Method for producing succinic acid through fermentation and application thereof

Abstract

The invention relates to a method for producing succinic acid by fermentation and application thereof, and provides a synthetic method of succinic acid, a genetic engineering strain and application thereof. According to the invention, a new path for synthesizing the succinic acid is opened up by increasing the conversion rate of the precursor, a new precursor is found for synthesizing the succinic acid, and a new method for producing the succinic acid by fermentation is provided.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biochemical engineering, and relates to a synthesis method of succinic acid, a genetically engineered strain, a preparation method thereof and application of the genetically engineered strain. BACKGROUND

[0002] Fumaric acid, also known as fumaric acid, trans-butenedioic acid and corydalis acid, has a chemical formula of C4H4O4, is a carboxylic acid derived from butene, and is a colorless and flammable crystal. In food, it is mainly used in meat products, fish products and other processing. Fumaric acid can be used as acidity regulator, acidifying agent, antioxidant aid, pickling accelerator and spice. It has strong buffering effect to maintain the pH of aqueous solution at about 3.0, and has important effect on antibacterial and mold prevention. At the same time, it has astringency, is one of the strongest solid acids, has low absorption rate, and helps to extend the shelf life of powder products. When fumaric acid becomes sodium fumarate, the water solubility and flavor are better.

[0003] Succinic acid is a C4-dicarboxylic acid, also known as succinic acid, which is an important intermediate in the tricarboxylic acid cycle in organisms. As an important dicarboxylic acid in industrial production, succinic acid is widely used in biological medicine, food, agriculture and other fields. The chemical synthesis of succinic acid mainly relies on electrochemistry and catalytic hydrogenation method, and maleic anhydride and n-hexane, which are petroleum chemical raw materials, are the key substrates of succinic acid. Although the conversion rate of chemical synthesis method is high, due to the limited production scale and strict operating conditions, the cost of succinic acid is high, which lacks market competitiveness. Compared with chemical processing, the raw materials for biosynthesis of succinic acid are more extensive and the cost is lower. As a model organism, Escherichia coli has the advantages of clear genetic background, perfect genetic operation system, clear metabolic network and rapid growth, so it is widely used as a cell factory for biological manufacturing of chemicals, which is also an advantage platform for biosynthesis of succinic acid. In the aerobic fermentation process, the endogenous regulation of TCA cycle in the natural pathway of Escherichia coli leads to less accumulation of succinic acid; therefore, the two-stage fermentation mode of increasing cell concentration in aerobic fermentation and accumulating succinic acid in anaerobic fermentation is often used in industrial production. In the fermentation process, the synthesis of by-products such as acetic acid and lactic acid will seriously affect the conversion rate of succinic acid. Therefore, finding new precursors for succinic acid synthesis and improving the conversion rate of precursors and opening up new synthesis paths to solve the defects of succinic acid fermentation process are still the difficulties to be solved in the field. SUMMARY

[0004] L-aspartate oxidase encoded by nadB gene in E. coli is the first enzyme in the de novo synthesis of NAD+ in bacteria. This FAD-dependent enzyme catalyzes the oxidation of L-aspartate to produce imino succinate and reduced flavin. To achieve the recycling of aspartate, we introduced imino succinate reductase encoded by bhcD gene from Paracoccus denitrificans aspartate cycle to catalyze the reduction of imino succinate to L-aspartate. Thus, the combination of nadB and bhcD genes in the process of catalyzing the conversion of fumarate to succinate ( Figure 1 ), bypasses the endogenous regulation of TCA cycle. The specific embodiments of the present invention include:

[0005] The first aspect of the present invention provides a method for synthesizing succinate, the method comprising the following steps:

[0006] 1) L-aspartate and fumarate are converted into succinate and imino succinate under the catalysis of L-aspartate oxidase (EC 1.4.3.16);

[0007] 2) imino succinate is recycled to produce L-aspartate.

[0008] In the specific embodiments of the present invention, the step 2) is carried out under the catalysis of ornithine cyclodeaminase / μ-crystallin superfamily protein; preferably, under the catalysis of imino succinate reductase.

[0009] In the specific embodiments of the present invention, the step 2) further comprises the step of spontaneous decomposition of imino succinate into OAA (oxaloacetate).

[0010] In the specific embodiments of the present invention, the method further comprises: 3) the step of ammonolysis of L-aspartate to produce fumarate; more preferably, the ammonolysis of L-aspartate is carried out under the catalysis of aspartate deaminase (EC 4.3.1.1) or methylaspartate deaminase (EC 4.3.1.2).

[0011] In the specific embodiments of the present invention, the method further comprises the step of producing aspartate from OAA (oxaloacetate); preferably, the step is carried out under the catalysis of aspartate aminotransferase (EC 2.6.1.1) or aspartate dehydrogenase (EC 1.4.1.21).

[0012] In the specific embodiments of the present invention, the method further comprises the step of promoting the production or accumulation of OAA (oxaloacetate).

[0013] In a particular embodiment of the application, the method further comprises a step of dehydration of β-hydroxyaspartate (BHA) to form imino succinate; and further preferably, a step of condensation of glycolate and glycine to form β-hydroxyaspartate.

[0014] In a particular embodiment of the application, the method further comprises a step of conversion of ethylene glycol into glycolate.

[0015] In a particular embodiment of the application, the step of conversion of ethylene glycol into glycolate comprises steps of conversion of ethylene glycol into glycolate via glycolate, glycolic acid and glyoxylic acid, in this order; preferably, ethylene glycol is converted into glycolate under the catalysis of fucO, a propandiol oxidoreductase or gldA; glycolate is converted into glycolic acid under the catalysis of aldA; and glycolic acid is converted into glyoxylic acid under the catalysis of glcDEF.

[0016] In a particular embodiment of the application, the L-aspartate oxidase (EC 1.4.3.16) is CAA31217 from E. coli, AAA21614 from B. subtilis or AAN67048 from P. putida; or an enzyme having at least 70%, or at least 80% or at least 90% sequence identity with the above-mentioned enzymes.

[0017] In the detailed description of the application, the iminosuccinate reductase is selected from an enzyme having at least 70%, or at least 80% or at least 90% sequence identity with WP_011750151.1; preferably the iminosuccinate reductase is WP_011750151.1, 6RQA_A, WP_104490941.1, WP_285049113.1, WP_028712349.1, WP_147428862.1, RQP04981.1, WP_230377319.1, WP_058099149.1, WP_024844791.1, WP_243784211.1, WP_114535439.1, WP_036753902.1, WP_214273870.1, WP_205294920.1, SFX01780.1, WP_289893705.1, WP_116221022.1, WP_323008703.1, WP_074966157.1, WP_199259739.1, WP_018001114.1, WP_115755144.1, WP_062562426.1, WP_149763389.1, WP_085502250.1, WP_288948763.1, WP_010395300.1, or WP_263568140; further preferably the amino acid sequence of the iminosuccinate reductase is as shown in any one of SEQ ID NOs: 7-18.

[0018] In the detailed description of the application, the aspartate dehydrogenase is CAA26173 from E. coli or AAG08814 from P. aeruginosa; the methylaspartate dehydrogenase is AAO37022 from Clostridium tetani.

[0019] In the detailed description of the application, the aspartate aminotransferase is CAA27279 from E. coli.

[0020] In the detailed description of the application, the aspartate dehydrogenase is AAG06893 from P. aeruginosa.

[0021] In the detailed description of the application, any step of the method for synthesizing succinic acid is optionally performed in or outside of the microbial cell, preferably in the microbial cell.

[0022] In the detailed description of the application, the microorganism is a genetically engineered strain.

[0023] In the detailed description of the application, the genetically engineered strain expresses or overexpresses the gene of the following enzyme or a combination of two or more enzyme genes:

[0024] 1) L-aspartate oxidase (NadB) (EC 1.4.3.16) gene;

[0025] 2) imino succinate reductase gene;

[0026] 3) aspartate ammonia lyase (AspA) (EC 4.3.1.1) gene or methyl aspartate ammonia lyase (EC 4.3.1.2) gene;

[0027] 4) aspartate aminotransferase (AspC) (EC 2.6.1.1) gene or aspartate dehydrogenase (NadX) (EC 1.4.1.21) gene;

[0028] 5) any gene of the pathway that promotes OAA production or accumulation, such as phosphoenolpyruvate carboxylase (Ppc or PckA) (EC 4.1.1.31 or EC 4.1.1.49) gene, and / or, pyruvate carboxylase (Pyc) (EC 6.4.1.1) gene;

[0029] 6) β-hydroxyaspartate aldolase (BhcC) gene;

[0030] 7) β-hydroxyaspartate (BHA) dehydratase (BhcB) gene;

[0031] 8) any enzyme or combination thereof that promotes the conversion of ethanediol to glyoxylate.

[0032] In the specific embodiments of the present application, the fumarate hydration and / or succinate dehydrogenation pathways in the genetically engineered strain are inhibited or blocked; preferably, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, attenuated or eliminated:

[0033] 1) fumarase gene fumA, fumC or a combination thereof; and / or

[0034] 2) succinate dehydrogenase gene sdhA, sdhB or a combination thereof.

[0035] In the specific embodiments of the present application, the expression of fumarase genes fumA and fumC in the genetically engineered strain is inhibited, attenuated or eliminated.

[0036] In the specific embodiments of the present application, the expression of succinate dehydrogenase genes sdhA and sdhB in the genetically engineered strain is inhibited, attenuated or eliminated.

[0037] In the detailed description of the present application, the expression of the succinate dehydrogenase genes sdhA and sdhB and the fumarase gene fumA is inhibited, weakened or eliminated in the genetically engineered strain.

[0038] In the detailed description of the present application, the expression of the succinate dehydrogenase genes sdhA and sdhB and the fumarase gene fumC is inhibited, weakened or eliminated in the genetically engineered strain.

[0039] In the detailed description of the present application, the expression of the succinate dehydrogenase genes sdhA and sdhB and the fumarase genes fumA and fumC is inhibited, weakened or eliminated in the genetically engineered strain.

[0040] In the detailed description of the present application, the method is performed with or without the addition of exogenous fumaric acid to the culture medium of the genetically engineered strain.

[0041] The second aspect of the present application provides a genetically engineered strain expressing or overexpressing a combination of two or more of the following genes:

[0042] 1) L-aspartate oxidase (EC 1.4.3.16) gene;

[0043] 2) imino succinate reductase gene.

[0044] In the detailed description of the present application, the genetically engineered strain also expresses or overexpresses: 3) aspartate ammonia-lyase (EC 4.3.1.1) gene (AspA) or methylaspartate ammonia-lyase (EC 4.3.1.2) gene.

[0045] In the detailed description of the present application, the genetically engineered strain also expresses or overexpresses: 4) aspartate aminotransferase (EC 2.6.1.1) gene or aspartate dehydrogenase (EC 1.4.1.21) gene (NadX).

[0046] In the detailed description of the present application, the genetically engineered strain also expresses or overexpresses: 5) any gene that promotes the OAA accumulation pathway, such as phosphoenolpyruvate carboxylase (Ppc or PckA) (EC 4.1.1.31 or EC 4.1.1.49) gene, and / or, pyruvate carboxylase (Pyc) (EC 6.4.1.1) gene.

[0047] In the detailed description of the present application, the genetically engineered strain also expresses or overexpresses: 6) β-hydroxyaspartate aldolase (BhcC) gene.

[0048] In the detailed description of the application, the genetically engineered strain further expresses or overexpresses: 7) a β-hydroxyaspartate (BHA) dehydratase (BhcB) gene;

[0049] In the detailed description of the application, the genetically engineered strain further expresses or overexpresses: 8) a gene of any enzyme that facilitates the conversion of ethanediol to glyoxylate.

[0050] In the detailed description of the application, the genes are introduced into the host strain via a plasmid or integrated into the genome of the host strain via genetic engineering.

[0051] In the detailed description of the application, the host strain is selected from a bacterium or a fungus; alternatively, the host strain is selected from a wild-type or genetically engineered Escherichia coli, Bacillus, Corynebacterium, Saccharomyces or Streptomyces; alternatively, the host strain is selected from a wild-type or genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; alternatively, the host strain is selected from a wild-type or genetically engineered Escherichia coli.

[0052] In the detailed description of the application, the fumarate hydration and / or succinate dehydrogenation pathways in the genetically engineered strain are inhibited or blocked; preferably, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, attenuated or eliminated:

[0053] 1) a fumarase gene fumA, fumC or a combination thereof; and / or

[0054] 2) a succinate dehydrogenase gene sdhA, sdhB or a combination thereof.

[0055] In the detailed description of the application, the expression of the fumarate dehydrogenase genes fumA and fumC in the genetically engineered strain is inhibited, attenuated or eliminated.

[0056] In the detailed description of the application, the expression of the succinate dehydrogenase genes sdhA and sdhB in the genetically engineered strain is inhibited, attenuated or eliminated.

[0057] In the detailed description of the application, the expression of the succinate dehydrogenase genes sdhA and sdhB and the fumarase gene fumA is inhibited, attenuated or eliminated in the genetically engineered strain.

[0058] In the detailed description of the application, the expression of the succinate dehydrogenase genes sdhA and sdhB and the fumarase gene fumC is inhibited, attenuated or eliminated in the genetically engineered strain.

[0059] In the detailed description of the application, the expression of the succinate dehydrogenase genes sdhA and sdhB and the fumarase genes fumA and fumC is inhibited, attenuated or eliminated in the genetically engineered strain.

[0060] In another aspect, the present application provides the use of the aforementioned genetically engineered strain for the production of succinic acid or a salt thereof.

[0061] In another aspect, the present application provides a method for the production of succinic acid or a salt thereof using the aforementioned genetically engineered strain, said method comprising the steps of:

[0062] The fermentation is carried out in the presence or absence of fumaric acid / fumarate to obtain succinic acid or a salt thereof.

[0063] In the detailed description of the application, the fermentation is aerobic or anaerobic fermentation.

[0064] In another aspect, the present application also provides a recombinant vector or a combination thereof comprising an L-aspartate oxidase (EC 1.4.3.16) gene and an imino succinate reductase gene.

[0065] In the detailed description of the application, the recombinant vector or a combination thereof further comprises: 3) an aspartate ammonia-lyase (EC 4.3.1.1) gene (AspA) or a methylaspartate ammonia-lyase (EC 4.3.1.2) gene.

[0066] In the detailed description of the application, the recombinant vector or a combination thereof further comprises: 4) an aspartate aminotransferase gene (AspC) or an aspartate dehydrogenase (EC 1.4.1.21) gene (NadX).

[0067] In the detailed description of the application, the recombinant vector or a combination thereof further comprises: 5) any gene that promotes the accumulation of OAA, such as a phosphoenolpyruvate carboxylase (Ppc or PckA) (EC 4.1.1.31 or EC 4.1.1.49) gene and / or a pyruvate carboxylase (Pyc) (EC 6.4.1.1) gene.

[0068] In the detailed description of the application, the recombinant vector or the combination thereof further comprises: 6) a β-hydroxyaspartate aldolase (BhcC) gene.

[0069] In the detailed description of the application, the recombinant vector or the combination thereof further comprises: 7) a β-hydroxyaspartate dehydratase (BhcB) gene.

[0070] In the detailed description of the application, the recombinant vector or the combination thereof further comprises: 8) a gene of any enzyme or the combination thereof that promotes the conversion of ethylene glycol to glyoxylate.

[0071] In the detailed description of the application, the recombinant vector or the combination thereof further comprises an element that knocks out the fumarase genes fumA and fumC and the succinate dehydrogenase genes sdhA and sdhB of the E. coli genome; preferably, the element is a CRISPR / Cas9 element or a λ-Red element.

[0072] In the detailed description of the application, the recombinant vector is a plasmid, a phagemid, an artificial chromosome, a genomic integration expression vector, a bacteriophage or an animal virus.

[0073] In another aspect, the application also provides an E. coli strain in which the fumarate hydration and / or succinate dehydrogenation pathway is inhibited or blocked;

[0074] Preferably, the fumarase genes fumA, fumC or the combination thereof are inhibited or eliminated; and / or

[0075] Preferably, the succinate dehydrogenase genes sdhA, sdhB or the combination thereof are inhibited or eliminated.

[0076] In the detailed description of the application, the expression of the succinate dehydrogenase genes fumA and fumC in the genetically engineered strain is inhibited or eliminated.

[0077] In the detailed description of the application, the expression of the succinate dehydrogenase genes sdhA and sdhB in the genetically engineered strain is inhibited or eliminated.

[0078] In the detailed description of the application, the expression of the succinate dehydrogenase genes sdhA and sdhB and the fumarase gene fumA in the genetically engineered strain is inhibited or eliminated.

[0079] In the detailed description of the application, the expression of the succinate dehydrogenase genes sdhA and sdhB and the fumarase gene fumC in the genetically engineered strain is inhibited or eliminated.

[0080] In the specific embodiments of the present application, the expression of succinate dehydrogenase genes sdhA and sdhB and fumarase genes fumA and fumC in the genetically engineered strain is inhibited or eliminated.

[0081] Advantages

[0082] The present application opens up a new path for synthesizing succinic acid by improving the precursor conversion rate, and finds a new precursor (fumaric acid) for succinic acid synthesis, and provides a new method for fermentative production of succinic acid. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1 Schematic diagram of the roles of the combination of nadB and bhcD genes in the process of catalyzing the conversion of fumaric acid to succinic acid. DETAILED DESCRIPTION

[0084] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology achieved based on the above description of the present application is covered within the scope of protection intended by the present application.

[0085] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0086] Definitions

[0087] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the present application will be further described below in combination with specific examples.

[0088] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. And the nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation steps used herein are the terms and conventional steps widely used in the corresponding field. At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided as follows.

[0089] As used herein, the term "amplification" refers to the intracellular activity of one or more enzymes encoded by appropriate DNA in a microorganism, 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.

[0090] Unless specifically stated, the terms "first", "second" do not indicate any order or importance, but the terms first, second, etc. are used to distinguish one object from another object.

[0091] As used herein, the term "gene synthesis" refers to the production using recombinant DNA technology or the use of synthetic DNA or amino acid sequence technology available and well known in the art. "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules having a defined sequence of nucleotides or of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both a nucleotide sequence which encodes a protein in conjunction with the mRNA sequence, and the complementary strand of either, provided in the coding strand in the sequence listing, and the non-coding strand which serves as the template for transcription and translation of the gene or cDNA, can be said to encode the protein or other product of the gene or cDNA.

[0092] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter, and the terms "overexpression" or "enhanced expression" refer to an elevated level of mRNA encoding a protein, and / or to an elevated level of a protein in a cell compared to the basal level of expression of the mRNA or to the basal level of the protein of a corresponding unmodified cell. For example, the introduction of an endogenous or exogenous gene into a microorganism to increase the expression level of the corresponding enzyme, or the replacement of a strong promoter or the introduction of an enhancer, etc. can also be used to increase the expression of an endogenous or exogenous gene.

[0093] As used herein, the term "vector" or "recombinant vector" is a composition of genetic material that includes an isolated nucleic acid and that can be used to transfer the isolated nucleic acid into a cell. The transferred nucleic acid is typically linked to, e.g., inserted into, a vector nucleic acid molecule. The vector can contain sequences that direct autonomous replication in the cell or can contain sequences sufficient to permit integration into the host cell DNA. Numerous vectors are known in the art, including but not limited to plasmids, phagemids, artificial chromosomes, bacteriophages, and animal viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The host can be not only a wild strain, but also a mutant strain or an artificial genetic recombinant; in some embodiments of the present application, the Escherichia coli K-12 strain is used as the host.

[0094] The inhibition, reduction or elimination of gene expression described in the present application includes the usual gene knockout methods, as well as other methods known to those skilled in the art for inhibiting gene expression or reducing / eliminating the functional activity of a gene, such as promoter knockout, introduction of enzyme inactivating / deactivating 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 directs enzyme synthesis, introduction of mutations or protein tags that accelerate the degradation / decomposition / inactivation of enzymes, inhibition of signal activation, RNA interference and gene silencing, etc.

[0095] As used herein, the sequence "identity" has the art-recognized meaning and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. While there are a number of methods for measuring identity between two polynucleotides or polypeptides, the term "identity" is art-recognized (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0096] Ornithine cyclodeaminase / mu-crystallin (OCD / CRYM) superfamily

[0097] The ornithine cyclodeaminase / mu-crystallin (OCD / CRYM) superfamily consists of enzymes involved in amino acid metabolism, most of which are classified as oxidoreductases.

[0098] Iminosuccinate reductase

[0099] Iminosuccinate reductase belongs to the ornithine cyclodeaminase / mu-crystallin (OCD / CRYM) superfamily members (Current Research in Biotechnology 4 (2022) 402-419), is an enzyme that plays a key role in the glycine assimilation pathway, which catalyzes the NADH-dependent reduction of iminosuccinate to L-aspartate.

[0100] Iminosuccinate reductase from Paracoccus has been studied and reported, the complex crystal formed with NAD+ was resolved (PDB Entry-6RQA), and was found to be involved in promoting microbial metabolism of ethylene glycol (see Metabolic Engineering 76 (2023) 97-109, etc.).

[0101] Non-limiting examples of imidiosuccinate reductase enzymes that can be used in the present application include: WP_011750151.1, 6RQA_A, WP_104490941.1, WP_285049113.1, WP_028712349.1, WP_147428862.1, RQP04981.1, WP_230377319.1, WP_058099149.1, WP_024844791.1, WP_243784211.1, WP_114535439.1, WP_036753902.1, WP_214273870.1, WP_205294920.1, SFX01780.1, WP_289893705.1, WP_116221022.1, WP_323008703.1, WP_074966157.1, WP_199259739.1, WP_018001114.1, WP_115755144.1, WP_062562426.1, WP_149763389.1, WP_085502250.1, WP_288948763.1, WP_010395300.1, WP_263568140.1, WP_145398930.1, WP_213352638.1, WP_013167434.1, WP_114347414.1, WP_247030082.1, WP_247198867.1, WP_312528369.1, MDN3711407.1, WP_036741495.1, WP_276189190.1, WP_028719068.1, WP_112888547.1, WP_221830521.1, WP_099649653.1, WP_150350833.1, WP_080622551.1, WP_278874544.1, WP_192337498.1, WP_155040858.1, AGT08751.1, WP_041527219.1, WP_325692736.1, WP_314188323.1, WP_311760970.1, WP_120441467.1, WP_155065854.1, WP_142665167.1, WP_134681356.1, WP_155096256.1, WP_241622078.1, WP_167621787.1, WP_154494501.1, WP_119899562.1, WP_216034624.1, WP_268940846.1, PHQ70216.1, WP_191312444.1, WP_260276782.1, WP_200689557.1, HRO14648.1, WP_311160206.1, WP_305961442.1, WP_076525216.1, WP_209055401.1, WP_294923239.1, WP_119751549.1, WP_155043695.1, WP_122111431.1, WP_207099778.1, WP_234853430.1, WP_272857984.1, WP_090616798.1, WP_207465699.1, WP_126154591.1, WP_306753659.1, WP_154763700.1, WP_306605604.1, WP_130989607.1, WP_305291801.1, WP_119751826.1, WP_255329844.1, WP_089388981.1, WP_275104026.1, WP_299841304.1, WP_235755037.1, WP_292978977.1, WP_293014677.1, WP_101751470.1, WP_271882549.1, WP_295044940.1 or WP_187792217.1.

[0102] Further preferred are enzymes having more than 90% sequence identity to WP_011750151.1 : WP_011750151.1, 6RQA_A, WP_104490941.1, WP_285049113.1, WP_028712349.1, WP_147428862.1, RQP04981.1, WP_230377319.1, WP_058099149.1, WP_024844791.1, WP_243784211.1, WP_114535439.1, WP_036753902.1, WP_214273870.1, WP_205294920.1, SFX01780.1, WP_289893705.1, WP_116221022.1, WP_323008703.1, WP_074966157.1, WP_199259739.1, WP_018001114.1, WP_115755144.1, WP_062562426.1, WP_149763389.1, WP_085502250.1, WP_288948763.1, WP_010395300.1 or WP_263568140.

[0103] Preferably, the imidosuccinate reductase disclosed in WO2021023801A1 can also be used in the present application, for example its disclosed SEQ ID NO:1-299; its preferred sequences (SEQ ID NO:1, 7, 22, 25, 26, 39, 47, 58, 75, 123, 135 and 160) are shown as SEQ ID NO:7-18 of the present application.

[0104] Bioavailability of beta-hydroxyaspartate cycle (BHAC) and ethylene glycol

[0105] Literature [2] The beta-hydroxyaspartate cycle (BHAC) is reported in detail in Nature 575, 500-504 (2019), see Figure 1 In the beta-hydroxyaspartate cycle (BHAC) cycle, BhcC catalyzes the condensation of glyoxylate with glycine to produce beta-hydroxyaspartate, and BhcB catalyzes the dehydration of beta-hydroxyaspartate to produce iminosuccinate.

[0106] Microorganisms can use ethylene glycol to synthesize glyoxylate, and these pathways are disclosed in the prior art, such as EP1748076A1, which reports that the conversion of ethylene glycol to glyoxylate in E. coli requires the participation of three enzymes to catalyze the 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 to lactaldehyde, glycolate, and glyoxylate, respectively. Non-patent literature (JOURNAL OF BACTERIOLOGY, Vol. 153, No. 1, p. 134-139) also reports that propylene glycol oxidoreductase can catalyze the oxidation of ethylene glycol to lactaldehyde. The present inventors have found that the NAD-dependent glycerol dehydrogenase encoded by the gldA gene in E. coli and Klebsiella pneumoniae can also react with ethylene glycol as a substrate to produce lactaldehyde (see Chinese patent application CN202311231991.X, which is incorporated herein by reference in its entirety).

[0107] L-aspartate oxidase

[0108] L-aspartate oxidase is a class of flavoprotein that uses CH-NH2 as a donor and usually uses O2 as an acceptor to generate H2O2, and can also use fumaric acid as an acceptor to generate succinic acid, see Structure 1999, Vol 7 No 7: 745-756.

[0109] Materials and methods

[0110] LB medium: 10 g / L peptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0111] Fermentation medium: glucose 20 g / L, yeast powder 5 g / L, M9 salt 11.3 g / L, magnesium sulfate 2 mM, calcium chloride 0.1 mM, sodium bicarbonate 2 g / L, VB1 5 mg / L, the medium is adjusted to pH 7.3-7.4 with concentrated ammonia water, sterilized at 115℃ for 15 min.

[0112] Strain fermentation method:

[0113] Single colonies were picked from LB plates and inoculated into LB medium (colonies with pZE plasmid were inoculated into LB medium containing 100 ug / L ampicillin) and cultured overnight at 37℃ with shaking; the overnight culture was inoculated into 250 ml shake flasks containing 20 ml fermentation medium at a ratio of 5%, and was fermented at 37℃ with shaking at 220 rpm, and samples were taken at regular intervals.

[0114] Succinic acid, glucose and fumaric acid concentration determination:

[0115] The standards used were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). 1 mL of the catalytic reaction liquid was centrifuged at 10000 r / min for 5 min to remove the bacterial bodies, and the resulting filtrate was filtered through a filter membrane with a pore size of 0.22 um. After dilution to an appropriate multiple, the sample filtrate was measured for the concentration of the above-mentioned products in the sample using high performance liquid chromatography (HPLC). The high performance liquid chromatograph was Shimadzu Nexera LC-40, and the chromatographic column was Bio-Rad Aminex HPX-87H 300x7.8mm. The column oven was set to 40℃; the detector was a RID refractive index detector (detector set to constant temperature 40℃). The mobile phase was 5mM sulfuric acid solution, and the flow rate was 0.6ml / min.

[0116] The information of the enzymes involved in the present application is shown in Table 1:

[0117] Table 1 Information of enzymes involved

[0118]

[0119] In the context of the present application, the enzymes mentioned include mutants that maintain enzyme activity, which have an amino acid sequence having at least 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number in the table.

[0120] The biological materials constructed by the present application are as follows:

[0121] Table 2 Information of biological materials involved

[0122]

[0123] Example 1: Construction of succinic acid bioconversion chassis strain

[0124] The phage was made using the corresponding single gene knockout strain in the E. coli Keio Knockout Collection Library 1 (Horizon Discovery, CO, USA), and the fumA deletion fragment with kanamycin (Kan) resistance was introduced into the starting strain E. coli W3110 by P1 phage transfection method, and then coated on LB plates containing 50 mg / L of kanamycin and cultured at 37°C overnight to obtain a deletion strain with Kan resistance; then the plasmid pCP20 was transformed into the above Kan-resistant transformant, and coated on LB plates containing 100 mg / L of ampicillin and cultured at 30°C for 24 hours, and then the correct transformant in which the kanamycin resistance gene Kan in the target gene was removed was identified by PCR, thereby obtaining an antibiotic-free deletion strain; then the correct transformant in which the Kan gene was removed was streaked on LB plates and cultured at 37°C or 42°C, and finally an antibiotic-free deletion fumA gene E. coli strain was obtained in which the pCP20 plasmid was removed. Similarly, the fumB, sdhA and sdhB deletion fragments were introduced into the fumA deletion strain or wild-type strain to construct the deletion strains SUC00 (W3110 AfumAC), SUC01 (W3110 AsdhAB), SUC02 (W3110 AsdhAB AfumA), SUC03 (W3110 AsdhAB AfumC) and SUC04 (W3110 AsdhAB AfumAC) based on E. coli W3110.

[0125] Example 2: Construction of recombinant plasmid pZE-aspC-aspA / mal-nadB-bhcD

[0126] The genes bhcD and mal were cloned from Paracoccus denitrificans and Clostridium pseudotetani, respectively, by PCR, and the genes aspA and nadB were cloned from Pseudomonas aeruginosa and E. coli MG1655, respectively, by PCR, and the genes aspC, aspA and nadB were cloned from E. coli MG1655 by PCR, and the Gibson Assembly 2 The DNA fragments obtained above were combined and ligated to the lac promoter of the plasmid pZEIac 3 in different ways, respectively, to obtain the following recombinant plasmids (details of the combination method are shown in Table 2): pSUC01: pZE-aspC-ec.aspA-nadB-bhcD, pSUC02: pZE-aspC-pa.aspA-nadB-bhcD and pSUC03: pZE-aspC-mal-nadB-bhcD.

[0127] Example 3: Fermentation of the chassis strain

[0128] The chassis strain obtained in Example 1 was fermented for 24 h and sampled for hplc detection, and the results are shown in Table 3. Compared with the wild-type W3110 strain, the fumAC and sdhAB knockout SUC04 strain accumulated 1.9 g / L fumaric acid, and no succinic acid was detected in all strains, which indicated that the chassis strain constructed in Example 1 could not accumulate succinic acid but could produce fumaric acid without overexpressing related genes.

[0129] Table 3 Fermentation data of chassis strains

[0130]

[0131]

[0132] Example 4: Fermentation of recombinant strains

[0133] The pSUC01 plasmid obtained in Example 2 was transformed into the strain obtained in Example 1 to obtain recombinant strains SUC05-SUC10 (see Table 2 for details), and the recombinant strains were fermented in fermentation medium for 24 h, and the data are shown in Table 4. The SUC05 strain with the W3110 wild-type strain as the background was used as the control group, and no succinic acid and fumaric acid accumulation was detected. The SUC07 strain with the sdhAB gene knockout could detect 1.2 g / L succinic acid; further knockout of the fumA, fumC and fumAC genes in the SUC06 strain could increase the accumulation of succinic acid, and the SUC10 strain could accumulate 2.8 g / L succinic acid, and no fumaric acid accumulation was detected. This indicates that overexpression of the succinic acid synthesis pathway constructed by the pSUC01 plasmid in Example 2 in the background of fumAC and sdhAB gene knockout helps to accumulate succinic acid, and at the same time consumes the endogenous produced fumaric acid.

[0134] Table 4 Fermentation data of recombinant strains

[0135]

[0136] Example 5: Fermentation experiment of recombinant strains with exogenous addition of fumaric acid

[0137] In Example 4, it has been verified that strains SUC06-SUC10 can produce succinic acid under fermentation conditions, and this example further demonstrates that exogenously added fumaric acid can also be used as a precursor to synthesize succinic acid through a new pathway. Fermentation was used to study the ability of strains to synthesize succinic acid by adding exogenous fumaric acid as a variable using strains SUC07-SUC10 described in Example 4, and after adding 3 g / L fumaric acid (actual concentration 3.1 g / L) to the fermentation medium, fermentation was carried out, and after 24 hours, samples were taken for HPLC detection, and the results are shown in Table 5. The control strain SUC05 is a W3110 chassis strain, and although 1.9 g / L of fumaric acid was consumed, no accumulation of succinic acid could be detected. Strains SUC08-SUC10 completely consumed the 3.1 g / L fumaric acid in the medium and produced succinic acid, and among them, the succinic acid yield of strain SUC10 was as high as 8.2 g / L. These data show that the succinic acid synthesis pathway we constructed in Example 2 can efficiently synthesize succinic acid using endogenous synthesis and exogenously added fumaric acid as precursors.

[0138] Table 5 Fermentation data of exogenous fumaric acid

[0139]

[0140] Example 6: Screening of aspartate ammonia lyases from different sources

[0141] In Example 2, the two key precursors of the reconstructed succinic acid synthesis pathway are aspartate and fumaric acid, and their ratio is regulated by aspartate ammonia lyase in E. coli cells, so in this example, different sources of aspartate ammonia lyase or methyl aspartate ammonia lyase constructed in Example 2 were expressed in the chassis strain SUC04, and recombinant strains SUC11 and SUC12 were further constructed to test their effect on succinic acid synthesis (results after 24 hours of fermentation are shown in Table 6). Compared with the control strain SUC05, the mal gene from Clostridium tetani, the ec.aspA gene from the endogenous E. coli, and the pa.aspA gene from Pseudomonas aeruginosa can all produce succinic acid.

[0142] Table 6 Fermentation data of recombinant strains expressing different sources of ammonia lyase

[0143]

[0144]

[0145] The above describes embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

[0146] References

[0147] 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.

[0148] 2. Gibson, D. G.; Young, L.; Chuang, R.-Y.; Venter, J. C; Hutchison, C. A.; Smith, H. O., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 2009, 6 (5), 343-345.

[0149] 3. Zhang, K.; Li, H.; Cho, K. M.; Liao, J. C, Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine. Proceedings of the National Academy of Sciences 2010, 107 (14), 6234-6239.

[0150] The nucleotide sequences of SEQ ID NOs: 1-6 in Table 1 are shown below:

[0151] SEQ ID NO: 1

[0152]

[0153]

[0154] SEQ ID NO: 2

[0155]

[0156] SEQ ID NO: 3

[0157]

[0158]

[0159] SEQ ID NO:4

[0160]

[0161] SEQ ID NO:5

[0162]

[0163]

[0164] SEQ ID NO:6

[0165]

[0166] SEQ ID NO:7

[0167]

[0168] SEQ ID NO:8

[0169]

[0170] SEQ ID NO:9

[0171]

[0172] SEQ ID NO:10

[0173]

[0174]

[0175] SEQ ID NO:11

[0176]

[0177] SEQ ID NO:12

[0178]

[0179] SEQ ID NO:13

[0180]

[0181] SEQ ID NO:14

[0182]

[0183] SEQ ID NO:15

[0184]

[0185] SEQ ID NO: 16

[0186]

[0187] SEQ ID NO: 17

[0188]

[0189]

[0190] SEQ ID NO: 18

[0191] .

Claims

1. A method for the synthesis of succinic acid, said method comprising the following steps: 1) L-aspartate and fumarate are converted into succinic acid and iminosuccinate under the catalysis of L-aspartate oxidase (EC 1.4.3.16); 2) iminosuccinate is recycled into L-aspartate.

2. The method of claim 1, wherein said step 2) is carried out under the catalysis of an ornithine cyclodeaminase / mu-crystallin superfamily protein; preferably, under the catalysis of an iminosuccinate reductase; Preferably, said step 2) further comprises a step of spontaneous decomposition of iminosuccinate into OAA (oxaloacetate); Preferably, the method further comprises: 3) a step of ammonolysis of L-aspartate into fumarate; more preferably, said ammonolysis of L-aspartate is carried out under the catalysis of aspartate deaminase (EC 4.3.1.1) or methylaspartate deaminase (EC 4.3.1.2); Preferably, said method further comprises a step of conversion of OAA (oxaloacetate) into aspartate; more preferably, said step is carried out under the catalysis of aspartate aminotransferase (EC 2.6.1.1) or aspartate dehydrogenase (EC 1.4.1.21); Preferably, said method further comprises a step of promoting the production or accumulation of OAA (oxaloacetate); Preferably, said method further comprises a step of dehydration of beta-hydroxyaspartate (BHA) into iminosuccinate; further preferably, it further comprises a step of condensation of glycolate and glycine into beta-hydroxyaspartate; Preferably, said method further comprises a step of conversion of ethylene glycol into glycolate; further preferably, said step of conversion of ethylene glycol into glycolate comprises the steps of conversion of ethylene glycol into glycolate, glycolate into glycolaldehyde and glycolaldehyde into glycolate, in this order; further preferably, said step of conversion of ethylene glycol into glycolate comprises the conversion of ethylene glycol into glycolaldehyde under the catalysis of fucO, propandiol oxidoreductase or gldA; the conversion of glycolaldehyde into glycolate under the catalysis of aldA; the conversion of glycolate into glycolaldehyde under the catalysis of glcDEF; Preferably, said L-aspartate oxidase (EC 1.4.3.16) is CAA31217 from Escherichia coli, AAA21614 from Bacillus subtilis or AAN67048 from Pseudomonas putida; or an enzyme having at least 70%, or at least 80% or at least 90% sequence identity with the above-mentioned enzymes. Preferably, the iminosuccinate reductase is selected from an enzyme having at least 70%, or at least 80% or at least 90% sequence identity to WP_011750151.1; preferably the iminosuccinate reductase is WP_011750151.1, 6RQA_A, WP_104490941.1, WP_285049113.1, WP_028712349.1, WP_147428862.1, RQP04981.1, WP_230377319.1, WP_058099149.1, WP_024844791.1, WP_243784211.1, WP_114535439.1, WP_036753902.1, WP_214273870.1, WP_205294920.1, SFX01780.1, WP_289893705.1, WP_116221022.1, WP_323008703.1, WP_074966157.1, WP_199259739.1, WP_018001114.1, WP_115755144.1, WP_062562426.1, WP_149763389.1, WP_085502250.1, WP_288948763.1, WP_010395300.1, or WP_263568140; further preferably the amino acid sequence of the iminosuccinate reductase is as set forth in any one of SEQ ID NOs: 7-18; Preferably, the aspartate deaminase is CAA26173 from E. coli or AAG08814 from P. aeruginosa; Preferably, the methylaspartate deaminase is AAO37022 from Clostridium tetani; Preferably, the aspartate aminotransferase is CAA27279 from E. coli; Preferably, the aspartate dehydrogenase is AAG06893 from P. aeruginosa.

3. The method of claim 1 or 2, wherein the steps of the method are optionally performed in or outside of microbial cells, preferably in microbial cells; Preferably, the microorganism is a genetically engineered strain; Preferably, the genetically engineered strain expresses or overexpresses the gene of: 1) L-aspartate oxidase (NadB) (EC 1.4.3.16); 2) iminosuccinate reductase; 3) aspartate deaminase (AspA) (EC 4.3.1.1) or methylaspartate deaminase (EC 4.3.1.2); 4) aspartate aminotransferase (AspC) (EC 2.6.1.1) or aspartate dehydrogenase (NadX) (EC 1.4.1.21); 5) any gene of the pathway that promotes OAA generation or accumulation, such as phosphoenolpyruvate carboxylase (Ppc or PckA) (EC 4.1.1.31 or EC 4.1.1.49) gene, and / or, pyruvate carboxylase (Pyc) (EC 6.4.1.1) gene; 6) β-hydroxyaspartate aldolase (BhcC) gene; 7) β-hydroxyaspartate dehydratase (BhcB) gene; 8) any gene of the enzyme or combination thereof that promotes the conversion of ethanediol to oxaloacetate; Preferably, the fumarate hydration and / or succinate dehydrogenation pathway in the genetically engineered strain is inhibited or blocked; more preferably, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, attenuated or eliminated: 1) fumarase genes fumA, fumC or a combination thereof; and / or 2) succinate dehydrogenase genes sdhA, sdhB or a combination thereof; More preferably, the expression of fumarase genes fumA and fumC in the genetically engineered strain is inhibited, attenuated or eliminated; More preferably, the expression of succinate dehydrogenase genes sdhA and sdhB in the genetically engineered strain is inhibited, attenuated or eliminated; More preferably, the expression of succinate dehydrogenase genes sdhA and sdhB and fumarase gene fumA in the genetically engineered strain is inhibited, attenuated or eliminated; More preferably, the expression of succinate dehydrogenase genes sdhA and sdhB and fumarase gene fumC in the genetically engineered strain is inhibited, attenuated or eliminated; More preferably, the expression of succinate dehydrogenase genes sdhA and sdhB and fumarase genes fumA and fumC in the genetically engineered strain is inhibited, attenuated or eliminated.

4. The method of claim 3, wherein the genetically engineered bacteria culture medium is supplemented with or without exogenous fumarate.

5. A genetically engineered strain expressing or overexpressing a combination of two or more of the following genes of enzymes: 1) L-aspartate oxidase (EC 1.4.3.16) gene; 2) imino succinate reductase gene; Preferably, the genetically engineered strain further expresses or overexpresses: 3) aspartate deaminase (EC 4.3.1.1) gene (AspA) or methylaspartate deaminase (EC 4.3.1.2) gene; Preferably, the genetically engineered strain further expresses or overexpresses: 4) aspartate aminotransferase (EC 2.6.1.1) gene or aspartate dehydrogenase (EC 1.4.1.21) gene; Preferably, the genetically engineered strain further expresses or overexpresses: 5) any gene of the pathway that promotes OAA accumulation, such as phosphoenolpyruvate carboxylase (Ppc or PckA) (EC 4.1.1.31 or EC 4.1.1.49) gene, and / or, pyruvate carboxylase (Pyc) (EC 6.4.1.1) gene; Preferably, the genetically engineered strain further expresses or overexpresses: 6) a β-hydroxyaspartate aldolase (BhcC) gene; Preferably, the genetically engineered strain further expresses or overexpresses: 7) a β-hydroxyaspartate (BHA) dehydratase (BhcB) gene; Preferably, the genetically engineered strain further expresses or overexpresses: 8) a gene of any enzyme or combination thereof that facilitates the conversion of ethylene glycol to oxaloacetate.

6. The genetically engineered strain of claim 5, wherein the genes are introduced into the host strain via a plasmid or integrated into the genome of the host strain by genetic engineering means. Preferably, the host strain is selected from a bacterium or a fungus; alternatively, the host strain is selected from a wild-type or genetically engineered Escherichia coli, Bacillus, Corynebacterium, Saccharomyces or Streptomyces; alternatively, the host strain is selected from a wild-type or genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; alternatively, the host strain is selected from a wild-type or genetically engineered Escherichia coli.

7. The genetically engineered strain of claim 5 or 6, wherein the fumarate hydration and / or succinate dehydrogenation pathway is inhibited or blocked in the genetically engineered strain; preferably, the expression of one, two or more of the following genes is inhibited, attenuated or eliminated in the genetically engineered strain: 1) fumarase genes fumA, fumC or a combination thereof; and / or 2) succinate dehydrogenase genes sdhA, sdhB or a combination thereof; More preferably, the expression of fumarase genes fumA and fumC is inhibited, attenuated or eliminated in the genetically engineered strain; More preferably, the expression of succinate dehydrogenase genes sdhA and sdhB is inhibited, attenuated or eliminated in the genetically engineered strain; More preferably, the expression of succinate dehydrogenase genes sdhA and sdhB and fumarase gene fumA is inhibited, attenuated or eliminated in the genetically engineered strain; More preferably, the expression of succinate dehydrogenase genes sdhA and sdhB and fumarase gene fumC is inhibited, attenuated or eliminated in the genetically engineered strain; More preferably, the expression of succinate dehydrogenase genes sdhA and sdhB and fumarase genes fumA and fumC is inhibited, attenuated or eliminated in the genetically engineered strain.

8. Use of the genetically engineered strain according to any one of claims 5-7 for the production of succinic acid or a salt thereof.

9. A method for the production of succinic acid or a salt thereof by the genetically engineered strain according to any one of claims 5-7, said method comprising the steps of: fermenting to obtain succinic acid or a salt thereof in the presence or absence of fumaric acid / fumarate; Preferably, the fermentation is aerobic or anaerobic fermentation.

10. A recombinant vector or combination thereof comprising a L-aspartate oxidase (EC 1.4.3.16) gene and an imino succinate reductase gene; Preferably, the recombinant vector or combination thereof further comprises: 3) an aspartate ammonia-lyase (EC 4.3.1.1) gene (AspA) or a methylaspartate ammonia-lyase (EC 4.3.1.2) gene; Preferably, the recombinant vector or combination thereof further comprises: 4) an aspartate aminotransferase gene (AspC) or an aspartate dehydrogenase (EC 1.4.1.21) gene (NadX); Preferably, the recombinant vector or combination thereof further comprises: 5) any gene that promotes OAA accumulation pathway, such as a phosphoenolpyruvate carboxylase (Ppc or PckA) (EC 4.1.1.31 or EC 4.1.1.49) gene, and / or, a pyruvate carboxylase (Pyc) (EC 6.4.1.1) gene; Preferably, the genetically engineered strain further expresses or overexpresses: 6) a beta-hydroxyaspartate aldolase (BhcC) gene; Preferably, the genetically engineered strain further expresses or overexpresses: 7) a beta-hydroxyaspartate (BHA) dehydratase (BhcB) gene; Preferably, the genetically engineered strain further expresses or overexpresses: 8) any gene of an enzyme or combination thereof that promotes the conversion of ethylene glycol to glyoxylate; Preferably, the recombinant vector or combination thereof further comprises elements for knocking out fumarase genes fumA and fumC and succinate dehydrogenase genes sdhA and sdhB of the E. coli genome; preferably, the elements are CRISPR / Cas9 elements or lambda-Red elements; Preferably, the recombinant vector is a plasmid, a phagemid, an artificial chromosome, a genomic integration expression vector, a bacteriophage or an animal virus.

11. An E. coli strain wherein the fumarate hydration and / or succinate dehydrogenation pathway is inhibited or blocked; Preferably, the fumarase genes fumA, fumC or a combination thereof are inhibited or eliminated; and / or Preferably, the succinate dehydrogenase genes sdhA, sdhB or a combination thereof are inhibited or eliminated; More preferably, the expression of the succinate dehydrogenase genes fumA and fumC is inhibited or eliminated in the genetically engineered strain; More preferably, the expression of the succinate dehydrogenase genes sdhA and sdhB is inhibited or eliminated in the genetically engineered strain; More preferably, the expression of the succinate dehydrogenase genes sdhA and sdhB and the fumarase gene fumA is inhibited or eliminated in the genetically engineered strain; More preferably, the expression of the succinate dehydrogenase genes sdhA and sdhB and the fumarase gene fumC in the genetically engineered strain is inhibited or eliminated; More preferably, the expression of the succinate dehydrogenase genes sdhA and sdhB and the fumarase genes fumA and fumC in the genetically engineered strain is inhibited or eliminated.

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