Genetically engineered bacteria for producing l-valine and construction method and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered bacterium for producing L-valine and a construction method and application thereof. The genetically engineered bacterium takes Escherichia coli as a host, heterologously overexpresses alss genes of Bacillus subtilis for coding acetyl-lactate synthetase and bcd genes for coding leucine dehydrogenase, brnFE genes of Corynebacterium glutamicum for coding branched-chain amino acid membrane transport proteins, and overexpresses ilvC genes of Escherichia coli for coding ketol-acid reductoisomerase mutants L67E / R68F / K75E, ilvD genes for coding dihydroxy-acid dehydratase, gapA genes for coding 3-phosphoglyceraldehyde dehydrogenase and pncB genes for coding nicotinate phosphoribosyltransferase; and does not express mdh genes for coding malate dehydrogenase; and replaces thioctamide dehydrogenase with a mutant A321T. A genetically engineered bacterium for stably and efficiently producing L-valine is obtained.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a genetically engineered bacterium that produces L-valine, its construction method, and its application. Background Technology

[0002] L-valine is one of the eight essential amino acids for mammals. Together with leucine and isoleucine, it forms the branched-chain amino acid family. It possesses various physiological functions and is widely used in animal feed, cosmetics, pharmaceuticals, food, and pesticides. The molecular formula of L-valine is C5H₂O. 11 NO2 has a relative molecular weight of 117.46 and a water solubility of 88.5 g / L at 25°C.

[0003] The main production methods for L-valine include protein hydrolysis extraction, chemical synthesis, and microbial fermentation. With increasing global emphasis on environmental protection, sustainable green biosynthesis technologies are gradually replacing traditional chemical synthesis techniques. Meanwhile, microbial fermentation offers advantages such as mild reaction conditions, low cost, and high purity, making de novo synthesis of L-valine from glucose the mainstream production method. Currently, the main domestic suppliers of L-valine are Shenyang CJ, Ningxia Yipin, Anhui Huaheng, and Langfang Meihua, with annual production capacities of 50,000, 20,000, 10,000, and 11,000 tons, respectively. With rapid market demand growth, L-valine is expected to enter the ranks of high-value-added bulk chemicals. Therefore, improving industrial indicators such as L-valine yield, conversion rate, and production intensity has become a key focus for research institutions.

[0004] To obtain efficient L-valine-producing strains, researchers have made numerous attempts. Park et al. (Park JH, Lee KH, Kim TY, Lee SY. Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico geneknockout simulation. Proc Natl Acad Sci US A. 2007, 104(19):7797-802.) used Escherichia coli as the starting strain, site-directedly mutated acetylhydroxy acid synthase to relieve the feedback inhibition of L-valine, overexpressed the ilvBNEDA gene of the synthesis pathway and the lrp and ygaZH genes responsible for the L-valine efflux system using the strong promoter tac, and deleted the aceF, mdh and pfkA genes. The resulting recombinant strain produced 7.55 g / L L-valine using 20 g / L glucose, with a glucose-to-acid conversion rate of 37.8%. Based on this, Park et al. constructed a recombinant strain using E. coli W as the chassis cell, which synthesized 60.7 g / L L-valine within 29.5 h (Park JH, Jang YS, Lee JW, Lee SY. Escherichia coli W as a new platform strain for the enhanced production of L-valine by systems metabolic engineering. Biotechnol Bioeng. 2011, 108(5):1140-7.).Hasegawa et al. site-directedly mutated acetylhydroxy acid reductase and introduced leucine dehydrogenase from Bacillus spheroidis to convert the coenzyme required for L-valine synthesis from NADPH to NADH, solving the cofactor imbalance problem. This enabled the pre-cultured recombinant Corynebacterium glutamicum strain to synthesize 172 g / L L-valine under hypoxic conditions for 24 h, with a sugar-acid conversion rate of 41% (Hasegawa S, Uematsu K, Natsuma Y, Suda M, Hiraga K, Jojima T, Inui M, Yukawa H. Improvement of the redox balance increase L-valine production by Corynebacterium glutamicum under oxygen deprivation conditions. Appl Environ Microbiol. 2012, 78(3): 865-75.). Blombach et al. replaced the natural promoter of aceE with the promoter dapA-A16 to reduce the expression of pyruvate dehydrogenase, enabling the recombinant Corynebacterium glutamicum strain to synthesize 85.6 g / L L-valine with a sugar-acid conversion rate of 23.4% (Buchholz J, Schwentner A, Brunnenkan B, Gabris C, Grimm S, Gerstmeir R, Takors R, Eikmanns BJ, Blombach B. Platform engineering of Corynebacterium glutamicum with reduced pyruvatedehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate. Appl Environ Microbiol. 2013, 79(18): 5566-75.).Huang Jianzhong et al. used ultraviolet mutagenesis combined with high-throughput screening to breed a *Brevibacterium flavum* mutant strain with a yield of 70.1 g / L L-valine and a sugar-acid conversion rate of 36.5% (Huang QG, Zeng BD, Liang L, Wu SG, Huang JZ. Genomeshuffling and high-throughput screening of *Brevibacterium flavum* MDV1 for enhanced L-valine production. World J Microbiol Biotechnol. 2018, 34(8): 121-130.). Zhang Xueli et al. used anaerobic adaptive evolution to breed an *Escherichia coli* mutant strain with a yield of 80 g / L L-valine and a sugar-acid conversion rate of 60% within 48 hours (CN 114457122 A). Rao Zhiming et al. discovered new metabolic modification targets by comparing the transcriptional levels of valine-producing strains under different dissolved oxygen conditions, resulting in a yield of 92 g / L and a sugar-acid conversion rate of 34% in recombinant Escherichia coli strains (Hao Y, Pan X, Xing R, You J, Hu M, Liu Z, Li X, Xu M, Rao Z. High-level production of L-valine in Escherichia coli using multi-modular engineering. Bioresour Technol. 2022, 359:127461).

[0005] Despite numerous attempts, the performance of existing L-valine-producing strains still needs improvement. Pyruvate is a precursor in L-valine synthesis and a link between the glycolysis pathway and the tricarboxylic acid cycle. Meanwhile, the tricarboxylic acid cycle provides a large amount of energy, reducing power, and intermediate metabolites for *E. coli* growth. Therefore, the current method of producing L-valine using microbial fermentation faces the problem of "competition between growth and production for carbon flux," leading to challenges such as long fermentation cycles and low sugar-acid conversion rates in large-scale industrial production. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to construct a genetically engineered Escherichia coli strain that produces L-valine. This strain has undergone systematic modification of the L-valine synthesis module and the cofactor regeneration module, enabling efficient and stable production of L-valine in Escherichia coli under anaerobic and / or hypoxic conditions.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a genetically engineered bacterium that uses *Escherichia coli* as a host and heterologously overexpresses the *alss* gene encoding acetolactate synthase and the *bcd* gene encoding leucine dehydrogenase from *Bacillus subtilis*, the *brnFE* gene encoding a branched-chain amino acid membrane transporter from *Corynebacterium glutamicum*, and overexpresses the *ilvC* gene encoding the ketolate reductase mutant L67E / R68F / K75E, the *ilvD* gene encoding dihydroxy acid dehydratase, the *gapA* gene encoding glyceraldehyde-3-phosphate dehydrogenase, and the *pncB* gene encoding nicotinic acid phosphoribosyltransferase from *E. coli*; and does not express the *mdh* gene encoding malate dehydrogenase; and replaces lipoamide dehydrogenase with the mutant A321T.

[0009] Secondly, the present invention provides a method for constructing the genetically engineered bacteria as described above, comprising: introducing and overexpressing the alss and bcd genes of Bacillus subtilis in a host Escherichia coli; introducing and overexpressing the brnFE gene of Corynebacterium glutamicum; overexpressing the ilvC gene encoding the ketool acid reductase mutant L67E / R68F / K75E, the ilvD gene encoding dihydroxy acid dehydratase, the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase, and the pncB gene encoding nicotinic acid phosphoribosyltransferase; knocking out or inactivating the mdh gene; and replacing the lipoamide dehydrogenase gene lpd with the gene encoding the mutant A321T.

[0010] Thirdly, the present invention provides the application of the genetically engineered bacteria described above in the efficient production of L-valine.

[0011] Fourthly, the present invention provides a method for producing L-valine using the genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-valine; and collecting the L-valine from the genetically engineered bacteria and / or the culture medium.

[0012] The beneficial effects of this invention are as follows:

[0013] This invention uses *Escherichia coli* as the starting strain and systematically modifies the L-valine synthesis and cofactor regeneration modules through metabolic engineering to obtain a genetically engineered strain, VAL9, with a clear genetic background capable of stably and efficiently producing L-valine. This strain can produce 24.36 g / L of L-valine in shake-flask fermentation at 60 r / min for 20 h; and 92 g / L of L-valine in a 5 L fermenter under fed-batch fermentation with limited oxygen for 32 h, achieving a conversion rate of 60.4% with no significant accumulation of other byproducts. The L-valine fermentation production method provided by this invention achieves the highest reported L-valine yield, production intensity, and conversion rate to date, and the fermentation process is largely automated, significantly reducing production costs. Attached Figure Description

[0014] Figure 1 Fermentation curve of VAL9 5L fermenter with batch feeding. Detailed Implementation

[0015] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0016] In a first aspect, the present invention provides a genetically engineered bacterium that uses *Escherichia coli* as a host and heterologously overexpresses the *alss* gene encoding acetolactate synthase and the *bcd* gene encoding leucine dehydrogenase from *Bacillus subtilis*, the *brnFE* gene encoding a branched-chain amino acid membrane transporter from *Corynebacterium glutamicum*, and overexpresses the *ilvC* gene encoding the ketolate reductase mutant L67E / R68F / K75E, the *ilvD* gene encoding dihydroxy acid dehydratase, the *gapA* gene encoding glyceraldehyde-3-phosphate dehydrogenase, and the *pncB* gene encoding nicotinic acid phosphoribosyltransferase from *E. coli*; and does not express the *mdh* gene encoding malate dehydrogenase; and replaces lipoamide dehydrogenase with the mutant A321T.

[0017] According to the present invention, the identification of mutants is as follows: amino acid residues are identified using the recognized IUPAC nomenclature, and DNA nucleic acid sequences are identified using the recognized IUPAC nomenclature. The mutated amino acid in the protein mutant is represented by "the amino acid replaced at the original amino acid position". For example, A321T indicates that the amino acid at position 321 of the amino acid sequence of lipoamide dehydrogenase (NCBI-ProteinID: NP_414658.1) is replaced by threonine (T), and its encoding gene is represented as lpd A321T. According to a specific embodiment of the present invention, the base at position 961 of the nucleotide sequence of its encoding gene (NCBI-GeneID: 944854) is replaced by guanine (G) with adenine (A), represented as lpd. G961A express.

[0018] For example, L67E / R68F / K75E indicates that amino acids at positions 67, 68, and 75 of the amino acid sequence of ketool acid reductase (NCBI-ProteinID: NP_418222.1) have been replaced, and its coding gene is represented as ilvC L67E / R68F / K75E. According to a specific embodiment of the present invention, in the nucleotide sequence (NCBI-GeneID: 948286) encoding the gene, the base at position 199 is replaced by cytosine (C) with guanine (G), the base at position 200 is replaced by thymine (T) with adenine (A), the base at position 202 is replaced by cytosine (C) with thymine (T), the base at position 203 is replaced by guanine (G) with thymine (T), and the base at position 223 is replaced by adenine (A) with guanine (G), i.e., ilvC C199G / T200A / C202T / G203T / A223G express.

[0019] According to the present invention, the host used to construct the genetically engineered bacteria can be any Escherichia coli, such as E. coli W3110, E. coli BL21, E. coli BW25113 and other model strains commonly used as genetic engineering hosts in the art, as long as the genes that need to be overexpressed or heterologously overexpressed can be expressed on them; the selection of each gene of the above-mentioned E. coli is based on the selected host, but all of them are genes that perform the same function.

[0020] According to a preferred embodiment of the present invention, the host is E. coli MG1655.

[0021] According to the present invention, the above-mentioned gene can be not expressed by conventional means in the art, for example, by inactivating or knocking out the gene by conventional means in the art.

[0022] According to the present invention, "non-expression" means that the amount of the gene expression product is significantly lower than the original level, for example, significantly reduced by at least 50%, 60%, 70%, 80%, 90%, or 100%.

[0023] According to the present invention, overexpression means that the amount of the gene expression product is significantly higher than the original level, for example, increased by 150% or more, 200% or more, 300% or more.

[0024] According to the present invention, the overexpression of the above-mentioned gene can be achieved by introducing and / or increasing the copy number of the gene into the Escherichia coli genome (e.g., by increasing the copy number of the gene through self-replicating plasmids such as pET28a, pTrc99a, pSTV28, or by increasing the copy number of the gene in the Escherichia coli chromosome), or by modifying the expression regulatory sequence of the gene (e.g., promoter, ribosome binding site, etc.), or a combination of the above methods.

[0025] According to a preferred embodiment of the present invention, the overexpression of the above-mentioned gene is achieved by linking the gene to a strong promoter and integrating it into the Escherichia coli genome. The integration site can be selected based on conventional knowledge of those skilled in the art, choosing pseudogene sites that will not significantly affect bacterial growth and basal metabolism, such as yeeP, ygiP, yghX, ygaY, yjiT, yjiP, ycjV, ycgH, ygaY, yeeL, ilvG, and rph gene sites. The strong promoter can be P... trc Any one of BBa-J23100 and T7, preferably P. trc .

[0026] Secondly, the present invention provides a method for constructing the genetically engineered bacteria as described above, comprising: introducing the alss and bcd genes of Bacillus subtilis and the brnFE gene of Corynebacterium glutamicum into the host Escherichia coli; overexpressing the ilvC gene encoding the ketool acid reductase mutant L67E / R68F / K75E, the ilvD gene encoding dihydroxy acid dehydratase, the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase, and the pncB gene encoding nicotinic acid phosphoribosyltransferase of Escherichia coli; knocking out or inactivating the mdh gene; and replacing the lipoamide dehydrogenase gene lpd with the gene encoding the mutant A321T.

[0027] The selection of the aforementioned genes and the selection of the host have been described in detail in the first aspect of this invention, and the content already described in the first aspect will not be repeated here.

[0028] According to a specific embodiment of the present invention, the method includes:

[0029] (1) Enhance the L-valine synthesis pathway

[0030] The alss gene (encoding acetolactate synthase) from Bacillus subtilis, the bcd gene (encoding leucine dehydrogenase), the ilvC gene (encoding the keto-alcoholic acid reductase mutant L67E / R68F / K75E), and the ilvD gene (encoding dihydroxy acid dehydratase) from E. coli were respectively integrated into the genome of the host *E. coli*, and were generated by the promoter P. trc Controlling its expression; this step can enhance the L-valine synthesis pathway, enabling E. coli to over-synthesize L-valine.

[0031] (2) Enhance the extracellular transport of L-valine

[0032] The brnEF gene derived from Corynebacterium glutamicum was integrated into the E. coli genome and directed by the promoter P. trc By controlling its expression, this step can enhance the extracellular transport of L-valine.

[0033] (3) Increase the supply of cofactor NADH

[0034] It integrates its own gapA gene, which encodes glyceraldehyde-3-phosphate dehydrogenase, and pncB gene, which encodes nicotinic acid phosphoribosyltransferase, into the genome of the host *Escherichia coli*, and is activated by the promoter P. trc Controlling its expression and knocking out the mdh gene encoding malate dehydrogenase can enhance the supply of the coenzyme NADH, thereby promoting the synthesis of L-valine.

[0035] (4) Improve the strain's tolerance to oxygen-limited environments

[0036] Replacing the lpd gene encoding lipoamide dehydrogenase with the gene encoding the mutant A321T can improve the strain's tolerance to oxygen-limited environments and further enhance the strain's L-valine production performance under oxygen-limited fermentation conditions.

[0037] Thirdly, the present invention provides the application of the genetically engineered bacteria described above in the efficient production of L-valine.

[0038] Fourthly, the present invention provides a method for producing L-valine using the genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-valine; and collecting the L-valine from the genetically engineered bacteria and / or the culture medium.

[0039] According to a fourth aspect of the invention, the culture of the genetically engineered bacteria can be carried out using methods conventional in the art. The culture medium used for the production of L-valine can be a synthetic or natural culture medium, such as a typical culture medium containing a carbon source, nitrogen source, sulfur source, inorganic ions, and other required organic and inorganic components.

[0040] The genetically engineered bacteria can be cultured under limited and / or hypoxic conditions for 16 to 72 hours, or 20 to 60 hours, or 24 to 48 hours; the culture temperature can be controlled within 30 to 45°C, or 30 to 37°C; and the pH can be adjusted between 5.0 and 8.0, or 6.0 and 7.5, or 6.8 and 7.2. The pH can be adjusted by using inorganic or organic acidic or alkaline substances, as well as ammonia.

[0041] After cultivation, solids, such as cells and cell debris, can be removed from the liquid culture medium using conventional techniques (e.g., centrifugation, membrane filtration). L-valine can then be recovered from the fermentation broth using any combination of conventional techniques (e.g., concentration, ion exchange chromatography, crystallization).

[0042] According to a preferred embodiment of the present invention, the preferred culture medium composition is as follows: glucose 10-30 g / L, yeast extract 0.5-3 g / L, (NH4)2SO4 3-6 g / L, K2HPO4 2-6 g / L, MgSO4·7H2O 0.1-3 g / L, sodium citrate 1-3 g / L, FeSO4·7H2O 10-200 mg / L, MnSO4·7H2O 5-20 mg / L, VB1, VB3, VB5, and VB12 each 1-6 mg / L, VH 0.1-20 mg / L, and the remainder is water.

[0043] According to another preferred embodiment of the present invention, the preferred culture medium composition is as follows: glucose 10-30 g / L, yeast extract 1-6 g / L, sodium citrate 1-5 g / L, (NH4)2SO4 3-6 g / L, KH2PO4 2-6 g / L, MgSO4·7H2O 0.1-3 g / L, FeSO4·7H2O 10-100 mg / L, MnSO4·7H2O 5-20 mg / L, VB1, VB3, VB5, VB12, and VH each 1-3 mg / L, with an additional 2% (v / v) phenol red solution, and the remainder being water.

[0044] The present invention will now be described in more detail through specific embodiments. In the following embodiments:

[0045] Unless otherwise specified, the gene editing method involved in the embodiments of this invention refers to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing. Metabolic engineering, 2015, 31:13-21.). The pREDCas9 carries the elimination system of the gRNA expression plasmid pGRB, the Red recombination system of λ phage, and the Cas9 protein expression system, with micranthin resistance (working concentration: 100 mg / L), and is cultured at 32℃; pGRB uses pUC18 as its backbone, including the promoter J23100, the gRNA-Cas9 binding region sequence, and the terminator sequence, with ampicillin resistance (working concentration: 100 mg / L), and is cultured at 37℃. This method achieves the knockout or knock-in of recombinant fragments by transforming the pGRB plasmid and recombinant DNA fragments, screening for positive strains, and then eliminating the plasmid. The recombinant fragment used for knockout consists of upstream and downstream homologous arms of the gene to be knocked out (upstream homologous arm - downstream homologous arm), while the recombinant fragment used for integration consists of upstream and downstream homologous arms of the integration site and the gene fragment to be integrated (upstream homologous arm - target gene - downstream homologous arm). Other specific molecular biology and genetic engineering techniques involved can be implemented using readily available technical manuals, textbooks, or literature reports, and need not be described in detail here. Furthermore, specific bacterial strains were selected as hosts in the following examples, and therefore specific gene integration sites, target genes, and primers were chosen based on the host. However, this does not mean that the purpose of the present invention can only be achieved through these specific selections, and should not be used to limit the scope of the invention. The essence and scope of the invention are defined only by the claims.

[0046] Example 1: Construction of Escherichia coli VAL9 genetically engineered strain

[0047] The primers used in the construction of strain 1 are shown in the table below:

[0048]

[0049]

[0050]

[0051] Construction process of strain 2

[0052] 2.1 alss gene integration

[0053] Using the E. coli MG1655 genome as a template, primers for the upstream homologous arm (UP-mbhA-S, UP-mbhA-A) and the downstream homologous arm (DN-mbhA-S, DN-mbhA-A) were designed based on the upstream and downstream sequences of its mbhA gene, and the upstream and downstream homologous arm fragments were amplified by PCR. Primers alss-S and alss-A, required for amplifying the alss fragment, were designed based on the alss gene sequence of Bacillus subtilis 168 (NCBI-GeneID: 936852), and the promoter P... trc The design involves the downstream primer of the upstream homologous arm and the upstream primer of the alss gene. These fragments are then fused using overlap PCR to obtain the integrated fragment of the alss gene (upstream homologous arm - P). trc The DNA fragment containing the target sequence used to construct plasmid pGRB-mbhA (downstream homologous arm of alss-) was prepared by annealing with primers gRNA-mbhA-S and gRNA-mbhA-A. This recombinant fragment and plasmid pGRB-mbhA were electroporated into competent E. coli MG1655 cells. Positive strains were screened, and the plasmid was then removed to obtain strain VAL1. Successful integration of the alss gene was verified by PCR and electrophoresis in the positive strain.

[0054] 2.2 bcd gene integration

[0055] Using the E. coli MG1655 genome as a template, upstream homologous arm primers (UP-yjiv-S, UP-yjiv-A) and downstream homologous arm primers (DN-yjiv-S, DN-yjiv-A) were designed based on the upstream and downstream sequences of its yjiv gene, and the upstream and downstream homologous arm fragments were amplified by PCR. Based on the bcd gene sequence of Bacillus subtilis 168 (NCBI-GeneID: 938670), primers bcd-S and bcd-A were designed to amplify the bcd fragment, and promoter P... trc The design involves the downstream primer of the upstream homologous arm and the upstream primer of the bcd gene. These fragments are then fused using overlap PCR to obtain the integrated fragment of the bcd gene (upstream homologous arm - P). trc The DNA fragment containing the target sequence used to construct plasmid pGRB-yjiv (downstream homologous arm of bcd) was prepared by annealing with primers gRNA-yjiv-S and gRNA-yjiv-A. This recombinant fragment and plasmid pGRB-yjiv were electroporated into competent VAL1 cells. Positive strains were screened, and the plasmid was then removed to obtain strain VAL2. Successful integration of the bcd gene was verified by PCR and electrophoresis in the positive strain.

[0056] 2.3 brnFE gene integration

[0057] Using the E. coli MG1655 genome as a template, primers for the upstream homologous arm (UP-yjiT-S, UP-yjiT-A) and the downstream homologous arm (DN-yjiT-S, DN-yjiT-A) were designed based on the upstream and downstream sequences of its yjiT gene, and the upstream and downstream homologous arm fragments were amplified by PCR. Primers brnEF-S and brnEF-A were designed based on the brnE and brnF gene sequences (KEGG-ID: cg0315, cg0314) of Corynebacterium glutamicum ATCC 13032 to amplify the brnEF fragment, and the promoter P... trc The design involves the downstream primer of the upstream homologous arm and the upstream primer of the brnEF gene. These fragments are then fused using overlap PCR to obtain the integrated fragment of the brnEF gene (upstream homologous arm - P). trc The DNA fragment containing the target sequence used to construct plasmid pGRB-yjiT (downstream homologous arm of brnEF) was prepared by annealing with primers gRNA-yjiT-S and gRNA-yjiT-A. This recombinant fragment and plasmid pGRB-yjiT were electroporated into competent VAL2 cells. Positive strains were screened, and the plasmid was then removed to obtain strain VAL3. Successful integration of the brnEF gene into the positive strain was verified by PCR and electrophoresis.

[0058] 2.4 ilvC C199G / T200A / C202T / G203T / A223G Gene integration

[0059] Using the E. coli MG1655 genome as a template, primers for the upstream homologous arm (UP-gapC-S, UP-gapC-A) and the downstream homologous arm (DN-gapC-S, DN-gapC-A) were designed based on the upstream and downstream sequences of the gapC gene, and the upstream and downstream homologous arm fragments were amplified by PCR. Based on the ilvC gene sequence (NCBI-GeneID: 948286), primers were designed to amplify ilvC. C199G / T200A / C202T / G203T / A223G The required primers for the fragment are ilvC*-S1, ilvC*-A1, ilvC*-S2, ilvC*-A2, and ilvC*-S3. First, primers ilvC*-S1, ilvC*-A1, ilvC*-S2, and ilvC*-A2 are used to obtain the upstream and downstream homologous arms containing the mutant sequence, respectively. These are then fused using overlap PCR to obtain the fragment containing the mutant sequence. Finally, primers ilvC*-S3 and ilvC*-A2 are used to obtain ilvC*-S3. * Gene fragment. Promoter P trc The primers were designed into the downstream primer UP-gapC-A of the upstream homologous arm and the upstream primer ilvC*-S3 of the ilvC* gene. These fragments were then fused using overlap PCR to obtain ilvC*. C199G / T200A / C202T / G203T / A223GIntegration fragment of gene (upstream homologous arm - P) trc -ilvC C199G / T200A / C202T / G203T / A223G The DNA fragment containing the target sequence used to construct plasmid pGRB-gapC (downstream homologous arm) was prepared by annealing with primers gRNA-gapC-S and gRNA-gapC-A. This recombinant fragment and plasmid pGRB-gapC were electroporated into competent VAL3 cells. Positive strains were screened, and the plasmid was then removed to obtain strain VAL4. Positive strains were verified by PCR and electrophoresis. C199G / T200A / C202T / G203T / A223G Mutation integration was successful.

[0060] 2.5ilvD gene integration

[0061] Using the E. coli MG1655 genome as a template, upstream homologous arm primers (UP-ydeU-S, UP-ydeU-A) and downstream homologous arm primers (DN-ydeU-S, DN-ydeU-A) were designed based on the upstream and downstream sequences of the ydeU gene, and the upstream and downstream homologous arm fragments were amplified by PCR. Based on the ilvD gene sequence (NCBI-GeneID: 948277), primers ilvD-S and ilvD-A were designed to amplify the ilvD fragment, and the promoter P... trc The design involves the downstream primer of the upstream homologous arm and the upstream primer of the ilvD gene. The above fragments are fused using overlap PCR to obtain the integrated fragment of the ilvD gene (upstream homologous arm - P). trc The DNA fragment containing the target sequence used to construct plasmid pGRB-ydeU (downstream homologous arm of ilvD) was prepared by annealing with primers gRNA-ydeU-S and gRNA-ydeU-A. This recombinant fragment and plasmid pGRB-ydeU were electroporated into competent VAL4 cells. Positive strains were screened, and the plasmid was then removed to obtain strain VAL5. Successful integration of the ilvD gene was verified by PCR and electrophoresis in the positive strain.

[0062] 2.6gapA gene integration

[0063] Using the E. coli MG1655 genome as a template, upstream homologous arm primers (UP-rph-S, UP-rph-A) and downstream homologous arm primers (DN-rph-S, DN-rph-A) were designed based on the upstream and downstream sequences of its rph gene. The upstream and downstream homologous arm fragments were amplified by PCR. Primers gapA-S and gapA-A were designed based on the gapA gene sequence (NCBI-GeneID: 947679) to amplify the gapA fragment. Promoter P... trc The design involves the downstream primer of the upstream homologous arm and the upstream primer of the gapA gene. The above fragments are then fused using overlap PCR to obtain the integrated fragment of the gapA gene (upstream homologous arm - P).trc The DNA fragment containing the target sequence used to construct plasmid pGRB-rph was prepared by annealing primers gRNA-rph-S and gRNA-rph-A (downstream homologous arm of gapA). This recombinant fragment and plasmid pGRB-rph were electroporated into competent VAL5 cells. Positive strains were screened, and the plasmid was then removed to obtain strain VAL6. Successful integration of the gapA gene into the positive strain was verified by PCR and electrophoresis.

[0064] 2.7 Integration of the pncB gene

[0065] Using the E. coli MG1655 genome as a template, upstream homologous arm primers (UP-yghE-S, UP-yghE-A) and downstream homologous arm primers (DN-yghE-S, DN-yghE-A) were designed based on the upstream and downstream sequences of the yghE gene, and the upstream and downstream homologous arm fragments were amplified by PCR. Based on the pncB gene sequence (NCBI-GeneID: 946648), primers pncB-S and pncB-A were designed to amplify the pncB fragment, and the promoter P... trc The design involves the downstream primer of the upstream homologous arm and the upstream primer of the pncB gene. The above fragments are fused using overlap PCR to obtain the integrated fragment of the pncB gene (upstream homologous arm - P). trc The DNA fragment containing the target sequence used to construct plasmid pGRB-yghE (downstream homologous arm of pncB) was prepared by annealing with primers gRNA-yghE-S and gRNA-yghE-A. This recombinant fragment and plasmid pGRB-yghE were electroporated into competent VAL6 cells. Positive strains were screened, and the plasmid was then removed to obtain strain VAL7. Successful integration of the pncB gene into the positive strain was verified by PCR and electrophoresis.

[0066] 2.8mdh gene knockout

[0067] Using the E. coli MG1655 genome as a template, upstream homologous arm primers (UP-Δmdh-S, UP-Δmdh-A) and downstream homologous arm primers (DN-Δmdh-S, DN-Δmdh-A) were designed based on the upstream and downstream sequences of its mdh gene (NCBI-GeneID: 947854). The upstream and downstream homologous arm fragments were amplified by PCR. These fragments were then fused using overlap PCR to obtain the integrated fragment of the Δmdh gene (upstream homologous arm - downstream homologous arm). The DNA fragment containing the target sequence used to construct the plasmid pGRB-mdh was prepared by annealing with primers gRNA-mdh-S and gRNA-mdh-A. This recombinant fragment and plasmid pGRB-mdh were electroporated into competent VAL7 cells. Positive strains were screened, and the plasmid was then removed to obtain strain VAL8. The successful knockout of the mdh gene was verified by PCR and electrophoresis in the positive strain.

[0068] 2.9lpd gene mutation

[0069] Using the E. coli MG1655 genome as a template, upstream homologous arm primers (UP-Δlpd-S, UP-Δlpd-A) and downstream homologous arm primers (DN-Δlpd-S, DN-Δlpd-A) were designed based on the upstream and downstream sequences of its lpd gene (NCBI-GeneID: 944854). The upstream and downstream homologous arm fragments were amplified by PCR, and the above fragments were fused by overlapping PCR to obtain the Δlpd gene fragment (upstream homologous arm-downstream homologous arm). The DNA fragment containing the target sequence used to construct pGRB-lpd was prepared by annealing primers gRNA-Δlpd-S and gRNA-Δlpd-A. Competent cells of VAL8 were prepared. Following the gene editing method described above, a partial fragment of the original lpd gene in the VAL8 engineered bacteria was knocked out, and a new gRNA cleavage sequence (gRNA-lpd*-S) was introduced. The new gRNA cleavage sequence was designed into the UP-Δlpd-A primer and the DN-Δlpd-S primer. After screening for positive strains, the plasmid pGRB-lpd was eliminated to obtain strain VAL8:Δlpd. The successful knockout of the lpd gene was verified by PCR and electrophoresis in the positive strains.

[0070] Using the E. coli MG1655 genome as a template, upstream homologous arm primers (UP-lpd*-S, UP-lpd*-A) and downstream homologous arm primers (DN-lpd*-S, DN-lpd*-A) were designed based on the upstream and downstream sequences of its lpd gene. The upstream and downstream homologous arm fragments were amplified by PCR, and these fragments were fused using overlap PCR to obtain lpd. G961A Gene fragment (upstream homologous arm - downstream homologous arm); construct pGRB-lpd G961AThe DNA fragment containing the target sequence was prepared by annealing with primers gRNA-lpd*-S and gRNA-lpd*-A. VAL8:Δlpd competent cells were prepared, and the recombinant fragment and plasmid pGRB-lpd were then combined. G961A Electroporation was performed on competent VAL8:Δlpd cells, and positive strains were screened. The plasmid was then removed to obtain strain VAL9. Positive strains were verified by PCR and electrophoresis to confirm lpd. G961A The mutated gene was successfully integrated.

[0071] Example 2: Production of L-valine by shake-flask fermentation of genetically engineered bacteria VAL8 and VAL9

[0072] The method for producing L-valine using the genetically engineered strain constructed above via shake-flask fermentation is as follows:

[0073] Slant activation culture: Streaking the -80℃ preserved bacterial strain onto the activation slant and incubating at 37℃ for 12 hours.

[0074] Seed culture: Use an inoculation loop to scrape a loop of slanted seeds and inoculate them into a 500mL Erlenmeyer flask containing 30mL of seed culture medium. Seal the flask with nine layers of gauze and incubate at 37℃ and 200rpm for 8-10 hours.

[0075] Fermentation culture: Inoculate 10-15% of the seed culture volume into a 500mL Erlenmeyer flask containing fermentation medium (final volume 30mL), seal with nine layers of gauze, and culture at 37℃ with shaking at 60r / min. During fermentation, add ammonia water in small amounts multiple times according to the phenol red indicator to control the pH of the medium at around 7.0. When the glucose in the medium is consumed, add 1mL of 60% (m / v) glucose solution until fermentation is complete. The fermentation cycle is 20h.

[0076] Activated slant culture medium: glucose 1g / L, peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, NaCl 5g / L, agar powder 25g / L, the remainder being water.

[0077] Seed culture medium: glucose 20 g / L, yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, the remainder being water.

[0078] Shake-flask fermentation medium: glucose 20 g / L, yeast extract 2 g / L, sodium citrate 1 g / L, (NH4)2SO4 3 g / L, KH2PO4 3 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 40 mg / L, MnSO4·7H2O 10 mg / L, VB1, VB3, VB5, VB12, and VH each 2 mg / L, plus 2% (v / v) phenol red solution, the remainder being water.

[0079] VAL8, after 20 hours of shake-flask fermentation, can produce 21.54 g / L of L-valine and OD. 600 The value was 12.60; VAL9, after 20 hours of shake-flask fermentation, could produce 24.36 g / L of L-valine, with an OD of 12.60. 600 The result was 14.15. These results indicate that mutating the lpd gene encoding lipoamide dehydrogenase improved the strain's growth performance by 12.30% and yield by 13.09% under limited oxygen conditions.

[0080] Example 3: Production of L-valine by fermentation of genetically engineered bacteria VAL9 in a fermenter

[0081] The method for producing L-valine using the genetically engineered strain constructed above in a 5L fermenter is as follows:

[0082] Slant activation culture: Scrape a loopful of inoculum from the -80℃ refrigerator preservation tube, spread it evenly on the activation slant, incubate at 37℃ for 12 hours, then transfer to a flask and continue incubation for 12 hours.

[0083] Fermentation culture: Wash the bacterial cells from the flask with an appropriate amount of sterile water, and inoculate the bacterial suspension into a 5L fermenter containing 3L of fermentation medium to begin fermentation. During fermentation, maintain the pH at approximately 7.0, the temperature at 35℃, the aeration rate at 0.2L / min, and the rotation speed at 300r / min. After fermentation begins and the base sugar in the medium is depleted, maintain the glucose concentration in the fermentation medium at 0.1-2g / L by adjusting the feed flow rate.

[0084] The composition of the slant and flask culture media was: glucose 1 g / L, peptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, NaCl 5 g / L, agar 25 g / L, and the remainder was water;

[0085] The fermentation medium consisted of: 10 g / L glucose, 2 g / L yeast extract, 1 g / L sodium citrate, 3 g / L (NH4)2SO4, 3 g / L KH2PO4, 1 g / L MgSO4·7H2O, 40 mg / L FeSO4·7H2O, 10 mg / L MnSO4·7H2O, 2 mg / L each of VB1, VB3, VB5, VB12, and VH, with the remainder being water.

[0086] After culturing the genetically engineered strain VAL9 in a 5L fermenter for 32 hours, the accumulation of L-valine reached 92 g / L, with a conversion rate of 60.4%. No significant accumulation of other amino acids or organic acids as byproducts was observed. The fermentation process curve is attached. Figure 1 .

[0087] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A genetically engineered bacterium for producing L-valine, characterized in that: The genetically engineered bacteria used *Escherichia coli* as a host and heterologously overexpressed *Bacillus subtilis* encoding acetolactate synthase. alss Genes and encoding leucine dehydrogenase bcd The gene, and the branched-chain amino acid membrane transporter encoded by Corynebacterium glutamicum. brnFE The gene, and overexpression of the *E. coli* mutant L67E / R68F / K75E encoding keto-olate reductase. ilvC Gene, encoding dihydroxy acid dehydratase ilvD The gene encodes glyceraldehyde-3-phosphate dehydrogenase. gapA Genes and encoding nicotinic acid phosphoribosyltransferase pncB Gene; and does not express the gene encoding malate dehydrogenase. mdh Gene; and replace lipoamide dehydrogenase with mutant A321T; The alss The nucleotide sequence of the gene is shown in the sequence with accession number 936852 on NCBI. The bcd The nucleotide sequence of the gene is shown in the sequence with accession number 938670 on NCBI. The brnFE Genes are brnE Genes and brnF Gene; The brnE The nucleotide sequence of the gene is shown in the KEGG accession number cg0315; brnF The nucleotide sequence of the gene is shown in the KEGG accession number cg0314. The ketool acid reductase mutant L67E / R68F / K75E is a protein mutant obtained by replacing leucine with glutamic acid at position 67, arginine with phenylalanine at position 68, and lysine with glutamic acid at position 75 of the ketool acid reductase amino acid sequence; wherein, the amino acid sequence of the ketool acid reductase is shown in the sequence with accession number NP_418222.1 on NCBI; The ilvD The nucleotide sequence of the gene is shown in the sequence with accession number 948277 on NCBI. The gapA The nucleotide sequence of the gene is shown in the sequence with accession number 947679 on NCBI. The pncB The nucleotide sequence of the gene is shown in the sequence with accession number 946648 on NCBI. The mdh The nucleotide sequence of the gene is shown in the sequence with accession number 947854 on NCBI. The mutant A321T is a protein mutant obtained by replacing the amino acid at position 321 of the amino acid sequence of lipoamide dehydrogenase with threonine instead of alanine; wherein the amino acid sequence of the lipoamide dehydrogenase is shown as the sequence with accession number NP_414658.1 on NCBI.

2. The genetically engineered bacteria according to claim 1, characterized in that: The alss gene is linked to the promoter P. trc ; and / or The bcd Gene linkage has promoter P trc ; and / or The brnFE Gene linkage has promoter P trc ; and / or The ilvC Gene linkage has promoter P trc ; and / or The ilvD Gene linkage has promoter P trc ; and / or The gapA Gene linkage has promoter P trc ; and / or The pncB Gene linkage has promoter P trc .

3. The genetically engineered bacteria according to claim 1, characterized in that: The host is one of E. coli MG1655, E. coli W3110, E. coli BL21 or E. coli BW25113.

4. A method for constructing the genetically engineered bacteria according to any one of claims 1-3, comprising: Introducing Bacillus subtilis, which encodes acetolactate synthase, into the host Escherichia coli. alss Genes and encoding leucine dehydrogenase bcd Genes and overexpression; Furthermore, it introduces the branched-chain amino acid membrane transport protein encoded by Corynebacterium glutamicum. brnFE Genes and overexpression; Furthermore, overexpression of the *E. coli* mutant encoding keto-olate reductase L67E / R68F / K75E ilvC Gene, encoding dihydroxy acid dehydratase ilvD The gene encodes glyceraldehyde-3-phosphate dehydrogenase. gapA Genes and encoding nicotinic acid phosphoribosyltransferase pncB Gene; And will encode malate dehydrogenase mdh Gene knockout or inactivation; And the lipoamide dehydrogenase gene lpd Replace it with the gene encoding the mutant A321T; The alss The nucleotide sequence of the gene is shown in the sequence with accession number 936852 on NCBI. The bcd The nucleotide sequence of the gene is shown in the sequence with accession number 938670 on NCBI. The brnFE Genes are brnE Genes and brnF Gene; The brnE The nucleotide sequence of the gene is shown in the KEGG accession number cg0315; brnF The nucleotide sequence of the gene is shown in the KEGG accession number cg0314. The ketool acid reductase mutant L67E / R68F / K75E is a protein mutant obtained by replacing leucine with glutamic acid at position 67, arginine with phenylalanine at position 68, and lysine with glutamic acid at position 75 of the ketool acid reductase amino acid sequence; wherein, the amino acid sequence of the ketool acid reductase is shown in the sequence with accession number NP_418222.1 on NCBI; The ilvD The nucleotide sequence of the gene is shown in the sequence with accession number 948277 on NCBI. The gapA The nucleotide sequence of the gene is shown in the sequence with accession number 947679 on NCBI. The pncB The nucleotide sequence of the gene is shown in the sequence with accession number 946648 on NCBI. The mdh The nucleotide sequence of the gene is shown in the sequence with accession number 947854 on NCBI. The mutant A321T is a protein mutant obtained by replacing the amino acid at position 321 of the amino acid sequence of lipoamide dehydrogenase with threonine instead of alanine; wherein the amino acid sequence of the lipoamide dehydrogenase is shown as the sequence with accession number NP_414658.1 on NCBI.

5. The use of the genetically engineered bacteria according to any one of claims 1-3 in the fermentation production of L-valine.

6. A method for producing L-valine using the genetically engineered bacteria according to any one of claims 1-3, comprising: The genetically engineered bacteria were cultured in a culture medium to produce L-valine; In addition, the L-valine is collected from the genetically engineered bacteria and / or the culture medium.

7. A lipoamide dehydrogenase mutant, the amino acid sequence of which is obtained by replacing alanine at position 321 of the amino acid sequence shown in NCBI-ProteinID:NP_414658.1 with threonine.

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