Recombinant escherichia coli for improving yield of L-homoserine and preparation method of recombinant escherichia coli

Abstract

The invention discloses recombinant escherichia coli capable of increasing the yield of L-homoserine and a preparation method of the recombinant escherichia coli, and belongs to the technical field of biology. By constructing a multi-way glucose transport system, L-homoserine synthesis precursor supply is enhanced, high-energy-consumption genes are knocked out, ATP regeneration is enhanced, ATP availability is improved, an acetic acid conversion way is established, and an L-homoserine degradation way is blocked, so that a metabolic flow of a synthesis way is further enhanced; a pyruvate carboxylase mutant and a designed promoter are introduced to weaken expression of citrate synthase and reconstruct key metabolic nodes, and fed-batch fermentation conditions of the strain in a 5L bioreactor are optimized according to metabolic characteristics of the strain; the genetically engineered bacterium which is clear in genetic background, does not carry plasmids, does not need induction and can stably and efficiently produce the L-homoserine is obtained, the yield of the L-homoserine reaches 141.5 g / L after the L-homoserine is fermented for 56h in a 5L fermentation tank, the saccharic acid conversion rate reaches 40%, and the genetically engineered bacterium has a certain application prospect.

Description

Technical Field

[0001] The present invention relates to a recombinant Escherichia coli for increasing the production of L-homoserine and a preparation method thereof, belonging to the field of biotechnology. Background Art

[0002] L-homoserine is a valuable non-protein amino acid, having important physiological functions and application values. It is also a precursor for the biosynthesis of L-threonine, L-lysine, and L-methionine. It has important functions such as antioxidant, immunomodulatory, promoting growth and development, etc., and has wide applications in the fields of chemical industry, medicine, agriculture, etc. At present, L-homoserine is mainly synthesized by chemical methods. The microbial fermentation method has advantages such as low cost, simple process, mild conditions, and relatively small environmental impact, and is more suitable for large-scale industrial production of L-homoserine.

[0003] Currently, the research on microbial fermentation for the production of L-homoserine mainly focuses on Corynebacterium glutamicum and Escherichia coli. Li et al. (Ning L, Lihong L, Shiqin Y, et al. Dual-channel glycolysis balances cofactor supply for L-homoserine biosynthesis in Corynebacterium glutamicum. Bioresource Technology, 2023, 369128473-128473.) studied the method for increasing the biosynthesis of L-homoserine in Corynebacterium glutamicum by constructing dual-channel glycolysis to balance cofactor supply. By enhancing the endogenous pentose phosphate pathway and introducing the heterologous Entner-Doudoroff pathway, the supply of NADPH was increased. Further, by co-expressing NADPH- and NADH-dependent glyceraldehyde-3-phosphate dehydrogenases, dual-channel glycolysis was constructed to balance intracellular cofactors, resulting in a 48.6% increase in the production of L-homoserine. Finally, the engineered strain accumulated 63.5 g / L of L-homoserine in a 5 L bioreactor. Vo et al. (Minh T V, Sunghoon P. Metabolic engineering of Escherichia coli W3110 for efficient production of homoserine from glucose. Metabolic Engineering, 2022, 73104-113.) conducted an in-depth exploration of the L-homoserine synthesis pathway in Escherichia coli W3110. By strengthening and redistributing the carbon flux, the production efficiency of L-homoserine was significantly improved. The strain W-H18 / pM2 / pR1 constructed by them was able to produce at 1.82 g·L-1 ·h -1 The production intensity accumulation is 110.8 g·L -1 of L-homoserine, while achieving a sugar-acid conversion rate of 0.64 g·g glucose -1 All of the above strains use plasmids as vectors to express key genes. Multiple copies of plasmids during fermentation can impose a certain burden on the growth of the bacteria, and plasmid expression vectors are prone to loss during the production process, resulting in unstable fermentation. Or a certain selection pressure needs to be added to maintain the plasmid, causing too high production costs, and ultimately making it difficult for these L-homoserine strains to be put into industrial production. Cai et al. (Mengmeng C, Zhenqiang Z, Xiangfei L, et al. Development of a nonauxotrophic L-homoserine hyperproducer in Escherichia coli by systems metabolic engineering. Metabolic engineering, 2022, 73:270-279.) constructed a non-inducible, non-auxotrophic, plasmid-free L-homoserine-producing strain in Escherichia coli, and the final yield reached 85.29 g / L.

[0004] Although existing research has significantly increased the yield of L-homoserine in engineered strains through systems metabolic engineering strategies, the current strategies mainly focus on strengthening the metabolic flux of the L-homoserine synthesis pathway, and lack coordinated regulation of the glucose transport system and energy metabolism pathway of existing strains, resulting in difficulties in simultaneously improving the substrate utilization rate and product synthesis efficiency. On the other hand, a single gene editing method is prone to trigger a compensatory response of the metabolic network, instead reducing the production stability of the strain. To further improve the performance of engineered strains, it is necessary to comprehensively consider the above factors and conduct more refined regulation and optimization of the metabolic network of the strain through a multi-module coordination method. Summary of the Invention

[0005] The present invention first provides a glucose transporter mutant, which has an amino acid mutation at one or more positions among the 162nd, 165th, 316th, 383rd, or 458th positions on the basis of the amino acid sequence corresponding to the parental glucose transporter;

[0006] The amino acid sequence of the glucose transporter has at least 80% identity with the amino acid shown in SEQ ID NO.11 and has glucose transport activity;

[0007] or a glucose transporter having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO. 11.

[0008] In one embodiment, the at least one mutation includes a substitution, deletion, or addition.

[0009] In one embodiment, corresponding to the amino acid sequence shown in SEQ ID NO. 11, the mutants are: A165L, V162S, W383S, A165L / V162S, A165L / W383S, V162S / W383S;

[0010] The present invention also provides a polynucleotide encoding the above-mentioned glucose transporter mutant.

[0011] The present invention also provides a recombinant vector carrying the polynucleotide.

[0012] In one embodiment, the recombinant vector uses a pTrc series vector, a pET series vector, a pRSF series vector, or a pGEX series vector as the expression vector.

[0013] In one embodiment, the recombinant vector uses pTrc-99a, pET-21(a), pRSF-Duet-1, or pGEX-6P-1 as the expression vector.

[0014] The present invention also provides a recombinant cell expressing the glucose transporter mutant.

[0015] In one embodiment, the recombinant cell uses bacteria or fungi as the expression host.

[0016] In one embodiment, the recombinant cell uses Escherichia coli, Bacillus subtilis, Bacillus licheniformis, or Pichia pastoris as the host cell.

[0017] The present invention also provides a method for synthesizing a polynucleotide, the method being to use a glucose transporter mutant; wherein the synthesis method includes in vitro transcription synthesis, isothermal amplification, or transcription-mediated amplification.

[0018] The present invention also provides a polynucleotide synthesis kit containing the glucose transporter mutant.

[0019] The present invention also provides a method for improving the glucose transport ability of a glucose transporter. The method is to have amino acid mutations at one or more sites among the 162nd, 165th, 316th, 383rd, or 458th positions based on the amino acid sequence corresponding to the parental glucose transporter; the amino acid sequence of the glucose transporter has at least 80% identity with the amino acid shown in SEQ ID NO.11 and has glucose transport activity;

[0020] SEQ ID NO.11:

[0021] MSSESSQGLVTRLALIAAIGGLLFGYDSAVIAAIGTPVDIHFIAPRHLSATAAASLSGMVVVAVLVGCVTGSLLSGWIGIRFGRRGGLLMSSICFVAAGFGAALTEKLFGTGGSALQIFCFFRFLAGLGIGVVSTLTPTYIAEIAPPDKRGQMVSGQQMAIVTGALTGYIFTWLLAHFGSIDWVNASGWCWSPASEGLIGIAFLLLLLTAPDTPHWLVMKGRHSEASKILARLEPQADPNLTIQKIKAGFDKAMDKSSAGLFAFGITVVFAGVSVAAFQQLVGINAVLYYAPQMFQNLGFGADTALLQTISIGVVNFIFTMIASRVVDRFGRKPLLIWGALGMAAMMAVLGCCFWFKVGGVLPLASVLLYIAVFGMSWGPVCWVVLSEMFPSSIKGAAMPIAVTGQWLANILVNFLFKVADGSPALNQTFNHGFSYLVFAALSILGGLIVARFVPETKGRSLDEIEEMWRSQK

[0022] Preferably, the method is that corresponding to the amino acid sequence shown in SEQ ID NO.11, the mutants are: A165L, V162S, W383S, A165L / V162S, A165L / W383S, V162S / W383S.

[0023] The nucleotide sequence of the parental glucose transporter is as shown in SEQ ID NO.12.

[0024] The present invention enhances the supply of PEP (phosphoenolpyruvate), a precursor for L-homoserine synthesis, by constructing a multi-pathway glucose transport system, improves ATP availability by knocking out energy-consuming genes and enhancing ATP regeneration, further strengthens the metabolic flux of the L-homoserine synthesis pathway by establishing an acetic acid conversion pathway and blocking the L-homoserine degradation pathway, reconstructs key metabolic nodes by introducing a pyruvate carboxylase mutant and weakening the expression of citrate synthase (gltA) through promoter design, and optimizes the fed-batch fermentation conditions of the strain in a 5 L bioreactor according to the metabolic characteristics of the strain, resulting in a genetically engineered bacterium with a clear genetic background, no plasmid, no induction required, and capable of stably and efficiently producing L-homoserine. The yield of L-homoserine reaches 141.5 g / L after 56 h of fermentation in a 5 L fermenter, and the sugar-acid conversion rate reaches 40%, showing certain application prospects.

[0025] The present invention provides a recombinant Escherichia coli for improving the yield of L-homoserine, and the recombinant Escherichia coli expresses the above-mentioned glucose transporter mutant.

[0026] In one embodiment, the recombinant Escherichia coli uses Escherichia coli HOM10 as the starting strain.

[0027] The Escherichia coli HOM10 is described in CN115109738B. Specifically, using Escherichia coli W3110 as the starting strain, the lactose operon repressor encoding gene lacI is knocked out, the expression of the homoserine kinase encoding gene thrB is weakened, the aspartokinase I / homoserine dehydrogenase I encoding gene thrA, the phosphoenolpyruvate carboxylase encoding gene ppc, the aspartate aminotransferase encoding gene aspC, the aspartate ammonia-lyase encoding gene aspA, the threonine and homoserine efflux system encoding gene rhtA, and the pyridine nucleotide transhydrogenase encoding gene pntAB are overexpressed, and the heterologous aspartokinase encoding gene lysC, the aspartate-semialdehyde dehydrogenase encoding gene asd, and the aspartate dehydrogenase encoding gene aspdh are introduced; among them, by replacing the original promoter of thrB with promoter P fliC weaken the expression of the homoserine kinase encoding gene thrB; the aspartokinase I / homoserine dehydrogenase I encoding gene thrA, the phosphoenolpyruvate carboxylase encoding gene ppc, the aspartate aminotransferase encoding gene aspC, the aspartate ammonia-lyase encoding gene aspA, the aspartokinase encoding gene lysC, the pyridine nucleotide transhydrogenase encoding gene pntAB, the aspartate-semialdehyde dehydrogenase encoding gene asd, and the aspartate dehydrogenase encoding gene aspdh are regulated by promoter P trc The threonine and homoserine efflux system encoding gene rhtA is regulated by promoter P lppRegulatory expression; integrating thrA into the gene loci of ycgH, ydeU, yjhE, and tfaD respectively; integrating ppc into the yeeL gene locus; integrating aspC into the ylbE gene locus; integrating aspA into the ycdN gene locus; integrating lysC into the ycjV gene locus; integrating pntAB into the ilvG and ygaY gene loci respectively; integrating rhtA into the yjiP gene locus; integrating asd into the yeeP gene locus; integrating aspdh into the yghX gene locus.

[0028] In one embodiment, the recombinant strain provided by the present invention is:

[0029] Knock out the lactate dehydrogenase encoding gene ldhA, pyruvate formate lyase encoding gene pflB, pyruvate oxidase encoding gene poxB, alcohol dehydrogenase encoding gene adhE, homoserine kinase encoding gene thrB, and flagellar synthesis regulatory factor encoding gene flhC of the starting strain HOM10; and in the starting strain, overexpress the above-mentioned glucose transporter mutant, glucose kinase encoding gene glk, galactose permease encoding gene galP, methylgalactoside transport system encoding gene mglABC, phosphoglycerate kinase encoding gene pgk, phosphoenolpyruvate carboxylase mutant encoding gene and phosphoenolpyruvate carboxylase encoding gene ppc, acetyl-CoA synthetase encoding gene acs; replace the original promoter of the citrate synthase encoding gene gltA with a synthetic weak promoter.

[0030] In one embodiment, the glucose transporter mutant encoding gene, the pyc mutant of phosphoenolpyruvate carboxylase from Corynebacterium glutamicum, phosphoenolpyruvate carboxylase encoding gene ppc, glucose kinase encoding gene glk, galactose permease encoding gene galP, methylgalactoside transport system encoding gene mglABC, phosphoglycerate kinase encoding gene pgk, acetyl-CoA synthetase encoding gene acs are regulated and expressed by promoter P trc Regulatory expression;

[0031] In one embodiment, the nucleotide sequence of promoter P trc is shown in SEQ ID NO.1.

[0032] In one embodiment, the citrate synthase encoding gene gltA is regulated and expressed by promoter P dps Regulatory expression, and the nucleotide sequence of promoter P dps is shown in SEQ ID NO.2.

[0033] In one embodiment, promoter Ptrc The controlled ppc was integrated into the yjiT gene locus; the promoter P trc The controlled pyc mutant was integrated into the yncK gene locus; the promoter P trc The controlled glf mutant was integrated into the ptsG gene locus; the promoter P trc The controlled glk was integrated into the galR gene locus; the promoter P trc The controlled pgk was integrated into the flhC gene locus; the promoter P trc The controlled acs was integrated into the poxB gene locus; the promoter P trc The controlled galP was integrated into the ldhA gene locus; the promoter P trc The controlled mglABC was integrated into the pflB gene locus.

[0034] In one embodiment, the nucleotide sequence of the gene glf A165L / W383S is shown in SEQ ID No.3, the NCBI accession number of the gene glk is NP_416889.1, the nucleotide sequence of the gene pyc mutant is shown in SEQ ID No.5, and the NCBI accession number of the gene ppc is NP_418391.1; the NCBI accession number of the gene ldhA is NP_415898.1, the NCBI accession number of the gene pflB is NP_415423.1, the NCBI accession number of the gene adhE is NP_415757.1, the NCBI accession number of the gene thrB is NP_414544.1, the NCBI accession number of the gene gltA is NP_415248.1, the NCBI accession number of the gene yjiT is YP_009518834.1, the NCBI accession number of the gene yncK is ABE-0285110, the NCBI accession number of the gene ptsG is NP_415619.1, the NCBI accession number of the gene galR is NP_417314.1, the NCBI accession number of the gene poxB is NP_415392.1, the NCBI accession number of the gene acs is NP_418493.1, the NCBI accession number of the gene pgk is NP_417401.1, the NCBI accession number of the gene flhC is NP_416405.1, the NCBI accession number of the gene galP is NP_417418.1, the NCBI accession number of the gene mglA is NP_416654.1, the NCBI accession number of the gene mglB is NP_416655.1, and the NCBI accession number of the gene mglC is NP_416653.1.

[0035] Among them, the pyc mutant is derived from Corynebacterium glutamicum; the glf mutant is derived from Zymomonas mobilis.

[0036] The present invention provides a method for producing L-homoserine using the above recombinant Escherichia coli; the method is to use glucose as a substrate and ferment L-homoserine through the above recombinant Escherichia coli.

[0037] In one embodiment, the temperature condition for the fermentation is: the reaction is carried out at 30-40 °C; preferably, the reaction is carried out at 35-37 °C; preferably, the reaction is carried out at 30-31 °C; preferably, the reaction is carried out at 32-33 °C; preferably, the reaction is carried out at 34-35 °C; preferably, the reaction is carried out at 36-37 °C; preferably, the reaction is carried out at 38-39 °C.

[0038] In one embodiment, the pH condition for the fermentation is: the reaction is carried out at a pH of 6.8-7.5; preferably, the reaction is carried out at a pH of 6.8-7.0; preferably, the pH is 7.2-7.3; preferably, the pH is 7.0-7.2.

[0039] In one embodiment, the fermentation time is at least 24 h; preferably, the fermentation time is 24-96 h; preferably, the fermentation time is 24-36 h; preferably, the fermentation time is 36-48 h; preferably, the fermentation time is 48-52 h; preferably, the fermentation time is 52-56 h; preferably, the fermentation time is 56-96 h.

[0040] The present invention also provides the application of the above glucose transporter in improving the glucose utilization efficiency of strains.

[0041] The present invention also provides the application of the above recombinant Escherichia coli or the above method in the preparation of L-homoserine or products containing L-homoserine.

[0042] Beneficial effects

[0043] (1) The present invention provides a glucose transporter mutant, which can enhance the consumption rate of glucose in the medium by the strain, cooperate with overexpression of glucokinase to relieve the inhibition of glucokinase activity by excessive glucose in the cell, enhance the carbon flux of L-homoserine synthesis by reconstructing the key node metabolic flux, and ultimately improve the yield and conversion rate of L-homoserine.

[0044] (2) The present invention provides a genetically engineered strain for producing L-homoserine. The product concentration of the engineered strain H13 reaches 59.1 g / L after 36 h of shake flask fermentation, and reaches 141.5 g / L after 56 h of fermentation in a 5 L fermenter. The sugar-acid conversion rate reaches 40%, showing certain application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Shake flask fermentation results of the L-homoserine genetically engineered strain;

[0046] Figure 2 Batch-fed fermentation process curve of the strain E. coli H13-A165L / W383S in a 5 L fermenter. DETAILED DESCRIPTION OF THE INVENTION

[0047] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0048] Some definitions or terms involved in the present invention:

[0049] Corresponds to: As used herein, the term "corresponds to" refers to the manner of determining a specific amino acid in a sequence (where a specific amino acid sequence is referred to). For example, for the purposes of the present invention, when referring to a specific amino acid position, a person skilled in the art can align another amino acid sequence with the amino acid sequence that has been referred to, so as to determine which specific amino acid may be of interest in the other amino acid sequence. Alternative alignment methods can be used, and these methods are well known to those skilled in the art.

[0050] Mutant: As used herein, when referring to the use of variants of the present invention, the term "mutant" means a polypeptide having glucose transporter mutant activity and containing alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "parent" glucose transporter mutant. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a certain position. In describing the variants of the present invention, for ease of reference, the following nomenclature has been adapted. The accepted IUPAC single-letter or three-letter amino acid abbreviations are used. Substitution: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the substitution of tryptophan at position 383 by serine is represented as "W383S". Multiple mutations are separated by the symbol (" / "), such as the substitution of tryptophan at position 383 by serine and the substitution of alanine at position 165 by leucine A165L / W383S.

[0051] Parent or parental glucose transporter: As used herein, the term "parent" glucose transporter refers to the glucose transporter that is modified to produce the glucose transporter mutants of the present invention. This term also refers to the polypeptide to which the mutants of the present invention are compared. The parent can be a naturally occurring (wild-type) polypeptide, or it can even be a variant thereof prepared by any suitable means. For example, the parent protein can be a variant of a naturally occurring polypeptide that has been modified or altered in its amino acid sequence. Thus, the parent glucose transporter can have one or more (or one or several) amino acid substitutions, deletions, and / or insertions. Thus, the glucose transporter can be a variant of the parent glucose transporter. The parent can also be an allelic variant, which is a polypeptide encoded by any one of two or more alternative forms of a gene that occupy the same chromosomal locus.

[0052] Wild-type enzyme: When referring to an amino acid sequence or a nucleic acid sequence, the term "wild-type" means that the amino acid sequence or the nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, an amino acid, or a nucleic acid sequence). In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in the laboratory, or modifications of wild-type sequences). When the parent enzyme is not a variant enzyme, the terms "wild-type enzyme" and "parent enzyme" can be used interchangeably.

[0053] Sequence identity: The degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0054] For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) is used to determine sequence identity between two amino acid sequences, which is implemented as in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later). The parameters used can be a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of "longest identity" marked by Needle (obtained using the -nobrief option) is used as the percentage identity and is calculated as follows:

[0055] (Number of identical residues x 100) / (Alignment length - Total number of gaps in the alignment)

[0056] Alternatively, the parameters used can be a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of "longest identity" marked by Needle (obtained using the -nobrief option) is used as the percentage identity and is calculated as follows:

[0057] (Number of identical deoxyribonucleotides x 100) / (Alignment length - Total number of gaps in the alignment)

[0058] Expression: As used herein, the term "expression" refers to any step involved in the production of a variant, including but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0059] Expression vector: As used herein, the term "expression vector" refers to a linear or circular DNA molecule that contains a polynucleotide encoding a variant and is operably linked to control sequences providing for its expression.

[0060] Host cell: The term "host cell" means any cell type that is readily transformable, transfectable, transducible, etc. with a nucleic acid construct or expression vector containing a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication, together with recombinant host cells, isolated host cells (e.g., isolated recombinant host cells), and heterologous host cells.

[0061] Recombination: When used in reference to a cell, nucleic acid, protein, or vector, the term "recombinant" means that it has been modified from its natural state. Thus, for example, a recombinant cell expresses a gene that is not found in a cell in its natural (non-recombinant) form, or expresses a natural gene at a different level or under different conditions compared to that found in nature. A recombinant nucleic acid differs from a natural sequence by one or more nucleotides and / or is operably linked to a heterologous sequence (e.g., a heterologous promoter in an expression vector). A recombinant protein differs from a natural sequence by one or more amino acids and / or is fused to a heterologous sequence. A vector containing a nucleic acid encoding a polypeptide is a recombinant vector. The terms "recombinant" are synonymous with "genetically modified" and "transgenic".

[0062] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0063] It will be appreciated from the foregoing that the present disclosure can be embodied in various ways, including but not limited to the following:

[0064] Example 1: A glucose transporter mutant that has an amino acid mutation at one or more of positions 162, 165, 316, 383, or 458 based on the amino acid sequence of a parental glucose transporter.

[0065] The amino acid sequence of the glucose transporter has at least 80% identity with the amino acid shown in SEQ ID NO. 11 and has glucose transport activity.

[0066] Example 2: According to the method of Example 1, the mutant is, corresponding to the amino acid sequence shown in SEQ ID NO. 11, the mutant is: A165L, V162S, W383S, A165L / V162S, A165L / W383S, V162S / W383S.

[0067] Example 3: A polynucleotide encoding the glucose transporter mutant of Example 1 or 2.

[0068] Example 4: A recombinant vector carrying the polynucleotide of Example 3.

[0069] Example 5: According to the recombinant vector of Example 4, the recombinant vector uses a pTrc series vector, a pET series vector, a pRSF series vector, or a pGEX series vector as an expression vector.

[0070] Example 6: The recombinant vector according to Example 5, wherein the recombinant vector uses pTrc-99a, pET-21(a), pRSF-Duet-1 or pGEX-6P-1 as the expression vector.

[0071] Example 7: A recombinant cell expressing the glucose transporter mutant of Example 1 or 2.

[0072] Example 8: The recombinant cell according to Example 7, wherein the recombinant cell uses bacteria or fungi as the expression host.

[0073] Example 9: The recombinant cell according to Example 8, wherein the recombinant cell uses Escherichia coli, Bacillus subtilis, Bacillus licheniformis or Pichia pastoris as the host cell.

[0074] Example 10: A polynucleotide synthesis kit comprising the glucose transporter mutant of Example 1 or 2.

[0075] Example 11: A method for improving the glucose transport ability of a glucose transporter, wherein, corresponding to the amino acid sequence shown in SEQ ID NO.11, the mutants are: A165L, V162S, W383S, A165L / V162S, A165L / W383S, V162S / W383S;

[0076] Example 12: A recombinant Escherichia coli expressing the mutant of Example 1 or 2.

[0077] Example 13: The recombinant Escherichia coli according to Example 12, wherein the recombinant Escherichia coli uses Escherichia coli as the starting strain, and knocks out the lactate dehydrogenase encoding gene ldhA, the pyruvate formate-lyase encoding gene pflB, the pyruvate oxidase encoding gene poxB, the alcohol dehydrogenase encoding gene adhE, the homoserine kinase encoding gene thrB, and the flagellar synthesis regulatory factor encoding gene flhC; overexpresses the glucose transporter mutant of Claim 1 or 2, the glucose kinase encoding gene glk, the galactose permease encoding gene galP, the methylgalactoside transport system encoding gene mglABC, the phosphoglycerate kinase encoding gene pgk, the phosphoenolpyruvate carboxylase mutant encoding gene and the phosphoenolpyruvate carboxylase encoding gene ppc, the acetyl-CoA synthetase encoding gene acs; weakly expresses the citrate synthase encoding gene gltA;

[0078] Preferably, the recombinant Escherichia coli is based on Escherichia coli as the starting strain, knocking out the lactate dehydrogenase-encoding gene ldhA, pyruvate formate-lyase-encoding gene pflB, pyruvate oxidase-encoding gene poxB, alcohol dehydrogenase-encoding gene adhE, homoserine kinase-encoding gene thrB, and flagellar synthesis regulatory factor-encoding gene flhC; overexpressing the glucose transporter mutant glf A165L / W383S , glucose kinase-encoding gene glk, galactose permease-encoding gene galP, methylgalactoside transport system-encoding gene mglABC, phosphoglycerate kinase-encoding gene pgk, phosphoenolpyruvate carboxylase mutant-encoding gene and phosphoenolpyruvate carboxylase-encoding gene ppc, acetyl-CoA synthetase-encoding gene acs; weakly expressing the citrate synthase-encoding gene gltA.

[0079] Preferably, the gene galP is integrated into the ldhA locus on the Escherichia coli genome, the gene mglABC is integrated into the pflB locus on the Escherichia coli genome, the mutant gene is integrated into the ptsG locus on the Escherichia coli genome, the gene glk is integrated into the galR locus on the Escherichia coli genome, the gene pgk is integrated into the flhC locus on the Escherichia coli genome, the gene is integrated into the ynck locus on the Escherichia coli genome, the gene acs is integrated into the poxB locus on the Escherichia coli genome, and the gene ppc is integrated into the yjiT locus on the Escherichia coli genome;

[0080] Preferably, the overexpression is regulated by the promoter P trc to regulate the expression of galP, mglABC, glf A165L / W383S , glk, pgk, acs, and ppc; the weak expression is regulated by the promoter P dps to regulate the expression of gltA;

[0081] Preferably, the nucleotide sequence of the promoter P trc is shown in SEQ ID NO.1, and the nucleotide sequence of the promoter P dps is shown in SEQ ID NO.2; the mutants are A165L and W383S, and the nucleotide sequence of the mutant gene glf A165L / W383S is shown in SEQ ID NO.3, the nucleotide sequence of the gene glk is shown in SEQ ID NO.4, the gene The nucleotide sequence of [gene name] is shown in SEQ ID NO.5, the nucleotide sequence of gene ppc is shown in SEQ ID NO.6, the nucleotide sequence of gene pgk is shown in SEQ ID NO.7, the nucleotide sequence of gene acs is shown in SEQ ID NO.8, the nucleotide sequence of gene galP is shown in SEQ ID NO.9, and the nucleotide sequence of gene mglABC is shown in SEQ ID NO.10;

[0082] Preferably, the starting strain of the recombinant Escherichia coli is Escherichia coli HOM10.

[0083] Example 14: A method for producing L-homoserine using the recombinant Escherichia coli described in Example 12 or 13 above; the method is to use glucose as a substrate and ferment L-homoserine through the above recombinant Escherichia coli.

[0084] Example 15: According to the method described in Example 14, the temperature condition for the fermentation is: the reaction is carried out at 30-40 °C; preferably, the reaction is carried out at 35-37 °C; preferably, the reaction is carried out at 30-31 °C; preferably, the reaction is carried out at 32-33 °C; preferably, the reaction is carried out at 34-35 °C; preferably, the reaction is carried out at 36-37 °C; preferably, the reaction is carried out at 38-39 °C.

[0085] The pH condition for the fermentation is: the reaction is carried out at a pH of 6.8-7.5; preferably, the reaction is carried out at a pH of 6.8-7.0; preferably, the pH is 7.2-7.3; preferably, the pH is 7.0-7.2;

[0086] The fermentation time is at least 24 h; preferably, the fermentation time is: 24-96 h; preferably, the fermentation time is: 24-36 h; preferably, the fermentation time is: 36-48 h; preferably, the fermentation time is: 48-52 h; preferably, the fermentation time is: 52-56 h; preferably, the fermentation time is: 56-96 h.

[0087] Example 16: According to the method described in Example 14 or 15, during shake flask fermentation, the activated strain is cultured at 35-37 °C and 180-250 r / min to obtain a seed liquid, and the seed liquid is inoculated into the fermentation medium at an inoculation amount of 10-20% (v / v). The fermentation temperature is 35-37 °C, the rotation speed is 180-250 r / min, and the pH is controlled at 7.0-7.2. When the glucose in the medium is exhausted, the fermentation is maintained by adding a 60% (m / v) glucose solution.

[0088] Among them, the fermentation time is preferably 24 - 48 h, more preferably 36 h.

[0089] The seed culture medium composition is: 20 - 30 g / L glucose, 5 - 10 g / L yeast powder, 1 - 5 g / L (NH4)2SO4, 1 - 5 g / L KH2PO4, 1 - 5 g / L MgSO4·7H2O, 1 - 5 g / L sodium citrate, 5 - 15 mg / L FeSO4·7H2O, 0.5 - 2 g / L L-threonine, 0.5 - 2 mg / L V H and 0.5 - 2 mg / L V B1 。

[0090] The fermentation culture medium composition is: 10 - 20 g / L glucose, 1 - 5 g / L yeast powder, 1 - 5 g / L (NH4)2SO4, 1 - 5 g / L KH2PO4, 1 - 5 g / L MgSO4·7H2O, 1 - 5 g / L sodium citrate, 20 - 30 mg / L FeSO4·7H2O, 0.5 - 2 g / L L-threonine, 0.5 - 2 mg / L V H and 0.5 - 2 mg / L V B1 。

[0091] Example 17: According to the method described in any one of Examples 14 - 16, when fermenting in a fermenter, after activating the recombinant Escherichia coli, it is cultured in a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into the fermentation culture medium at an inoculation amount of 10 - 20% (v / v) for fermentation culture. Among them, when culturing in the seed culture medium, the culture temperature is 35 - 37 °C, the pH is 7.0 - 7.2, and the dissolved oxygen is controlled at 25 - 30% by adjusting the stirring speed and ventilation volume; during the fermentation culture process, the fermentation temperature is 35 - 37 °C, the pH is 7.0 - 7.2, the dissolved oxygen is controlled at 25 - 30%, and batch feeding is used to control the glucose residual sugar concentration in the fermentation broth to be 1 - 10 g / L.

[0092] The fermentation time is preferably 48 - 60 h, more preferably 56 h.

[0093] The seed culture medium composition is: 25 - 35 g / L glucose, 5 - 10 g / L yeast powder, 1 - 5 g / L peptone, 1 - 5 g / L KH2PO4, 0.5 - 2 g / L MgSO4·7H2O, 1 - 5 g / L citric acid or sodium citrate, 5 - 10 mg / L FeSO4·7H2O, 5 - 10 mg / L MnSO4·H2O, 0.5 - 2 g / L L-threonine, 0.2 - 2 mg / L V H and 0.5 - 2 mg / L V B1 。

[0094] The composition of the fermentation medium is as follows: 10 - 20 g / L glucose, 10 - 15 g / L corn steep liquor, 1 - 5 g / L yeast extract, 1 - 5 g / L peptone, 1 - 5 g / L KH2PO4, 0.5 - 3 g / L MgSO4·7H2O, 1 - 5 g / L citric acid or citrate, 10 - 30 mg / L FeSO4·7H2O, 10 - 20 mg / L MnSO4·H2O, 0.5 - 2 g / L L-threonine, 0.2 - 2 mg / L V H and 0.3 - 1 mg / L V B1 , with or without betaine, preferably, 1 - 3 g / L betaine is added.

[0095] During fermentation culture, the medium is fed or not fed. The composition of the medium is as follows: 1 - 10 g / L yeast extract, 1 - 10 g / L peptone, 1 - 10 g / L KH2PO4, 1 - 5 g / L MgSO4·7H2O, 1 - 5 g / L citric acid, 5 - 15 mg / L FeSO4·7H2O, 5 - 15 mg / L MnSO4·H2O, 0.1 - 1 mg / L V H and 0.1 - 1 mg / L V B1 .

[0096] The medium is fed during fermentation for 14 - 20 h.

[0097] Example 18: A method for constructing a recombinant Escherichia coli for producing L-homoserine, comprising the following steps in any order: (1) knocking out the adhE and thrB genes of Escherichia coli HOM10; (2) integrating the P trc -pyc mutant into the ynck gene locus; (3) integrating the P trc -glf mutant into the ptsG gene locus; (4) integrating the P trc -ppc into the yjiT gene locus; (5) integrating the P trc -pgk into the flhC gene locus; (6) integrating the P trc -acs into the poxB gene locus; (7) integrating the P trc -galP into the ldhA gene locus; (8) integrating the P trc -mglABC into the pflB gene locus.

[0098] In this example, Escherichia coli with a clear metabolic pathway and simple genetic manipulation is used as the starting strain. Starting from the genetic engineering transformation of glucose uptake, energy supply, and the L-homoserine biosynthesis pathway, the overall metabolic pathway is analyzed and reconstructed to obtain a genetically engineered strain with a clear genetic background, without carrying a plasmid, and capable of stably and efficiently producing L-homoserine.

[0099] The genetically engineered bacterium obtained in this example enriches the glucose uptake mode, enhances the availability of ATP, increases the synthesis flux and precursor supply of L-homoserine, reconstructs the carbon flow of the metabolic central node, and weakens the metabolic flux entering the TCA cycle by introducing a synthetic weak promoter, thereby effectively improving the production of L-homoserine.

[0100] Example 19: Application of the recombinant Escherichia coli described in Example 12 or 13 above in the preparation of L-homoserine or a product containing L-homoserine.

[0101] The expression method, vector construction method, and gene knockout method adopted in the present invention are only examples. Those skilled in the art can adopt any other method as long as they can achieve the gene expression, vector construction, and gene knockout of the present invention, and can implement the technical solution of the present invention, which is within the protection scope of the present invention.

[0102] The detection methods involved in the following examples are as follows:

[0103] Detection method for the glucose utilization ability of the strain

[0104] The remaining glucose content and consumption rate in the culture medium are detected by an SBA-40E biosensor analyzer (the calculation method is: sample once every 2 h before the initial sugar in the shake flask fermentation is completely consumed (this time can be determined by offline measurement with an SBA-40E biosensor analyzer combined with the color of phenol red in the culture medium no longer changing. The chassis strain of the present invention is usually 6 h, and the error within 30 min does not exceed 1 g / L). Perform a linear fit on the glucose concentration-time curve, and the slope is the average consumption rate. The formula is:

[0105] C t = C0 - k·t.

[0106] Where the slope k is the glucose consumption rate (Glucose Consumption Rate, GCR); C0 is the initial glucose concentration; C t is the remaining glucose concentration at time t). Under the same culture conditions, a strain with a higher glucose consumption rate and a higher final sugar-acid conversion rate is considered to have a higher ability to utilize glucose from the culture medium.

[0107] Detection of L-homoserine yield

[0108] The yield of L-homoserine is calculated by high performance liquid chromatography. The concentrations of L-homoserine and other amino acids are determined by pre-column derivatization with orthophthalaldehyde (OPA). The liquid chromatography conditions are: column temperature 40 °C, ultraviolet detector wavelength 338 nm, C18 AAA chromatographic column, and mobile phase flow rate 1 mL / min.

[0109] The genotypes of the strains involved in the following examples are shown in Table 1 below:

[0110] Table 1: Summary of Strain Genetic Modification

[0111]

[0112] The primer sequences involved in the following examples are shown in Table 2 below.

[0113] Table 2: Primers Involved in the Present Invention

[0114]

[0115]

[0116]

[0117] Example 1: Plasmid Construction Containing glf Mutant and Mutant

[0118] 1. Plasmid Construction Containing glf Mutant

[0119] Using the genome of Zymomonas mobilis as a template, amplification primers (pTrc-99a-EcoRI-glf-1, pTrc-99a-BamHI-glf-4) and mutant primers (pTrc-99a-glf K458V -2, pTrc-99a-glf K458V -3, pTrc-99a-glf A165L -2, pTrc-99a-glf A165L -3, pTrc-99a-glf V162S -2, pTrc-99a-glf V162S -3, pTrc-99a-glf W383S -2, pTrc-99a-glf W383S -3, pTrc-99a-glf N316T -2, pTrc-99a-glf N316T -3, pTrc-99a-glf A165L / V162S -2, pTrc-99a-glf A165L / V162S -3) were designed according to the sequences of the glf gene (SEQ ID NO.12) and pTrc-99a, and the corresponding fragments were amplified by PCR.

[0120] Then, the above fragments were fused by the method of overlap PCR to obtain glf K458V gene, glfA165L Gene, glf V162S Gene, glf W383S Gene, glf N316T Gene, glf A165L / V162S Fragments of the gene. The gene fragments were respectively ligated with plasmid pTrc-99a to construct plasmid pTrc-99a-glf K458V , pTrc-99a-glf A165L , pTrc-99a-glf V162S , pTrc-99a-glf W383S , pTrc-99a-glf N316T , pTrc-99a-glf A165L / V162S .

[0121] According to the above method, using pTrc-99a-glf A165L -2, pTrc-99a-glf A165L -3, pTrc-99a-glf / W383S -2, pTrc-99a-glf W383S -3 primers to obtain fragments of the glf A165L / W383S gene. The gene fragments were ligated with plasmid pTrc-99a to construct plasmid pTrc-99a-glf A165L / W383S . Using pTrc-99a-glf V162S -2, pTrc-99a-glf V162S -3, pTrc-99a-glf W383S -2, pTrc-99a-glf W383S -3 primers to obtain fragments of the glf V162S / W383S gene. The gene fragments were ligated with plasmid pTrc-99a to construct plasmid pTrc-99a-glf V162S / W383S .

[0122] 2、 Construction of mutants

[0123] Using the genome of Corynebacterium glutamicum as a template, amplification primers and mutant primers were designed according to the sequence of the pyc gene (NCBI number: WP_011013816.1) and the sequence of pTrc-99a, and the corresponding fragments were amplified by PCR. Then, the above fragments were fused by the method of overlap PCR to obtain fragments of the gene (SEQ ID NO.5). The gene fragments were ligated with plasmid pTrc-99a to construct plasmid

[0124] Example 2: Construction of a Strain Containing a Glucose Transporter Mutant

[0125] The specific steps are as follows:

[0126] I. Construction of a Strain Containing Wild-Type Glucose Transporter

[0127] 1. Construction of the Genetically Engineered Bacterium E. coli W3110 H1

[0128] This example is a further modification based on the previously constructed strain HOM10 (the strain HOM10 is the genetically engineered bacterium E. coli W3110 HOM10, and the construction process is described in the Chinese invention patent text with the publication number CN115109738B). Since the strain HOM10 is obtained by modifying E. coli W3110, it is named E. coli W3110 HOM10 in the present invention.

[0129] Knock out the ldhA gene of E. coli W3110 HOM10:

[0130] Using the E. coli W3110 genome (NCBI number: NZ_CP165600.1) as a template, upstream homologous arm primers (ldhA-1, ldhA-2) and downstream homologous arm primers (ldhA-3, ldhA-4) are designed according to the upstream and downstream sequences of its ldhA gene (NCBI number: NP_415898.1), and the upstream and downstream homologous arm fragments are amplified by PCR. Then, the above fragments are fused by the method of overlap PCR to obtain the ldhA gene knockout fragment. The DNA fragment obtained by annealing primers gRNA-ldhA-1 and gRNA-ldhA-2 is ligated to plasmid pGRB to construct plasmid pGRB-ldhA. Plasmid pGRB-ldhA and the ldhA gene knockout fragment are simultaneously electrotransformed into the electrocompetent cells of E. coli W3110 HOM10 containing pREDCas9 to obtain positive transformants, and the plasmid is eliminated to obtain the E. coli W3110 H1 (E. coli W3110 HOM10ΔldhA) strain.

[0131] 2. Construction of the Genetically Engineered Bacterium E. coli W3110 H2

[0132] Knock out the pflB gene of E. coli W3110 HOM10:

[0133] Using the E. coli W3110 genome as a template, upstream homologous arm primers (pflB-1, pflB-2) and downstream homologous arm primers (pflB-3, pflB-4) were designed according to the upstream and downstream sequences of its pflB gene (NCBI accession number: NP_415423.1), and the upstream and downstream homologous arm fragments were amplified by PCR. Then, the above fragments were fused by the method of overlap PCR to obtain the knockout fragment of the pflB gene. The DNA fragment obtained by annealing primers gRNA-pflB-1 and gRNA-pflB-2 was ligated to plasmid pGRB to construct plasmid pGRB-pflB. Plasmid pGRB-pflB and the knockout fragment of the pflB gene were simultaneously electrotransformed into electrocompetent cells of E. coli W3110 HOM10 containing pREDCas9 to obtain positive transformants, and after eliminating the plasmid, E. coli W3110 H2 (E. coli W3110 HOM10ΔpflB) strain was obtained.

[0134] 3. Construction of genetically engineered bacterium E. coli W3110 H3

[0135] Knock out the adhE gene of E. coli W3110 HOM10:

[0136] Using the E. coli W3110 genome as a template, upstream homologous arm primers (adhE-1, adhE-2) and downstream homologous arm primers (adhE-3, adhE-4) were designed according to the upstream and downstream sequences of its adhE gene (NCBI accession number: NP_415757.1), and the upstream and downstream homologous arm fragments were amplified by PCR. Then, the above fragments were fused by the method of overlap PCR to obtain the knockout fragment of the adhE gene. The DNA fragment obtained by annealing primers gRNA-adhE-1 and gRNA-adhE-2 was ligated to plasmid pGRB to construct plasmid pGRB-adhE. Plasmid pGRB-adhE and the knockout fragment of the adhE gene were simultaneously electrotransformed into electrocompetent cells of E. coli W3110 HOM10 containing pREDCas9 to obtain positive transformants, and after eliminating the plasmid, E. coli W3110 H3 (E. coli W3110 HOM10ΔadhE) strain was obtained.

[0137] 4. Construction of genetically engineered bacterium E. coli W3110 H4

[0138] Knock out the thrB gene of E. coli W3110 H3 (E. coli W3110 HOM10ΔadhE):

[0139] Using the E. coli W3110 genome as a template, upstream homologous arm primers (thrB-1, thrB-2) and downstream homologous arm primers (thrB-3, thrB-4) were designed according to the upstream and downstream sequences of its thrB gene (NCBI accession number: NP_414544.1), and the upstream and downstream homologous arm fragments were amplified by PCR. Then, the above fragments were fused by overlapping PCR to obtain the thrB gene knockout fragment. The DNA fragment obtained by annealing primers gRNA-thrB-1 and gRNA-thrB-2 was ligated to plasmid pGRB to construct plasmid pGRB-thrB. Through shake flask fermentation screening, it was found that the L-homoserine yield of strain H3 was higher than that of strains HOM10, H1, and H2. Therefore, iterative modification was continued based on strain H3.

[0140] Plasmid pGRB-thrB and the thrB gene knockout fragment were simultaneously electrotransformed into electrocompetent cells of E. coli W3110 H3 containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, E. coli W3110 H4 strain (E. coli W3110 HOM10ΔadhEΔthrB) was obtained.

[0141] 5. Construction of genetically engineered bacteria containing glucose transporters

[0142] To verify whether the glucose transporter can improve the L-homoserine production performance of E. coli W3110 H4 strain, the glucose transporter encoding gene (SEQ ID NO.12) from Zymomonas mobilis was integrated into the ptsG locus (NCBI accession number: NP_415619.1).

[0143] Using the E. coli W3110 genome as a template, upstream homologous arm primers (ptsG-1, ptsG-2) and downstream homologous arm primers (ptsG-5, ptsG-6) were designed according to the ptsG gene sequence. Then, using the Zymomonas mobilis genome as a template, primers ptsG-3 and ptsG-4 were designed according to the glf gene sequence. Among them, the P trc promoter sequence was designed on primers ptsG-2 and ptsG-3. Each fragment was amplified by PCR, and then using them as templates, fusion PCR was carried out to obtain the P trc -glf gene integration fragment. The DNA fragment obtained by annealing primers gRNA-ptsG-1 and gRNA-ptsG-2 was ligated to plasmid pGRB to construct plasmid pGRB-ptsG. Plasmid pGRB-ptsG and P trcThe integrated fragment of the -glf gene was co-electroporated into electrocompetent cells of H4 (E. coli W3110 HOM10ΔadhEΔthrB) containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, the E. coli W3110 H5 (E. coli W3110 HOM10ΔadhEΔthrB ptsG::P trc -glf) strain was obtained.

[0144] II. Construction and Screening of Glucose Transporter Mutants

[0145] To further improve the ability of the engineered strain to absorb and utilize glucose, point mutations were introduced into the gene encoding the glucose transporter (nucleotide sequence shown in SEQ ID NO. 12), and a recombinant plasmid containing the corresponding mutant gene fragment of glf was constructed on the pTrc99a plasmid as a template for facilitating the subsequent acquisition of recombinant fragments.

[0146] 1. Construction of Genetically Engineered Strains E. coli W3110 H5-1 to E. coli W3110 H5-8

[0147] The genes encoding the glucose transporters glf K458V mutant, glf A165L mutant, glf V162S mutant, glf W383S mutant, glf N316T mutant, glf A165L / V162S mutant, glf A165L / W383S mutant, glf V162S / W383S mutant from Zymomonas mobilis were respectively integrated into the ptsG locus (NCBI accession number: NP_415619.1).

[0148] Using the E. coli W3110 genome as a template, upstream homologous arm primers (ptsG-1, ptsG-2) and downstream homologous arm primers (ptsG-5, ptsG-6) were designed based on the ptsG gene sequence. Then, using the pTrc99a-glf K458V constructed in Example 1, pTrc99a-glf A165L pTrc99a-glf V162S pTrc99a-glf W383S pTrc99a-glf N316T pTrc99a-glf A165L / V162S pTrc99a-glf A165L / W383S pTrc99a-glf V162S / W383SUsing the plasmid as a template, primers ptsG-3 and ptsG-4 were designed. Among them, the P trc promoter sequence was designed on primers ptsG-2 and ptsG-3. Each fragment was obtained by PCR amplification. Then, using them as templates, fusion PCR was carried out to obtain the P trc -glf K458V gene, P trc -glf A165L gene, P trc -glf V162S gene, P trc -glf W383S gene, P trc -glf N316T gene, P trc -glf A165L / V162S gene, P trc -glf A165L / W383S gene, P trc -glf V162S / W383S gene integration fragments. The DNA fragment obtained by annealing primers gRNA-ptsG-1 and gRNA-ptsG-2 was ligated with plasmid pGRB to construct plasmid pGRB-ptsG. Plasmid pGRB-ptsG was respectively combined with P trc -glf K458V gene, P trc -glf A165L gene, P trc -glf V162S gene, P trc -glf W383S gene, P trc -glf N316T gene, P trc -glf A165L / V162S gene, P trc -glf A165L / W383S gene, P trc -glf V162S / W383S gene integration fragments were simultaneously electrotransformed into electrocompetent cells of H4 (E. coli W3110 HOM10ΔadhEΔthrB) containing pREDCas9 to obtain positive transformants. After eliminating the plasmids, E. coli W3110 H5-1 (E. coli W3110 HOM10ΔadhEΔthrB ptsG::P trc -glf K458V ), E. coli W3110 H5-2 (E. coli W3110HOM10ΔadhEΔthrB ptsG::P trc -glf A165L)、E.coli W3110 H5-3 (E.coli W3110 HOM10ΔadhEΔthrB ptsG::P trc -glf V162S )、E.coli W3110 H5-4 (E.coli W3110 HOM10ΔadhEΔthrB ptsG::P trc -glf W383S )、E.coli W3110 H5-5 (E.coli W3110 HOM10ΔadhEΔthrB ptsG::P trc -glf N316T )、E.coli W3110 H5-6 (E.coli W3110 HOM10ΔadhEΔthrB ptsG::P trc -glf A165L / V162S )、E.coli W3110 H5-7 (E.coli W3110 HOM10ΔadhEΔthrB ptsG::P trc -glf A165L / W383S )、E.coli W3110 H5-8 (E.coli W3110 HOM10ΔadhEΔthrB ptsG::P trc -glf V162S / W383S ) strains.

[0149] 2. Verification of the mutant effect

[0150] Respectively inoculate the strains prepared in step 1 on the slant medium, culture at 37 °C for 12 h, and subculture once. Then, scrape a loop of slant seeds with an inoculation loop and inoculate them into a 500 mL round-bottom Erlenmeyer flask containing 30 mL of seed medium, seal it with nine layers of gauze, and culture at 37 °C and 220 rmp for 8 - 10 h; prepare the seed liquid. The composition of the seed medium is: 30 g / L glucose, 10 g / L yeast powder, 4 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 5 mg / L FeSO4·7H2O, 0.5 - 2 g / L L-threonine, 0.5 mg / L V H and 0.5 mg / L V B1 , the rest is water, pH 7.0 - 7.2.

[0151] The seed liquid was inoculated into a 500 mL baffled Erlenmeyer flask containing 30 mL of fermentation medium at an inoculum size of 15% (v / v), sealed with nine layers of gauze, and cultured at 37 °C and 240 r / min. During the fermentation process, phenol red was used as an indicator, and the pH was controlled at 7.0 - 7.2 by adding 25% ammonia water. When the glucose in the medium was exhausted, the fermentation was maintained by adding a 60% (m / v) glucose solution to a final concentration of 5 g / L; the fermentation cycle was 36 h; and the fermentation broth was obtained. The composition of the fermentation medium was: 10 g / L glucose, 5 g / L yeast extract, 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 30 mg / L FeSO4·7H2O, 0.5 - 2 g / L L-threonine, 0.5 mg / L V H , 0.5 mg / L V B1 and 8 mg / L phenol red, with the rest being water, pH 7.0 - 7.2.

[0152] The remaining glucose content, consumption rate, and sugar-to-acid conversion rate in the obtained fermentation broth were measured respectively; among them, the sugar-to-acid conversion rate (SACR) was defined as the percentage of unit mass of carbohydrate converted into L-homoserine, and the calculation method was:

[0153] SACR = (M sugar / M L-Homoserine ) × 100%

[0154] where: M L-Homoserine : the mass (g) of L-homoserine produced; M sugar : the mass (g) of carbohydrate consumed, which was the difference between the total mass of added glucose and the mass of residual glucose in the system at the end of fermentation.

[0155] After the strain construction was completed, the effect of introducing the glf mutant protein on the glucose consumption rate and sugar-to-acid conversion rate of the strain was verified by shake-flask fermentation, and the results are shown in Table 3.

[0156] Table 3: Effect of glf mutant protein on the glucose consumption rate and sugar-to-acid conversion rate of the strain

[0157] Strain Glucose consumption rate (g / L / h) Sugar-acid conversion rate (g / g glucose) H4 4.02 0.2643 <![CDATA[H5(H4,ptsG::P trc -glf)]]> 4.11 0.2682 <![CDATA[H5-1(H4,ptsG::P trc -glf K458V )]]> 3.83 0.2551 <![CDATA[H5-2(H4,ptsG::P trc -glf A165L )]]> 4.84 0.275 <![CDATA[H5-3(H4,ptsG::P trc -glf V162S )]]> 4.27 0.2694 <![CDATA[H5-4(H4,ptsG::P trc -glf W383S )]]> 4.31 0.2717 <![CDATA[H5-5(H4,ptsG::P trc -glf N316T )]]> 4.05 0.2639 <![CDATA[H5-6(H4,ptsG::P trc -glf A165L / V162S )]]> 4.96 0.2941 <![CDATA[H5-7(H4,ptsG::P trc -glf A165L / W383S )]]> 5.02 0.3249 <![CDATA[H5-8(H4,ptsG::P trc -glf V162S / W383S )]]> 4.20 0.2554

[0158] The results showed that the highest glucose consumption rate of the engineered strain H5-7 reached 5.02 g / L / h, and the highest sugar-to-acid conversion rate reached 0.3249 g / g glucose, proving that the L-homoserine production level of the strain could be effectively improved by combinatorial mutation of the glucose transporter.

[0159] Example 3: Construction of a Strain with High Yield of L-Homoserine

[0160] The specific steps are as follows:

[0161] 1. Construction of the genetically engineered bacterium E. coli W3110 H6

[0162] Integrate the P trc -glk gene into the galR locus.

[0163] Using the E. coli W3110 genome as a template, design upstream homologous arm primers (galR-1, galR-2) and downstream homologous arm primers (galR-5, galR-6) according to the sequence of the galR gene (NCBI accession number: NP_417314.1). Then, design primers galR-3 and galR-4 according to the sequence of the glk gene (NCBI accession number: NP_416889.1). Among them, the P trc promoter sequence is designed on primers galR-2 and galR-3. Amplify each fragment by PCR, and then use them as templates for fusion PCR to obtain the integrated fragment of the P trc -glk gene. Anneal the DNA fragments obtained from primers gRNA-galR-1 and gRNA-galR-2 and ligate them to plasmid pGRB to construct plasmid pGRB-galR. Co-electroporate plasmid pGRB-galR and the integrated fragment of the P trc -glk gene into the electrocompetent cells of H5 containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, obtain the E. coli W3110H6 (E. coli W3110 HOM10ΔadhEΔthrB ptsG::P trc -glf A165L / W383S galR::P trc -glk) strain.

[0164] 2. Construction of the genetically engineered bacterium E. coli W3110 H7

[0165] Integrate the P trc -galP gene into the ldhA locus.

[0166] Using the E. coli W3110 genome as a template, design upstream homologous arm primers (ldhA-1, ldhA-galP-2) and downstream homologous arm primers (ldhA-galP-5, ldhA-4) according to the sequence of the ldhA gene (NCBI accession number: NP_415898.1). Then, design primers ldhA-galP-3 and ldhA-galP-4 according to the sequence of the galP gene (NCBI accession number: NP_417418.1). Among them, the Ptrc The promoter sequence was designed on primers ldhA-galP-2 and ldhA-galP-3. Each fragment was obtained by PCR amplification. Then, using them as templates, fusion PCR was performed to obtain the integrated fragment of the P trc -galP gene. The DNA fragment obtained by annealing primers gRNA-ldhA-1 and gRNA-ldhA-2 was ligated with plasmid pGRB to construct plasmid pGRB-ldhA. Plasmid pGRB-ldhA and the integrated fragment of the P trc -galP gene were simultaneously electrotransformed into the electrocompetent cells of H6 containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, E. coli W3110 H7 (E. coli W3110 HOM10ΔadhEΔthrBptsG::P trc -glf A165L / W383S galR::P trc -glk ldhA::P trc -galP) strain was obtained.

[0167] 3. Construction of genetically engineered bacterium E. coli W3110 H8

[0168] Integrate the P trc -mglABC gene into the pflB locus (NCBI accession number: NP_415423.1).

[0169] Using the E. coli W3110 genome as a template, upstream homologous arm primers (pflB-1, pflB-mglABC-2) and downstream homologous arm primers (pflB-mglABC-5, pflB-4) were designed according to the mglABC gene (NCBI accession numbers: NP_416654.1, NP_416655.1, and NP_416653.1) sequence. Then, primers pflB-mglABC-3 and pflB-mglABC-4 were designed according to the mglABC gene sequence. Among them, the P trc promoter sequence was designed on primers pflB-mglABC-2 and pflB-mglABC-3. Each fragment was obtained by PCR amplification. Then, using them as templates, fusion PCR was performed to obtain the integrated fragment of the P trc -mglABC gene. The DNA fragment obtained by annealing primers gRNA-pflB-1 and gRNA-pflB-2 was ligated with plasmid pGRB to construct plasmid pGRB-pflB. Plasmid pGRB-pflB and the P trcThe integrated fragment of the -mglABC gene was simultaneously electrotransformed into the electrocompetent cells of H7 containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, the E. coli W3110 H8 (E. coli W3110HOM10ΔadhEΔthrB ptsG::P trc -glf A165L / W383S galR::P trc -glk ldhA::P trc -galP pflB::P trc -mglABC) strain was obtained.

[0170] 4. Construction of the genetically engineered bacterium E. coli W3110 H9

[0171] The P trc -pgk gene was integrated into the flhC locus (the NCBI number of flhC is: NP_416405.1, and the NCBI number of the pgk gene is: NP_417401.1)

[0172] Using the E. coli W3110 genome as a template, upstream homologous arm primers (flhC-1, flhC-2) and downstream homologous arm primers (flhC-5, flhC-6) were designed according to the flhC gene sequence. Then, primers flhC-3 and flhC-4 were designed according to the pgk gene sequence. Among them, the P trc promoter sequence was designed on primers flhC-2 and flhC-3. Each fragment was obtained by PCR amplification, and then fusion PCR was carried out using them as templates to obtain the integrated fragment of the P trc -pgk gene. The DNA fragment obtained by annealing primers gRNA-flhC-1 and gRNA-flhC-2 was ligated with plasmid pGRB to construct plasmid pGRB-flhC. Plasmid pGRB-flhC and the integrated fragment of the P trc -pgk gene were simultaneously electrotransformed into the electrocompetent cells of H8 containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, the E. coli W3110 H9 (E. coli W3110 HOM10ΔadhEΔthrBptsG::P trc -glf A165L / W383S galR::P trc -glk ldhA::P trc -galP pflB::P trc -mglABC flhC::P trc -pgk) strain was obtained.

[0173] 5. Construction of the genetically engineered bacterium E. coli W3110 H10

[0174] Integrate the P trc -acs gene into the poxB locus (NCBI accession number: NP_415392.1)

[0175] Using the E. coli W3110 genome as a template, upstream homologous arm primers (poxB-1, poxB-2) and downstream homologous arm primers (poxB-5, poxB-6) were designed according to the poxB gene sequence. Then, primers poxB-3 and poxB-4 were designed according to the acs gene (NCBI accession number: NP_418493.1) sequence. Among them, the P trc promoter sequence was designed on primers poxB-2 and poxB-3. Each fragment was obtained by PCR amplification, and then fusion PCR was performed using them as templates to obtain the P trc -acs gene integration fragment. The DNA fragment obtained by annealing primers gRNA-poxB-1 and gRNA-poxB-2 was ligated to plasmid pGRB to construct plasmid pGRB-poxB. Plasmid pGRB-poxB and the P trc -acs gene integration fragment were simultaneously electrotransformed into electrocompetent cells of H9 containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, E. coli W3110 H10 (E. coli W3110 HOM10ΔadhEΔthrB ptsG::P trc -glf A165L / W383S galR::P trc -glk ldhA::P trc -galP pflB::P trc -mglABC flhC::P trc -pgk poxB::P trc -acs) strain was obtained.

[0176] 6. Construction of genetically engineered bacterium E. coli W3110 H11

[0177] Integrate the pyc mutant gene from Corynebacterium glutamicum (SEQ ID NO.5) into the ynck locus (NCBI accession number: ABE-0285110).

[0178] Using the E. coli W3110 genome as a template, upstream homologous arm primers (ynck-1, ynck-2) and downstream homologous arm primers (ynck-5, ynck-6) were designed according to the ynck gene sequence. Then, using the plasmid as a template, primers ynck-3 and ynck-4 were designed according to the pyc mutant gene sequence. Among them, the Ptrc The promoter sequence was designed on primers ynck-2 and ynck-3. Each fragment was obtained by PCR amplification, and then fusion PCR was performed using them as templates to obtain the integrated fragment of the gene. The DNA fragment obtained by annealing primers gRNA-ynck-1 and gRNA-ynck-2 was ligated to plasmid pGRB to construct plasmid pGRB-ynck. Plasmid pGRB-ynck and the integrated fragment of the gene were simultaneously electrotransformed into electrocompetent cells of H10 containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, E. coli W3110 H11 strain was obtained.

[0179] 7. Construction of genetically engineered bacterium E. coli W3110 H12

[0180] Integrate P trc -ppc into the yjiT gene locus (NCBI number: YP_009518834.1);

[0181] Using the E. coli W3110 genome as a template, upstream homologous arm primers (yjiT-1, yjiT-2) and downstream homologous arm primers (yjiT-5, yjiT-6) were designed according to the yjiT gene sequence, and primers yjiT-3 and yjiT-4 were designed according to the ppc gene sequence (NCBI number: NP_418391.1). Among them, the P trc promoter sequence was designed on primers yjiT-2 and yjiT-3. Each fragment was obtained by PCR amplification, and then fusion PCR was performed using them as templates to obtain P trc -ppc gene integrated fragment. The DNA fragment obtained by annealing primers gRNA-yjiT-1 and gRNA-yjiT-2 was ligated to plasmid pGRB to construct plasmid pGRB-yjiT. Plasmid pGRB-yjiT and P trc -ppc gene integrated fragment were simultaneously electrotransformed into electrocompetent cells of H11 containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, E. coli W3110 H12 strain was obtained.

[0182] 8. Construction of genetically engineered bacterium E. coli W3110 H13

[0183] Replace the original promoter P gltA of the gltA gene (NP_415248.1) with P dps ; P gltAThe sequence (SEQ ID NO.13) is as follows:

[0184] aggtcacgataacaacatttatttaatttttaatcatctaatttgacaatcattcaacaaagttgttacaaacattaccaggaaaagcatataatgcgtaaaagttatgaagtcggtatttcacctaagattaacttatgtaacagtgtggaagtattgaccaattcattcgggacagttattagtggtagacaagtttaataattcggattgctaagtacttgattcgccatttattcgtcatcaatggatcctttacctgcaagcgcccagagctctgtacccaggttttcccctctttcacagagcggcgagccaaataaaaaacgggtaaagccaggttgatgtgcgaaggcaaatttaagttccggcagtcttacgcaataaggcgctaaggagaccttaa

[0185] Using the E. coli W3110 genome as a template, upstream homologous arm primers (P gltA -1, P gltA -2) and downstream homologous arm primers (P gltA -3, P gltA -4) were designed according to the upstream and downstream sequences of the promoter region of its gltA gene. The P dps sequence (SEQ ID NO.2) was designed on primers P gltA -2 and P gltA -3, and the upstream and downstream homologous arm fragments were amplified by PCR. Then, using them as templates, fusion PCR was performed to obtain the replacement fragment of the P gltA gene. The DNA fragment obtained by annealing primers gRNA-P gltA -1 and gRNA-P gltA -2 was ligated to plasmid pGRB to construct plasmid pGRB-P gltA . Plasmid pGRB-P gltA and the replacement fragment of the P gltA gene were simultaneously electrotransformed into electrocompetent cells of E. coli W3110 H12 containing pREDCas9 to obtain positive transformants, and after eliminating the plasmid, E. coli W3110 H13 was obtained A strain, named E. coli W3110H13 (or E. coli W3110H13-A165L / W383S).

[0186] According to the above method, the difference is that the glucose transporter is adjusted to the wild-type glucose transporter, and the prepared strain is named: E. coli W3110H13-WT.

[0187] Example 4: Shake flask fermentation of L-homoserine using genetically engineered bacteria

[0188] (1) Respectively take the preserved strains of E. coli W3110 H14-WT and E. coli W3110 H13-A165L / W383S at -80 °C and inoculate them on the slant medium, culture at 37 °C for 12 h and subculture once. Then, scrape a loop of slant seeds with an inoculation loop and inoculate them into a 500 mL round-bottomed Erlenmeyer flask containing 30 mL of seed medium, seal it with nine layers of gauze, and culture at 37 °C and 220 rmp for 8 - 10 h; the seed liquid is prepared. The composition of the seed medium is: 30 g / L glucose, 10 g / L yeast powder, 4 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 5 mg / L FeSO4·7H2O, 0.5 - 2 g / L L-threonine, 0.5 mg / L V H and 0.5 mg / L V B1 , and the rest is water, pH 7.0 - 7.2.

[0189] (2) Inoculate the seed liquid into a 500 mL baffled Erlenmeyer flask containing 30 mL of fermentation medium at an inoculation amount of 15% (v / v), seal it with nine layers of gauze, culture at 37 °C and 240 r / min. During the fermentation process, use phenol red as an indicator, control the pH at 7.0 - 7.2 by adding 25% ammonia water. When the glucose in the medium is exhausted, add a 60% (m / v) glucose solution to a final concentration of 5 g / L to maintain the fermentation; the fermentation cycle is 36 h; the fermentation broth is prepared. The composition of the fermentation medium is: 10 g / L glucose, 5 g / L yeast powder, 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 30 mg / L FeSO4·7H2O, 0.5 - 2 g / L L-threonine, 0.5 mg / L V H , 0.5 mg / L V B1 and 8 mg / L phenol red, and the rest is water, pH 7.0 - 7.2.

[0190] The results showed that in the shake flask stage, the yield of E. coli W3110 H13-A165L / W383S was 12.1% higher than that of E. coli W3110 H14-WT, reaching 59.1 g / L.

[0191] In addition, as a common metabolic by-product, the content of acetic acid will affect the metabolic growth of bacteria. 1.5 g / L, 2 g / L and 1.2 g / L of acetic acid were detected in the fermentation broths of H1, H2 and H3 strains respectively. After knocking out the relevant genes, no acetic acid accumulation was detected in the fermentation broth of H13 strain, proving that the relevant operation successfully blocked the synthesis pathway of acetic acid and effectively promoted the synthesis of L-homoserine.

[0192] Example 5: Fermentation production of L-homoserine by genetically engineered strain H13 in a 5 L fermenter

[0193] Prepare L-homoserine using E. coli W3110 H13-A165L / W383S and E. coli W3110 H13-WT respectively

[0194] (1) Seed culture: Pour an appropriate amount of sterile water into the slant, suspend the bacteria with an inoculation loop, and then inoculate the bacterial suspension into the seed medium for culture. The culture temperature is 37 °C, the initial aeration rate is 2 L / min, the initial stirring speed is 200 r / min, and the pH of the medium is controlled at 7.0 - 7.2 by automatically adding 25% ammonia water. The dissolved oxygen is controlled at 25 - 30% by stirring and ventilation. When OD 600 reaches 15 - 20, it is ready to be inoculated into the fermentation medium. The composition of the seed medium is: 30 g / L glucose, 5 g / L yeast powder, 3 g / L peptone, 1.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 1 g / L sodium citrate, 1 g / L L-threonine, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·H2O, 1 mg / L V H and 0.5 mg / L V B1 , and the rest is water, pH 7.0 - 7.2.

[0195] (2) Fermentation culture: Inoculate the seed liquid into 2 L of fermentation medium at an inoculation amount of 15% (v / v). The culture temperature is 37 °C. Control the pH of the medium at 7.0 - 7.2 by automatically adding 25% ammonia water. Control the dissolved oxygen at 25 - 30% by stirring and ventilation. When the glucose in the medium is exhausted, automatically add 80% glucose solution to control the residual glucose concentration in the fermentation broth at 1 - 10 g / L. The composition of the fermentation medium is: 10 g / L glucose, 10 g / L corn steep liquor, 4 g / L yeast powder, 3 g / L peptone, 4 g / L KH₂PO₄, 1 g / L MgSO₄·7H₂O, 2 g / L sodium citrate, 1 g / L L-threonine, 10 mg / L FeSO₄·7H₂O, 10 mg / L MnSO₄·H₂O, 0.2 mg / L V H and 0.3 mg / L V B1 , and the rest is water, pH 7.0 - 7.2.

[0196] Ferment in a 5 L fermenter for 58 h. The yield of E. coli W3110 H13-A165L / W383S is 13.2% higher than that of E. coli W3110 H13-WT, reaching 119.3 g / L. No other amino acid and organic acid by-products are detected, nor is acetic acid accumulation detected.

[0197] Example 6: Optimization of the conditions for fermenting and producing L-homoserine by genetically engineered bacterium H13-A165L / W383S in a 5 L fermenter

[0198] (1) Seed culture: Pour an appropriate amount of sterile water into the slant, suspend the bacteria with an inoculation loop, and then inoculate the bacterial suspension into the seed medium for culture. The culture temperature is 37 °C, the initial ventilation volume is 2 L / min, the initial stirring speed is 200 r / min. Control the pH of the medium at 7.0 - 7.2 by automatically adding 25% ammonia water. Control the dissolved oxygen at 25 - 30% by stirring and ventilation. When OD 600 reaches 15 - 20, prepare to inoculate into the fermentation medium. The composition of the seed medium is: 35 g / L glucose, 5 g / L yeast powder, 3 g / L peptone, 1.5 g / L KH₂PO₄, 0.5 g / L MgSO₄·7H₂O, 1 g / L citric acid, 1 g / L L-threonine, 10 mg / L FeSO₄·7H₂O, 10 mg / L MnSO₄·H₂O, 1 mg / L V H and 0.5 mg / L V B1 , and the rest is water, pH 7.0 - 7.2.

[0199] (2) Fermentation culture: Inoculate the seed liquid into 2 L of fermentation medium at an inoculation amount of 20% (v / v). The culture temperature is 37 °C. Control the pH of the medium at 7.0 - 7.2 by automatically adding 25% ammonia water. Control the dissolved oxygen at 25 - 30% by stirring and ventilation. When the glucose in the medium is exhausted, automatically add an 80% glucose solution and 1 g / L betaine, and control the residual glucose concentration in the fermentation broth at 1 - 10 g / L. Feed the feeding medium at a flow rate of 14 mL / h between 14 - 20 h of fermentation.

[0200] The fermentation medium composition is as follows: 10 g / L glucose, 12 g / L corn steep liquor, 4 g / L yeast powder, 3 g / L peptone, 4 g / L KH2PO4, 1 g / L MgSO4·7H2O, 2 g / L citric acid, 1 g / L L-threonine, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·H2O, 0.2 mg / L V H and 0.3 mg / L V B1 ; the rest is water, and the pH is 7.0 - 7.2. The feeding medium composition is as follows: 4 g / L yeast powder, 3 g / L peptone, 4 g / L KH2PO4, 1 g / L MgSO4·7H2O, 2 g / L citric acid, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·H2O, 0.5 mg / L V H and 0.5 mg / L V B1 .

[0201] Ferment in a 5 L fermenter for 56 h. The fermentation process curve is shown in Figure 2 , where the highest yield of L-homoserine reaches 141.5 g / L, and the highest sugar-acid conversion rate reaches 40%. No other amino acid and organic acid by-products are detected, nor is the accumulation of acetic acid detected.

[0202] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A glucose transporter mutant, characterized in that, The glucose transporter mutant is based on the amino acid sequence of the parental glucose transporter shown in SEQ ID NO. 11, where alanine at position 165 is mutated to leucine, or valine at position 162 is mutated to serine, or tryptophan at position 383 is mutated to serine, or alanine at position 165 is mutated to leucine while valine at position 162 is mutated to serine, or alanine at position 165 is mutated to leucine while tryptophan at position 383 is mutated to serine.

2. A polynucleotide encoding the glucose transporter mutant according to claim 1, or a vector carrying the polynucleotide, or a cell expressing the glucose transporter mutant according to claim 1.

3. A polynucleotide synthesis kit, characterized in that, Comprising the glucose transporter mutant according to claim 1.

4. A method for improving the glucose transport ability of glucose transporters, characterized in that, The method is that, based on the amino acid sequence of the parental glucose transporter shown in SEQ ID NO. 11, alanine at position 165 is mutated to leucine, or valine at position 162 is mutated to serine, or tryptophan at position 383 is mutated to serine, or alanine at position 165 is mutated to leucine while valine at position 162 is mutated to serine, or alanine at position 165 is mutated to leucine while tryptophan at position 383 is mutated to serine.

5. A method for improving the glucose consumption rate and sugar-acid conversion rate of a recombinant strain, characterized in that, The method is that the recombinant strain expresses the glucose transporter mutant according to claim 1, or carries the polynucleotide or vector according to claim 2.

6. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli expresses the mutant according to claim 1; Preferably, the recombinant Escherichia coli is an Escherichia coli as the starting strain, in which the lactate dehydrogenase-encoding gene ldhA, pyruvate formate-lyase-encoding gene pflB, pyruvate oxidase-encoding gene poxB, alcohol dehydrogenase-encoding gene adhE, homoserine kinase-encoding gene thrB, and flagellar synthesis regulatory factor-encoding gene flhC are knocked out; the glucose transporter mutant described in claim 1, glucose kinase-encoding gene glk, galactose permease-encoding gene galP, methylgalactoside transport system-encoding gene mglABC, phosphoglycerate kinase-encoding gene pgk, phosphoenolpyruvate carboxylase mutant-encoding gene and phosphoenolpyruvate carboxylase-encoding gene ppc, acetyl-CoA synthetase-encoding gene acs are overexpressed; the citrate synthase-encoding gene gltA is weakly expressed; Preferably, the recombinant Escherichia coli is based on Escherichia coli as the starting strain, knocking out the lactate dehydrogenase-encoding gene ldhA, pyruvate formate lyase-encoding gene pflB, pyruvate oxidase-encoding gene poxB, alcohol dehydrogenase-encoding gene adhE, homoserine kinase-encoding gene thrB, and flagellar synthesis regulatory factor-encoding gene flhC; overexpressing the glucose transporter mutant glf A165L / W383S , glucose kinase-encoding gene glk, galactose permease-encoding gene galP, methylgalactoside transport system-encoding genes mglABC, phosphoglycerate kinase-encoding gene pgk, phosphoenolpyruvate carboxylase mutant-encoding gene and phosphoenolpyruvate carboxylase-encoding gene ppc, acetyl-CoA synthetase-encoding gene acs; weakly expressing the citrate synthase-encoding gene gltA.

7. The recombinant Escherichia coli according to claim 6, characterized in that, Integrate the gene galP into the ldhA locus on the Escherichia coli genome, integrate the gene mglABC into the pflB locus on the Escherichia coli genome, and the mutant is glf A165L / W383S , integrate the mutant glf A165L / W383S gene into the ptsG locus on the Escherichia coli genome, integrate the gene glk into the galR locus on the Escherichia coli genome, integrate the gene pgk into the flhC locus on the Escherichia coli genome, and integrate the gene into the ynck locus on the Escherichia coli genome, integrate the gene acs into the poxB locus on the Escherichia coli genome, and integrate the gene ppc into the yjiT locus on the Escherichia coli genome; Preferably, the overexpression is achieved by promoter P trc regulating the expression of galP, mglABC, and glf A165L / W383S , glk, pgk, acs, and ppc; the weak expression is achieved by promoter P dps regulating the expression of gltA; Preferably, the promoter P trc has the nucleotide sequence shown in SEQ ID NO.1, and the promoter P dps has the nucleotide sequence shown in SEQ ID NO.2; the mutant is A165L / W383S, and the nucleotide sequence of the mutant gene glf A165L / W383S is shown in SEQ ID NO.3, the nucleotide sequence of the gene glk is shown in SEQ ID NO.4, the nucleotide sequence of the gene is shown in SEQ ID NO.5, the nucleotide sequence of the gene ppc is shown in SEQ ID NO.6, the nucleotide sequence of the gene pgk is shown in SEQ ID NO.7, the nucleotide sequence of the gene acs is shown in SEQ ID NO.8, the nucleotide sequence of the gene galP is shown in SEQ ID NO.9, and the nucleotide sequence of the gene mglABC is shown in SEQ ID NO.10; Preferably, the starting strain of the recombinant Escherichia coli is Escherichia coli HOM10.

8. A method for preparing L-homoserine, characterized in that, L-homoserine is prepared by fermentation using the recombinant Escherichia coli according to claim 6 or 7; Preferably, the temperature condition for the fermentation is: the reaction is carried out at 30 - 40 °C; or at 35 - 37 °C; or at 30 - 31 °C; or at 32 - 33 °C; or at 34 - 35 °C; or at 36 - 37 °C; or at 38 - 39 °C; Preferably, the pH condition for the fermentation is: the reaction is carried out at a pH of 6.8 - 7.5; or at a pH of 6.8 - 7.0; or at a pH of 7.2 - 7.3; or at a pH of 7.0 - 7.2; Preferably, the fermentation time is at least 24 h.

9. Use of the glucose transporter mutant according to claim 1 in improving the glucose utilization efficiency of a strain.

10. Use of the recombinant Escherichia coli according to claims 6 - 7 or the method according to claim 8 in the preparation of L-homoserine or a product containing L-homoserine.

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