Construction and application of escherichia coli engineering strain for synthesizing L-threonine by using ethylene glycol

Abstract

The invention relates to escherichia coli engineering bacteria co-utilizing glucose and ethylene glycol and a method for producing L-threonine through fermentation of the escherichia coli engineering bacteria, a genetic engineering method is adopted to obtain a genetic engineering strain, and the genetic engineering strain substantially expresses ethylene glycol metabolism genes including glycerol dehydrogenase, lactaldehyde reductase and lactaldehyde dehydrogenase; the amino acid sequence of the aspartic acid transferase, the aspartic acid kinase, the aspartic acid semialdehyde dehydrogenase, the homoserine dehydrogenase 1, the homoserine kinase, the threonine synthase, the threonine transport protein, the galactose synthase, the phosphoenolpyruvate decarboxylase and the phosphoenolpyruvate synthase are shown in the description; the gene engineering strain is an E.coli BW25113 strain which is characterized in that ldhA, pta, poxB, adhE, pflB, mgsA, frdA, ptsG, tdcB, tdh, ltaE, yiaY, yqhD and ycdW genes are substantially inactivated, or the gene engineering strain is an E.coli BW25113 strain which is obtained by substantially inactivating ldhA, pta, poxB, adhE, pflB, mgsA, frdA, ptsG, tdcB, tdh, ltaE, yiaY, yqhD and ycdW genes. And carrying out fermentation culture on the genetic engineering strain by taking glucose and ethylene glycol as carbon sources, and separating the L-threonine from the culture after the culture is finished.

Description

Technical Field

[0001] The present invention belongs to the field of biochemical engineering, and relates to a method for constructing an Escherichia coli engineering bacterium using ethylene glycol and its fermentation for producing L-threonine. Background Art

[0002] Ethylene glycol has extensive industrial application value and can be used to manufacture polyester plastics, such as polyethylene terephthalate. As a common industrial fermentation substrate, glucose has a high degree of overlap between the processes of participating in biomass synthesis and synthesizing high-value chemicals during the metabolic conversion of microbial cell factories, which limits the efficiency of synthesizing target products using glucose. Ethylene glycol can minimize the interaction between the biomass and chemical synthesis pathways and improve the substrate utilization rate. In addition, ethylene glycol is relatively economical in price and can be obtained through the electrochemical conversion of CO 2 , hydrolysis of plastic waste or hydrogenolysis of cellulose waste, so it is a potentially abundant renewable substrate and can be used as an advantageous substrate for microorganisms to produce high-value chemicals.

[0003] L-threonine belongs to the category of non-essential amino acids and is an important intermediate metabolite for the synthesis of various essential amino acids in organisms. It is also an important precursor for energy metabolism, amino acid metabolism, and neurotransmitter synthesis. Currently, L-threonine mainly comes from chemical synthesis, but its large-scale production and application are limited by low synthesis efficiency and difficulty in separation and purification. Compared with chemical synthesis methods, microbial synthesis of L-threonine 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 L-threonine biosynthesis metabolic network and culture conditions. Escherichia coli has been widely used in the research and production practice of L-threonine biosynthesis due to its clear genetic background and fast growth rate. Summary of the Invention

[0004] 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 three 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. 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 glycerol to react to form dihydroxyacetone, and the GldA protein subunit requires Zn 2+Catalyze reactions as cofactors. Meanwhile, GldA also has a wide substrate specificity and can react with ethylene glycol as a substrate to produce glycolaldehyde. In addition, inactivating glycolaldehyde reductase YqhD and glyoxylate / hydroxypyruvate reductase YcdW can improve the conversion rate of ethylene glycol.

[0005] Specific aspects of the present invention are as follows:

[0006] The present invention first provides a method for increasing the fermentation yield of L-threonine. 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.

[0007] The ethylene glycol utilization genes include: a) genes encoding glycerol dehydrogenase gldA or lactaldehyde reductase fucO; and b) genes encoding lactaldehyde dehydrogenase (aldA).

[0008] Another aspect of the present invention provides a genetically engineered bacterium for producing threonine. The genetically engineered strain contains at least one endogenous or exogenous threonine synthesis pathway or transport 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).

[0009] In a specific embodiment of the present invention, the genetically engineered strain overexpresses c) at least one enzyme in the threonine synthesis pathway or transport pathway.

[0010] In a specific embodiment of the present invention, the genetically engineered strain overexpresses at least one enzyme in the following pathways:

[0011] 1) Glucose transport or metabolism pathway and aspartic acid synthesis pathway, preferably one, two or more of galactose symporter galP, phosphoenolpyruvate synthase ppsA, phosphoenolpyruvate decarboxylase ppc and aspartate aminotransferase aspC;

[0012] Or

[0013] 2) Homoserine synthesis pathway, preferably one, two or more of aspartate kinase lysC, aspartate semialdehyde dehydrogenase asd and homoserine dehydrogenase 1 thrA;

[0014] Or

[0015] 3) Threonine synthesis and transport pathway, preferably one, two or more of homoserine kinase thrB, threonine synthase thrC and threonine transporter rhtA.

[0016] 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.

[0017] In a further preferred embodiment of the present invention, the generation pathways of metabolic by-products succinic acid, lactic acid, ethanol, acetic acid and formic acid and the L-threonine degradation pathway in the genetically engineered strain are inhibited or blocked.

[0018] 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: phosphoacetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvaldehyde synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA, threonine dehydrogenase tdh, threonine dehydratase tdcB, specific phosphoglucose transporter ptsG, threonine aldolase ltaE or threonine dehydrogenase yiaY.

[0019] In a specific embodiment of the present invention, the lactaldehyde reductase fucO is AAA23825 derived from Escherichia coli, preferably its mutant FucO I6L,L7V .

[0020] In a specific embodiment of the present invention, 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. It can also be a propanediol / glycerol bifunctional dehydrogenase MTU07760.1 derived from the genus Parasutterella excrementihominis, as well as natural or artificial mutants of enzymes from the same species.

[0021] 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.

[0022] 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.

[0023] 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).

[0024] The second aspect of the present invention provides a method for fermentative production of L-threonine, the method comprising: the step of culturing the above-mentioned genetically engineered bacterium in the presence of ethylene glycol.

[0025] Preferably, ethylene glycol accounts for 0.01%-20% (weight ratio) of the culture medium.

[0026] 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 Escherichia coli strain is inhibited, weakened or eliminated: glyoxylate / hydroxypyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD.

[0027] In a further preferred embodiment of the present invention, the generation pathways of metabolic by-products succinic acid, lactic acid, ethanol, acetic acid and formic acid and the L-threonine decomposition pathway in the strain are inhibited or blocked.

[0028] 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: phosphoacetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, acetolactaldehyde synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA, threonine dehydrogenase tdh, threonine dehydratase tdcB, specific phosphoglucose transporter ptsG, threonine aldolase ltaE or threonine dehydrogenase yiaY.

[0029] Beneficial technical effects

[0030] The genetically engineered strain of the present application ferments to produce threonine using glucose and ethylene glycol as carbon sources. Specific embodiments

[0031] The following will further elaborate on the synthesis method of the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative explanation 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 intended to be protected by the present invention.

[0032] Technical terms

[0033] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory operation procedures used herein are all widely used terms and conventional procedures in the corresponding fields. At the same time, to better understand the present disclosure, the definitions and explanations of relevant terms are provided below.

[0034] 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 enhanced 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.

[0035] Unless specifically stated, the terms "first" and "second" do not denote any order or importance, but are used to distinguish one object from another object.

[0036] As used herein, the term "gene synthesis" refers to being produced using recombinant DNA technology or obtained using available and well-known synthetic DNA or amino acid sequence technologies 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 synthesizing 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, if the transcription and translation of mRNA corresponding to that gene produce a protein in a cell or other biological system, the gene encodes the protein. Both the nucleotide sequence equivalent to the mRNA sequence and usually the coding strand provided in the sequence listing, and the non-coding strand used as the template for transcribing the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0037] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter. The terms "overexpression" or "enhanced expression" refer to an elevated level of the mRNAs encoding a protein, and / or to an elevated level of the protein in a cell as compared to the basal level of the expressed mRNAs or to a corresponding unmodified cell having a basal level of the protein. For example, an endogenous or exogenous gene of a microorganism is introduced to increase the expression level of the corresponding enzyme. The expression of an endogenous or exogenous gene can also be increased by means of replacing a strong promoter or introducing an enhancer, etc.

[0038] As used herein, the term "vector" or "recombinant vector" is a composition of genetic material that includes an isolated nucleic acid and can be used to transfer the isolated nucleic acid into a cell interior. The transferred nucleic acid is usually ligated, for example, inserted into the vector nucleic acid molecule. The vector can contain sequences that direct autonomous replication in a cell or can 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.

[0039] As used herein and unless otherwise specified, the term "about", when referring to measurable values such as amounts, time periods, etc., means including variations of ±10% from a given value, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, as long as such variations are suitable for implementing the disclosed methods.

[0040] In the present invention, serial numbers are used to represent combinations of reaction steps, DNA molecules, biological materials, or enzymes. For example, a)-c) includes a combination of 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 a limitation on the manner of carrying out each step reaction. Each reaction step can be carried out step by step or simultaneously, can be carried out inside a cell or outside a cell, 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 also 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. However, in the present invention, it is preferred that the combination of DNA molecules and enzymes is expressed in the same organism.

[0041] In the present invention, "at least one" means at least one of the same or similar referents, and does not necessarily exist. For example, the combination includes "at least one DNA molecule encoding threonine synthase thrC", which means including one or more DNA molecules encoding threonine synthase thrC. However, when there is an expression such as "optionally including" or "optionally combined", the "DNA molecule encoding threonine synthase thrC" can be absent.

[0042] 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 a change in a nucleotide sequence or an amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

[0043] In the present invention, the term "natural state" refers to the activity of a polypeptide in a microorganism in an unmodified state, i.e., the activity in the natural state.

[0044] 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.

[0045] Therefore, it is obvious to further mutate the enzyme of the present invention to obtain a further mutant that still has 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 such a 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.

[0046] 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.

[0047] 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 (such as lysine, arginine, and histidine), acidic side chains (such as aspartic acid and glutamic acid), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched side chains (such as threonine, valine, and isoleucine), and aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0048] As used in the present invention, "conservative substitution" generally exchanges one amino acid at one or more positions of a protein. Such substitutions can be conservative. Exemplary substitutions that are considered conservative substitutions include 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, strain differences, species differences, etc. from which the gene is derived.

[0049] As used in the present invention, the term "corresponding to" has the meaning generally 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 alignment 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.

[0050] In a specific embodiment, the homology or sequence identity may be 90% or more, preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology.

[0051] 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 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.

[0052] 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.

[0053] 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 / deactivating 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%.

[0054] As used herein, the term "vector" or "recombinant vector" is a composition of genetic material that includes isolated nucleic acid and can be used to transfer the isolated nucleic acid into a cell interior. The transferred nucleic acid is usually 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; in some specific embodiments of the present invention, the Escherichia coli mutant strain BW25113 is used as the host.

[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: 20 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). 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, kanamycin, and streptomycin 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 48 h, then stop fermentation and take samples.

[0059] Determination of the concentrations of L - threonine, glucose, and ethylene glycol (EG): All standard products used were purchased from Sigma - Aldrich (www.sigmaaldrich.cn). Take 1 mL of the fermentation broth, centrifuge at 10000 r / min for 5 min to remove the bacteria, filter the obtained filtrate through a filter membrane with a pore size of 0.22 μm. After diluting the sample filtrate by an appropriate multiple, use high - performance liquid chromatography (HPLC) to determine the concentrations of the above - mentioned products in the sample.

[0060] Determination conditions for glucose and ethylene glycol (EG): The high - performance liquid chromatograph is Shimadzu Nexera LC - 40, and the chromatographic column is Bio - Rad Aminex HPX - 87H 300×7.8 mm. Set the column oven temperature to 40°C; the detector is a refractive index detector (RID) (set the detector temperature to 40°C). The mobile phase is 5 mM sulfuric acid solution, and the flow rate is 0.6 ml / min.

[0061] Determination conditions for L - threonine: The high - performance liquid chromatograph is Shimadzu LC - 40D; the chromatographic column is Welch UltimateAQ - C18, 4.6×250 mm; set the column oven temperature to 40°C; the detector is an ultraviolet detector (set the detector temperature to 40°C), and the detection wavelength is 338 nm; the mobile phase is an aqueous sodium acetate solution and methanol, with gradient elution.

[0062] The information about the enzymes involved in the present invention is as follows:

[0063]

[0064] 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.

[0065] The biological materials constructed in the present invention are as follows in the table:

[0066]

[0067]

[0068] Example

[0069] Example 1: Construction of L-threonine synthesis chassis strain

[0070] Using the single-gene knockout strains corresponding in the E. coli Keio Knockout Collection library 1 (Horizon Discovery, CO, USA), phages were prepared from the corresponding single-gene knockout strains. The DNA target gene deletion fragment with kanamycin (Kan) resistance was introduced into the starting strain Escherichia coli BW25113 by P1 phage transfection method, and spread on an LB plate containing 50 mg / L kanamycin and cultured overnight at 37 °C to obtain a deletion strain with Kan resistance; then the plasmid pCP20 was transformed into the above Kan-resistant transformant, spread on an LB plate containing 100 mg / L ampicillin, and cultured at 30 °C for 24 hours. The correct transformant with the kanamycin resistance gene Kan removed from the target gene was identified by PCR, thus obtaining a non-resistant deletion strain; then the correct transformant with the Kan gene removed was streaked for single colonies on an LB plate and cultured at 37 or 42 °C, and finally a non-resistant Escherichia coli strain without the pCP20 plasmid was obtained. By repeating this process, a deletion strain Thr00 (ldhAΔptaΔpoxBΔpflBΔmgsAΔfrdAΔlysAΔadhEΔptsGΔtdcBΔtdhΔltaEΔyiaY) based on BW25113 was constructed, in which the production pathways of metabolic by-products (succinic acid, lactic acid, ethanol, acetic acid and formic acid) and the partial decomposition pathway of L-threonine were blocked and knocked out to avoid waste of carbon sources and reduce the decomposition of L-threonine.

[0071] Based on the Thr00 strain, after combinatorial knockout of the yqhD and ycdW genes, strains Thr01 (Thr00ΔyqhD), Thr02 (Thr00ΔycdW) and Thr03 (Thr00ΔyqhDΔycdW) were obtained.

[0072] Example 2: Construction of recombinant plasmids pZE-ec.gldA, pZE-fucO, pZE-aldA, pZE-fucO-aldA, pZE-ec.gldA-aldA, pZE-kp.gldA-aldA, pZA-thrA S345F BC-rhtA-galP-ppc-ppsA and pZS-aspC-lysC T352I -asd

[0073] The kp.gldA gene was cloned from the Klebsiella pneumoniae genome by PCR; the genes fucO, ec.gldA, and aldA were cloned from the Escherichia coli BW25113 genome, and Gibson Assembly was used. 2 The DNA fragments obtained above were ligated to the plasmid pZElac in different combinations. 3 After the lac promoter of pZElac, the following recombinant plasmids were obtained (for details of the combination methods, 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.

[0074] The genes thrA S345F 、thrB, thrC, galP, ppc, ppsA, and rhtA in the E. coli BW25113 genome were cloned by PCR. The thrA gene was cloned in two fragments, and the S345F mutation was introduced by primers near the fragment interface. The cloned fragments obtained were ligated to the lac promoter of the plasmid pZAlac by Gibson Assembly to obtain the recombinant plasmid pZA-thrA S345F BC-rhtA-galP-ppc-ppsA (pTHR01).

[0075] The genes aspC, lysC T352I and asd in the E. coli BW25113 genome were cloned by PCR. The lysC gene was cloned in two fragments, and the T352I mutation was introduced by primers near the fragment interface. The cloned fragments obtained were ligated to the lac promoter of the plasmid pZAlac by Gibson Assembly to obtain the recombinant plasmid pZS-aspC-lysC T352I -asd (pTHR02).

[0076] The above recombinant plasmids were transformed into the Thr00-03 strain. For details of the specific plasmid-strain combinations, please refer to the Materials and Methods section.

[0077] Example 3: Shake-flask fermentation of strains Thr04, Thr05, and Thr06

[0078] Using strain Thr04 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. After 48 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 L-threonine production of Thr05 and Thr06 compared to the control strain (the threonine-producing strain Thr04 without overexpression of EG metabolic pathway-related genes).

[0079] Table 1: Flask fermentation data of L-threonine

[0080]

[0081] Example 4: Flask fermentation of strains Thr04, Thr07, Thr08 and Thr09

[0082] Using strain Thr04 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. After 48 hours of fermentation, the fermentation data are shown in Table 2. The results show 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 L-threonine production of the strain overexpressing gldA / fucO-aldA increased significantly, and the L-threonine increase of the strain overexpressing ec.gldA (Thr09) was significantly higher than that of the strain overexpressing fucO (Thr08). In addition, the single overexpression of the aldA gene (Thr07) can also slightly increase the consumption of ethylene glycol and the production of L-threonine.

[0083] Table 2: Flask fermentation data of L-threonine

[0084]

[0085] Example 5: Effects of deletion of yqhD and / or ycdW genes on L-threonine fermentation

[0086] To explore the roles of the side reaction enzymes YqhD and YcdW in the EG utilization pathway, using strain Thr09 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. After 48 hours of fermentation, the fermentation data are shown in Table 3. The results show 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 L-threonine production of the single knockout strain and the double knockout strain were improved compared to the control group. 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 at the same time, the production of L-threonine also increased.

[0087] Table 3: Flask fermentation data of L-threonine

[0088]

[0089] Example 6: Influence of gldA from Other Sources on L-Threonine Fermentation

[0090] Using the Thr12 strain as a control, the strain Thr13 carrying the Klebsiella pneumoniae kp.gldA gene was subjected to shake-flask fermentation together. The composition of the fermentation medium and the fermentation conditions can be found in the Materials and Methods section. After 48 hours of fermentation, the fermentation data are shown in Table 4. The gldA gene from Klebsiella pneumoniae has better activity, resulting in an increase in the yield of L-threonine.

[0091] Table 4: Shake-Flask Fermentation Data of L-Threonine

[0092]

[0093] References

[0094] 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.

[0095] 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.

[0096] 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。

Claims

1. A method for increasing the fermentation yield of L-threonine, 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 threonine, the genetically engineered strain comprising at least one endogenous or exogenous threonine synthesis pathway or transport 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 threonine according to claim 2, wherein the genetically engineered strain overexpresses c) at least one enzyme in the threonine synthesis pathway or transport pathway; Preferably, the genetically engineered strain overexpresses at least one enzyme in the following pathways: 1) The glucose transport or metabolism pathway and the aspartic acid synthesis pathway, preferably one, two or more of galactose symporter galP, phosphoenolpyruvate synthase ppsA, phosphoenolpyruvate decarboxylase ppc and aspartate aminotransferase aspC; Or 2) The homoserine synthesis pathway, preferably one, two or more of aspartate kinase lysC, aspartate semialdehyde dehydrogenase asd and homoserine dehydrogenase 1 thrA; Or 3) The threonine synthesis and transport pathway, preferably one, two or more of homoserine kinase thrB, threonine synthase thrC and threonine transporter rhtA.

4. The genetically engineered bacterium for producing threonine according to claim 2 or 3, wherein the expression of one or two of the following genes in the genetically engineered strain is inhibited, weakened or eliminated: glyoxylate / hydroxypyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD; Preferably, in the genetically engineered strain, the production pathways of metabolic by-products such as succinic acid, lactic acid, ethanol, acetic acid and formic acid and the partial decomposition pathway of L-threonine are inhibited or blocked; Preferably, the expression of one, two or more of the following genes in the genetically engineered strain is inhibited, weakened or eliminated: phosphoacetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, acetolactaldehyde synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA, threonine dehydrogenase tdh, threonine dehydratase tdcB, specific phosphoglucose transporter ptsG, threonine aldolase ltaE or threonine dehydrogenase yiaY.

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

6. The genetically engineered bacterium for producing threonine 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 can 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 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.

7. The genetically engineered bacterium for producing threonine according to any one of claims 2-6, 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).

8. A method for fermentatively producing L-threonine, the method comprises: culturing the genetically engineered bacterium according to any one of claims 1-7 in the presence of ethylene glycol; Preferably, ethylene glycol accounts for 0.01%-20% (weight ratio) of the culture medium.

9. A chassis bacterium, which is an Escherichia coli strain, wherein the expression of one or both of the following genes in the Escherichia coli strain is inhibited, weakened or eliminated: glyoxylate / hydroxypyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD; Preferably, the metabolic by-product production pathways of succinic acid, lactic acid, ethanol, acetic acid and formic acid and the L-threonine degradation pathway 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, weakened or eliminated: phosphoacetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, acetolactaldehyde synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA, threonine dehydrogenase tdh, threonine dehydratase tdcB, specific phosphoglucose transporter ptsG, threonine aldolase ltaE or threonine dehydrogenase yiaY.

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