Recombinant escherichia coli for inactivating or weakly expressing SPMB family inner membrane protein and application of recombinant escherichia coli in production of L-amino acid

Abstract

The invention discloses recombinant escherichia coli for inactivating or weakly expressing SPMB family inner membrane protein and application of the recombinant escherichia coli in production of L-amino acid. The invention relates to the technical field of gene engineering, and provides recombinant escherichia coli, and SPMB family inner membrane protein in the recombinant escherichia coli is inactivated or reduced in activity. According to the invention, the yjiG gene (coding gene of SPMB family inner membrane protein) in escherichia coli is knocked out, so that the expression intensity of the yjiG gene is weakened, and fermentation production strains of L-threonine, L-tryptophan, L-arginine and L-valine, which are more cost-effective, can be produced. The invention has important significance for improving the fermentation yield of bacteria, namely L-threonine, L-tryptophan, L-arginine and L-valine.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to recombinant Escherichia coli inactivating or weakly expressing SPMB family inner membrane proteins and application thereof in producing L-amino acids. Background Art

[0002] L-threonine is one of the eight essential amino acids and is an amino acid that humans and animals cannot synthesize by themselves. L-threonine can enhance grain absorption, regulate metabolic balance in the body, and promote growth and development of the body. It is widely used in feed, medicine and food industries. L-tryptophan plays an important role in the growth, development and metabolism of humans and animals. It is called the second essential amino acid. Its role is manifested in that it can directly affect the physiological and biochemical processes such as growth and metabolism of animals. On the other hand, it can be metabolized and transformed into nicotinamide, nicotinic acid and 5-hydroxytryptamine (5-HT) in the body of animals. These transformation products play an important role in the body. As an essential amino acid, L-tryptophan is one of the important raw materials for protein synthesis. With the development of aquaculture and medical and health products, the market demand for tryptophan is also increasing. The main methods for producing tryptophan are chemical synthesis, enzyme method and fermentation method. The fermentation method for producing tryptophan has the advantages of mild reaction conditions, low cost, green, clean and environmentally friendly. Therefore, in the context of low-carbon and environmental protection, it is of great significance to use fermentation to produce tryptophan, optimize the fermentation process of tryptophan, and further increase the yield of tryptophan. L-arginine is a semi-essential basic amino acid in humans and animals. It is an important raw material for synthesizing protein creatine and an important intermediate metabolite in the urea cycle of organisms. Therefore, it has a wide range of uses in the pharmaceutical and food industries. L-valine is a branched-chain amino acid that promotes the development of mammalian mammary cells, improves the immune status of animals, and accelerates the repair of muscle tissue. It is widely used in the pharmaceutical, food, and feed industries. At present, L-valine is mainly produced in industry through microbial fermentation. With the increasing market demand for L-valine, researchers are increasingly paying attention to continuously optimizing the fermentation performance of production strains and developing more economical and efficient fermentation processes to improve the production efficiency of valine.

[0003] Breeding efficient strains with L-amino acid production capacity is the key to the industrial application of microbial fermentation. At present, there are two main methods for breeding L-amino acid production strains: 1) Rational metabolic engineering: This method mainly uses efficient gene editing technology to systematically transform the L-amino acid synthesis network in the chassis microorganisms to maximize the redirection of carbon metabolic flux to the L-amino acid synthesis pathway, mainly including blocking the L-amino acid degradation pathway, removing the feedback inhibition regulation mechanism of key enzymes in the synthesis pathway, enhancing the supply of precursors, optimizing the balance of chassis cell coenzyme supply, modifying the transmembrane transport system, etc. 2) Irrational adaptive evolution: Adaptive evolution refers to a breeding strategy that changes the growth environment of microorganisms under laboratory conditions, allowing microorganisms to spontaneously mutate in the process of environmental adaptation and complete the evolution process, and then rationally analyzes the evolutionary results. Since this strategy obtains mutant strains in an irrational way and then analyzes them in a rational way, it is a semi-rational breeding strategy that combines rationality and irrationality. Compared with mutagenesis breeding, the spontaneous mutations in the adaptive evolution process are screened and strengthened generation by generation due to the limitation of specific culture conditions, so it is more directional and greatly reduces the number of irrelevant mutation samples.

[0004] yjiG The gene encodes the inner membrane protein of the SPMB family, and the gene expression level has decreased significantly during the anaerobic adaptive evolution process. yjiG The effect of reduced gene expression on L-amino acid production has not been studied. In particular, there is no research on the effect of reduced gene expression on L-amino acid production. yjiG Genes for the production of L-threonine, L-tryptophan, L-arginine and L-valine in Enterobacteriaceae have been reported. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant Escherichia coli in which an SPMB family inner membrane protein is inactivated or weakly expressed, and an application of the recombinant Escherichia coli in producing L-amino acids.

[0006] In a first aspect, the present invention claims a recombinant Escherichia coli.

[0007] The inner membrane protein of the SPMB family in the recombinant Escherichia coli claimed by the present invention is inactivated or has reduced activity.

[0008] Furthermore, the recombinant Escherichia coli is obtained by inhibiting the expression and / or activity of the SPMB family inner membrane protein in the recipient Escherichia coli.

[0009] Among them, inhibiting the expression and / or activity of the SPMB family inner membrane protein refers to inhibiting the expression and / or activity of the target gene (the coding gene of the SPMB family inner membrane protein, i.e. yjiGCompared with the original expression intensity of the target gene, the expression intensity of the target gene is significantly weakened and / or the activity is significantly reduced after modification, and the specific method includes but is not limited to reducing the copy number of the target gene by using CRISPRi technology or RNAi interference technology. The same below.

[0010] Specifically, the inhibition of the expression and / or activity of the SPMB family inner membrane protein can be achieved by at least one of the following six regulatory levels: 1) inhibiting the coding gene of the SPMB family inner membrane protein at the gene transcription level (i.e. yjiG 1) inhibiting the expression of the coding gene of the SPMB family inner membrane protein at the post-transcriptional level (i.e., inhibiting the expression of the SPMB family inner membrane protein by splicing or processing the primary transcript of the relevant gene); 2) inhibiting the expression of the coding gene of the SPMB family inner membrane protein at the post-transcriptional level (i.e., inhibiting the expression of the SPMB family inner membrane protein by regulating the transport of the mRNA of the relevant gene from the nucleus to the cytoplasm); 3) inhibiting the expression of the SPMB family inner membrane protein at the RNA transport level of the gene (i.e., inhibiting the expression of the SPMB family inner membrane protein by regulating the transport of the mRNA of the relevant gene from the nucleus to the cytoplasm); 4) inhibiting the expression of the SPMB family inner membrane protein by regulating at the translation level of the gene; 5) inhibiting the expression of the SPMB family inner membrane protein by regulating at the mRNA degradation level of the gene; 6) inhibiting the activity of the SPMB family inner membrane protein by regulating at the post-translational level of the gene. The same below.

[0011] Furthermore, inhibiting the expression and / or activity of the SPMB family inner membrane protein in the recipient bacteria can be achieved by any of the following methods: knocking out or reducing the encoding gene of the SPMB family inner membrane protein in the genome of the recipient bacteria (i.e. yjiG gene) expression.

[0012] In a second aspect, the present invention claims protection for the use of the recombinant Escherichia coli described in the first aspect above in the fermentation production of L-amino acids.

[0013] In a third aspect, the present invention claims protection for any of the following applications: (A1) Application of inhibiting the expression and / or activity of SPMB family inner membrane proteins in increasing L-amino acid production; (A2) Use of a substance that inhibits the expression and / or activity of an inner membrane protein of the SPMB family for increasing L-amino acid production.

[0014] Furthermore, inhibiting the expression and / or activity of the SPMB family inner membrane protein can be achieved by any of the following methods: knocking out or reducing the expression of the gene encoding the SPMB family inner membrane protein in the genome of the recipient bacteria.

[0015] Furthermore, the substance is a substance that can knock out or reduce the expression of the gene encoding the SPMB family inner membrane protein in the genome of the recipient bacteria.

[0016] Wherein, the recipient bacteria may be bacteria, such as Enterobacteriaceae bacteria.

[0017] In one embodiment of the present invention, the recipient bacteria is Escherichia coli.

[0018] In a fourth aspect, the present invention claims a method for producing L-amino acids.

[0019] The method for producing L-amino acids claimed in the present invention may include the following steps: culturing the recombinant Escherichia coli described in the first aspect above, and obtaining L-amino acids from the fermentation products.

[0020] Wherein, the cultivation of the recombinant Escherichia coli can be carried out by a person skilled in the art using methods in the prior art, or by optimizing and improving the fermentation method through conventional experiments. The fermentation culture can be carried out in a suitable culture medium under fermentation conditions known in the art. The culture medium can contain: a carbon source, a nitrogen source, trace elements and combinations thereof. During the cultivation, the pH of the culture can be adjusted. During the cultivation, the temperature of the culture can be controlled. During the cultivation, the rotation speed can be controlled. During the cultivation, the fermentation time can be controlled. During the cultivation, the dissolved oxygen can be controlled.

[0021] In the implementation case of the present invention, the specific culture medium formula and fermentation process used refer to the relevant part of Example 3.

[0022] In each of the above related aspects, the SPMB family inner membrane protein comprises or is any of the following: (B1) a protein containing or having the amino acid sequence shown in SEQ ID No.6; (B2) A protein derived from bacteria and having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the protein defined in (B1) and associated with the production of L-amino acids.

[0023] In the above proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using an identity search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0024] The 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or more identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or more identity may be at least 95%, 96%, 97%, 98%, or 99% identity.

[0025] In each of the above-mentioned related aspects, the coding gene of the SPMB family inner membrane protein (i.e. yjiG Gene) contains or is any of the following: (C1) the DNA molecule shown in SEQ ID No.5; (C2) A DNA molecule derived from bacteria that has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the DNA sequence defined in (C1) and encodes the SPMB family inner membrane protein. In the above genes, the identity of the nucleotide sequence can be determined using an identity search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default value), respectively, and searching for a pair of nucleotide sequences to calculate the identity, and then the identity value (%) can be obtained.

[0026] In the above genes, the above 95% identity may be at least 96%, 97%, 98% identity. The above 90% identity may be at least 91%, 92%, 93%, 94% identity. The above 85% identity may be at least 86%, 87%, 88%, 89% identity. The above 80% identity may be at least 81%, 82%, 83%, 84% identity.

[0027] In each of the above related aspects, the L-amino acid may be L-threonine, L-tryptophan, L-arginine and / or L-valine.

[0028] In a fifth aspect, the present invention claims a method for constructing an engineered bacterium for producing L-amino acids.

[0029] The method for constructing an engineered bacterium for producing L-amino acids claimed in the present invention may include the following steps: inhibiting the expression and / or activity of the SPMB family inner membrane protein in the recipient bacterium.

[0030] Furthermore, inhibiting the expression and / or activity of the SPMB family inner membrane protein in the recipient bacteria can be achieved by any of the following methods: knocking out or reducing the expression of the gene encoding the SPMB family inner membrane protein in the genome of the recipient bacteria.

[0031] Furthermore, the recipient bacteria may be bacteria, such as Enterobacteriaceae.

[0032] In one embodiment of the present invention, the recipient bacteria is Escherichia coli.

[0033] In each of the above-mentioned related aspects, the expression and / or activity of the SPMB family inner membrane protein can be inhibited by introducing into the recipient bacteria a gene encoding a target for the SPMB family inner membrane protein (i.e. yjiG Accordingly, the substance may be a gene encoding a target protein of the SPMB family (i.e. yjiG Gene editing tools such as siRNA or shRNA.

[0034] In one embodiment of the present invention, the gene editing tool is a CRISPR / Cas9 editing tool. Specifically, the CRISPR / Cas9 editing tool targets the gene encoding the inner membrane protein of the SPMB family (i.e. yjiG gene), whose target sequence is shown in SEQ ID No.1.

[0035] In each of the above related aspects, the bacterium is a bacterium capable of producing L-amino acids.

[0036] "Bacteria capable of producing L-amino acids" means that the bacteria have the following abilities: the ability to produce and accumulate L-amino acids in the bacteria using external substances (such as culture medium), and further the ability to secrete L-amino acids into the culture system. Thus, L-amino acids can be collected when the bacteria are cultured in the culture medium.

[0037] The recombinant bacteria can be used to produce a variety of products, such as glutamic acid, glycine, alanine, leucine, isoleucine, methionine, proline, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, aspartic acid, histidine, shikimic acid, protocatechuic acid, succinic acid, alpha-ketoglutaric acid, citric acid, ornithine, citrulline, etc.

[0038] The bacteria may be wild-type bacteria collected naturally or modified bacteria.

[0039] "Modified bacteria" refers to modified bacteria obtained by artificially mutating and / or inducing mutations in naturally collected wild-type bacteria.

[0040] Furthermore, the bacteria may be from the genus Escherichia ( Escherichia sp. ), Erwinia (Erwinia sp. ), Agrobacterium ( Agrobacterium sp. ), Flavobacterium ( Flavobacterium sp. )、Alcaligenes spp. Alcaligenes sp. ), Pseudomonas ( Pseudomonas sp. ), Bacillus ( Bacillus sp. ), Brevibacterium ( Brevibacterium sp. ), Corynebacterium ( Corynebacterium sp. ), Aerobacter spp. Aerobacter sp. ), Enterobacter spp. Enterobacteria sp. ), Micrococcus ( Micrococcus sp. ), Serratia spp. Serratia sp. ), Salmonella spp. Salmonella sp. ), Streptomyces ( Streptomyces sp. ), Providencia spp. Providencia sp. ), etc., but not limited to.

[0041] Furthermore, the bacteria may be Escherichia coli (also known as Escherichia coli) ( Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum )、Brevibacillus lactofermentans( Brevibacterium lactofermentum )、Breibacterium flavum( Brevibacterium flavum )、Corynebacterium pekinensis( Corynebacterium pekinense )、Brevibacterium ammoniaphagus( Brevibacterium ammoniagenes )、Corynebacterium obtusiformis( Corynebacterium crenatum ) or Pantoea ( Pantoea ), but not limited to this.

[0042] In the implementation case of the present invention, the bacteria is specifically Escherichia coli.

[0043] In a specific implementation case of the present invention, the receptor Escherichia coli used in the fermentation production of L-threonine is Escherichia coli W3110 or Escherichia coli CGMCC No. 25404 (reference biological material: YP0158; depository: General Microbiological Center of China National Microbiological Culture Collection Committee; abbreviation of depository: CGMCC; address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: July 25, 2022; depository registration number: CGMCC No. 25404). Accordingly, the recombinant Escherichia coli (or the engineered bacteria) is Escherichia coli W3110-Δ yjiG or E. coli YPThr-Δ yjiG ; The Escherichia coli W3110-Δ yjiG To knock out the Escherichia coli W3110 genome yjiG The recombinant bacteria obtained by recombining the gene (i.e., the coding gene of the SPMB family inner membrane protein mentioned above, the same below); the Escherichia coli YPThr-Δ yjiG To knock out the yjiG The recombinant bacteria obtained after gene expression.

[0044] In a specific implementation case of the present invention, the receptor Escherichia coli used in the fermentation production of L-tryptophan is Escherichia coli W3110 or Escherichia coli CGMCC No. 25403 (reference biological material: YP006D; depository: General Microbiological Center of China National Microbiological Culture Collection Committee; abbreviation of depository: CGMCC; address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: July 25, 2022; depository registration number: CGMCC No. 25403). Accordingly, the recombinant Escherichia coli (or the engineered bacteria) is the Escherichia coli W3110-Δ yjiG or E. coli YPTrp-Δ yjiG ; The Escherichia coli YPTrp-Δ yjiG To knock out the E. coli CGMCC No.25403 genome yjiG The recombinant bacteria obtained after gene expression.

[0045] In a specific implementation case of the present invention, the receptor Escherichia coli used in the fermentation production of L-arginine is Escherichia coli W3110 or Escherichia coli CGMCC No.25402 (reference biological material: YP004-8; depository institution: General Microbiological Center of China National Microbiological Culture Collection Committee; abbreviation of depository institution: CGMCC; address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: July 25, 2022; depository registration number: CGMCC No.25402); accordingly, the recombinant Escherichia coli (or the engineered bacteria) is the Escherichia coli W3110-Δ yjiG or E. coli YPR-Δ yjiG ; The E. coli YPR-Δ yjiG To knock out the yjiG The recombinant bacteria obtained after gene expression.

[0046] In a specific implementation case of the present invention, the receptor Escherichia coli used in the fermentation production of L-valine is Escherichia coli W3110 or Escherichia coli CGMCC No.22721 (reference biological material: YP045; depository institution: General Microbiological Center of China National Microbiological Culture Collection Committee; abbreviation of depository institution: CGMCC; address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: June 15, 2021; depository registration number: CGMCC No.22721); accordingly, the recombinant Escherichia coli (or the engineered bacteria) is the Escherichia coli W3110-Δ yjiG or E. coli YPV-Δ yjiG ; The Escherichia coli YPV-Δ yjiG To knock out the yjiG The recombinant bacteria obtained after gene expression.

[0047] Experiments have shown that the inactivation or reduction of the activity of the inner membrane protein of the SPMB family can produce more cost-effective L-threonine, L-tryptophan, L-arginine and L-valine fermentation production strains. The present invention is of great significance for increasing the fermentation yield of L-threonine, L-tryptophan, L-arginine and L-valine. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the pREDCas9 plasmid map.

[0049] Figure 2 This is the map of pGRB-yjiG gRNA plasmid.

[0050] Collection Instructions 1 Classification name: Escherichia coli (Escherichia coli ); Reference biological material: YP0158; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Abbreviation of depository institution: CGMCC; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: July 25, 2022; The registration number of the Collection Center is: CGMCC No.25404.

[0051] In the present invention, the strain is referred to as Escherichia coli CGMCC No.25404.

[0052] Collection Notes 2 Classification name: Escherichia coli ( Escherichia coli ); Reference biological material: YP006D; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Abbreviation of depository institution: CGMCC; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: July 25, 2022; The registration number of the Collection Center is: CGMCC No.25403.

[0053] In the present invention, the strain is referred to as Escherichia coli CGMCC No.25403.

[0054] Collection Instructions 3 Classification name: Escherichia coli ( Escherichia coli ); Reference biological materials: YP004-8; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Abbreviation of depository institution: CGMCC; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: July 25, 2022; The registration number of the Collection Center is: CGMCC No.25402.

[0055] In the present invention, the strain is referred to as Escherichia coli CGMCC No.25402.

[0056] Collection Instructions 4 Classification name: Escherichia coli ( Escherichia coli ); Reference biological materials: YP045; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Abbreviation of depository institution: CGMCC; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: June 15, 2021; The registration number of the Collection Center is: CGMCC No.22721.

[0057] In the present invention, the strain is referred to as Escherichia coli CGMCC No.22721. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0059] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0060] The amino acid sequence of the SPMB family inner membrane protein involved in the following examples is shown in SEQ ID No.6, and its coding gene sequence in the Escherichia coli genome (i.e. yjiG The gene sequence is shown in SEQ ID No.5.

[0061] The primers used in the construction of each engineering bacteria involved in the following examples are summarized in Table 1.

[0062]

[0063] Example 1. Construction of engineered strains for producing L-amino acids In this example, an engineered bacterium W3110-Δ was constructed for producing L-amino acids (including L-threonine, L-tryptophan, L-arginine and L-valine). yjiG , and the L-threonine high-producing strain YPThr-Δ yjiG , L-tryptophan high-producing strain YPTrp-Δ yjiG , L-arginine high-yielding strain YPR-Δ yjiG and L-valine high-yielding strain YPV-Δ yjiG .

[0064] The above-mentioned engineering strains were generated using the CRISPR / Cas9 gene editing method (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.) Knockout of different receptors in the Escherichia coli genome yjiG The two plasmids (pREDCas9 and pGRB-yjiG gRNA) used in this method are shown in Figure 1 and Figure 2 The pREDCas9 plasmid carries the elimination system of the expression plasmid pGRB, the Red recombination system of λ phage and the Cas9 protein expression system, spectinomycin resistance (working concentration: 100 mg / L), and is cultured at 32°C; the pGRB-yjiG gRNA plasmid uses pUC18 as the backbone, including yjiG gRNA sequence, promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100 mg / L), culture at 37°C.

[0065] 1. Engineering bacteria W3110-Δ yjiG Construction 1. Construction of pGRB-yjiG gRNA plasmid The purpose of constructing the plasmid pGRB-yjiG gRNA is to transcriptionally target the Escherichia coli genome yjiG The gRNA of the gene forms a complex with the Cas9 protein, and recognizes the target gene target site through base pairing and PAM to achieve double-strand break of the target DNA. The pGRB-yjiG gRNA plasmid is constructed by recombination of the DNA fragment containing the target sequence with the linearized vector fragment.

[0066] (1) Target sequence design Design target sequences using CRISPR RGEN Tools as follows: 5'-GGCAACCCGGTGCAAAATGT-3' (SEQ ID No. 1).

[0067] (2) Preparation of DNA fragments containing target sequences Design primers: 5'-linearized vector end sequence (15 bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector end sequence (15 bp)-3' and its reverse complementary primer (i.e. gRNA-yjiG -S and gRNA- yjiG -A), prepare a DNA fragment containing the target sequence (i.e., the insert fragment in step (4) below) by annealing the single-stranded DNA. Reaction conditions: pre-denaturation at 95°C for 5 min; annealing at 30-50°C for 1 min. The annealing system is shown in Table 2.

[0068]

[0069] (3) Preparation of linear vector Using pGRB plasmid (product of Addgene, catalog number #71539) as a template, primers pGRB-PF and pGRB-PR (for specific sequences, see Table 1) were used for PCR amplification to obtain a linear vector.

[0070] (4) Recombination reaction The recombination system is shown in Table 3. The recombinant enzyme used was NEBuilder enzyme (purchased from NEB Company), and the recombination conditions were: 50°C, 30min.

[0071]

[0072] (5) Conversion Take 4 μL of the recombinant reaction solution and add it to 100 mL of E. coli DH5α transfection competent cells (purchased from TAKARA). Mix gently and place on ice for 20 min. Heat shock at 42℃ for 45-90 s. Immediately place on ice for 2-3 min. Add 900 μL of SOC medium (purchased from TAKARA) and recover at 37℃ for 1 h. Centrifuge at 8000 rpm for 2 min, discard part of the supernatant, keep about 200 μL, resuspend the bacteria and apply it to a plate containing 100 mg / L ampicillin. Invert the plate and culture it at 37℃ overnight. After a single colony grows on the plate, perform colony PCR identification using primers sgRNA-PF / sgRNA-PR (specific sequences are shown in Table 1). The 457 bp (SEQ ID No.3) is a positive recombinant, that is, the pGRB-yjiG gRNA plasmid is obtained.

[0073] 2. Preparation of donor DNA fragments Used for yjiG The recombinant fragment of gene knockout is yjiG The upstream and downstream homology arms of the gene are composed of upstream homology arm and downstream homology arm. yjiG The upstream and downstream sequences of the gene were used as templates to design upstream and downstream homology arm primers (the primers for amplifying the upstream homology arm are UP in Table 1 -yjiG- S and UP -yjiG- A, the primers for amplifying the upstream homology arm are the DNA in Table 1-yjiG- S and DN -yjiG- A) The upstream and downstream homology arms were amplified by PCR using the KAPA HiFi HotStart High-Fidelity Amplification Kit, and then the donor DNA fragment was prepared by overlapping PCR. The PCR system and method are shown in Table 4.

[0074]

[0075] The overlapping PCR system is shown in Table 5.

[0076]

[0077] Note: The template consists of equimolar amounts of the amplified fragments of the upstream and downstream homology arms and the target gene, and the total amount does not exceed 10 ng.

[0078] PCR reaction conditions (KAPA HiFi HotStart High-Fidelity Amplification Kit): pre-denaturation (95°C) for 5 min; then 30 cycles: denaturation (98°C) for 10 s, annealing ((Tm-3 / 5)°C) for 15 s, extension at 72°C (this enzyme activity extends about 1 kb in 1 min); continue extension at 72°C for 10 min; maintain (4°C).

[0079] The sequence of the final amplified product (i.e., the donor DNA fragment) is shown in SEQ ID No.2.

[0080] 3. Transformation of plasmids and donor DNA fragments (1) Transformation with pREDCas9 The pREDCas9 plasmid (product of Addgene, catalog number #71541) was electroporated into the electroporation competent state of the starting strain (Escherichia coli W3110), and the cells were revived and cultured and then plated on LB plates containing spectinomycin and cultured overnight at 32°C. Single colonies growing on the resistant plates were subjected to colony PCR using identification primers pRedCas9-PF / pRedCas9-PR, and the positive recombinant containing the pREDCas9 plasmid was obtained with 943 bp (SEQ ID No. 4).

[0081] (2) Preparation of electroporation competent cells of target strain containing pREDCas9 The positive strain containing the pREDCas9 plasmid obtained in step (1) was cultured at 32°C until the OD 600 =0.1~0.2, add 0.1 M IPTG (to make the final concentration 0.1 mM), and continue to culture until OD 600=0.6-0.7 for competent cell preparation. The purpose of adding IPTG is to induce the expression of the recombinase on the pREDCas9 plasmid. The culture medium and preparation process required for the preparation of competent cells refer to conventional standard operations.

[0082] (3) Transformation of pGRB-yjiG gRNA and donor DNA fragment The pGRB-yjiG gRNA plasmid constructed in step 1 and the donor DNA fragment prepared in step 2 were simultaneously electroporated into the electroporation competent cells containing pREDCas9. The revived bacteria after electroporation were spread on LB plates containing ampicillin and spectinomycin and cultured at 32°C overnight. -yjiG- S / DN- yjiG -A (see Table 1) was verified by colony PCR, and the amplified fragment with a size of 606 bp (SEQ ID No. 2) was selected as the positive recombinant and maintained.

[0083] 4. Elimination of plasmids (1) Elimination of pGRB-yjiG gRNA plasmid The positive recombinants obtained in the previous step were placed in LB medium containing 0.2% arabinose for overnight culture, and then appropriately diluted and spread on LB plates containing spectinomycin resistance, and cultured at 32°C overnight. Single colonies were picked and streaked one by one on LB plates containing ampicillin and spectinomycin resistance, and single colonies that did not grow on the ampicillin plate but grew on the spectinomycin resistance plate were selected to continue the elimination of the pREDCas9 plasmid.

[0084] (2) Elimination of pREDCas9 plasmid The positive recombinant was transferred to LB liquid medium without resistance, cultured at 42℃ overnight, and then appropriately diluted and spread on LB plates without resistance, and cultured at 37℃ overnight. Single colonies were picked and streaked one by one on LB plates containing spectinomycin resistance and no resistance, and single colonies that did not grow on spectinomycin resistance plates and grew on non-resistance plates were selected to preserve the bacteria, thus obtaining the engineered bacteria W3110-Δ yjiG .

[0085] The final engineered bacteria W3110-Δ yjiG To knock out the Escherichia coli W3110 genome yjiG The recombinant bacteria of gene (SEQID No.5) are specifically obtained by deleting the sequence between the upstream homology arm (positions 1-284 of SEQ ID No.2) and the downstream homology arm (positions 285-606 of SEQ ID No.2) in the genome of Escherichia coli W3110, and keeping other sequences unchanged.

[0086] 2. L-threonine high-producing strain YPThr-Δ yjiG Construction See step 1. The only difference from step 1 is that the recipient bacteria is replaced by Escherichia coli W3110 with Escherichia coli CGMCC No. 25404.

[0087] The final engineered bacteria YPThr-Δ yjiG To knock out the E. coli CGMCC No.25404 genome yjiG The recombinant bacteria of gene (SEQ ID No.5) are specifically obtained by deleting the sequence between the upstream homology arm (positions 1-284 of SEQ ID No.2) and the downstream homology arm (positions 285-606 of SEQ ID No.2) in the genome of Escherichia coli CGMCC No.25404, and keeping other sequences unchanged.

[0088] 3. L-tryptophan high-producing strain YPTrp-Δ yjiG Construction See step 1. The only difference from step 1 is that the recipient bacteria is replaced by Escherichia coli W3110 with Escherichia coli CGMCC No. 25403.

[0089] The final engineered bacteria YPTrp-Δ yjiG To knock out the E. coli CGMCC No.25403 genome yjiG The recombinant bacteria of gene (SEQ ID No.5) are specifically obtained by deleting the sequence between the upstream homology arm (positions 1-284 of SEQ ID No.2) and the downstream homology arm (positions 285-606 of SEQ ID No.2) in the genome of Escherichia coli CGMCC No.25403, and keeping other sequences unchanged.

[0090] IV. L-arginine high-yielding strain YPR-Δ yjiG Construction See step 1. The only difference from step 1 is that the recipient bacteria is replaced by Escherichia coli W3110 with Escherichia coli CGMCC No. 25402.

[0091] The final engineered bacteria YPR-Δ yjiG To knock out the E. coli CGMCC No.25402 genome yjiG The recombinant bacteria of gene (SEQ ID No.5) are specifically obtained by deleting the sequence between the upstream homology arm (positions 1-284 of SEQ ID No.2) and the downstream homology arm (positions 285-606 of SEQ ID No.2) in the genome of Escherichia coli CGMCC No.25402, and keeping other sequences unchanged.

[0092] 5. L-valine high-yielding strain YPV-Δ yjiG Construction See step 1. The only difference from step 1 is that the recipient bacteria is replaced by Escherichia coli W3110 with Escherichia coli CGMCC No. 22721.

[0093] The final engineered bacteria YPV-Δ yjiG To knock out the E. coli CGMCC No.22721 genome yjiG The recombinant bacteria of gene (SEQ ID No.5) are specifically obtained by deleting the sequence between the upstream homology arm (positions 1-284 of SEQ ID No.2) and the downstream homology arm (positions 285-606 of SEQ ID No.2) in the genome of Escherichia coli CGMCC No.22721, and keeping other sequences unchanged.

[0094] Example 2: Construction of plasmid containing overexpression yjiG Genetically engineered strains One , yjiG Gene expression vector pET28(a)- yjiG Construction To build yjiG Gene overexpression plasmids require wild-type yjiG The gene (sequence as SEQ ID No. 5) was cloned into the expression vector pET28 (a). Escherichia coli )W3110 genome sequence was used as a template and PCR amplification was performed with primers yjiG-PF / yjiG-PR (sequence shown in Table 1) to obtain the wild-type yjiG Gene. After recovery and BamH I / Xho The expression vector pET28(a) (purchased from BioVector, containing kanamycin resistance) was digested with I and ligated with NEBuilder enzyme (purchased from NEB) at 50°C for 30 min. The ligation product was transformed into DH5α and spread on 2-YT agar plates containing kanamycin (50 mg / L) and cultured at 37°C to obtain yjiG pET28(a) transformants of genes yjiG The clones grown in culture were identified using primers T7 / T7t (sequences in Table 1) and r Taq PCR. The fragment containing 749 bp (sequence as SEQ ID No. 7) amplified by PCR was identified as the clone containing yjiG pET28(a) positive transformant of gene yjiG .

[0095] 2. Overexpression yjiG Construction of genetically engineered strains To verify yjiG Effect of gene overexpression on the production of L-amino acids such as L-threonine, L-tryptophan, L-valine and L-arginine by strains. yjiG The overexpression of the gene was studied in high-yield strains by introducing it into L-threonine-producing strain CGMCC No.25404, L-tryptophan-producing strain CGMCC No.25403, L-arginine-producing strain CGMCC No.25402 and L-valine-producing strain CGMCC No.22721. yjiG Effects of genes on the production of L-amino acids such as L-threonine, L-tryptophan, L-valine and L-arginine.

[0096] Prepare competent cells of E. coli of L-threonine CGMCC No.25404, L-tryptophan CGMCC No.25403, L-arginine CGMCC No.25402 and L-valine CGMCC No.22721. 600 =0.6, collect the bacteria to prepare competent cells, and transform the plasmid pET28(a)- yjiG , spread on 2-YT agar plates containing kanamycin (50 mg / L) and culture at 37°C. The single colonies produced by the culture were identified by primers T7 / T7t and r Taq PCR. The PCR amplified fragment containing 749 bp (such as SEQ ID No. 7) was a positive transformant, and the fragment that could not be amplified was the original bacterium.

[0097] PCR amplification system: 2×Premix r Taq (purchased from TAKARA) 12.5 μL, upstream and downstream primers (10 pM) 1 μL each, supplemented with ddH2O, total volume 25 μL.

[0098] PCR amplification program: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 90 s (30 cycles), and over-extension at 72°C for 10 min.

[0099] The overexpression plasmid pET28(a)- yjiG The recombinant bacteria obtained by introducing L-threonine producing bacteria CGMCC No.25404, L-tryptophan producing bacteria CGMCC No.25403, L-arginine producing bacteria CGMCC No.25402 and L-valine producing bacteria CGMCC No.22721 were named YPThr-pET28(a)- yjiG、YPTrp-pET28(a)- yjiG 、YPR-pET28(a)- yjiG 、YPV-pET28(a)- yjiG .

[0100] Recombinant bacteria YPThr-pET28(a)- yjiG 、YPTrp-pET28(a)- yjiG 、YPR-pET28(a)- yjiG and YPV-pET28(a)- yjiG The expression plasmid pET28(a)- yjiG Containing the yjiG Gene, pET28(a)- yjiG Overexpression recombinant bacteria can significantly and stably increase the expression level of SPMB family inner membrane proteins.

[0101] Example 3, L-amino acid fermentation test 1. L-threonine fermentation experiment Escherichia coli W3110, Escherichia coli CGMCC No. 25404, and the engineered bacteria W3110-Δ yjiG and YPThr-Δ yjiG , the engineered bacteria YPThr-pET28 (a) constructed in Example 2 yjiG The fermentation experiments were carried out in a 5L fermentation tank of BLBIO-5GC-4-H model (Shanghai Bailun Biotechnology Co., Ltd.) using L-threonine fermentation medium and culture conditions, and each strain was repeated three times. yjiG The strain contains the pET28(a) expression vector. IPTG induction is required during the fermentation process. The specific induction method is as follows: add IPTG at a final concentration of 0.1 mM after inoculation to induce yjiG Gene overexpression. After the fermentation, the L-threonine content was detected by high performance liquid chromatography (HPLC), and the results were averaged over three replicates, as shown in Table 6. Experimental results P<0.05 (*) indicates a significant difference, and P<0.01 (**) indicates an extremely significant difference.

[0102] L-threonine fermentation medium: the solvent is water, the solutes and their concentrations are glucose 13 g / L, (NH4)2SO4 1 g / L, H3PO4 0.5 g / L, KCl 0.8 g / L, MgSO4•7H2O 0.8 g / L, FeSO4•7H2O 0.01 g / L, MnSO4•H2O 0.01 g / L, FM902 yeast powder 1.5 g / L, corn syrup 5 g / L, molasses 17 g / L, and the pH is adjusted to 7.0 by adding ammonia.

[0103] L-Threonine fermentation conditions: Calibrate DO 100%: temperature 37℃, air volume 5L / min, speed 800rpm, tank pressure 0Mpa, calibrate after 5min; Inoculum size 10% (v / v); Initial conditions: pH 7.0, culture temperature 37°C, tank pressure 0 MPa, air volume 0.5 L / min, rotation speed 400 rpm; Full process control: 1. When dissolved oxygen is less than 30%, the rotation speed is increased in sequence: 500rpm→600rpm→air volume 1L / min→700rpm→800rpm; 2. After 8 hours of fermentation, the tank pressure is increased by 0.01Mpa; after 12 hours of fermentation, the tank pressure is increased by 0.02Mpa→0.03Mpa→0.04Mpa→0.05Mpa; Residual sugar control: 0.1-0.5% before F12h; after F12h, the residual sugar should be controlled at 0.1-0.3% according to DO requirements; Feed materials: 25% ammonia water, 55% concentrated sugar, 10% sodium bicarbonate; Fermentation cycle: about 30 hours, the control process uses 20-30% dissolved oxygen as the standard for increasing and decreasing air volume.

[0104]

[0105] Note: ns means YPThr-pET28(a)- yjiG Compared with CGMCC No.25404, there was no significant difference in the yield of L-threonine (P>0.05).

[0106] As shown in the above fermentation results, both the high-yield L-threonine strain CGMCC No.25404 and the model strain W3110, yjiG Gene knockout can help increase L-threonine production, while overexpression yjiG The gene has no effect on improving the production of L-threonine.

[0107] 2. L-Tryptophan Fermentation Experiment Escherichia coli W3110, Escherichia coli CGMCC No. 25403, and the engineered bacteria W3110-ΔyjiG and YPTrp-Δ yjiG , the engineered bacteria YPTrp-pET28 (a) constructed in Example 2 yjiG The fermentation experiments were carried out in a 5L fermentation tank of BLBIO-5GC-4-H model (Shanghai Bailun Biotechnology Co., Ltd.) using L-tryptophan fermentation medium and culture conditions, and each strain was repeated three times. yjiG The strain contains the pET28(a) expression vector. IPTG induction is required during the fermentation process. The specific induction method is as follows: add IPTG at a final concentration of 0.1 mM after inoculation to induce yjiG Gene overexpression. After the fermentation, the L-tryptophan content was detected by high performance liquid chromatography (HPLC), and the results were averaged over three replicates, as shown in Table 7. Experimental results P<0.05 (*) indicates a significant difference, and P<0.01 (**) indicates an extremely significant difference.

[0108] L-tryptophan fermentation medium: the solvent is water, the solutes and their concentrations are glucose 7g / L, FM902 yeast powder 1g / L, (NH4)2SO41.2g / L, citric acid 1.2g / L, MgSO4•7H2O 1.5g / L, K2HPO4•3H2O 5.5g / L, defoaming agent 0.2mL / L, and ammonia is added to adjust the pH to 7.0.

[0109] L-Tryptophan Culture Conditions: Calibration DO 100%: temperature 35°C, speed 800rpm, air volume 5L / min, tank pressure 0.00Mpa; Inoculum size 10% (v / v); Initial conditions: temperature 35°C, pH 7.0, air volume 1.0 L / min, speed 350 rpm; Full process control: when dissolved oxygen is ≤20% before the end of the basal sugar, increase →400rpm→450rpm in sequence; when the basal sugar is consumed, add sugar to control the dissolved oxygen at 15-30%; pH F7.0 before 24h, F6.7 after 24h; Residual sugar control: 0.1-0.5% before F12h; after F12h, the residual sugar should be controlled at 0.1-0.3% according to DO requirements; Feed materials: 25% ammonia water, 55% concentrated sugar, 10% sodium bicarbonate; Fermentation cycle: about 34 hours, the control process uses 15-30% dissolved oxygen as the standard for increasing and decreasing air volume.

[0110]

[0111] Note: ns means YPTrp-pET28(a)- yjiGCompared with CGMCC No.25403, there was no significant difference in the production of L-tryptophan (P>0.05).

[0112] As shown in the above fermentation results, both the high-yield L-tryptophan strain CGMCC No.25403 and the model strain W3110, yjiG Gene knockout can help increase L-tryptophan production, while overexpression yjiG The gene has no effect on improving the production of L-tryptophan.

[0113] 3. L-arginine fermentation experiment Escherichia coli W3110, Escherichia coli CGMCC No. 25402, and the engineered bacteria W3110-Δ yjiG and YPR-Δ yjiG , the engineered bacteria YPR-pET28 (a) constructed in Example 2 yjiG The fermentation experiments were carried out in a 5L fermentation tank of BLBIO-5GC-4-H model (Shanghai Bailun Biotechnology Co., Ltd.) with L-arginine fermentation medium and culture conditions, and each strain was repeated three times. yjiG The strain contains the pET28(a) expression vector. IPTG induction is required during the fermentation process. The specific induction method is as follows: add IPTG at a final concentration of 0.1 mM after inoculation to induce yjiG Gene overexpression. After the fermentation, the L-arginine content was detected by high performance liquid chromatography (HPLC), and the results were averaged over three replicates, as shown in Table 8. Experimental results P<0.05 (*) indicates a significant difference, and P<0.01 (**) indicates an extremely significant difference.

[0114] L-arginine fermentation medium: the solvent is water, the solutes and their concentrations are glucose 8g / L, FM902 yeast powder 3g / L, K2HPO4•3H2O 6g / L, MgSO4•7H2O 1g / L, FeSO4•7H2O 0.05g / L, betaine 0.5g / L, VB 12 0.005g / L, defoamer 0.3mL / L, ammonium sulfate 3g / L, pH 7.2.

[0115] L-arginine fermentation conditions: Calibration DO 100%: temperature 35°C, pH 7.2, speed 100 rpm, air volume 6 L / min, tank pressure 0.00 MPa; Inoculum size 10% (v / v); Initial conditions: temperature 35°C, pH 7.2, tank pressure 0.01 MPa, air volume 1.5 L / min, speed 350 rpm; Full process control: control DO at 20-30%; when dissolved oxygen ≤ 25%, increase the speed each time: 300rpm→400rpm→2.0L / min→500rpm→0.02Mpa→600rpm→3.0L / min→0.03Mpa→700rpm→3.5L / min→0.04Mpa→800rpm→900rpm→4.0L / min→0.05Mpa→1000rpm; Residual sugar control: The residual sugar is controlled at 0.05-0.1% throughout the process; Feed materials: 25% ammonia water, 80% concentrated sugar, 10% sodium bicarbonate; Fermentation cycle: about 50h, the control process uses 20-30% dissolved oxygen as the standard for increasing and decreasing air volume.

[0116]

[0117] Note: ns means YPR-pET28(a)- yjiG Compared with CGMCC No.25402, there was no significant difference in the production of L-arginine (P>0.05).

[0118] As shown in the above fermentation results, whether it is the high-yield L-arginine strain CGMCC No.25402 or the model strain W3110, yjiG Gene knockout can help increase L-arginine production, while overexpression yjiG The gene has no effect on improving the production of L-arginine.

[0119] IV. L-valine fermentation experiment Escherichia coli W3110, Escherichia coli CGMCC No. 22721, and the engineered bacteria W3110-Δ yjiG and YPV-Δ yjiG , the engineered bacteria YPV-pET28 (a) constructed in Example 2 yjiG The fermentation experiments were carried out in a 5L fermentation tank of BLBIO-5GC-4-H model (Shanghai Bailun Biotechnology Co., Ltd.) using L-valine fermentation medium and culture conditions, and each strain was repeated three times. yjiG The strain contains the pET28(a) expression vector. IPTG induction is required during the fermentation process. The specific induction method is as follows: add IPTG at a final concentration of 0.1 mM after inoculation to induce yjiGGene overexpression. After the fermentation, the L-valine content was detected by high performance liquid chromatography (HPLC), and the results were averaged over three replicates, as shown in Table 9. Experimental results P<0.05 (*) indicates a significant difference, and P<0.01 (**) indicates an extremely significant difference.

[0120] L-valine fermentation medium: the solvent is water, the solutes and their concentrations are yeast extract powder 4g / L, corn steep liquor powder 2g / L, peptone 4g / L, methionine 2g / L, KH2PO4•3H2O 7g / L, MgSO4•7H2O 2g / L, CoCl220mg / L, (NH4)2SO43g / L, citric acid 2g / L, FeSO4•7H2O 50mg / L, MnSO4•7H2O 30mg / L, VH 20mg / L, VB11.5mg / L, VB31.5mg / L VB 12 1.5g / L, defoamer 0.3mL / L, (NH4)2SO43g / L, pH value 7.0.

[0121] L-valine fermentation conditions: Fermentation control temperature: 35℃; Fermentation period: 30h; Dissolved oxygen electrode calibration method: calibrate the zero point in saturated sodium sulfite solution and calibrate the hundred point in air; L-valine fermentation includes two stages of aerobic-limited oxygen fermentation. The cells are first cultured under aerobic fermentation. The air volume, speed and sugar addition rate are adjusted in the early stage to control the dissolved oxygen at about 25%. 600 When the value reaches 50-60, the rotation speed is reduced to 400rpm and the air volume is reduced to 2 L / min; and the aerobic fermentation is converted to oxygen-limited fermentation.

[0122]

[0123] Note: ns means YPV-pET28(a)- yjiG Compared with CGMCC No.22721, there was no significant difference in the yield of L-valine (P>0.05).

[0124] The above fermentation results show that for both the high-yield L-valine strain CGMCC No.22721 and the model strain W3110, yjiG Gene knockout can help increase L-valine production, while overexpression yjiG The gene has no effect on improving the production of L-valine.

[0125] From all the above fermentation results, it can be seen that for both the strains with high production of corresponding L-amino acids and the model strain W3110, yjiGThe knockout of genes all contributed to the increase of L-threonine, L-tryptophan, L-arginine and L-valine production, while overexpression yjiG The gene has no effect on improving the production of L-threonine, L-tryptophan, L-arginine and L-valine.

[0126] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A recombinant Escherichia coli, characterized in that: The SPMB family inner membrane protein in the recombinant Escherichia coli is inactivated or has reduced activity; The SPMB family inner membrane protein comprises any of the following: (B1) a protein comprising the amino acid sequence shown in SEQ ID No.6; (B2) A protein derived from bacteria and having 99% or more identity with the protein defined in (B1) and associated with the production of L-amino acids.

2. Use of the recombinant Escherichia coli according to claim 1 in fermentation production of L-amino acids.

3. The use according to claim 2, characterized in that: The L-amino acid is L-threonine, L-tryptophan, L-arginine and / or L-valine.

4. Any of the following applications: (A1) Application of inhibiting the expression and / or activity of SPMB family inner membrane proteins in increasing L-amino acid production; (A2) Use of a substance that inhibits the expression and / or activity of an inner membrane protein of the SPMB family for increasing L-amino acid production; The SPMB family inner membrane protein comprises any of the following: (B1) a protein comprising the amino acid sequence shown in SEQ ID No.6; (B2) A protein derived from bacteria and having 99% or more identity with the protein defined in (B1) and associated with the production of L-amino acids.

5. The use according to claim 4, characterized in that: Inhibiting the expression and / or activity of the SPMB family inner membrane protein is achieved by any of the following methods: knocking out or reducing the expression of the gene encoding the SPMB family inner membrane protein in the recipient bacterial genome; The substance is a substance that can knock out or reduce the expression of the coding gene of the SPMB family inner membrane protein in the genome of the recipient bacteria.

6. The use according to any one of claims 4 or 5, characterized in that: The L-amino acid is L-threonine, L-tryptophan, L-arginine and / or L-valine.

7. A method for producing L-amino acids, comprising the following steps: culturing the recombinant Escherichia coli according to claim 1, and obtaining L-amino acids from the fermentation products.

8. The method according to claim 7, characterized in that: The L-amino acid is L-threonine, L-tryptophan, L-arginine and / or L-valine.

9. A method for constructing an engineered bacterium for producing L-amino acids, comprising the steps of: inhibiting the expression and / or activity of an inner membrane protein of the SPMB family in a recipient bacterium; The SPMB family inner membrane protein comprises any of the following: (B1) a protein comprising the amino acid sequence shown in SEQ ID No.6; (B2) A protein derived from bacteria and having 99% or more identity with the protein defined in (B1) and associated with the production of L-amino acids.

10. The method according to claim 9, characterized in that The recipient bacteria are bacteria.

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