneumoniae is ABR79620 (kp.gldA); enzymes that maintain the above enzyme activity and have an amino acid sequence with at least 98% or at least 99% sequence identity to the amino acid sequence represented by the protein accession number are also applicable to the present invention.

[0122] The present invention further provides a biological method for producing tartaric acid, comprising the following steps:

[0123] a) Glyoxylate reacts with glycolyl-CoA in an enzyme that catalyzes the Claisen condensation (EC 2.3.3.-) to produce tartaric acid.

[0124] In a specific embodiment of the present invention, the malate synthase (GlcB) is a mutant having enhanced catalytic activity towards glycolyl-CoA.

[0125] In a specific embodiment of the present invention, the parent of the mutant is Escherichia coli malate synthase G, and the protein sequence accession number is CAA52639; preferably, the mutant contains 454A and / or 633G, has enhanced catalytic activity for ethanolyl-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 enzyme is also applicable to the present invention.

[0126] 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).

[0127] In a specific embodiment of the present invention, the method further comprises: b) synthesizing glycolyl-CoA from glycolaldehyde by propionaldehyde dehydrogenase (PduP).

[0128] 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.

[0129] 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).

[0130] 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).

[0131] The present invention realizes the conversion of ethylene glycol into tartaric acid in recombinant Escherichia coli.

[0132] 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.

[0133] The starting strain of the present invention is Escherichia coli BW25113 (K12 strain). Those skilled in the art know that other similar Escherichia coli strains such as other K strains (such as MG1665 or W3110), W strains (such as ATCC 9637), Crooks strains (such as ATCC 8739), B strains (such as ATCC 11303, BL21 (DE3) or REL606), C strains (such as ATCC 23461) and Escherichia coli probiotic Nissle 1917 (EcN / DSM 6601) and their derivative strains, as well as Escherichia coli of other genetic lineages can also be used in the present invention.

[0134] The present invention first constructs an Escherichia coli strain with a knockout gene encoding a metabolic enzyme that is 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 succinic acid, lactic acid, ethanol, acetic acid and formic acid are blocked or knocked out to avoid waste of carbon sources.

[0135] The present invention uses gene knockout to inhibit, reduce or eliminate the expression of the corresponding enzyme. Those skilled in the art 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 of gene transcription / translation, introduction of mutations or nucleic acid sequences that accelerate the degradation of mRNA that guides enzyme synthesis, introduction of mutations or protein tags that accelerate enzyme degradation / decomposition / inactivation, inhibition of signal activation, RNA interference and gene silencing.

[0136] In a specific embodiment of the present invention, the expression of one, two or more of the following genes in the microorganism is inhibited, weakened or eliminated:

[0137] i) NADPH-dependent aldehyde reductase yqhD;

[0138] ii) glyoxylate / hydroxypyruvate reductase ycdW;

[0139] iii) glyoxylate-aldehyde ligase gcl;

[0140] iv) malate synthase A, aceB and malate synthase G, glcB;

[0141] v) tartrate dehydratase α subunit, tartrate dehydratase β subunit ttdAB.

[0142] 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.

[0143] The present invention uses two recombinant plasmids expressing gldA-aldA-glcDEF and glcB mutant-pduP to transfer the enzyme that uses ethylene glycol to synthesize tartaric acid into Escherichia coli for expression. Those skilled in the art know that any other plasmid combination is also applicable to the present invention as long as it expresses the above enzyme.

[0144] 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 a regulatory element.

[0145] Those skilled in the art also know that other strain construction methods such as genome integration are also applicable to the present invention. For example, random integration or site-specific integration technology can be used to construct the corresponding strain.

[0146] Enzymes that catalyze the Claisen Condensation reaction (EC 2.3.3.-)

[0147] Enzymes that can catalyze the Claisen condensation reaction are known to those skilled in the art, and 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 in the present invention.

[0148] Biological pathway for the conversion of ethylene glycol to glycoaldehyde

[0149] Glycerol dehydrogenase (GDH) mainly catalyzes glycerol to produce dihydroxyacetone (DHA), but some glycerol dehydrogenases (GDH) also have a certain activity of catalyzing ethylene glycol. The preferred glycerol dehydrogenases (GDH) of the present invention include: glycerol dehydrogenases or propylene glycol / glycerol bifunctional dehydrogenases derived from the genus Escherichia, such as AAC43051 derived from Escherichia coli, or glycerol dehydrogenases derived from Escherichia albertii such as OSL28309.1; or glycerol dehydrogenases derived from Klebsiella pneumoniae such as ABR79620, or glycerol dehydrogenases derived from Shigella such as Shigella boydii or Shigella dysenteriae (such as ABB68416.1 or ABB63725.1), or glycerol dehydrogenases derived from Streptococcus pneumoniae (such as 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 AAC44426. glutamicum), such as SJM54452.1; it can also be a propanediol / glycerol bifunctional dehydrogenase MTU07760.1 derived from Parasutterella excrementihominis, and a natural or artificial mutant of an enzyme from the same species.

[0150] In addition, lactaldehyde reductase fucO or propanediol oxidoreductase can also be used to catalyze the conversion of ethylene glycol to glycolaldehyde.

[0151] Materials and Methods

[0152] The information of the enzyme involved in the present invention is as follows:

[0153]

[0154] In the context of the present application, the enzyme referred to includes 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.

[0155] The biomaterials constructed by the present invention are shown in the following table:

[0156]

[0157] Example 1: Construction of DA7 and DT13 strains

[0158] Using the E. coli Keio Knockout Collection 1 (Horizon Discovery, CO, USA) was used to prepare phages, and the DNA target gene deletion fragment with kanamycin (Kan) resistance was introduced into the starting strain Escherichia coli BW25113 using the P1 phage transfection method, and then coated on an LB plate containing 50 mg / L kanamycin, and cultured overnight at 37 degrees to obtain a deletion strain with Kan resistance; then the plasmid pCP20 was transformed into the above Kan-resistant transformant, coated on an LB plate containing 100 mg / L ampicillin, and cultured at 30 degrees for 24 hours. The correct transformant with the kanamycin resistance gene Kan removed in the target gene was identified by PCR to obtain a non-resistant deletion strain. By repeating this process, a deletion strain DA7 based on BW25113 was constructed, in which the production pathways of metabolic byproducts (succinic acid, lactic acid, ethanol, acetic acid and formic acid) were blocked and knocked out to avoid waste of carbon sources.

[0159] The same method was used to knock out Δgcl, ΔglcB, ΔaceB, ΔycdW and ΔyqhD based on DA7 to obtain the DT12 strain.

[0160] Using λ-Red recombination technology 2 , according to the literature 1 Describe the conditions: Use primer pairs seqID01 and seqID02 to perform PCR 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 2The prepared DT12 strain competent cells were placed in LB medium at 37 degrees for half an hour for recovery and then coated on LB plates containing 50 mg / L kanamycin. After culturing at 37 degrees overnight, the correct positive transformant was identified, and the ttdAB gene was replaced by the kanamycin resistance gene to obtain the strain DT13K. Then PCP20 was transformed into DT13K, coated on an LB plate containing 100 mg / L ampicillin, and after culturing at 30 degrees for 24 hours, the correct transformant with the kanamycin resistance gene Kan removed from the ttdAB site was identified by PCR to obtain the strain DT13. The metabolic enzyme encoding genes that are not conducive to tartaric acid accumulation and can divert EG metabolic intermediates in strain DT13 were knocked out.

[0161] 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)

[0162] 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.

[0163] As above, ec.gldA, aldA and glcDEF were cloned from the genome of Escherichia coli BW25113 by PCR, and the cloned fragments were connected to the plasmid pZElac (ampicillin resistance, ColE1 replicon, lac promoter) by Gibson Assembly to obtain 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 shown.

[0164] The malate synthase gene glcB in the genome of E. coli BW25113 and the pduP gene in the genome of Salmonella typhimurium LT2 were cloned by PCR, and the obtained gene fragments were connected to the plasmid pZAlac (kanamycin resistance, P15A replicon, lac promoter) by Gibson Assembly to obtain the recombinant plasmid pZA-glcB-pduP (plasmid EG00).

[0165] The best substrates of malate synthase (encoded by glcB gene) are glyoxylate and acetyl-CoA. The present invention realizes the synthesis of malate synthase by modifying the acetyl-CoA binding region of malate synthase so that it can accept glycolyl-CoA. Figure 1 Synthesis of tartaric acid as shown. Using the recombinant plasmid pZA-glcB-pduP (EG00) as a template, a PCR product in which the leucine at position 454 in GlcB was mutated to alanine (using primers seqID03 and seqID04), or alanine at position 633 was mutated to glycine (using primers seqID05 and seqID06) was obtained by circular plasmid PCR site-directed mutagenesis technology. The above two PCR products were digested with restriction endonuclease Dpn I to remove the template, and then transformed into Escherichia coli respectively, and finally the recombinant plasmid pZA-glcB was obtained. 454A -pduP (plasmid EG01) and pZA-glcB 633G -pduP (plasmid EG02). The plasmid structures of EG00, EG01 and EG02 are as follows Figure 3 shown.

[0166] Example 3: DA7-EG strain shake flask fermentation

[0167] First, plasmids EG00, EG01 and EG02 were electroporated into the DA7 (BW25113ΔldhAΔptaΔpoxBΔadhEΔpflBΔmgsAΔfrdA) recipient strain containing EG08 plasmid, and strains DA7-EG00, DA7-EG01 and DA7-EG02 were obtained. Shake flask fermentation was carried out with DA7 strain as the control strain, and the fermentation medium formula was as follows: glucose 30g / L, yeast powder 5g / L, M9 salt 11.3g / L, magnesium sulfate 2mM, calcium chloride 0.1mM, ethylene glycol 10g / L, VB1 5mg / L, calcium carbonate 20g / L, IPTG0.5mM. Fermentation was carried out at 30°C for 24 hours, with a rotation speed of 220rpm. The 24-hour fermentation results are shown in Table 1.

[0168] Table 1 Fermentation data of DA7-EG series strains

[0169]

[0170] The fermentation broth of the recombinant strain was analyzed by LC-MS. Since the tartaric acid production was very low, only qualitative analysis of tartaric acid was performed here (+; -). Figure 4 and Figure 5 The figure shows the LC-MS peak diagram of L-tartaric acid standard and fermentation broth (taking DA7-EG01 as an example; the results of DA7-EG02 are similar to DA7-EG01 and are not shown). A small amount of tartaric acid can be detected in the 24h fermentation broth of DA7-EG01 strain (qualitative analysis by LC-MS), while no tartaric acid was detected in the fermentation broth samples of the control strain DA7 and DA7-EG00 overexpressing wild-type GlcB. Comprehensive analysis shows that the malate synthase mutants expressed by the EG01 and EG02 plasmids successfully catalyze the synthesis of tartaric acid from glyoxylate and glycolyl-CoA.

[0171] Example 4: DT13-TA strain shake flask fermentation

[0172] Plasmid EG01 was electroporated into the DT13 (BW25113ΔldhAΔptaΔpoxBΔadhEΔpflBΔmgsAΔfrdAΔgclΔglcBΔaceBΔycdWΔyqhDΔttdAB) recipient strain containing plasmid EG05 or EG08 to obtain strains DT13-TA1 and DT13-TA1. Shake flask fermentation was carried out, and the fermentation medium formula was as follows: glucose 30g / L, yeast powder 5g / L, M9 salt 11.3g / L, magnesium sulfate 2mM, calcium chloride 0.1mM, ethylene glycol 10g / L, VB1 5mg / L, calcium carbonate 20g / L, IPTG 0.5mM. Fermentation was carried out at 30°C for 24 hours, with a rotation speed of 220rpm. 24-hour fermentation results: DT13-TA1 produced 6.3 mg / L of tartaric acid, and DT13-TA2 produced 4.1 mg / L of tartaric acid.

[0173] References:

[0174] 1.Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, KA; Tomita, M.; Wanner, BL; 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.

[0175] 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.

[0176] Sequence information

[0177] seqID01 :

[0178]

[0179] seqID02 :

[0180]

[0181] seqID03 :

[0182]

[0183] seqID04 :

[0184]

[0185] seqID05 :

[0186]

[0187] seqID06 :

[0188]

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). More preferably, malate synthase (GlcB) catalyzes the synthesis of tartaric acid from glyoxylate 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), EJ0970635 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 a glycerol dehydrogenase from Corynebacterium glutamicum such as AAC44426. glutamicum), such as SJM54452.1, or glycerol dehydrogenase from Salmonella, such as CNU06578.1, and can also be propanediol / glycerol bifunctional dehydrogenase MTU07760.1 from Parasutterella excrementihominis, as well as natural or artificial mutants of enzymes 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 of a 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 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 glycolaldehyde 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: firstly obtaining glycolic acid by the method of claim 6, and then 3) converting the 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 production pathways of metabolic byproducts such as succinate, lactate, ethanol, acetate and formate 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, 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.

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.

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