Genetically engineered bacteria for producing homoserine and construction method and application thereof

Abstract

The present invention discloses a genetically engineered bacterium for producing homoserine and a construction method and application thereof. The method comprises enhancing the precursor and reducing power supply in the genetically engineered bacterium. pyc、ppc、 Ec thrA、tkt Gene, knockout or weakening of branching pathways gltA、 dapA、avtA、alaT、pqo , ddh , thrB Gene; Modification of transporter system lysE、 Ec rthA Gene; Introduce heterologous feedback-resistant aspartate kinase gene Sc lysC and homoserine dehydrogenase Bd home Gene; Introduction of heterologous NADPH production enzymes; Enhancement of osmotic pressure tolerance in genetically engineered bacteria cglK、 betP、grpE The present invention discloses a method for constructing a genetically engineered bacterium with improved L-homoserine production by utilizing the method, and a method for producing L-homoserine and other downstream products by utilizing the genetically engineered bacterium.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and compound production, and specifically to a genetically engineered bacterium for efficiently producing L-homoserine and a construction method and application thereof. Background Art

[0002] L-homoserine is a valuable non-protein amino acid that is widely used in food, feed, medicine, agriculture and other fields. It plays a key role in the synthesis of essential amino acids such as L-methionine and L-threonine. In recent years, the application prospects of L-homoserine have become increasingly broad. It is not only the basic raw material for platform compounds such as L-homoserine lactone and γ-butyrolactone, but also has potential immunomodulatory properties, which contributes to the healthy growth of animals and plants. In addition, L-homoserine has the same chemical structure as the non-selective herbicide glufosinate ammonium, and has important application value in the synthesis of optically pure L-glufosinate ammonium. Therefore, the development of green biomanufacturing of L-homoserine not only has important theoretical significance in the field of biosynthesis, but also provides a new path for sustainable development in modern agriculture and medicine.

[0003] Since the traditional chemical synthesis of L-homoserine is complex and costly, it is difficult to achieve large-scale commercialization. In recent years, microbial fermentation has become an alternative method that has attracted much attention. In particular, Escherichia coli is an effective host for the production of L-homoserine. Through genetic modification, the yield and production efficiency have been significantly improved. For example, researchers optimized the metabolic engineering of Escherichia coli to enable it to accumulate more than 100 g / L of L-homoserine during the fermentation process (Chen Y, et al. Balancing the AspC and AspA Pathways of Escherichia coli by SystematicMetabolic Engineering Strategy for High-Efficient l-Homoserine Production.ACS Synth Biol, 2024, 13:2457-2469; Liu Z, et al. High-efficient productionof L-homoserine in Escherichia coli through engineering synthetic pathwaycombined with regulating cell division. Bioresour Technol, 2023, 389: 129828;VO TM, et al. Metabolic engineering of Escherichia coliW3110 for efficient production of homoserine from glucose. MetabEng, 2022, 73: 104-113). Nevertheless, current research is still continuing to improve the fermentation characteristics of Escherichia coli and strive to achieve higher production efficiency in order to promote the biomanufacturing of L-homoserine towards industrialization.

[0004] In the field of large-scale fermentation of amino acids, Corynebacterium glutamicum has a long history of application and potential. Although significant progress has been made in the production of L-homoserine in Escherichia coli, relatively few studies have been conducted on Corynebacterium glutamicum, and its production is still lower than that of Escherichia coli. The latest research has achieved effective accumulation of L-homoserine in Corynebacterium glutamicum through genome editing and metabolic flux redistribution, with a maximum production of 63.5 g / L (Li N, et al. Dual-channel glycolysis balances cofactor supply for l-homoserine biosynthesis in Corynebacterium glutamicum . Bioresour Technol, 2023, 369: 128473). Although some progress has been made in the fermentation production of L-homoserine in Corynebacterium glutamicum, its overall titer is still far behind the level achieved in Escherichia coli. Given the strong potential of Corynebacterium glutamicum for efficient amino acid fermentation, further modification and optimization of its production performance in L-homoserine production has broad research prospects.

[0005] L-homoserine is a non-essential chiral amino acid, an important precursor for the biosynthesis of aspartate-derived amino acids, and a key intermediate for the synthesis of other renewable platform compounds. At present, homoserine is mainly produced by chemical synthesis, which has problems such as complex process, high technical barriers, low safety, and severe environmental pollution, which also limits the application of products in the food, medicine and other industries. In recent years, microbial fermentation has gradually become the focus of attention for the efficient production of L-homoserine as an economically feasible and environmentally friendly method, but there are also limiting factors such as the lack of strain resources and relatively backward production technology. Summary of the invention

[0006] The present invention is based on the homoserine metabolic pathway ( Figure 1) to conduct research, and then proposed that the primary technical problem to be solved is to provide a genetically engineered bacterium that can efficiently produce L-homoserine. The second technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned genetically engineered food safety bacteria that produces L-homoserine. The third technical problem to be solved by the present invention is to provide the application of the above-mentioned genetically engineered bacteria, and its fermentation method for producing L-homoserine.

[0007] In order to solve the above technical problems, the present invention first provides a genetically engineered bacterium for producing homoserine, which is in the genus Corynebacterium ( Corynebacterium ) or Escherichia coli ( Escherichia ) in bacteria thrB Gene, overexpression of aspartate kinase gene with feedback resistance lysC and homoserine dehydrogenase hom Gene.

[0008] Further modification of one or more of the following genes: overexpression pyc Gene, ppc Gene, thrA Gene, tkt Gene, cglK Gene , betP Gene , grpE Gene; Overexpression lysE Gene; Introduce heterologous NADPH production enzyme gene, aspartate kinase gene Sc lysC Homoserine dehydrogenase Bd hom Gene; weaken or knock out gltA, dapA, avtA, alaT, pqo , ddh , thrB Gene; Enhanced transporter system lysE, Ec rthA Gene.

[0009] Preferably, the genetic modification includes: enhancing the precursor and reducing power supply in the genetically engineered bacteria pyc, ppc, Ec thrA, tkt Gene, knockout or weakening of branching pathways gltA, dapA, avtA, alaT, pqo , ddh , thrB Gene; Enhanced transporter system lysE, Ec rthA Gene; Introduce heterologous feedback-resistant aspartate kinase gene Sc lysC and homoserine dehydrogenase Bd hom Gene; Introducing heterologous NADPH production enzyme Sp gapN or Ca gapN Gene; Enhancement of osmotic stress tolerance in genetically engineered bacteria cglK Gene.

[0010] Specifically, the EcthrA The GenBank accession number of the gene is NP_414543.1 or NP_415334.1 Sc lysC The gene is derived from Saccharomyces cerevisiae ( Saccharomyces cerevisiae ); the feedback resistance Bd hom The gene is derived from Brachypodium distachyon ( Brachypodium distachyon ); the coding gene Ca gapN From Clostridium acetobutylicum ( Clostridium acetobutylicum ); said Sp gapN The gene is derived from Streptococcus pyogenes serotype M1 ( Streptococcus pyogenes serotype M1 ).

[0011] Preferably, the genetic modification comprises: enhancing the functions of the genetically engineered bacteria involved in the supply of precursors and reducing power. pyc, ppc, Ec thrA, tkt Gene, knockout or weakening of branching pathways gltA, dapA, avtA, alaT, pqo , ddh , thrB Gene; Enhanced transporter system lysE, Ec rthA Gene; Introduce heterologous feedback-resistant aspartate kinase gene Sc lysC and homoserine dehydrogenase Bd hom Gene; Introducing heterologous NADPH production enzyme Sp gapN ; Enhancement of osmotic pressure tolerance in genetically engineered bacteria cglK .

[0012] Based on the teachings of the present invention and the common knowledge in the art, those skilled in the art know and can use genes from various sources to construct the above-mentioned genetically engineered bacteria. In a specific embodiment, the coding gene pyc [GenBank:CAF19394.1] , ppc [GenBank: CAF21593.1], tkt [GenBank: CAF21582.1], gltA [GenBank: CAF19535.1], dapA [GenBank: CAF20312.1], avtA [GenBank: CAF21261.1], alaT [GenBank: CAF20870.1], lysE [GenBank: CAF19965.1], pqo [GenBank: CAF21272.1], ddh [GenBank:CAF21279.1], thrB [GenBank:CAF19888.1], cglK [GenBank: BAB98170.1], betP [GenBank: CAF19598.1], grpE [GenBank: CAF20820.1], Derived from Corynebacterium glutamicum ( Corynebacterium glutamicum ) ATCC 13032; the feedback inhibition mutant gene Ec thrA [GenBank:NP_414543.1] and Ec rhtA [GenBank:NP_415334.1] From Escherichia coli ( Escherichia coli ) MG1655; the feedback inhibition mutant gene Sc lysC Derived from Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) S288c; the feedback inhibition mutant gene Bd hom From Brachypodium distachyon ( Brachypodiumdistachyon ); the coding gene Ca gapN From Clostridium acetobutylicum ( Clostridium acetobutylicum ) ATCC 824; the encoding gene Sp gapN From Streptococcus pyogenes serotype M1 ( Streptococcus pyogenes serotype M1 ).

[0013] In a specific embodiment, the encoding gene Ec thrA The sequence is shown in SEQ ID NO: 1, and the coding gene Sc lysC The sequence is shown in SEQ ID NO: 2, and the coding gene Bd hom The sequence is shown in SEQ ID NO: 3, and the coding gene Ca gapN The sequence is shown in SEQ ID NO:4; the coding gene Sp gapN The sequence is shown in SEQ ID NO: 5, and the other coding genes are all derived from Corynebacterium glutamicum ATCC 13032, and their nucleic acid and protein sequences can be obtained from public databases through GenBank numbers.

[0014] Specifically, attenuation refers to reducing the expression or activity of the gene in the genetically engineered bacteria, which can be achieved by adding inhibitors to the culture medium of the genetically engineered bacteria or modifying the gene in the genetically engineered bacteria to reduce its expression or activity;

[0015] Enhance the expression or activity of a gene by expressing the endogenous or heterologous gene using a strong promoter, and / or increasing the copy number of the gene, and / or modifying the promoter of the gene to enhance the transcription initiation rate, and / or modifying the translation regulatory region or rare codons of the messenger RNA carrying the gene to enhance the translation intensity, and / or modifying the gene itself to enhance mRNA stability and protein stability;

[0016] The introduction of heterologous genes can be achieved by introducing them into expression vector plasmids or by integrating heterologous genes into the genome.

[0017] Further, in a preferred embodiment, the "enhancement" refers to increasing the expression or activity of endogenous genes existing in the genetically engineered bacteria itself; or exogenously introducing genes that do not exist in the genetically engineered bacteria themselves, and allowing the exogenously introduced genes to express or function in the genetically engineered bacteria. In a preferred embodiment, the "weakening" or "attenuation" refers to reducing the expression or activity of one or more genes in the genetically engineered bacteria. In a preferred embodiment, the "weakening" or "attenuation" means that the expression or activity of the weakened gene is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% of the expression or activity of the non-weakened gene; preferably, the gene is completely inactivated. In a preferred embodiment, the "weakened gene expression or activity" can be achieved by, for example, adding an inhibitor to the culture medium of the genetically engineered bacteria or knocking out the gene in the genetically engineered bacteria.

[0018] In a preferred embodiment, the expression or activity of a gene can be enhanced by using a strong promoter to express the endogenous or heterologous gene, and / or increasing the copy number of the gene, and / or modifying the promoter of the gene to enhance the transcription initiation speed, and / or modifying the translation regulatory region or rare codons of the messenger RNA carrying the gene to enhance the translation intensity, and / or modifying the gene itself to enhance the mRNA stability and protein stability. As the most preferred solution, the strong endogenous promoter Ptuf is used to enhance the mRNA stability and protein stability. 、 Psod or artificially constructed strong promoter can achieve enhanced expression of target gene.

[0019] In a specific embodiment, the strong promoter Ptuf sequence is shown as SEQ ID NO:6, the strong promoter Psod sequence is shown as SEQ ID NO:7, and the strong promoter PH7 sequence is shown as SEQ ID NO:8.

[0020] Preferably, the starting bacteria is Corynebacterium glutamicum ( Corynebacterium glutamicum ) or Escherichia coli, more preferably Corynebacterium glutamicum ATCC 13032. The improvement of L-homoserine production by genetic modification of the present invention can refer to the original strain not producing L-homoserine, but having the ability to produce L-homoserine after being constructed by the method of the present invention; or it can refer to the original strain being able to produce L-homoserine, but having the ability to produce L-homoserine after being constructed by the method of the present invention. Therefore, the construction method of the present invention can construct an L-homoserine-producing strain from scratch, or can transform an existing L-homoserine-producing strain.

[0021] The present invention also provides a method for constructing the genetically engineered bacteria, which is obtained by knocking out or weakening the relevant genes in the starting bacteria through a gene editing method, or enhancing the expression of the relevant genes; and by introducing heterologous genes through a gene transformation method.

[0022] The invention also provides application of the genetically engineered bacteria in producing homoserine.

[0023] The present invention also provides a method for producing homoserine, which comprises the steps of culturing the genetically engineered bacteria and collecting the produced homoserine.

[0024] Specifically, in the fermentation medium, at 30-35°C, the dissolved oxygen is controlled at 15-25%, the stirring speed is coupled with the dissolved oxygen to ensure the stability of the dissolved oxygen, the pH is maintained at 6.5-7.5 by adding ammonia water, the fermentation is maintained by adding glucose solution, the sugar concentration is maintained at 5-10g / L, and the fermentation cycle is 60-72h.

[0025] The beneficial effect of the present invention is that a genetically engineered bacterium for efficient production of L-homoserine and its application are provided, and efficient fermentation production of L-homoserine is achieved. In the best embodiment, the genetically engineered bacteria of the present invention are fermented for 68 hours, and the L-homoserine content can reach 136.5 ± 4.2 g / L. The genetically engineered bacteria provided by the present invention are used to produce L-homoserine, which has many advantages such as high yield, non-toxicity, mild conditions, and less environmental pollution compared to traditional chemical synthesis methods, and has broad application prospects in the fields of food, medicine, agriculture, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of L-homoserine biosynthesis metabolism. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers, and the reagents and biological materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0028] Example 1. Construction of knockout / integration plasmid for metabolic modification of L-homoserine strains

[0029] In some specific embodiments of this example, the vector backbone used is pK18mobsacB shuttle plasmid, which is used to insert or delete the target gene at a specific site on the Corynebacterium glutamicum genome. All primers used are synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. Each component biological element is mainly obtained by purification after PCR amplification, and each fragment is spliced ​​with the plasmid backbone in a certain order by molecular assembly technology such as ClonExpress®II One Step Cloning Kit, Vazyme Biotech, China, to complete the construction of the recombinant plasmid required for metabolic transformation of L-homoserine strains in Corynebacterium glutamicum.

[0030] (1) Pyruvate carboxylase encoding gene pyc Enhanced expression

[0031] Pyruvate carboxylase (Pyc) is a key metabolic enzyme that catalyzes the reaction of pyruvate with carbon dioxide to produce oxaloacetate, and participates in carbon metabolism and energy balance. In bacteria such as Corynebacterium glutamicum, Pyc helps cells adapt to different environmental conditions by regulating the TCA cycle, amino acid synthesis, and carbon source distribution. Pyc expression and activity directly affect amino acid production and are key regulatory factors for improving product production during industrial fermentation.

[0032] In the specific implementation of this embodiment, the strong promoter Psod is preferably used to realize the pyruvate carboxylase gene pyc In situ enhanced expression, that is, on the chromosome pyc A strong promoter is inserted before the start codon of the gene. In order to achieve the purpose of this technology, it is necessary to construct a knock-in plasmid PK-Psod-pyc. The specific experimental steps are as follows: ① Design primers according to the sequence, use the genome of Corynebacterium glutamicum as a template, and use PCR amplification pyc The upstream homology arm of the gene; primers were designed based on the sequence, and the strong promoter Psod sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; primers were designed based on the sequence, and the genome of Corynebacterium glutamicum was used as a template to amplify the strong promoter Psod sequence by PCR pyc ② Double digest the pK18mobsacB shuttle plasmid with BamHI and XbaI to obtain a plasmid backbone with sticky ends and homologous regions at both ends; ③ Use the Novozymes one-step efficient seamless cloning kit to connect the plasmid backbone and the target fragment to obtain the knock-in plasmid PK-Psod-pyc; ④ Transform the obtained knock-in plasmid PK-Psod-pyc into Corynebacterium glutamicum, and use a two-step homologous recombination method to obtain a strain with enhanced expression of the pyruvate carboxylase gene, whose genotype is represented by P sod - pyc .

[0033] (2) Gene encoding phosphoenolpyruvate carboxylase ppc Enhanced expression

[0034] Phosphoenolpyruvate carboxylase (Ppc) is a key enzyme that is widely found in bacteria, plants, and eukaryotes. It is responsible for catalyzing the reaction of phosphoenolpyruvate (PEP) combining with carbon dioxide to produce oxaloacetate (OAA). In bacteria such as Corynebacterium glutamicum, Ppc plays an important role in carbon metabolism, especially in the fermentation process, regulating carbon source utilization and energy balance. Through the action of Ppc, bacteria can effectively fix carbon dioxide and produce important metabolic intermediates such as oxaloacetate, which is essential for the synthesis of amino acids, sugars, and other biomolecules.

[0035] In the specific implementation of this example, the strong promoter Ptuf is preferably used to realize the phosphoenolpyruvate carboxylase encoding gene ppc In situ enhanced expression, that is, on the chromosome ppc A strong promoter is inserted before the start codon of the gene. In order to achieve the purpose of this technology, it is necessary to construct a knock-in plasmid PK-Ptuf-ppc. The specific experimental steps are as follows: ① Design primers according to the sequence, use the genome of Corynebacterium glutamicum as a template, and use PCR amplification ppc The upstream homology arm of the gene; primers were designed based on the sequence, and the strong promoter Ptuf sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; primers were designed based on the sequence, and the genome of Corynebacterium glutamicum was amplified by PCR using the genome of Corynebacterium glutamicum as a template ppc ② Double digest the pK18mobsacB shuttle plasmid with BamHI and XbaI to obtain a plasmid backbone with sticky ends and homologous regions at both ends; ③ Use the Novozymes one-step efficient seamless cloning kit to connect the plasmid backbone and the target fragment to obtain the knock-in plasmid PK-Ptuf-ppc; ④ Transform the obtained knock-in plasmid PK-Ptuf-ppc into Corynebacterium glutamicum, and use a two-step homologous recombination method to obtain a strain with enhanced expression of phosphoenolpyruvate carboxylase gene, whose genotype is represented by P tuf - ppc .

[0036] (3) Transketolase gene tkt Enhanced expression

[0037] The transketolase gene tkt in bacteria such as Corynebacterium glutamicum encodes transketolase, which is one of the key enzymes in the pentose phosphate PPP pathway. PPP participates in the synthesis of nucleotides and amino acids by converting glucose into pentose sugars and their metabolic intermediates, and provides the reducing equivalent NADPH. This pathway not only plays an important role in the energy metabolism of bacteria, but is also closely related to the antioxidant capacity, metabolic regulation and stress response of cells. By regulating the tkt gene, bacteria can regulate the activity of PPP under different physiological conditions, thereby affecting their growth and the synthesis of metabolites.

[0038] In the specific implementation of this example, the strong promoter Ptuf is preferably used to realize the phosphoenolpyruvate carboxylase encoding gene tkt In situ enhanced expression, that is, on the chromosome tkt A strong promoter is inserted before the start codon of the gene. In order to achieve the purpose of this technology, it is necessary to construct a knock-in plasmid PK-Ptuf-tkt. The specific experimental steps are as follows: ① Design primers according to the sequence, use the genome of Corynebacterium glutamicum as a template, and use PCR amplification tkt The upstream homology arm of the gene; primers were designed based on the sequence, and the strong promoter Ptuf sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; primers were designed based on the sequence, and the genome of Corynebacterium glutamicum was amplified by PCR using the genome of Corynebacterium glutamicum as a template tkt ② Double digest the pK18mobsacB shuttle plasmid with BamHI and XbaI to obtain a plasmid backbone with sticky ends and homologous regions at both ends; ③ Use the Novozymes one-step efficient seamless cloning kit to connect the plasmid backbone and the target fragment to obtain the knock-in plasmid PK-Ptuf-tkt; ④ Transform the obtained knock-in plasmid PK-Ptuf-tkt into Corynebacterium glutamicum, and use a two-step homologous recombination method to obtain a transketolase gene expression enhanced strain, whose genotype is represented by P tuf - tkt .

[0039] (4) Amino acid transporter encoding genes lysE Enhanced expression

[0040] The importance of transporters to amino acid production has been convincingly demonstrated. When fermenting a certain amino acid, the amino acid molecules in the cell must be transported outside the cell in order to increase the yield of the amino acid. The efficient secretion of amino acids can effectively reduce the feedback inhibition and repression of the intracellular product concentration on important nodes in the metabolic pathway. Among the currently known outward transporters, LysE and YbjE can transport lysine, ArgO, LysE, and CgmA can transport arginine, and YeaS can transport histidine. In the production of amino acid fermentation, LysE can transport the most basic amino acids and is also the most widely used in engineering strain breeding. After extensive and in-depth research, the inventor unexpectedly found that the overexpression of LysE can improve the L-homoserine production capacity of the strain.

[0041] In the specific implementation of this example, the strong promoter Ptuf is preferably used to realize the phosphoenolpyruvate carboxylase encoding gene lysE In situ enhanced expression, that is, on the chromosome lysE A strong promoter is inserted before the start codon of the gene. In order to achieve the purpose of this technology, it is necessary to construct a knock-in plasmid PK-Ptuf-lysE. The specific experimental steps are as follows: ① Design primers according to the sequence, use the genome of Corynebacterium glutamicum as a template, and use PCR amplification lysE The upstream homology arm of the gene; primers were designed based on the sequence, and the strong promoter Ptuf sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; primers were designed based on the sequence, and the genome of Corynebacterium glutamicum was amplified by PCR using the genome of Corynebacterium glutamicum as a template lysE ② Double digest the pK18mobsacB shuttle plasmid with BamHI and XbaI to obtain a plasmid backbone with sticky ends and homologous regions at both ends; ③ Use the Novozymes one-step efficient seamless cloning kit to connect the plasmid backbone and the target fragment to obtain the knock-in plasmid PK-Ptuf-lysE; ④ Transform the obtained knock-in plasmid PK-Ptuf-lysE into Corynebacterium glutamicum, and use a two-step homologous recombination method to obtain an amino acid transporter encoding gene expression-enhanced strain, whose genotype is represented by P tuf - lysE .

[0042] (5) Alanine aminotransferase gene alaT and avtA Knockout

[0043] Alanine aminotransferase gene (alaT) and acetylalanine aminotransferase gene (avtA) are both involved in important enzyme catalysis reactions of amino acid metabolism. alaT The gene encodes alanine aminotransferase (ALT), an enzyme that plays a key role in amino acid metabolism and mainly catalyzes the transamination reaction between alanine and α-keto acid.avtA The gene encodes acetylalanine aminotransferase (AVT), which is involved in the synthesis of acetylalanine, which is an intermediate or final product in multiple metabolic pathways. Alanine aminotransferases AlaT and AvtA catalyze the production of L-alanine from pyruvate with the amino donors L-glutamate and L-valine, respectively. The pathway for producing L-alanine catalyzed by AlaT is the main pathway for producing L-alanine in cells, while the pathway for producing L-alanine catalyzed by AvtA is a secondary pathway. Both play an important role in the synthesis and metabolism of amino acids and are key genes for understanding the amino acid metabolic network of microorganisms and engineering applications.

[0044] In the specific implementation of this example, it is preferred to knock out alaT and avtA In order to achieve the purpose of this technology, it is necessary to construct knock-in plasmids PK-alaT and PK-avtA. The specific experimental steps are as follows: ① Design primers based on the sequence, use the genome of Corynebacterium glutamicum as a template, and use PCR amplification alaT The upstream homology arm of the gene; primers were designed according to the sequence, and PCR amplification was performed using the genome of Corynebacterium glutamicum as a template. alaT The downstream homology arm of the gene; primers were designed according to the sequence, and PCR amplification was performed using the genome of Corynebacterium glutamicum as a template. avtA The upstream homology arm of the gene; primers were designed according to the sequence, and PCR amplification was performed using the genome of Corynebacterium glutamicum as a template. avtA The downstream homologous arm of the gene; ② Use BamHI and XbaI to double-digest the pK18mobsacB shuttle plasmid to obtain a plasmid backbone with sticky ends and homologous regions at both ends; ③ Use the Novozymes one-step efficient seamless cloning kit to connect the plasmid backbone and the target fragment to obtain knock-in plasmids PK-alaT and PK-avtA; ④ Transform the obtained knock-in plasmid PK-alaT or PK-avtA into Corynebacterium glutamicum, and use a two-step homologous recombination method to obtain alanine aminotransferase gene knockout strains, whose genotypes are represented as follows: ∆alaT or ∆avtA .

[0045] (6) Dihydropyridine dicarboxylic acid synthase gene dapA Weakened expression

[0046] Dihydrodipicolinate synthase DapA is a key enzyme in the lysine biosynthesis pathway. The expression of dihydrodipicolinate synthase gene can be reduced by introducing site-directed mutagenesis in the promoter. dapAIn the specific implementation of this embodiment, it is preferred to use a mutant dapA promoter to replace its own natural promoter on the chromosome. In order to achieve this technical purpose, it is necessary to construct a knock-in plasmid PK-dapAmut, and the specific experimental steps used are: ① Design primers according to the sequence, use the genome of Corynebacterium glutamicum as a template, and use PCR amplification dapA The upstream homology arm of the gene; primers were designed according to the sequence, and PCR amplification was performed using the genome of Corynebacterium glutamicum as a template. dapA The downstream homology arms of the gene contain a promoter mutation site -16G→C in the middle of the two homology arms. ② The pK18mobsacB shuttle plasmid was double-digested with BamHI and XbaI to obtain a plasmid backbone with sticky ends and homology regions at both ends; ③ The plasmid backbone and the target fragment were connected using the Novozymes one-step efficient seamless cloning kit to obtain the knock-in plasmid PK-dapAmut; ④ The knock-in plasmid PK-dapAmut was transformed into Corynebacterium glutamicum, and a two-step homologous recombination method was used to obtain a strain with weakened expression of the dihydropicolinate synthase gene, and its genotype was expressed as P dapAmut .

[0047] (7) Citrate synthase gene gltA Weakened expression

[0048] Citrate synthase catalyzes the condensation of oxaloacetate and acetyl-CoA to form citric acid, and is the key enzyme controlling the glycolytic pathway to enter the TCA cycle. Therefore those skilled in the art can regulate the expression of citrate synthase to regulate the carbon flow entering the TCA cycle. In some aspects, the activity of citrate synthase is regulated by reducing the activity of endogenous citrate synthase gene, which can be achieved by using a transgenic encoding NADH-insensitive citrate synthase to replace the endogenous citrate synthase gene with a chromosome replacement or by using a transgenic encoding NADH-insensitive citrate synthase derived from bacillus subtilis, and can also be regulated (such as reducing) the activity of citrate synthase by replacing the endogenous citrate synthase gene promoter with a low expression promoter.

[0049] In the specific implementation of this example, it is preferred to weaken the citrate synthase gene by truncating the promoter sequence on the chromosome. gltA In order to achieve the purpose of this technology, it is necessary to construct a knock-in plasmid PK-P1gltA. The specific experimental steps are as follows: ① Design primers based on the sequence, use the genome of Corynebacterium glutamicum as a template, and use PCR amplification gltA The homologous arm of the promoter sequence after truncation of the gene upstream; primers were designed according to the sequence, and PCR amplification was performed using the genome of Corynebacterium glutamicum as a template. gltA②Use BamHI and XbaI to double-digest the pK18mobsacB shuttle plasmid to obtain a plasmid backbone with sticky ends and homologous regions at both ends; ③Use the Novozymes one-step efficient seamless cloning kit to connect the plasmid backbone and the target fragment to obtain the knock-in plasmid PK-P1gltA; ④Transform the obtained knock-in plasmid PK-P1gltA into Corynebacterium glutamicum, and use a two-step homologous recombination method to obtain a strain with a weakened and enhanced citrate synthase gene, and its genotype is expressed as ∆ P1 gltA .

[0050] (8) Heterologous aspartate kinase and efflux protein gene Ec thrA -Ec rhtA Enhanced expression

[0051] In Escherichia coli, glucose enters the citric acid cycle in the form of acetyl-CoA after glycolysis and is converted into oxaloacetate to participate in the synthesis of L-homoserine. Aspartate kinase / homoserine dehydrogenase catalyzes the first and third reactions of L-homoserine biosynthesis, which is the rate-limiting step in homoserine production. Its activity determines the proportion of metabolic flow to the L-homoserine synthesis pathway. It is encoded by the thrA gene and is subject to feedback inhibition by the end product threonine at the enzyme activity level. On the one hand, since the activity of aspartate kinase / homoserine dehydrogenase is strictly regulated by threonine, relieving the feedback inhibition of threonine on aspartate kinase / homoserine dehydrogenase is the only way to develop strains with high production of L-threonine and L-homoserine. On the other hand, in industrial breeding, the application of transporters can improve the environmental tolerance of engineered strains, improve the stereoselectivity of transport, and relieve growth inhibition. The outward transport proteins RhtA, RhtB and YeaS in Escherichia coli can transport L-threonine and L-homoserine to the extracellular space and can serve as important targets for strain metabolic modification.

[0052] In the specific implementation of this example, it is preferred to introduce the feedback inhibition-resistant aspartate kinase / homoserine dehydrogenase gene Ec from Escherichia coli into the chromosome. thrA and efflux protein gene Ec rhtA The selected integration site is the pyruvate quinone oxidoreductase gene on the chromosome pqo In order to achieve this technical purpose, it is necessary to construct a knock-in plasmid PK-ThrB-Ec thrA- Ec rhtA The specific experimental steps are as follows: ① Design primers based on the sequence, use the genome of Corynebacterium glutamicum as a template, and use PCR amplification pqoThe upstream homology arm of the gene; primers were designed based on the sequence, and the strong promoter Ptuf sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; primers were designed based on the sequence, and the strong promoter feedback inhibition Ec was amplified by PCR using the genome of Escherichia coli as a template thrA-C1034T Gene mutant sequence; primers were designed based on the sequence, and the E. coli genome was used as a template to amplify the strong promoter Ec rhtA Gene sequence; primers were designed based on the sequence, and PCR amplification was performed using the genome of Corynebacterium glutamicum as a template. pqo ②Use BamHI and XbaI to double-digest the pK18mobsacB shuttle plasmid to obtain a plasmid backbone with sticky ends and homologous regions at both ends; ③Use the Novozymes one-step efficient seamless cloning kit to connect the plasmid backbone and the target fragment to obtain the knock-in plasmids PK-ThrB and PK-ThrB-Ec thrA- Ec rhtA ④ The knock-in plasmid PK-ThrB or PK-ThrB-Ec thrA- Ec rhtA Transformation of Corynebacterium glutamicum to obtain pyruvate quinone oxidoreductase by two-step homologous recombination pqo The genotypes of the knockout strains and the strains with enhanced expression of aspartate kinase / homoserine dehydrogenase and efflux protein genes are ∆pqo and ∆ pqo ::Ec thrA- Ec rhtA .

[0053] (9) Heterologous aspartate kinase gene Sc lysC Enhanced expression

[0054] In the metabolic pathway of aspartate family amino acids synthesized by microbial fermentation, aspartate kinase is the rate-limiting enzyme that limits the flow rate of carbon and nitrogen sources, and is one of the keys to whether high-yield aspartate family amino acids can be obtained. In the inventor's previous patent CN115725537A, by mutating and screening the aspartate kinase encoding gene LysC from Saccharomyces cerevisiae, aspartate kinase mutants with different degrees of improved catalytic activity were obtained, laying the foundation for efficient fermentation production of L-homoserine and other downstream aspartate family metabolites.

[0055] In the specific implementation of this example, it is preferred to introduce the aspartate kinase gene from Saccharomyces cerevisiae into the chromosome, and the selected integration site is the alanine aminotransferase gene on the chromosome. avtA In order to achieve this technical purpose, it is necessary to construct a knock-in plasmid PK-AvtA-Sc lysCThe specific experimental steps that can be used are as follows: ① Design primers based on the sequence, use the genome of Corynebacterium glutamicum as a template, and use PCR amplification avtA The upstream homology arm of the gene was designed according to the sequence, and the strong promoter Psod sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; the codon-optimized Sc lysC Gene, primers were designed according to the sequence, and Sc lysC-T896C Gene mutant sequence; primers were designed based on the sequence, and PCR amplification was performed using the genome of Corynebacterium glutamicum as a template. avtA ②Use BamHI and XbaI to double-digest the pK18mobsacB shuttle plasmid to obtain a plasmid backbone with sticky ends and homologous regions at both ends; ③Use the Novozymes one-step efficient seamless cloning kit to connect the plasmid backbone and the target fragment to obtain the knock-in plasmid PK-AvtA-Sc lysC ④ The knock-in plasmid PK-AvtA-Sc lysC Transform Corynebacterium glutamicum and obtain an aspartate kinase gene expression-enhanced strain by two-step homologous recombination, the genotype of which is ∆avtA ::Sc lysC .

[0056] (10) Heterologous homoserine dehydrogenase gene Bd hom Enhanced expression

[0057] Homoserine dehydrogenase is a key enzyme in the aspartate metabolic pathway. It catalyzes the redox reaction between aspartate-β-semialdehyde and L-homoserine with NAD+ or NADP+ as cofactor. It is an enzyme that only exists in microorganisms and plants, and does not exist in mammals. In some bacteria, such as Escherichia coli, homoserine dehydrogenase not only plays the role of homoserine dehydrogenase, but also has aspartate kinase activity. By modifying homoserine dehydrogenase, the yield of downstream products can be changed. For example, the biological products produced by the aspartate metabolic pathway include homoserine, threonine, lysine, methionine, etc.

[0058] In the specific implementation of this example, it is preferred to introduce homoserine dehydrogenase from Brachypodium distachyon on the chromosome, and the selected integration site is the alanine aminotransferase gene on the chromosome. alaT In order to achieve this technical purpose, it is necessary to construct the knock-in plasmid PK-AlaT-Bd hom The specific experimental steps are as follows: ① Design primers based on the sequence, use the genome of Corynebacterium glutamicum as a template, and use PCR amplification alaTThe upstream homology arm of the gene; primers were designed according to the sequence, and the strong promoter Ptuf sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; the codon-optimized Bd hom Gene, primers were designed according to the sequence, and PCR amplification was used to obtain Bd hom Gene sequence; primers were designed based on the sequence, and PCR amplification was performed using the genome of Corynebacterium glutamicum as a template. alaT ②Use BamHI and XbaI to double-digest the pK18mobsacB shuttle plasmid to obtain a plasmid backbone with sticky ends and homologous regions at both ends; ③Use the Novozymes one-step efficient seamless cloning kit to connect the plasmid backbone and the target fragment to obtain the knock-in plasmid PK-AlaT-Bd hom ④ The knock-in plasmid PK-AlaT-Bd hom Transform Corynebacterium glutamicum and obtain an aspartate kinase gene expression-enhanced strain by two-step homologous recombination, the genotype of which is ∆alaT ::Bd hom .

[0059] (11) Heterologous NADPH production enzyme gene Ca gapN / Sp gapN Enhanced expression

[0060] In the construction of microbial cell factories, regulating the metabolic balance of cofactors to improve the efficiency of product synthesis pathways, thereby regulating cell growth and product production, and allowing metabolic flux to flow to the target product to the maximum extent, has become an important means of metabolic regulation. Another important factor limiting the high-yield production of L-homoserine by microbial fermentation is the problem of unbalanced reducing power in its metabolic pathway. The aspartate metabolic pathway requires a large amount of NADPH during the synthesis of L-homoserine. Therefore, introducing exogenous genes such as NADP+-dependent 3-phosphoglyceraldehyde dehydrogenase into the metabolic pathway of synthesizing the target product is an important strategy to achieve intracellular cofactor balance.

[0061] In the specific implementation of this example, it is preferred to introduce 3-phosphoglyceraldehyde dehydrogenase from Clostridium acetobutylicum or Streptococcus pyogenes into the chromosome. gapN Gene, promotes NADPH regeneration, the selected integration site is the diaminopimelate dehydrogenase gene on the chromosome ddh In order to achieve this technical purpose, it is necessary to construct the knock-in plasmid PK-Ddh-Ca gapN and PK-Ddh-Sp gapN The specific experimental steps are as follows: ① Design primers based on the sequence, use the genome of Corynebacterium glutamicum as a template, and use PCR amplification ddhThe upstream homology arm of the gene was designed according to the sequence, and the strong promoter Ptuf sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; the codon-optimized Ca gapN Gene, primers were designed according to the sequence, and Ca was obtained by PCR amplification. gapN Gene sequence; Sp after codon optimization synthesized by GenScript gapN Gene, primers were designed according to the sequence, and Sp gapN Gene sequence; primers were designed based on the sequence, and PCR amplification was performed using the genome of Corynebacterium glutamicum as a template. ddh ②Use BamHI and XbaI to double-digest the pK18mobsacB shuttle plasmid to obtain a plasmid backbone with sticky ends and homologous regions at both ends; ③Use the Novozymes one-step efficient seamless cloning kit to connect the plasmid backbone and the target fragment to obtain the knock-in plasmid PK-Ddh-Ca gapN and PK-Ddh-Sp gapN ; ④ The knock-in plasmid PK-Ddh-Ca gapN or PK-Ddh-Sp gapN Transform Corynebacterium glutamicum and obtain aspartate kinase gene expression enhanced strains by two-step homologous recombination. ∆ddh ::Ca gapN or ∆ddh ::Sp gapN .

[0062] (12) Genes related to strain osmotic tolerance cglK / betP / grpE Enhanced expression

[0063] When producing compounds, Corynebacterium glutamicum strains are often subjected to various adverse conditions, which reduce cell viability and production performance. During the fermentation process, high concentrations of nutrients and products in the fermentation broth may cause changes in osmotic pressure, thereby affecting the production performance of the cells. In the specific implementation of this embodiment, the inventors, after extensive and in-depth research, unexpectedly found that it is preferred to enhance the expression of a functional potassium uptake protein gene on the chromosome. cglK , compatible solute uptake system genes betP and molecular chaperone genes grpE , which helps to improve the ability of Corynebacterium glutamicum to produce L-homoserine. In order to achieve the technical purpose, the integration site selected is the homoserine kinase on the chromosome thrB Gene region, need to construct knock-in plasmids PK-ThrB-cglK, PK-ThrB-betP and PK-ThrB-grpE, the specific experimental steps are: ① according to the sequence design primers, with the glutamicum genome as template, use PCR amplificationthrB The upstream homology arm of the gene; primers were designed according to the sequence, and the strong promoter Ptuf sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; primers were designed according to the sequence, and the cglK gene sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; primers were designed according to the sequence, and the betP gene sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; primers were designed according to the sequence, and the grpE gene sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template; primers were designed according to the sequence, and the grpE gene sequence was amplified by PCR using the genome of Corynebacterium glutamicum as a template. thrB ②Use BamHI and XbaI to double-digest the pK18mobsacB shuttle plasmid to obtain a plasmid backbone with sticky ends and homologous regions at both ends; ③Use the Novozymes one-step efficient seamless cloning kit to connect the plasmid backbone and the target fragment to obtain knock-in plasmids PK-ThrB-cglK, PK-ThrB-betP and PK-ThrB-grpE; ④Transform the obtained knock-in plasmids PK-ThrB-cglK, PK-ThrB-betP or PK-ThrB-grpE into Corynebacterium glutamicum, and use a two-step homologous recombination method to obtain strains with enhanced expression of aspartate kinase genes, whose genotypes are respectively ∆thrB ::cglK, ∆thrB ::betP or ∆thrB ::grpE.

[0064] Example 2. Construction of genetically engineered bacteria producing homoserine

[0065] As an important food safety grade industrial microorganism, Corynebacterium glutamicum is widely used in industrial fermentation of various amino acids, as well as biosynthesis of organic acids, nucleotides and vitamins, showing significant economic value and broad application prospects. Studies have shown that the biosynthesis of L-homoserine in Corynebacterium glutamicum begins with the aspartate branch pathway. First, glucose generates oxaloacetate through the glycolysis pathway, pentose phosphate pathway and tricarboxylic acid cycle (TCA cycle), and then enters the aspartate branch pathway. Then, aspartate is converted into L-homoserine in sequence under the catalysis of aspartate kinase (encoded by lysC gene), aspartate semialdehyde dehydrogenase (encoded by asd gene) and homoserine dehydrogenase (encoded by hom gene), and finally L-homoserine is secreted into the extracellular environment through efflux transporters. As an important intermediate product of the aspartate family, L-homoserine is difficult to accumulate efficiently in wild-type strains, and is often directly used as a precursor for the biosynthesis of threonine and methionine.

[0066] In the implementation of this embodiment, wild-type Corynebacterium glutamicum ATCC 13032 was used as the starting strain, and a series of genetically engineered bacteria were constructed by the method described in Example 1 to improve the L-homoserine accumulation level of the strain. In a preferred specific embodiment, a chassis engineering bacterium HSE-5 with a certain L-homoserine production capacity was selected for metabolic modification, and the strain had the L-homoserine kinase encoding gene knocked out. thrB , and enhanced expression of the feedback-inhibiting aspartate kinase gene lysC mutant and the feedback-inhibiting homoserine dehydrogenase gene hom mutant, which can accumulate 2.0±0.3 g / L L-homoserine (see the applicant's aforementioned patent CN 112375726 B). The obtained genetically engineered strain for producing L-homoserine has at least one or more of the following genotype characteristic combinations: pyc, ppc, tkt, lysE The genes are strengthened, gltA, dapA, avtA, alaT, pqo, ddh, thrB The genes are weakened and contain exogenously introduced EcthrA, EcrhtA, SclysC, Bdhom, CagapN, SpgapN Isogenic, cglK, betP, grpE Osmotic stress-related genes were enhanced.

[0067] Example 3. Fermentation and detection of genetically engineered bacteria producing L-homoserine

[0068] In the specific implementation of this embodiment, a method for producing L-homoserine by fermentation using genetically engineered bacteria is provided, and the specific steps are as follows:

[0069] (1) Seed liquid culture: The activated genetically engineered bacteria producing L-homoserine are inoculated into a 250 mL round-bottom flask containing a seed medium, wherein 20 mL of the seed medium is contained, and cultured at 30-32° C. and 200-220 rpm in a shaking incubator for 16-20 h;

[0070] (2) Shake flask fermentation: Inoculate the seed culture of step (1) into a 500 mL round-bottomed Erlenmeyer flask containing 30 mL of fermentation medium at an inoculum size of 5-10%, and perform fermentation at 30-35° C. and 200-220 rpm. Maintain the pH by adding 20 g / L of calcium carbonate. The fermentation cycle is 60-72 h.

[0071] (3) Fermentation in a fermenter: Inoculate the seed culture of step (1) into a 5 L fermenter containing 2 L fermentation medium at a rate of 5-10%, maintain the temperature at 30-35°C, control the dissolved oxygen at about 20%, couple the stirring speed with the dissolved oxygen to ensure the stability of the dissolved oxygen, maintain the pH at 6.5-7.5 by adding ammonia water, and maintain the fermentation by adding glucose solution. The sugar concentration is maintained at 5-10 g / L. The fermentation period is 60-72 h.

[0072] As a preferred culture medium formula, the seed culture medium ingredients include: glucose 20-30g / L, corn steep liquor 20-30g / L, urea 2.5-5g / L, ammonium sulfate 2.5-5g / L, potassium dihydrogen phosphate 0.5-1g / L, magnesium sulfate 0.25-0.5g / L, calcium carbonate 10-20g / L, and the most preferred seed culture medium ingredients are: glucose 25g / L, corn steep liquor 30g / L, urea 5g / L, ammonium sulfate 5g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium carbonate 20g / L; the fermentation medium ingredients include: glucose 60-80g / L, corn steep liquor 20-30g / L, peptone 1-5g / L, ammonium sulfate 5-10g / L, potassium dihydrogen phosphate 0.5-1g / L, magnesium sulfate 0.25-0.5g / L, potassium acetate 0.25-0.5g / L, sodium citrate 1-2g / L, threonine 0.2-0.5 g / L, manganese sulfate 5-10mg / L, ferrous sulfate 5-10mg / L, VB1 0.5-1mg / L, biotin 0.05-0.1mg / L, calcium carbonate 10-20g / L, the most preferred seed culture medium composition is: glucose 80g / L, corn steep liquor 30g / L, peptone 5g / L, ammonium sulfate 10g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, sodium citrate 1 g / L, threonine 0.5 g / L, manganese sulfate 0.01g / L, ferrous sulfate 0.01g / L, VB11mg / L, biotin 0.1mg / L, calcium carbonate 20g / L. The above culture media can be prepared by standard methods.

[0073] In another specific embodiment of the present embodiment, a method for determining L-homoserine by high performance liquid chromatography (HPLC) is provided, and the specific steps are as follows: the fermentation sample is centrifuged to obtain the supernatant, a microinjection needle is used, the injection volume is 10 μL, an Agilent 1260 Infinity II Prime liquid chromatography system is used, and a ZORBAX Eclipse amino acid analysis (AAA) chromatographic column is equipped. O-phthalaldehyde (OPA) is used to perform pre-column online derivatization of amino acids, the column temperature is 30°C, the mobile phase A: 40 mM NaH2PO4 pH = 7.8, the mobile phase B: methanol: acetonitrile: water = 4.5:4.5:1, the flow rate is 2 mL / min, and the ultraviolet detection wavelength is 338 nm.

[0074] In this example, the shake flask fermentation culture system was first used to evaluate the homoserine genetic engineering bacteria obtained in Example 2 in the fermentation production of L-homoserine. The results are shown in Table 1.

[0075] Table 1 Detection of homoserine fermentation yield by genetically engineered bacteria

[0076]

[0077] The results showed that optimizing the metabolic flux of homoserine biosynthesis in the strain was helpful to improve the strain's ability to produce L-homoserine. Compared with the starting strain of Corynebacterium glutamicum producing L-homoserine, the gene encoding pyruvate carboxylase was expressed alone. pyc Increase the L-homoserine content of genetically engineered bacteria by 80% and enhance the expression of phosphoenolpyruvate carboxylase encoding gene ppc Increase the L-homoserine content of genetically engineered bacteria by 55% and enhance the expression of transketolase gene alone tkt The L-homoserine content of genetically engineered bacteria increased by 110%, while at the same time enhancing the expression pyc , ppc , tkt The gene can make the genetically engineered bacteria accumulate L-homoserine to 5.6±0.3 g / L, which is 2.8 times higher than the starting strain HSE5. The introduction of the amino acid transporter system is also very important for further improving the yield of the target product. The enhanced expression of its own LysE family protein alone increased the L-homoserine content of the genetically engineered bacteria by 11%, and the enhanced expression of the EcRhtA transporter from Escherichia coli alone increased the L-homoserine content of the genetically engineered bacteria by 14%. lysE and Ec rhtA The gene can further increase the L-homoserine accumulation of genetically engineered bacteria to 7.1±0.2 g / L, which is 1.27 times higher than that of the starting strain TIBHOM2. Knocking out or weakening the bypass metabolic pathway of the microorganism helps to concentrate the metabolic flux to the target product. Knocking out the alanine aminotransferase gene alone alaT or avtA , had little effect on the accumulation of L-homoserine in the strain, but knocking out the alanine aminotransferase gene alaT and avtA , the L-homoserine content of the genetically engineered bacteria increased slightly by 10%. In addition, knocking out the pyruvate quinone oxidoreductase gene alone pqo The strain has little effect on the L-homoserine content accumulated; the introduction of exogenous key enzymes in the metabolic pathway of aspartic acid derivatives can help to further significantly improve the strain's L-homoserine production level. alaT Integration site-enhanced expression of the feedback-resistant aspartate kinase gene Sc from Saccharomyces cerevisiae lysC , which increased the L-homoserine content of genetically engineered bacteria by 19%. avtA The integration site further enhances the expression of the feedback-resistant homoserine dehydrogenase gene Bd from Brachypodium distachyon hom , the L-homoserine content of genetically engineered bacteria increased by 76% to 12.5±0.4 g / L, using the chromosome pqoIntegration site enhanced expression of Escherichia coli-derived aspartate kinase / homoserine dehydrogenase gene Ec thrA and efflux protein gene Ec rhtA , the L-homoserine content of the genetically engineered bacteria can be further increased to 16.2±0.4 g / L, which is 2.28 times higher than that of the starting strain TIBHOM4; optimizing the key nodes of the TCA cycle and the metabolic flow of the lysine synthesis branch and enhancing the accumulation of precursors can also help to increase the accumulation of L-homoserine. dapA Gene expression can increase the L-homoserine content of genetically engineered bacteria by 7.4%, further reducing the key enzymes in the TCA cycle gltA Gene expression can further increase the L-homoserine content of genetically engineered bacteria by 7.5%, reaching 18.7±0.3 g / L; by adjusting the metabolic balance of cofactors and increasing the amount of NADPH generated, the metabolic flow can be maximized to the target product, and the exogenous introduction of Clostridium acetobutylicum 3-phosphoglyceraldehyde dehydrogenase Ca gapN The gene can increase the L-homoserine content of genetically engineered bacteria by 4.4%, and the exogenous introduction of Clostridium acetobutylicum 3-phosphoglyceraldehyde dehydrogenase Ca gapN The gene can increase the L-homoserine content of genetically engineered bacteria by 8.2% to 19.8±0.5 g / L; solving or alleviating the osmotic stress of microorganisms during fermentation can also help increase the accumulation of L-homoserine. Strengthening the expression of osmotic stress-related genes on chromosomes can improve the level of L-homoserine production in genetically engineered bacteria. In the optimal implementation scheme, strengthening the expression of functional potassium uptake protein genes cglK The best genetically engineered bacterium TIBHOM16 can be obtained. Under shake flask fermentation culture conditions, the engineered strain can accumulate 20.6±0.3 g / L L-homoserine.

[0078] Subsequently, we used a 5L fermenter for fed-batch fermentation, with 2L of fermentation medium in each tank, maintaining the culture condition at 32℃, controlling the dissolved oxygen at about 20%, coupling the stirring speed with the dissolved oxygen to ensure the stability of dissolved oxygen, maintaining the pH at 7.0 by adding ammonia water, and maintaining the fermentation by adding 60% glucose solution, with the sugar concentration maintained below 5 g / L, and the fermentation cycle was 72 h. The best genetically engineered bacteria TIBHOM16, under fed-batch fermentation conditions, can eventually accumulate 138.5±2.6 g / L L-homoserine, showing excellent L-homoserine fermentation performance and application potential.

[0079] The above detailed description of a genetically engineered bacterium for producing L-homoserine and its construction method and application with reference to specific embodiments is illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A genetically engineered bacterium for producing homoserine, characterized in that: It is knocked out or weakened in Corynebacterium glutamicum ATCC 13032 tB Gene, overexpression of feedback-resistant aspartate kinase lysC Homoserine dehydrogenase hom Gene; Further comprising the following gene modification: enhanced expression of phosphoenolpyruvate carboxylase ppc gene, transketolase gene tkt Pyruvate carboxylase encoding gene pyc , overexpression lysE Genes and Enhanced Transporter System Ec oeLh Gene.

2. The genetically engineered bacterium according to claim 1, characterized in that Further included are the following genetic modifications: knocking out or weakening the branch pathway aLaT and aV One or two 。 3. The genetically engineered bacterium according to claim 2, characterized in that: Further included is the following genetic modification: introduction of a heterologous aspartate kinase gene Sc having feedback resistance lysC and homoserine dehydrogenase Bd hom One or both genes 。 4. The genetically engineered bacterium according to claim 3, characterized in that Further included are the following genetic modifications: knocking out or weakening the branch pathway pq Gene.

5. The genetically engineered bacterium according to claim 4, characterized in that Further comprising the following genetic modification: enhancing the Ec involved in the supply of precursors and reducing power in the genetically engineered bacteria thrA Gene.

6. The genetically engineered bacterium according to claim 5, characterized in that Further included are the following genetic modifications: knocking out or weakening the branch pathway dapA Gene.

7. The genetically engineered bacterium according to claim 6, characterized in that Further included are the following genetic modifications: knocking out or weakening the branch pathway gltA Gene.

8. The genetically engineered bacterium according to claim 7, characterized in that: Further included are the following genetic modifications: knocking out or weakening the branch pathway dhdh Gene.

9. The genetically engineered bacterium according to claim 8, characterized in that Further included are the following genetic modifications: introduction of heterologous NADPH production enzyme Sp gapN or Ca gapN Gene.

10. The genetically engineered bacterium according to claim 9, characterized in that: Further included are the following genetic modifications: enhancing the osmotic pressure tolerance in genetically engineered bacteria cglK, betP, grpE Gene.

11. The genetically engineered bacterium according to claim 9, characterized in that: The Ec thrA The GenBank accession number of the gene is NP_414543.1 ; The Ec oeLh The GenBank accession number of the gene is NP_415334.1 Sc lysC The gene is derived from Saccharomyces cerevisiae ( Saccharomyces cerevisiae ); the feedback resistance Bd hom The gene is derived from Brachypodium distachyon ( Brachypodium distachyon ); the Ca gapN The gene is derived from Clostridium acetobutylicum ( Clostridium acetobutylicum ); said Sp gapN The gene is derived from Streptococcus pyogenes serotype M1 ( Streptococcus pyogenes serotype M1 ).

12. The genetically engineered bacterium according to any one of claims 1 to 11, characterized in that: Attenuation refers to reducing the expression or activity of the genes in the genetically engineered bacteria, which can be achieved by adding inhibitors to the culture medium of the genetically engineered bacteria or modifying the genes in the genetically engineered bacteria to reduce their expression or activity; Enhance the expression or activity of a gene by expressing the endogenous or heterologous gene using a strong promoter, and / or increasing the copy number of the gene, and / or modifying the promoter of the gene to enhance the transcription initiation rate, and / or modifying the translation regulatory region or rare codons of the messenger RNA carrying the gene to enhance the translation intensity, and / or modifying the gene itself to enhance mRNA stability and protein stability; The introduction of heterologous genes can be achieved by introducing them into expression vector plasmids or by integrating heterologous genes into the genome.

13. The method for constructing a genetically engineered bacterium according to any one of claims 1 to 12, characterized in that: It is obtained by knocking out or weakening the relevant genes in the starting bacteria through gene editing methods, or enhancing the expression of relevant genes; and by introducing heterologous genes through gene transformation methods.

14. Use of the genetically engineered bacterium according to any one of claims 1 to 12 in producing homoserine.

15. A method for producing homoserine, characterized in that: The method comprises the steps of culturing the genetically engineered bacteria as claimed in any one of claims 1 to 12, and collecting the produced homoserine.

16. The method according to claim 15, characterized in that In the fermentation medium, at 30-35°C, the dissolved oxygen is controlled at 15-25%, the stirring speed is coupled with the dissolved oxygen to ensure the stability of the dissolved oxygen, the pH is maintained at 6.5-7.5 by adding ammonia water, the fermentation is maintained by adding glucose solution, the sugar concentration is maintained at 5-10g / L, and the fermentation cycle is 60-72h.

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