Method for producing L-malic acid by adding ethylene glycol and construction and application of malic acid producing escherichia coli

Abstract

The invention relates to a method for increasing the yield of L-malic acid produced by fermentation by adding ethylene glycol, which comprises the following steps: constructing genetic engineering escherichia coli, and introducing ethylene glycol metabolism genes (overexpression) including glycerol dehydrogenase, lactaldehyde reductase, lactaldehyde dehydrogenase and the like; and overexpression of other genes involving malic acid production, such as malic acid synthase, phosphoenolpyruvate carboxylase and malic acid export protein. The gene engineering strain is an E.coli BW25113 strain of which ldhA, pta, poxB, adhE, pflB, mgsA, frdA, maeA, maeB, yqhD, ycdW and iclR genes are substantially inactivated, and the deleted genes are beneficial to reducing the generation of metabolic byproducts and increasing the accumulation of malic acid. The genetic engineering strain is subjected to fermentation culture by taking glucose and ethylene glycol as carbon sources, and L-malic acid is separated from a culture after culture is finished.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering and relates to a method for constructing an Escherichia coli engineering bacterium utilizing ethylene glycol and producing L-malic acid by fermentation. Background Art

[0002] Ethylene glycol has a wide range of industrial applications and can be used to make polyester plastics, such as polyethylene terephthalate. Glucose is a common industrial fermentation substrate. During the metabolic transformation of microbial cell factories, there is a high degree of overlap between the two processes involved in biomass synthesis and the synthesis of high-value chemicals, which limits the efficiency of using glucose to synthesize target products. Ethylene glycol can minimize the interaction between biomass and chemical synthesis pathways and improve substrate utilization. In addition, ethylene glycol is relatively economical and can be produced through CO 2 Electrochemical conversion of , obtained from the hydrolysis of plastic waste or hydrogenolysis of cellulosic waste, is therefore a potentially abundant renewable substrate that can serve as a dominant substrate for microbial production of high-value chemicals.

[0003] The molecular formula of malic acid is C 4 H 6 O 5 It is a C4 dicarboxylic acid produced by organisms and is an important intermediate in the cellular metabolic network. Malic acid has two stereoisomers, L and D, of which the L isomer is naturally present. L-malic acid is a compound with great economic value and is widely used in food, medicine, and chemical industries. At the same time, L-malic acid can also be used as a precursor of high-value chemicals with broad market prospects. For example, sunitinib malate, a tyrosine kinase inhibitor with anti-angiogenic and anti-tumor activity, can be used in patients with renal cell carcinoma and gastrointestinal stromal tumors. Calcium citrate malate can also be used as a calcium source to treat hyperammonemia and liver dysfunction because it improves bone strength on the basis of reducing the risk of kidney stones. C4 dicarboxylic acids (including malate, fumarate, and succinate) are considered to be potential bulk chemical precursors that can be produced from renewable resources through biological or chemical transformation.

[0004] At present, the global market demand for malic acid is about 80,000 tons per year, while the annual output of malic acid is 40,000 tons. The synthesis methods of malic acid mainly include chemical synthesis method, microbial synthesis method and enzymatic method. Among them, industrial production of malic acid is mainly through the hydration of maleic anhydride, and this process will produce D / L malic acid. Due to the partial physiological toxicity of D-type and DL-type malic acid, the US FDA restricts the application of D-type and DL-type malic acid in the fields of food and medicine. In addition, the chemical synthesis method also has problems such as low yield, high cost and environmental pollution. Compared with the chemical synthesis method, microbial synthesis of malic acid has the advantages of low cost, low energy consumption, high yield, strong selectivity and environmental friendliness. In recent years, a large amount of research work has been invested in optimizing the biological synthesis metabolic network and culture conditions of malic acid. Escherichia coli, as a model organism, has the advantages of clear genetic background, perfect genetic operation system, clear metabolic network and rapid growth, making it widely used as a cell factory in the biological manufacturing of chemicals and also an excellent platform for the biosynthesis of L-malic acid. Summary of the Invention

[0005] In Escherichia coli, ethylene glycol can be converted into glyoxylate, and then enter the metabolic network of Escherichia coli for utilization. It is found that 3 enzymes are required to catalyze the reaction of ethylene glycol to glyoxylate, namely lactaldehyde reductase, lactaldehyde dehydrogenase and glycolate oxidase, and the encoding genes are fucO, aldA and glcDEF respectively, which catalyze the sequential conversion of ethylene glycol into glycolaldehyde, glycolic acid, and finally into glyoxylate. The Fe 2+ -dependent propanediol oxidoreductase FucO is easily oxidized and inactivated, so it is sensitive to oxygen, and its mutant FucO I6L,L7V can improve the oxygen stability of FucO. In addition, the NAD-dependent glycerol dehydrogenase encoded by the gldA gene in Escherichia coli can catalyze the reaction of glycerol to produce dihydroxyacetone. The GldA protein subunit requires Zn2+ as a cofactor to catalyze the reaction. At the same time, GldA also has a wide range of substrate specificities and can react with ethylene glycol as a substrate to produce glycolaldehyde. In addition, inactivating the glycolaldehyde reductase YqhD and the glyoxylate / hydroxypyruvate reductase YcdW can improve the conversion rate of ethylene glycol.

[0006] The present invention relates to a method for increasing the yield of L - malic acid in fermentation by adding ethylene glycol. By constructing a genetically engineered Escherichia coli, ethylene glycol metabolism genes (over - expressed) are introduced, including genes such as glycerol dehydrogenase, lactaldehyde reductase, lactaldehyde dehydrogenase, etc.; and other genes involved in malic acid production such as malate synthase, phosphoenolpyruvate carboxylase, and malic acid exporter are over - expressed. And the genetically engineered strain is an E. coli BW25113 strain substantially inactivated for the ldhA, pta, poxB, adhE, pflB, mgsA, frdA, maeA, maeB, yqhD, ycdW, iclR genes. The above - deleted genes contribute to reducing the generation of metabolic by - products and increasing the accumulation of malic acid. The genetically engineered strain is fermented and cultured using glucose and ethylene glycol as carbon sources, and L - malic acid is isolated from the culture after the culture is completed.

[0007] Without being limited by any theory, after being metabolized by the genetically engineered strain, ethylene glycol can increase the accumulation and production of malic acid through multiple pathways. For example, it promotes the accumulation of oxaloacetate, a key metabolic intermediate in the tricarboxylic acid cycle, or directly promotes the accumulation of glyoxylate, an intermediate in the malic acid synthesis pathway.

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

[0009] The present invention first provides a method for increasing the fermentation yield of malic acid. The method includes adding ethylene glycol to the culture medium. Preferably, the cumulative addition amount of ethylene glycol is greater than 10 g / L, and / or introducing ethylene glycol - utilization genes into the fermentation strain.

[0010] The ethylene glycol - utilization genes include: a) genes encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO; and b) genes encoding lactaldehyde dehydrogenase (aldA).

[0011] The present invention further provides a genetically engineered bacterium for producing malic acid. The genetically engineered strain contains at least one endogenous or exogenous malic acid synthesis pathway and expresses: a) at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO, and / or b) at least one DNA molecule encoding lactaldehyde dehydrogenase (aldA)

[0012] In a specific embodiment of the present invention, the genetically engineered strain over - expresses c) at least one enzyme in the malic acid synthesis pathway or transport pathway; preferably, over - expresses at least one enzyme in the glyoxylate pathway; more preferably, over - expresses at least one DNA molecule encoding one, two, or three of the following enzymes: phosphoenolpyruvate carboxylase ppc, C4 - dicarboxylate transporter dcuA, or malate synthase G (glcB).

[0013] In a further preferred embodiment of the present invention, the expression of one or both of the following genes in the genetically engineered strain is inhibited, weakened or eliminated: glyoxylate / hydroxypyruvate reductase ycdW and / or NADPH-dependent aldehyde reductase yqhD.

[0014] In a further preferred embodiment of the present invention, the production pathways of metabolic by-products succinic acid, lactic acid, ethanol, acetic acid and formic acid and the malic acid decomposition pathway in the genetically engineered strain are inhibited or blocked.

[0015] In a further preferred embodiment of the present invention, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, weakened or eliminated: NAD + -dependent malate dehydrogenase maeA, NADP + -dependent malate dehydrogenase maeB, DNA-binding transcriptional repressor iclR, phosphoacetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, acetolactate synthase mgsA, D-lactate dehydrogenase ldhA or fumarate reductase flavoprotein subunit frdA.

[0016] In a specific embodiment of the present invention, the lactaldehyde reductase fucO is AAA23825 derived from Escherichia coli, preferably its mutant FucO I6L,L7V .

[0017] 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 derived from Escherichia albertii such as OSL28309.1; or a glycerol dehydrogenase derived from Klebsiella pneumoniae such as ABR79620, or a glycerol dehydrogenase derived from the genus Shigella such as Shigella boydii or Shigella dysenteriae (such as ABB68416.1 or ABB63725.1), or a glycerol dehydrogenase derived from Streptococcal pneumoniae such as VTQ29411.1, or a glycerol dehydrogenase derived from Bacillus stearothermophilus such as AAA22477, or a glycerol dehydrogenase (alcohol / choline dehydrogenase) derived from Bacillus subtilis such as CAB15083.2, or a glycerol dehydrogenase derived from Pseudomonas putida such as AAC44426, or a glycerol dehydrogenase derived from Clostridium saccharobutylicum such as AAA83520.1, or a glycerol dehydrogenase derived from Clostridium acetobutylicum such as AAK79593.1, or a glycerol dehydrogenase derived from the genus Salmonella such as CNU06578.1, or an alcohol dehydrogenase (glycerol dehydrogenase) derived from Corynebacterium glutamicum such as SJM54452.1, and may also be a propanediol / glycerol bifunctional dehydrogenase MTU07760.1 derived from the genus Parasutterella excrementihominis, as well as natural or artificial mutants of enzymes derived from the same species.

[0018] Preferably, the glycerol dehydrogenase derived from Escherichia coli has a protein sequence accession number of AAC43051 (ec.gldA); preferably, the glycerol dehydrogenase derived from Klebsiella pneumoniae has a protein sequence accession number of ABR79620 (kp.gldA); or an enzyme that maintains the above enzyme activity and has an amino acid sequence with at least 96%, or 97%, or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number AAC43051 or ABR79620.

[0019] In a specific embodiment of the present invention, the phosphoenolpyruvate carboxylase ppc is CAA29332 from Escherichia coli and has a K620S mutation.

[0020] In a specific embodiment of the present invention, the DNA molecule is introduced into the host strain through a plasmid or integrated into the genome of the host strain by genetic engineering means.

[0021] 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 (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 (Escherichia coli).

[0022] The second aspect of the present invention provides a method for fermentatively producing L-malic acid, the method comprising: culturing the above-mentioned genetically engineered bacterium in the presence of ethylene glycol.

[0023] Preferably, ethylene glycol accounts for 0.01%-20% (weight ratio) of the culture medium.

[0024] The third aspect of the present invention provides a chassis bacterium, which is an Escherichia coli strain, and the expression of one or two of the following genes in the chassis bacterium is inhibited, weakened or eliminated: glyoxylate / hydroxypyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD.

[0025] In a specific embodiment of the present invention, in the Escherichia coli strain, the generation pathways of metabolic by-products succinic acid, lactic acid, ethanol, acetic acid and formic acid and the malic acid decomposition pathway are inhibited or blocked.

[0026] Preferably, the expression of one, two or more of the following genes in the Escherichia coli strain is inhibited, weakened or eliminated: NAD + -dependent malate dehydrogenase maeA, NADP +Dependent on malate dehydrogenase maeB, DNA-binding transcriptional repressor iclR, phosphoacetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, methylglyoxal synthase mgsA, D-lactate dehydrogenase ldhA or fumarate reductase flavoprotein subunit frdA.

[0027] Beneficial technical effects

[0028] The genetically engineered strain of the present application produces L-malic acid using glucose and ethylene glycol as carbon sources. Description of the drawings:

[0029] Figure 1 : In vivo metabolic pathway from ethylene glycol to malic acid: Gray gene names represent gene deletions (dashed arrows with an X in non-bold), and black gene names represent gene overexpressions (bold solid arrows) Detailed implementation manners

[0030] The synthesis method of the present invention will be further described in detail below with reference to specific examples. It should be understood that the following examples are only for illustrative and explanatory purposes of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0031] In the present invention, unless otherwise specified, 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 all widely used terms and conventional steps in the corresponding fields. At the same time, for a better understanding of the present disclosure, the definitions and explanations of relevant terms are provided below.

[0032] As used herein, the term "amplification" refers to the intracellular activity of one or more enzymes encoded by appropriate DNA in a microorganism, such as being increased 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.

[0033] Unless specifically stated, the terms "first" and "second" do not indicate any order or importance, but are used to distinguish one object from another object.

[0034] As used herein, the term "gene synthesis" refers to the generation using recombinant DNA technology or the obtaining using synthetic DNA or amino acid sequence technologies available and known in the art. "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA to serve as a template for the synthesis of other polymers and macromolecules in biological processes, where the polymers and macromolecules have either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if the transcription and translation of the mRNA corresponding to that gene produce a protein in a cell or other biological system. Both the nucleotide sequence equivalent to the mRNA sequence and typically provided in the sequence listing as the coding strand, and the non-coding strand that serves as the template for transcribing the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0035] 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 terms "overexpression" or "enhanced expression" refer to an elevated level of the mRNAs encoding the protein, and / or to an elevated level of the protein in a cell as compared to a corresponding unmodified cell having a basal level of expression of the mRNAs or a basal level of the protein. For example, introducing an endogenous or exogenous gene into a microorganism to increase the expression level of the corresponding enzyme, or enhancing the expression of an endogenous or exogenous gene by replacing the promoter with a strong promoter or introducing an enhancer, etc.

[0036] 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 interior. The transferred nucleic acid is typically ligated, for example, inserted into the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in a cell or may contain sequences 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. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The host can be not only a wild strain, but also its mutant strain or an artificial genetic recombinant.

[0037] As used herein and unless otherwise specified, the term "about" when referring to a measurable value such as an amount, a time period, etc., means including a variation of ±10% from the given value, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, provided that such variation is suitable for practicing the disclosed method.

[0038] In the present invention, serial numbers are used to represent combinations of reaction steps, DNA molecules, biological materials or enzymes. For example, a)-c) represents a combination including three aspects numbered 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 does not represent the limitation on the reaction mode of each step. Each reaction step can be carried out step by step or simultaneously, can be carried out inside cells or outside cells, or can be carried out under cell-free enzyme catalysis. Unless otherwise stated, the combination of DNA molecules, the combination of biological materials or the combination of enzymes does not mean that the combination of DNA molecules, the combination of biological materials or the combination of enzymes is in the same organism or biological material, but the present invention preferably expresses the combination of the DNA molecules and enzymes in the same organism.

[0039] In the present invention, "at least one" means at least one of the same or similar referents, not necessarily existing. For example, the combination includes "at least one DNA molecule encoding malate synthase (GlcB)", which means including one or more DNA molecules encoding malate synthase (GlcB), but when there is an expression of "optionally included" or "optionally combined", the "DNA molecule encoding malate synthase (GlcB)" may not exist.

[0040] In the present invention, the terms "amino acid mutation" or "nucleotide mutation" used include "substituting, repeating, deleting or adding one or more amino acids or nucleotides". In the present invention, the term "mutation" refers to the change of a nucleotide sequence or an amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

[0041] In the present invention, the term "natural state" refers to the activity of a polypeptide in a microorganism in an unmodified state, that is, the activity in the natural state.

[0042] In the present invention, the term "retaining activity" has the same or similar meaning as that conventionally understood by those skilled in the art, and both refer to that the amino acid sequence of a certain fragment is a 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.

[0043] Therefore, it is obvious to further mutate the enzyme of the present invention to obtain a further mutant still having the corresponding function and activity. For example, it is well known to those skilled in the art that adding or subtracting several amino acid residues at either end of a polypeptide, for example, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue will not affect the function of the obtained mutant. For example, for the convenience of purification, technicians often attach a 6×His tag to 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.

[0044] In the present invention, the term "conservative mutation" refers to a mutation that can normally maintain the function of a protein. Representative examples of conservative mutations are conservative substitutions.

[0045] As used in the present invention, the term "conservative substitution" involves replacing 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 those 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 side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0046] As used in the present invention, "conservative substitution" generally exchanges one amino acid at one or more positions of a protein. Such substitution can be conservative. As substitutions regarded as conservative substitutions, by way of example, can be cited the substitution of Ala with Ser or Thr, the substitution of Arg with Gln, His, or Lys, the substitution of Asn with Glu, Gln, Lys, His, or Asp, the substitution of Asp with Asn, Glu, or Gln, the substitution of Cys with Ser or Ala, the substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, the substitution of Glu with Gly, Asn, Gln, Lys, or Asp, the substitution of Gly with Pro, the substitution of His with Asn, Lys, Gln, Arg, or Tyr, the substitution of Ile with Leu, Met, Val, or Phe, the substitution of Leu with Ile, Met, Val, or Phe, the substitution of Lys with Asn, Glu, Gln, His, or Arg, the substitution of Met with Ile, Leu, Val, or Phe, the substitution of Phe with Trp, Tyr, Met, Ile, or Leu, the substitution of Ser with Thr or Ala, the substitution of Thr with Ser or Ala, the substitution of Trp with Phe or Tyr, the substitution of Tyr with His, Phe, or Trp, and the substitution of Val with Met, Ile, or Leu. In addition, conservative mutations also include naturally occurring mutations resulting from individual differences, differences in strains, species, etc. from which the gene is derived.

[0047] As used herein, the term "corresponding to" has the meaning commonly understood by those of ordinary skill in the art. Specifically, "corresponding to" means the position in one sequence that corresponds to a specified position in another sequence after the two sequences are aligned by homology or sequence identity. Thus, for example, with respect to "the amino acid residue corresponding to position 40 of the amino acid sequence shown in Sequence 1", if a 6×His tag is added to one end of the amino acid sequence shown in Sequence 1, then the position corresponding to position 40 of the amino acid sequence shown in Sequence 1 in the resulting mutant may be position 46.

[0048] In a specific embodiment, the homology or sequence identity may be more than 90%, preferably more than 95%, more preferably 96%, 97%, 98%, 99% homology.

[0049] 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, A.M. ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W. ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G. eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J. eds., M Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods for determining identity seek to obtain the maximum match between the sequences being tested. Methods for determining identity are compiled in computer programs available to the public. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is available to the public 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.

[0050] 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 terms "overexpression" or "enhanced expression" refer to an elevated level of the mRNAs encoding a protein, and / or to an elevated level of the protein in a cell as compared to a corresponding unmodified cell having a basal level of the expressed mRNAs or a basal level of the protein. For example, introducing an endogenous or exogenous gene into a microorganism to increase the expression level of the corresponding enzyme, or enhancing the expression of an endogenous or exogenous gene by replacing a strong promoter or introducing an enhancer, etc.

[0051] The present invention inhibits, reduces or eliminates the expression of the corresponding enzyme by gene knockout. Those skilled in the art know that other methods for inhibiting enzyme expression or reducing / eliminating enzyme functional activity can also be applied to the construction of genetically engineered strains, such as promoter knockout or replacement, introduction of enzyme-inactivating / diminishing mutations, deletion (or partial deletion) of essential functional elements or regions for gene transcription / translation, introduction of mutations or nucleic acid sequences that accelerate the degradation of mRNA guiding enzyme synthesis, introduction of mutations or protein tags that accelerate enzyme degradation / decomposition / inactivation, inhibition of signal activation, RNA interference and gene silencing, CRISPRi, etc.; in the specific embodiments of the present invention, the expression level of the enzyme is reduced by more than 30% or the activity of the enzyme is reduced by more than 30%.

[0052] As used herein, the term "vector" or "recombinant vector" is a composition of genetic material that includes isolated nucleic acids and can be used to transfer the isolated nucleic acids into cells. The transferred nucleic acids are usually ligated, for example, inserted into the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in cells or may contain sequences 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. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The host can be not only a wild strain but also its mutant strain or an artificial genetic recombinant; in some specific embodiments of the present invention, the Escherichia coli mutant strain BW25113 is used as the host.

[0053] Four-carbon organic acid / malic acid synthesis pathway: Generally, in microorganisms, four-carbon organic acids are mainly generated through the following three pathways 1 : The reductive tricarboxylic acid cycle pathway located in the cytoplasm, the oxidative tricarboxylic acid cycle pathway located in the mitochondria, and the glyoxylate shunt pathway. The reductive tricarboxylic acid cycle starts with the carboxylation of pyruvate and is generally considered the main pathway for organic acid accumulation in eukaryotic microorganisms. In this pathway, pyruvate first reacts with ATP and CO 2It is converted into oxaloacetate by pyruvate carboxylase (PYC) located in the cytoplasm. Subsequently, oxaloacetate is successively converted into malate, fumarate, and succinate under the action of malate dehydrogenase (MDH), fumarase (FUM), and fumarate reductase (FRD). The second major pathway for the production of four-carbon organic acids is the oxidative tricarboxylic acid cycle. In this pathway, oxaloacetate and acetyl-CoA are converted into citrate by citrate synthase, and then two oxidation reactions occur in mitochondria under aerobic conditions to generate succinate, which is then converted into fumarate and malate through the action of succinate dehydrogenase and fumarase. The glyoxylate pathway is also a potential pathway for the production of four-carbon organic acids. In this pathway, isocitrate formed through the oxidative tricarboxylic acid cycle is decomposed into succinate and glyoxylate under the catalysis of isocitrate lyase. Subsequently, glyoxylate combines with acetyl-CoA and is synthesized into malate under the catalysis of malate synthase. This pathway involves five enzymes: pyruvate carboxylase (PC), citrate synthase (CS), aconitase (ACN), isocitrate lyase (ICL), and malate synthase (MS).

[0054] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains.

[0055] Materials and Methods

[0056] LB medium: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0057] Fermentation medium: 10 g / L glucose, 5 g / L yeast extract, 11.3 g / L M9 salts, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 10 g / L ethylene glycol, 5 mg / L VB1, 10 g / L calcium carbonate. The pH of the medium is adjusted to 7.3 - 7.4 with concentrated ammonia water and sterilized at 115 °C for 15 min.

[0058] Flask fermentation method: Pick monoclonal colonies from the activated strains on an LB plate (containing an appropriate concentration of antibiotics) at 37 °C and inoculate them into an LB liquid medium (containing an appropriate concentration of antibiotics), and culture at 37 °C for 12 - 16 h at a rotation speed of 220 rpm. Inoculate the LB overnight culture into a 250 ml flask containing 25 ml of fermentation medium (containing 10 g / L CaCO3 as a pH stabilizer) with appropriate concentrations of ampicillin and kanamycin at an inoculation amount of 1%; after sealing with a breathable membrane, culture at 30 °C and 220 rpm. When the OD600 reaches 1, add IPTG (final concentration 0.2 mM) to induce plasmid expression, continue culturing for 12 or 24 h, then stop fermentation and take samples.

[0059] Determination of the concentrations of malic acid, glucose, and ethylene glycol (EG): All the standard products used were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). Take 1 mL of the fermentation broth, centrifuge at 10,000 r / min for 5 min to remove the cells, and filter the obtained filtrate through a filter membrane with a pore size of 0.22 μm. After the sample filtrate is diluted by an appropriate multiple, the concentrations of the above products in the sample are determined by high-performance liquid chromatography (HPLC). The high-performance liquid chromatograph is Shimadzu Nexera LC-40, and the chromatographic column is Bio-Rad Aminex HPX-87H 300×7.8 mm. The column oven is set at 40 °C; the detector is a refractive index detector (RID) (the detector is set at a constant temperature of 40 °C). The mobile phase is 5 mM sulfuric acid solution, and the flow rate is 0.6 ml / min.

[0060] The information of the enzymes involved in the present invention is as follows:

[0061]

[0062]

[0063] In the context of the present application, the enzymes mentioned include mutants that maintain enzyme activity, and the mutants 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 in the table.

[0064] The biological materials constructed in the present invention are as follows in the table:

[0065]

[0066]

[0067] Examples

[0068] Example 1: Construction of a malic acid synthesis chassis strain

[0069] Using the E. coli Keio Knockout Collection library 2(Horizon Discovery, CO, USA) Prepare phages using the corresponding single-gene knockout strains, and use the P1 phage transfection method to introduce the DNA target gene deletion fragment with kanamycin (Kan) resistance into the starting strain Escherichia coli BW25113. Spread it on an LB plate containing 50 mg / L of kanamycin and culture it overnight at 37 °C to obtain a deletion strain with Kan resistance. Then, transform the plasmid pCP20 into the above-mentioned Kan-resistant transformant, spread it on an LB plate containing 100 mg / L of ampicillin, and culture it at 30 °C for 24 hours. Identify the correct transformant with the kanamycin resistance gene Kan removed from the target gene by PCR to obtain a deletion strain without resistance. Then, streak single colonies of the correct transformant with the Kan gene removed on an LB plate and culture it at 37 or 42 °C to finally obtain a kanamycin-free Escherichia coli strain without the pCP20 plasmid. By repeating this process, a deletion strain Mal00 (ldhA ΔptaΔpoxBΔadhEΔpflBΔmgsAΔfrdAΔiclRΔmaeAΔmaeB) based on BW25113 was constructed, in which the generation pathways of metabolic by-products (succinic acid, lactic acid, ethanol, acetic acid, and formic acid) and the partial decomposition pathway of malic acid were blocked and knocked out to avoid waste of carbon sources and reduce the decomposition of malic acid.

[0070] Based on the Mal00 strain, strains Mal01 (Mal00ΔyqhD), Mal02 (Mal00ΔycdW), and Mal03 (Mal00ΔyqhDΔycdW) were obtained by combinatorial knockout of the yqhD and ycdW genes.

[0071] Example 2: Construction of recombinant plasmids pZE-ec.gldA, pZE-fucO, pZE-aldA, pZE-fucO-aldA, pZE-ec.gldA-aldA, pZE-kp.gldA-aldA, and pZA-ppc K620S -dcuA-glcB

[0072] Use PCR to clone the kp.gldA gene from the Klebsiella pneumoniae genome respectively; clone the genes fucO, ec.gldA, and aldA from the Escherichia coli BW25113 genome, and use Gibson Assembly 3 Connect the above-obtained DNA fragments in different ways to the plasmid pZElac 4After the lac promoter, the following recombinant plasmids were obtained (for details of the combination method, please refer to the Materials and Methods section): pZE-ec.gldA, pZE-aldA, pZE-fucO, pZE-ec.gldA-aldA, pZE-kp.gldA-aldA, and pZE-fucO-aldA.

[0073] Genes ppc, dcuA, and glcB in the genome of E. coli BW25113 were cloned by PCR. The ppc gene was cloned in two fragments, and the K620S mutation was introduced by primers near the fragment interface. 5 Using Gibson Assembly, the cloned fragments obtained were ligated behind the lac promoter of plasmid pZElac to obtain the recombinant plasmid pZA-ppc K620S -dcuA-glcB (pMAL).

[0074] The above recombinant plasmids were transformed into the Mal00-03 strain. For details of the specific plasmid and strain combinations, please refer to the Materials and Methods section.

[0075] Example 3: Flask fermentation of strains Mal04, Mal05, and Mal06

[0076] Using the Mal04 strain as a control, flask fermentation was carried out. The composition of the fermentation medium and the fermentation conditions are shown in the Materials and Methods section. After 12 hours of fermentation, the fermentation data are shown in Table 1. The results show that the overexpression of the first gene fucO or gldA in the ethylene glycol (EG) metabolic pathway slightly increased the malic acid production of Mal05 and Mal06 compared to the control strain (Mal04 without overexpression of genes related to the EG metabolic pathway).

[0077] Table 1: Flask fermentation data of malic acid

[0078]

[0079] Example 4: Flask fermentation of strains Mal04, Mal07, Mal08, and Mal09

[0080] Using the Mal04 strain as a control, flask fermentation was carried out. The composition of the fermentation medium and the fermentation conditions can be found in the Materials and Methods section. The fermentation data after 12 hours of fermentation are shown in Table 2. The results showed that the introduction of aldA on the basis of overexpressing gldA / fucO significantly increased the consumption of EG; at the same time, compared with the control strain, the malic acid production of the strain overexpressing gldA / fucO-aldA increased significantly, and the increase in malic acid of the strain overexpressing ec.gldA (Mal09) was significantly higher than that of the strain overexpressing fucO (Mal08). In addition, the single overexpression of the aldA gene (Mal07) could also slightly increase the consumption of ethylene glycol and the production of malic acid.

[0081] Table 2: Flask fermentation data of malic acid

[0082]

[0083] Example 5: Effects of deletion of yqhD and / or ycdW genes on malic acid fermentation

[0084] To explore the roles of the side reaction enzymes YqhD and YcdW in the EG utilization pathway, using the Mal09 strain as a control, flask fermentation was carried out. The composition of the fermentation medium and the fermentation conditions can be found in the Materials and Methods section. The fermentation data after 12 hours of fermentation are shown in Table 3. The results showed that the knockout of the yqhD and / or ycdW genes had no obvious effect on the utilization of glucose, but the ethylene glycol utilization rate and malic acid production of the single knockout strains were slightly higher than those of the control group. The ethylene glycol utilization rate of the double knockout strain of YqhD and ycdW increased by 20.9%, and the malic acid production increased by 55.9%. In summary, the knockout of the yqhD and ycdW genes improved the ability of the strain to utilize ethylene glycol to a certain extent and significantly increased the malic acid production.

[0085] Table 3: Flask fermentation data of malic acid

[0086]

[0087] Example 6: Effects of gldA from other sources on malic acid fermentation

[0088] Using the Mal12 strain as a control, the strain Mal13 carrying the kp.gldA gene of Klebsiella pneumoniae was subjected to flask fermentation together. The composition of the fermentation medium and the fermentation conditions can be found in the Materials and Methods section. The fermentation data after 12 hours of fermentation are shown in Table 4. The kp.gldA gene from Klebsiella pneumoniae had better activity, resulting in an increase in malic acid production.

[0089] Table 4: Flask fermentation data of malic acid

[0090]

[0091] References

[0092] 1. Wang Yingshan, Guo Feng, Yan Wei, et al. Research progress on metabolic engineering of four-carbon organic acid biosynthesis. Chinese Journal of Biotechnology, 2021, 37(5): 1697-1720.

[0093] 2. 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.

[0094] 3. 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.

[0095] 4. 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.

[0096] 5. Trichez, D.; Auriol, C.; Baylac, A.; Irague, R.; Dressaire, C.; Carnicer-Heras, M.; Heux, S.; J.M.; Walther, T., Engineering of Escherichia coli for Krebs cycle-dependent production of malic acid. Microbial Cell Factories 2018, 17(1), 113。

Claims

1. A method for increasing the malic acid fermentation yield, the method comprising adding ethylene glycol to a culture medium, preferably, the cumulative addition amount of ethylene glycol is greater than 10 g / L, and / or introducing an ethylene glycol utilization gene into a fermentation strain.

2. A genetically engineered bacterium for producing malic acid, the genetically engineered strain comprising at least one endogenous or exogenous malic acid synthesis pathway and expressing: a) at least one DNA molecule encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO, and / or b) at least one DNA molecule encoding lactaldehyde dehydrogenase (aldA).

3. The genetically engineered bacterium for producing malic acid according to claim 2, the genetically engineered strain overexpressing c) at least one enzyme of the malic acid synthesis pathway or transport pathway; preferably, overexpressing at least one enzyme of the glyoxylate pathway; more preferably, overexpressing at least one DNA molecule encoding one, two or three of the following enzymes: phosphoenolpyruvate carboxylase ppc, C4-dicarboxylate transporter dcuA or malate synthase G (glcB).

4. The genetically engineered bacterium for producing malic acid according to claim 3, the expression of one or two of the following genes in the genetically engineered strain being inhibited, attenuated or eliminated: glyoxylate / hydroxypyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD.

5. The genetically engineered bacterium for producing malic acid according to claim 4, the production pathways of metabolic by-products succinic acid, lactic acid, ethanol, acetic acid and formic acid and the malic acid decomposition pathway in the genetically engineered strain being 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: NAD+-dependent malate dehydrogenase maeA, NADP+-dependent malate dehydrogenase maeB, DNA-binding transcriptional repressor iclR, phosphoacetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvaldehyde synthase mgsA, D-lactate dehydrogenase ldhA or fumarate reductase flavoprotein subunit frdA.

6. The genetically engineered bacterium for producing malic acid according to claim 2, wherein the lactaldehyde reductase fucO is AAA23825 derived from Escherichia coli; preferably, it is a mutant FucO thereof I6L,L7V .

7. The genetically engineered bacterium for producing malic acid according to claim 2, wherein the glycerol dehydrogenase is a glycerol dehydrogenase derived from the genus Escherichia or a propanediol / glycerol bifunctional dehydrogenase, such as AAC43051 derived from Escherichia coli, or a glycerol dehydrogenase derived from Escherichia albertii such as OSL28309.1; or a glycerol dehydrogenase derived from Klebsiella pneumoniae such as ABR79620, or a glycerol dehydrogenase derived from the genus Shigella such as Shigella boydii or Shigella dysenteriae (such as ABB68416.1 or ABB63725.1), or a glycerol dehydrogenase derived from Streptococcal pneumoniae such as VTQ29411.1, or a glycerol dehydrogenase derived from Bacillus stearothermophilus such as AAA22477, or a glycerol dehydrogenase (alcohol / choline dehydrogenase) derived from Bacillus subtilis such as CAB15083.2, or a glycerol dehydrogenase derived from Pseudomonas putida such as AAC44426, or a glycerol dehydrogenase derived from Clostridium saccharobutylicum such as AAA83520.1, or a glycerol dehydrogenase derived from Clostridium acetobutylicum such as AAK79593.1, or a glycerol dehydrogenase derived from the genus Salmonella such as CNU06578.1, or an alcohol dehydrogenase (glycerol dehydrogenase) derived from Corynebacterium glutamicum such as SJM54452.1, and may also be a propanediol / glycerol bifunctional dehydrogenase MTU07760.1 derived from the genus Parasutterella excrementihominis, and natural or artificial mutants of enzymes derived from the same species; Preferably, the glycerol dehydrogenase derived from Escherichia coli has a protein sequence accession number of AAC43051 (ec.gldA); preferably, the glycerol dehydrogenase derived from Klebsiella pneumoniae has a protein sequence accession number of ABR79620 (kp.gldA); or an enzyme having the above enzyme activity and having an amino acid sequence with at least 96%, or 97%, or 98%, or at least 99% sequence identity to the amino acid sequence represented by the protein accession number AAC43051 or ABR79620.

8. The genetically engineered bacterium for producing malic acid as claimed in claim 3, wherein the phosphoenolpyruvate carboxylase ppc has a K620S mutation.

9. The genetically engineered bacterium for producing malic acid as claimed in any one of claims 2-8, wherein the DNA molecule is introduced into the host strain through a plasmid or integrated into the genome of the host strain by genetic engineering means; 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 (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Bacillus megaterium (Bacillus megaterium), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Corynebacterium glutamicum (Corynebacterium glutamicum), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida utilis (Candida utilis) or Pichia pastoris (Pichia pastoris); optionally, the host strain is selected from wild or genetically engineered Escherichia coli (Escherichia coli).

10. A method for fermentatively producing L-malic acid, the method comprises: culturing the genetically engineered bacterium as claimed in any one of claims 2-9 in the presence of ethylene glycol; Preferably, the proportion of ethylene glycol in the culture medium is 0.01%-20% (weight ratio).

11. A chassis bacterium, which is an Escherichia coli strain, wherein the expression of one or two of the following genes in the chassis bacterium is inhibited, weakened or eliminated: glyoxylate / hydroxypyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD; Preferably, in the Escherichia coli strain, the production pathways of metabolic by-products succinic acid, lactic acid, ethanol, acetic acid and formic acid and a part of the malic acid decomposition pathway are inhibited or blocked; Preferably, the expression of one, two or more of the following genes in the Escherichia coli strain is inhibited, attenuated or eliminated: NAD + -dependent malate dehydrogenase maeA, NADP + -dependent malate dehydrogenase maeB, DNA-binding transcriptional repressor iclR, phosphoacetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, methylglyoxal synthase mgsA, D-lactate dehydrogenase ldhA or fumarate reductase flavoprotein subunit frdA.

Images