Recombinant Escherichia coli with inactivated or weakly expressed inner membrane protein of the SPMB family and its application in the production of L-amino acids

Abstract

The present invention discloses a recombinant Escherichia coli with inactivated or weakly expressed inner membrane proteins of the SPMB family and its application in the production of L-amino acids. The present invention relates to the technical field of genetic engineering. The present invention provides a recombinant Escherichia coli in which the inner membrane proteins of the SPMB family are inactivated or have reduced activity. The present invention knocks out the yjiG gene (the coding gene of the inner membrane protein of the SPMB family) in Escherichia coli to weaken the yjiG gene expression intensity, and can produce more cost-effective fermentation production strains of L-threonine, L-tryptophan, L-arginine and L-valine. The present invention is of great significance for improving the fermentation yields of bacterial L-threonine, L-tryptophan, L-arginine and L-valine.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a recombinant Escherichia coli with inactivated or weakly expressed inner membrane proteins of the SPMB family and its application in the production of L-amino acids. Background Art

[0002] L-threonine is one of the eight essential amino acids and is an amino acid that cannot be synthesized by humans and animals themselves. L-threonine can strengthen the absorption of grains, regulate the body's metabolic balance, and promote the growth and development of the body, and is widely used in the feed, pharmaceutical, and food industries. L-tryptophan plays an important role in the growth, development, and metabolism of humans and animals and is called the second essential amino acid. Its role is manifested in that it can directly affect the physiological and biochemical processes such as the growth and metabolism of animals, and on the other hand, it can be metabolically converted into nicotinamide, niacin, and 5-hydroxytryptamine (5-HT) in the animal body, and these conversion products play important roles in the body. As an essential amino acid, L-tryptophan is one of the important raw materials for protein synthesis. With the development of the aquaculture industry and pharmaceutical and health products, the market demand for tryptophan is also increasing continuously. The production methods of tryptophan mainly include chemical synthesis method, enzymatic method, and fermentation method. The fermentation method for producing tryptophan has the advantages of mild reaction conditions, low cost, green, clean, and environmental protection. Therefore, in the situation of low-carbon environmental protection, it is of great significance to use the fermentation method to produce tryptophan, optimize the fermentation process of tryptophan, and further improve the yield of tryptophan. L-arginine is a semi-essential basic amino acid in the human body and animals, is an important raw material for synthesizing protein creatine, and is also 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 belongs to branched-chain amino acids and has the functions of promoting the development of mammalian mammary gland cells, improving the immune state of animals, and accelerating the repair of muscle tissues, and is widely used in industries such as medicine, food, and feed. At present, L-valine is mainly produced by microbial fermentation in industry. With the continuous increase in the market demand for L-valine, continuously optimizing the fermentation performance of production strains and developing more economical and efficient fermentation processes to improve the production efficiency of valine have also increasingly attracted the attention of researchers.

[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 gene), the expression intensity of the gene is significantly weakened and / or the activity is significantly reduced after modification. The specific methods include, but are not limited to, using the CRISPRi technology or the RNAi interference technology to reduce the copy number of the target gene. The same applies hereinafter.

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

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

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

[0013] In a third aspect, the present invention claims any of the following uses:

[0014] (A1) The use of inhibiting the expression and / or activity of the inner membrane protein of the SPMB family in increasing the yield of L-amino acids;

[0015] (A2) The use of a substance that inhibits the expression and / or activity of the inner membrane protein of the SPMB family in increasing the yield of L-amino acids.

[0016] Furthermore, the inhibition of the expression and / or activity of the inner membrane protein of the SPMB family can be achieved by any of the following methods: knocking out or reducing the expression of the coding gene of the inner membrane protein of the SPMB family in the genome of the recipient bacterium.

[0017] Furthermore, the substance is a substance capable of knocking out or reducing the expression of the coding gene of the SPMB family inner membrane protein in the genome of the recipient bacterium.

[0018] Among them, the recipient bacterium can be a bacterium, such as a bacterium of the Enterobacteriaceae family.

[0019] In one embodiment of the present invention, the recipient bacterium is Escherichia coli.

[0020] Fourthly, the present invention claims to protect a method for producing L-amino acids.

[0021] The method for producing L-amino acids claimed by 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 product.

[0022] Among them, culturing the recombinant Escherichia coli can be carried out by those skilled in the art using methods in the prior art, and the fermentation method can also be optimized and improved through routine tests. The fermentation culture can be carried out in a suitable medium under fermentation conditions known in the art. The medium can contain: carbon source, nitrogen source, trace elements and their combinations. During the culture, the pH of the culture can be adjusted. During the culture, the temperature of the culture can be controlled. During the culture, the rotation speed can be controlled. During the culture, the fermentation time can be controlled. During the culture, the dissolved oxygen can be controlled.

[0023] In the embodiment of the present invention, for the specific medium formulation and fermentation process adopted, refer to the relevant part of Example 3.

[0024] In the above relevant aspects, the SPMB family inner membrane protein includes or is any one of the following:

[0025] (B1) a protein containing or being the amino acid sequence shown in SEQ ID No. 6;

[0026] (B2) a protein derived from bacteria and having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the protein defined in (B1) and being related to the production of L-amino acids.

[0027] For the above-mentioned proteins, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using identity search sites on the Internet, such as the BLAST web page on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0028] The above-mentioned identity of 80% or more can be an identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The above-mentioned identity of 85% or more can be an identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The above-mentioned identity of 90% or more can be an identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The above-mentioned identity of 95% or more can be an identity of at least 95%, 96%, 97%, 98% or 99%.

[0029] In each of the above-related aspects, the coding gene of the SPMB family inner membrane protein (i.e., yjiG gene) includes or is any one of the following:

[0030] (C1) The DNA molecule shown in SEQ ID No. 5;

[0031] (C2)A DNA molecule derived from bacteria that has an identity of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the DNA sequence defined by (C1) and encodes the inner membrane protein of the SPMB family. Among the above genes, the identity of nucleotide sequences can be determined using identity search sites on the Internet, such as the BLAST web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of nucleotide sequences, and then the identity value (%) can be obtained.

[0032] Among the above genes, the identity of more than 95% can be at least 96%, 97%, 98%. The identity of more than 90% can be at least 91%, 92%, 93%, 94%. The identity of more than 85% can be at least 86%, 87%, 88%, 89%. The identity of more than 80% can be at least 81%, 82%, 83%, 84%.

[0033] In the above related aspects, the L-amino acid can be L-threonine, L-tryptophan, L-arginine and / or L-valine.

[0034] In a fifth aspect, the present invention claims a method for constructing an engineered bacterium for producing L-amino acids.

[0035] The method for constructing an engineered bacterium for producing L-amino acids claimed by the present invention may include the following steps: inhibiting the expression and / or activity of the inner membrane protein of the SPMB family in the recipient bacterium.

[0036] Furthermore, inhibiting the expression and / or activity of the inner membrane protein of the SPMB family in the recipient bacterium can be achieved by any of the following methods: knocking out or reducing the expression of the coding gene of the inner membrane protein of the SPMB family in the genome of the recipient bacterium.

[0037] Furthermore, the recipient bacterium can be a bacterium, such as a bacterium of the Enterobacteriaceae family.

[0038] In one embodiment of the present invention, the recipient bacterium is Escherichia coli.

[0039] In the above related aspects, inhibiting the expression and / or activity of the inner membrane protein of the SPMB family can be achieved by introducing a gene editing tool targeting the coding gene of the inner membrane protein of the SPMB family (i.e., yjiG gene), or siRNA, shRNA, etc. into the recipient bacterium. Correspondingly, the substance can be a gene editing tool targeting the coding gene of the inner membrane protein of the SPMB family (i.e., yjiG gene), or siRNA, shRNA, etc.

[0040] 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 coding gene of the inner membrane protein of the SPMB family (i.e., yjiG gene), and its target sequence is shown as SEQ ID No.1.

[0041] In the above related aspects, the bacterium is a bacterium having the ability to produce L-amino acids.

[0042] "A bacterium having the ability to produce L-amino acids" means that the bacterium has the following ability: the ability to utilize external substances (such as a culture medium) to produce and accumulate L-amino acids in the bacterium, and further may include the ability to secrete L-amino acids into the culture system. Thus, L-amino acids can be collected when the bacterium is cultured in a culture medium.

[0043] The recombinant bacterium 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, α-ketoglutaric acid, citric acid, ornithine, citrulline, etc.

[0044] The bacterium can be a wild-type bacterium collected naturally or a modified bacterium.

[0045] "Modified bacterium" refers to a modified bacterium obtained by artificially mutating and / or mutagenizing a wild-type bacterium collected naturally.

[0046] Furthermore, the bacterium can be from the genus Escherichia ( Escherichia sp. ), the genus Erwinia (Erwinia sp. ), the genus Agrobacterium ( Agrobacterium sp. ), the genus Flavobacterium ( Flavobacterium sp. ), the genus Alcaligenes ( Alcaligenes sp. ), the genus Pseudomonas ( Pseudomonas sp. ), the genus Bacillus ( Bacillus sp. ), the genus Brevibacterium ( Brevibacterium sp. ), the genus Corynebacterium ( Corynebacterium sp.), Aerobacter Aerobacter sp. ), Enterobacter Enterobacteria sp. ), Micrococcus Micrococcus sp. ), Serratia Serratia sp. ), Salmonella Salmonella sp. ), Streptomyces Streptomyces sp. ), Providencia Providencia sp. ), etc., but not limited thereto.

[0047] Furthermore, the bacterium can be Escherichia coli (also known as E. coli) ( Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Brevibacterium lactofermentum ( Brevibacterium lactofermentum ), Brevibacterium flavum ( Brevibacterium flavum ), Corynebacterium pekinense ( Corynebacterium pekinense ), Brevibacterium ammoniagenes ( Brevibacterium ammoniagenes ), Corynebacterium crenatum ( Corynebacterium crenatum ), or Pantoea ( Pantoea ), but not limited thereto.

[0048] In the embodiment of the present invention, the bacterium is specifically Escherichia coli.

[0049] In a specific embodiment of the present invention, the recipient 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 institution: China General Microbiological Culture Collection Center; depository institution abbreviation: CGMCC; address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: July 25, 2022; deposit center registration number: CGMCC No. 25404). Correspondingly, the recombinant Escherichia coli (or the engineered bacterium) is Escherichia coli W3110-Δ yjiG or Escherichia coli YPThr-Δ yjiG ; the Escherichia coli W3110-Δ yjiG is the recombinant bacterium obtained by knocking out the yjiG gene in the genome of Escherichia coli W3110 (i.e., the coding gene of the SPMB family inner membrane protein described above, the same below); the Escherichia coli YPThr-Δ yjiG is the recombinant bacterium obtained by knocking out the yjiG gene in the genome of Escherichia coli CGMCC No. 25404.

[0050] In a specific embodiment of the present invention, the recipient 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 institution: China General Microbiological Culture Collection Center; abbreviated name of the depository institution: CGMCC; address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: July 25, 2022; registration number in the depository center: CGMCC No. 25403). Accordingly, the recombinant Escherichia coli (or the engineered bacterium) is the Escherichia coli W3110-Δ yjiG or Escherichia coli YPTrp-Δ yjiG ; the Escherichia coli YPTrp-Δ yjiG is a recombinant bacterium obtained by knocking out the yjiG gene in the genome of Escherichia coli CGMCC No. 25403.

[0051] In a specific embodiment of the present invention, the recipient 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: China General Microbiological Culture Collection Center; abbreviated name of the depository institution: CGMCC; address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: July 25, 2022; registration number in the depository center: CGMCC No. 25402); accordingly, the recombinant Escherichia coli (or the engineered bacterium) is the Escherichia coli W3110-Δ yjiG or Escherichia coli YPR-Δ yjiG ; the Escherichia coli YPR-Δ yjiG is a recombinant bacterium obtained by knocking out the yjiG gene in the genome of Escherichia coli CGMCC No. 25402.

[0052] In a specific embodiment of the present invention, the recipient 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: China General Microbiological Culture Collection Center; abbreviated name of the depository institution: CGMCC; address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: June 15, 2021; registration number in the depository center: CGMCC No. 22721); accordingly, the recombinant Escherichia coli (or the engineered bacterium) is the Escherichia coli W3110-Δ yjiG or Escherichia coli YPV-Δ yjiG ; the Escherichia coli YPV-Δ yjiGThe recombinant bacterium obtained by knocking out the yjiG gene in the genome of Escherichia coli CGMCC No.22721.

[0053] Experiments have proved that inactivating or reducing the activity of inner membrane proteins of the SPMB family in the present invention can produce more cost-effective fermentation production strains of L-threonine, L-tryptophan, L-arginine and L-valine. The present invention is of great significance for improving the fermentation yields of L-threonine, L-tryptophan, L-arginine and L-valine. Description of the Drawings

[0054] Figure 1 is the plasmid map of pREDCas9.

[0055] Figure 2 is the plasmid map of pGRB-yjiG gRNA.

[0056] Deposit Description 1

[0057] Taxonomic name: Escherichia coli ( Escherichia coli );

[0058] Reference biological material: YP0158;

[0059] Depositary institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms;

[0060] Abbreviation of the depositary institution: CGMCC;

[0061] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0062] Deposit date: July 25, 2022;

[0063] Registration number in the depositary center: CGMCC No.25404.

[0064] In the present invention, this strain is simply referred to as Escherichia coli CGMCC No.25404.

[0065] Deposit Description 2

[0066] Taxonomic name: Escherichia coli ( Escherichia coli );

[0067] Reference biological material: YP006D;

[0068] Depositary institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms;

[0069] Abbreviation of the depositary institution: CGMCC;

[0070] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0071] Date of deposit: July 25, 2022;

[0072] Accession number in the depositary: CGMCC No.25403.

[0073] In the present invention, this strain is simply referred to as Escherichia coli CGMCC No.25403.

[0074] Deposit description 3

[0075] Taxonomic name: Escherichia coli ( Escherichia coli );

[0076] Biological material referred to: YP004-8;

[0077] Depositary: China General Microbiological Culture Collection Center;

[0078] Abbreviation of the depositary: CGMCC;

[0079] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0080] Date of deposit: July 25, 2022;

[0081] Accession number in the depositary: CGMCC No.25402.

[0082] In the present invention, this strain is simply referred to as Escherichia coli CGMCC No.25402.

[0083] Deposit description 4

[0084] Taxonomic name: Escherichia coli ( Escherichia coli );

[0085] Biological material referred to: YP045;

[0086] Depositary: China General Microbiological Culture Collection Center;

[0087] Abbreviation of the depositary: CGMCC;

[0088] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0089] Date of deposit: June 15, 2021;

[0090] Accession number in the depositary: CGMCC No.22721.

[0091] In the present invention, this strain is simply referred to as Escherichia coli CGMCC No.22721. Detailed implementation mode

[0092] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0093] In the experimental methods in the following embodiments, unless otherwise specified, they are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0094] The amino acid sequences of the inner membrane proteins of the SPMB family involved in the following embodiments are shown in SEQ ID No. 6, and the coding gene sequences (i.e., yjiG gene sequences) in the Escherichia coli genome are shown in SEQ ID No. 5.

[0095] The primers used in the construction of each engineering bacterium involved in the following embodiments are summarized in Table 1.

[0096]

[0097] Example 1. Construction of each engineering strain for producing L-amino acids

[0098] In this example, engineering bacteria W3110-Δ for producing L-amino acids (including L-threonine, L-tryptophan, L-arginine and L-valine) were constructed yjiG , as well as high-yield L-threonine strain YPThr-Δ yjiG , high-yield L-tryptophan strain YPTrp-Δ yjiG , high-yield L-arginine strain YPR-Δ yjiG and high-yield L-valine strain YPV-Δ yjiG .

[0099] The above-mentioned engineering bacteria were constructed by using the CRISPR / Cas9 gene editing method (Li Y, Lin Z, Huang C, etal. Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing. Metabolic engineering, 2015, 31: 13-21.) to knock out the yjiGObtained after the gene (SEQ ID No.5), the maps of the two plasmids (pREDCas9 and pGRB-yjiG gRNA) used in this method are shown in Figure 1 and Figure 2 . Among them, the pREDCas9 plasmid carries the elimination system of the expression plasmid pGRB, the Red recombination system of phage λ and the Cas9 protein expression system, with chlortetracycline resistance (working concentration: 100 mg / L), and is cultured at 32 °C; the pGRB-yjiG gRNA plasmid is based on pUC18 and includes yjiG the gRNA sequence, promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, with ampicillin resistance (working concentration: 100 mg / L), and is cultured at 37 °C.

[0100] I. Construction of engineering bacterium W3110-Δ yjiG

[0101] 1. Construction of pGRB-yjiG gRNA plasmid

[0102] The purpose of constructing the plasmid pGRB-yjiG gRNA is to transcribe the gRNA targeting the yjiG gene in the Escherichia coli genome, so as to form a complex with the Cas9 protein, and identify the target site of the target gene through base pairing and PAM, realizing the double-strand break of the target DNA. The pGRB-yjiG gRNA plasmid was constructed by the method of recombining the DNA fragment containing the target sequence with the linearized vector fragment.

[0103] (1) Target sequence design

[0104] Use CRISPR RGEN Tools to design the target sequence as follows:

[0105] 5’- GGCAACCCGGTGCAAAATGT-3’ (SEQ ID No.1).

[0106] (2) Preparation of DNA fragment containing target sequence

[0107] Design primers: 5’-linearized vector terminal sequence (15 bp)-restriction enzyme site-target sequence (excluding PAM sequence)-linearized vector terminal sequence (15 bp)-3’ and its reverse complementary primer (i.e., gRNA- yjiG -S and gRNA- yjiG -A in Table 1), and prepare the DNA fragment containing the target sequence (i.e., the insert fragment in step (4) below) by annealing 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. ​

[0108]

[0109] (3)Preparation of linear vector

[0110] Using the pGRB plasmid (product of addgene, catalog number #71539) as a template, a linear vector was obtained by PCR amplification with primers pGRB-PF and pGRB-PR (specific sequences are shown in Table 1).

[0111] (4)Recombination reaction

[0112] The recombination system is shown in Table 3. The used recombinase is NEBuilder enzyme (purchased from NEB), and the recombination conditions are: 50 °C, 30 min.

[0113]

[0114] (5)Transformation

[0115] Take 4 μL of the recombination reaction solution and add it to 100 mL of Escherichia coli DH5α competent cells for transformation (purchased from TAKARA). After gently mixing, incubate on ice for 20 min, heat shock at 42 °C for 45 - 90 s, immediately incubate on ice for 2 - 3 min, add 900 μL of SOC medium (purchased from TAKARA), and recover at 37 °C for 1 h. Centrifuge at 8000 rpm for 2 min, discard part of the supernatant, leave about 200 μL, resuspend the cell pellet and spread it on a plate containing 100 mg / L ampicillin. Invert the plate and incubate overnight at 37 °C. After single colonies grow on the plate, perform colony PCR identification with primers sgRNA-PF / sgRNA-PR (specific sequences are shown in Table 1). The positive recombinant with 457 bp (SEQ ID No.3) is obtained, that is, the pGRB-yjiG gRNA plasmid is obtained.

[0116] 2. Preparation of donor DNA fragment

[0117] For yjiG The recombinant fragment for gene knockout consists of yjiG the upstream and downstream homologous arms of the gene (upstream homologous arm - downstream homologous arm). Using the primer design software primer5, with yjiG the upstream and downstream sequences of the gene as templates, upstream and downstream homologous arm primers were designed (the primers for amplifying the upstream homologous arm are UP -yjiG- S and UP -yjiG- A in Table 1, and the primers for amplifying the downstream homologous arm are DN -yjiG- S and DN -yjiG-A) After separately amplifying the upstream and downstream homologous arms by PCR using the KAPA HiFi HotStart High-Fidelity Amplification Kit, the donor DNA fragment was prepared by overlapping PCR. The system and method of PCR are shown in Table 4.

[0118]

[0119] The system of overlapping PCR is shown in Table 5.

[0120]

[0121] Note: The template consists of equimolar amplification fragments of the upstream and downstream homologous arms and the target gene, and the total amount does not exceed 10 ng.

[0122] 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 per minute); continue to extend at 72°C for 10 min; hold at (4°C).

[0123] The sequence of the finally obtained amplification product (i.e., the donor DNA fragment) is shown in SEQ ID No. 2.

[0124] 3. Transformation of plasmid and donor DNA fragment

[0125] (1) Transformation of pREDCas9

[0126] The pREDCas9 plasmid (product of addgene, catalog number #71541) was electrotransformed into the electrocompetent cells of the starting strain (E. coli W3110) by electroporation. After the cells were resuscitated and cultured, they were spread on an LB plate containing spectinomycin and cultured overnight at 32°C. Single colonies growing on the resistant plate were subjected to colony PCR with the identification primers pRedCas9-PF / pRedCas9-PR. Those with 943 bp (SEQ ID No. 4) were positive recombinants containing the pREDCas9 plasmid.

[0127] (2) Preparation of electrocompetent cells of the target strain containing pREDCas9

[0128] The positive strain containing the pREDCas9 plasmid obtained in step (1) was cultured at 32°C until OD 600 = 0.1 - 0.2, then 0.1 M IPTG was added (to make its final concentration 0.1 mM), and the culture was continued until OD 600When OD600 = 0.6 - 0.7, competent cells were prepared. The purpose of adding IPTG is to induce the expression of the recombinase on the pREDCas9 plasmid. The medium required for the preparation of competent cells and the preparation process refer to the conventional standard operation.

[0129] (3)Transformation of pGRB-yjiG gRNA and donor DNA fragment

[0130] The pGRB-yjiG gRNA plasmid constructed in step 1 above and the donor DNA fragment prepared in step 2 above were simultaneously electrotransformed into electrocompetent cells containing pREDCas9. The bacteria after electrotransformation and resuscitation culture were spread on an LB plate containing ampicillin and gentamycin, and cultured overnight at 32 °C. Using the primer UP -yjiG- S / DN- yjiG -A (see Table 1) for colony PCR verification, and the positive recombinants with an amplified fragment size of 606 bp (SEQ ID No. 2) were screened and the bacteria were preserved.

[0131] 4. Elimination of plasmids

[0132] (1)Elimination of pGRB-yjiG gRNA plasmid

[0133] The positive recombinants obtained in the previous step were cultured overnight in LB medium containing 0.2% arabinose, appropriately diluted and spread on an LB plate with gentamycin resistance, and cultured overnight at 32 °C. Single colonies were picked and streaked one by one onto LB plates containing ampicillin and gentamycin resistance respectively. The single colonies that did not grow on the ampicillin plate but grew on the gentamycin resistance plate were selected to continue the elimination of the pREDCas9 plasmid.

[0134] (2)Elimination of pREDCas9 plasmid

[0135] The positive recombinants were transferred to LB liquid medium without resistance and cultured overnight at 42 °C, appropriately diluted and spread on an LB plate without resistance, and cultured overnight at 37 °C. Single colonies were picked and streaked one by one onto LB plates with gentamycin resistance and without resistance respectively. The single colonies that did not grow on the gentamycin resistance plate but grew on the non-resistant plate were selected and the bacteria were preserved, namely the engineered strain W3110-Δ yjiG .

[0136] The finally obtained engineered strain W3110-Δ yjiG is the Escherichia coli W3110 genome in which yjiGThe 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.

[0137] 2. L-threonine high-producing strain YPThr-Δ yjiG Construction

[0138] 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.

[0139] 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.

[0140] 3. L-tryptophan high-producing strain YPTrp-Δ yjiG Construction

[0141] 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.

[0142] 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.

[0143] IV. L-arginine high-yielding strain YPR-Δ yjiG Construction

[0144] 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.

[0145] The final engineered bacteria YPR-ΔyjiG is a recombinant bacterium with the yjiG gene (SEQ ID No.5) knocked out from the genome of Escherichia coli CGMCC No.25402. Specifically, it is obtained by deleting the sequence between the upstream homologous arm (the 1st - 284th positions of SEQ ID No.2) and the downstream homologous arm (the 285th - 606th positions of SEQ ID No.2) in the genome of Escherichia coli CGMCC No.25402, while keeping other sequences unchanged.

[0146] V. Construction of high - yield L - valine strain YPV - Δ yjiG

[0147] Refer to Step 1. The difference from Step 1 is only that the recipient bacterium is replaced from Escherichia coli W3110 with Escherichia coli CGMCC No.22721.

[0148] The finally obtained engineered bacterium YPV - Δ yjiG is a recombinant bacterium with the yjiG gene (SEQ ID No.5) knocked out from the genome of Escherichia coli CGMCC No.22721. Specifically, it is obtained by deleting the sequence between the upstream homologous arm (the 1st - 284th positions of SEQ ID No.2) and the downstream homologous arm (the 285th - 606th positions of SEQ ID No.2) in the genome of Escherichia coli CGMCC No.22721, while keeping other sequences unchanged.

[0149] Example 2. Construction of an engineered strain containing over - expression of yjiG gene on a plasmid

[0150] One 、 yjiG Construction of gene expression vector pET28(a) - yjiG

[0151] To construct an over - expression plasmid of yjiG gene, the wild - type yjiG gene (the sequence is as SEQ IDNo.5) needs to be cloned into the expression vector pET28(a). Using the genomic sequence of Escherichia coli ( Escherichia coli ) W3110 published by NCBI as a template, PCR amplification is carried out with primers yjiG - PF / yjiG - PR (the sequences are shown in Table 1) to obtain the wild - type yjiG gene. After recovery, it is combined with the one treated with BamH I / Xho ​​The expression vector pET28(a) (purchased from BioVector Co., Ltd., containing kanamycin resistance) digested and recovered by I was ligated with NEBuilder enzyme (purchased from NEB Co., Ltd.) at 50 °C for 30 min. The ligation product was transformed into DH5α and spread on a 2-YT agar plate containing kanamycin (50 mg / L) and cultured at 37 °C to obtain the pET28(a) transformant pET28(a)- yjiG containing the yjiG gene. The monoclonal colonies grown from the culture were identified by primers T7 / T7t (sequences are shown in Table 1) and r Taq PCR. The pET28(a) positive transformant pET28(a)- yjiG containing the yjiG gene was the one with a 749-bp (sequence as SEQ ID No.7) fragment amplified by PCR.

[0152] II. Overexpression yjiG Construction of engineering strains overexpressing the

[0153] To verify the effect of overexpression of the yjiG gene on the production of L-amino acids such as L-threonine, L-tryptophan, L-valine, and L-arginine by the strain, the expression vector pET28(a)- yjiG constructed in step I was separately introduced into the L-threonine-producing bacterium CGMCC No.25404, the L-tryptophan-producing bacterium CGMCC No.25403, the L-arginine-producing bacterium CGMCC No.25402, and the L-valine-producing bacterium CGMCC No.22721, so as to deeply study the effect of overexpression of the yjiG gene on the yields of L-amino acids such as L-threonine, L-tryptophan, L-valine, and L-arginine in high-yield strains.

[0154] Competent cells of several Escherichia coli strains, namely L-threonine CGMCC No.25404, L-tryptophan CGMCC No.25403, L-arginine CGMCC No.25402, and L-valine CGMCC No.22721, were prepared. When OD 600 = 0.6, the bacteria were collected to prepare competent cells, which were separately transformed with the plasmid pET28(a)- yjiG constructed in step I and spread on a 2-YT agar plate containing kanamycin (50 mg / L) and cultured at 37 °C. The single colonies produced by the culture were identified by primers T7 / T7t and r Taq PCR. The positive transformant was the one with a 749-bp (such as SEQ ID No.7) fragment amplified by PCR, and the original bacterium was the one without the amplified fragment.

[0155] PCR amplification system: 12.5 μL of 2×Premix r Taq (purchased from TAKARA), 1 μL each of the upstream and downstream primers (10 pM), and ddH2O was added to make the total volume 25 μL.

[0156] 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 final extension at 72°C for 10 min.

[0157] The overexpression plasmid pET28(a)- yjiG was separately introduced into the L-threonine-producing bacterium CGMCC No.25404, the L-tryptophan-producing bacterium CGMCC No.25403, the L-arginine-producing bacterium CGMCC No.25402, and the L-valine-producing bacterium CGMCC No.22721. The resulting recombinant bacteria were named YPThr-pET28(a)- yjiG 、YPTrp-pET28(a)- yjiG 、YPR-pET28(a)- yjiG 、YPV-pET28(a)- yjiG respectively.

[0158] The expression plasmid pET28(a)- yjiG in the recombinant bacteria YPThr-pET28(a)- yjiG 、YPTrp-pET28(a)- yjiG 、YPR-pET28(a)- yjiG and YPV-pET28(a)- yjiG contains the gene shown in SEQ ID No.5. yjiG The recombinant bacteria overexpressing pET28(a)- yjiG can significantly and stably increase the expression level of the inner membrane protein of the SPMB family.

[0159] Example 3. L-Amino Acid Fermentation Test

[0160] I. L-Threonine Fermentation Experiment

[0161] Escherichia coli W3110, Escherichia coli CGMCC No.25404, the engineered bacterium W3110-Δ yjiG constructed in Example 1, and YPThr-Δ yjiG , and the engineered bacterium YPThr-pET28(a)- yjiGThey were separately inoculated into a 5L fermenter of model BLBIO-5GC-4-H (Shanghai Bailun Biotechnology Co., Ltd.) for fermentation experiments with an L-threonine fermentation medium and culture conditions, and each strain was repeated three times. Among them, YPThr-pET28(a)- yjiG is a strain containing the pET28(a) expression vector, and IPTG induction is required during the fermentation process. The specific induction method is as follows: After inoculation in the fermentation culture, IPTG with a final concentration of 0.1 mM is added to induce yjiG the overexpression of the gene. After the fermentation is completed, the L-threonine content is detected by high performance liquid chromatography (HPLC), and the results are the average values of three repetitions, as shown in Table 6. The experimental results with P<0.05 (*) indicate significant differences, and P<0.01 (**) indicate extremely significant differences.

[0162] L-threonine fermentation medium: The solvent is water, and the solutes and their concentrations are 13 g / L of glucose, 1 g / L of (NH4)2SO4, 0.5 g / L of H3PO4, 0.8 g / L of KCl, 0.8 g / L of MgSO4•7H2O, 0.01 g / L of FeSO4•7H2O, 0.01 g / L of MnSO4•H2O, 1.5 g / L of FM902 yeast powder, 5 g / L of corn steep liquor, 17 g / L of molasses, and ammonia is introduced to adjust the pH to 7.0.

[0163] L-threonine fermentation culture conditions:

[0164] Calibrate DO to 100%: Temperature 37°C, air volume 5 L / min, rotation speed 800 rpm, tank pressure 0 Mpa, and calibrate after 5 min;

[0165] Inoculation amount 10% (v / v);

[0166] Initial conditions: pH 7.0, culture temperature 37°C, tank pressure 0 Mpa, air volume 0.5 L / min, rotation speed 400 rpm;

[0167] Full-process control: 1. When the dissolved oxygen < 30%, successively increase the rotation speed to 500 rpm → 600 rpm → air volume 1 L / min → 700 rpm → 800 rpm; 2. Increase the tank pressure by 0.01 Mpa at 8 h of fermentation; increase the tank pressure by 0.02 Mpa → 0.03 Mpa → 0.04 Mpa → 0.05 Mpa at 12 h;

[0168] Residual sugar control: Before 12 h of fermentation, it is 0.1 - 0.5%; after 12 h of fermentation, control the residual sugar at 0.1 - 0.3% in combination with the DO requirement;

[0169] Feed materials: 25% ammonia water, 55% concentrated sugar, 10% antifoaming agent;

[0170] Fermentation cycle: about 30 h. During the control process, the standard for increasing or decreasing the air volume is based on the dissolved oxygen of 20-30%.

[0171]

[0172] Note: ns represents YPThr-pET28(a)- yjiG Compared with CGMCC No.25404, there was no significant difference in the yield of L-threonine (P>0.05).

[0173] As shown by the above fermentation results, for both the high-yield L-threonine strain CGMCC No.25404 and the model strain W3110, yjiG the knockout of the gene contributed to the increase in the yield of L-threonine, while the overexpression of yjiG the gene had no improvement effect on the production of L-threonine.

[0174] II. L-tryptophan fermentation experiment

[0175] Escherichia coli W3110, Escherichia coli CGMCC No.25403, and the engineered strain W3110-Δ constructed in Example 1 yjiG and YPTrp-Δ yjiG and the engineered strain YPTrp-pET28(a)- constructed in Example 2 yjiG were respectively inoculated into a 5L fermenter of the BLBIO-5GC-4-H model (Shanghai Bailun Biotechnology Co., Ltd.) for fermentation experiments with L-tryptophan fermentation medium and culture conditions, and each strain was repeated three times. Among them, YPTrp-pET28(a)- yjiG is a strain containing the pET28(a) expression vector, and IPTG induction is required during the fermentation process. The specific induction method is: add IPTG with a final concentration of 0.1 mM after inoculation in fermentation culture to induce yjiG the overexpression of the gene. After the fermentation is completed, the content of L-tryptophan is detected by high performance liquid chromatography (HPLC), and the results are the average values of three repetitions, as shown in Table 7. The experimental results with P<0.05 (*) indicate significant differences, and P<0.01 (**) indicate extremely significant differences.

[0176] L-tryptophan fermentation medium: The solvent is water, and the solutes and their concentrations are glucose 7 g / L, FM902 yeast powder 1 g / L, (NH4)2SO4 1.2 g / L, citric acid 1.2 g / L, MgSO4•7H2O 1.5 g / L, K2HPO4•3H2O 5.5 g / L, antifoaming agent 0.2 mL / L, and ammonia is introduced to adjust the pH to 7.0.

[0177] L-tryptophan culture conditions:

[0178] Calibration DO 100%: Temperature 35°C, rotation speed 800 rpm, air volume 5 L / min, tank pressure 0.00 Mpa;

[0179] Inoculation amount 10% (v / v);

[0180] Initial conditions: Temperature 35°C, pH 7.0, air volume 1.0 L / min, rotation speed 350 rpm;

[0181] Full process control: When the dissolved oxygen ≤ 20% before the bottom sugar is consumed, increase the rotation speed step by step to 400 rpm → 450 rpm in sequence; after the bottom sugar is consumed, control the dissolved oxygen at 15 - 30% during sugar supplementation; pH is 7.0 before F24h and 6.7 after F24h;

[0182] Residual sugar control: 0.1 - 0.5% before F12h; after F12h, control the residual sugar at 0.1 - 0.3% in combination with the DO requirement;

[0183] Feed - in materials: 25% ammonia water, 55% concentrated sugar, 10% antifoaming agent;

[0184] Fermentation cycle: About 34h, and use the dissolved oxygen of 15 - 30% as the standard for increasing and decreasing the air volume during the control process.

[0185]

[0186] Note: ns represents YPTrp - pET28(a) - yjiG Compared with CGMCC No.25403, there is no significant difference in the yield of L - tryptophan (P > 0.05).

[0187] As shown by the above fermentation results, for both the high - yield L - tryptophan - producing strain CGMCC No.25403 and the model strain W3110, yjiG the knockout of the gene yjiG is helpful for the improvement of the L - tryptophan yield, while the over - expression of the gene

[0188] III. Fermentation experiment of L - arginine

[0189] Inoculate Escherichia coli W3110, Escherichia coli CGMCC No.25402, and the engineered strain W3110 - Δ yjiG constructed in Example 1 and YPR - Δ yjiG and the engineered strain YPR - pET28(a) - yjiG constructed in Example 2 into a 5L fermenter of model BLBIO - 5GC - 4 - H (Shanghai Bailun Biotechnology Co., Ltd.) respectively, and conduct fermentation experiments with the L - arginine fermentation medium and culture conditions, and each strain is repeated three times. Among them, YPR - pET28(a) - yjiGThe strain contains the pET28(a) expression vector and requires IPTG induction during the fermentation process. The specific induction method is as follows: After inoculating for fermentation culture, add IPTG with a final concentration of 0.1 mM to induce yjiG gene overexpression. After the fermentation is completed, the L-arginine content is detected by high performance liquid chromatography (HPLC). The results are the average of three replicates, as shown in Table 8. The experimental results with P<0.05 (*) indicate significant differences, and P<0.01 (**) indicate extremely significant differences.

[0190] L-arginine fermentation medium: The solvent is water, and the solutes and their concentrations are 8 g / L of glucose, 3 g / L of FM902 yeast powder, 6 g / L of K2HPO4•3H2O, 1 g / L of MgSO4•7H2O, 0.05 g / L of FeSO4•7H2O, 0.5 g / L of betaine, VB 12 0.005 g / L, 0.3 mL / L of antifoaming agent, 3 g / L of ammonium sulfate, pH 7.2.

[0191] L-arginine fermentation culture conditions:

[0192] Calibrate DO to 100%: Temperature 35°C, pH 7.2, rotation speed 100 rpm, air volume 6 L / min, tank pressure 0.00 Mpa;

[0193] Inoculation amount 10% (v / v);

[0194] Initial conditions: Temperature 35°C, pH 7.2, tank pressure 0.01 mpa, air volume 1.5 L / min, rotation speed 350 rpm;

[0195] Full process control: Control DO at 20 - 30%; When the dissolved oxygen ≤ 25%, increase the rotation speed by 300 rpm → 400 rpm → 2.0 L / min → 500 rpm → 0.02 Mpa → 600 rpm → 3.0 L / min → 0.03 Mpa → 700 rpm → 3.5 L / min → 0.04 Mpa → 800 rpm → 900 rpm → 4.0 L / min → 0.05 Mpa → 1000 rpm each time;

[0196] Residual sugar control: Control the residual sugar at 0.05 - 0.1% throughout the process;

[0197] Feed materials: 25% ammonia water, 80% concentrated sugar, 10% antifoaming agent;

[0198] Fermentation cycle: About 50 h, and control the process with the dissolved oxygen at 20 - 30% as the standard for increasing and decreasing the air volume.

[0199]

[0200] Note: ns represents YPR-pET28(a)- yjiG There was no significant difference in the yield of L-arginine compared with CGMCC No.25402 (P>0.05).

[0201] As shown by the above fermentation results, for both the L-arginine high-yielding strain CGMCC No.25402 and the model strain W3110, yjiG the knockout of the gene contributed to the increase in the yield of L-arginine, while the overexpression of yjiG the gene had no enhancing effect on the production of L-arginine.

[0202] IV. L-valine fermentation experiment

[0203] Escherichia coli W3110, Escherichia coli CGMCC No.22721, and the engineered strain W3110-Δ constructed in Example 1 yjiG and YPV-Δ yjiG 、the engineered strain YPV-pET28(a)- constructed in Example 2 yjiG were respectively inoculated into a 5L fermenter of model BLBIO-5GC-4-H (Shanghai Bailun Biotechnology Co., Ltd.) for fermentation experiments with an L-valine fermentation medium and culture conditions, and each strain was repeated three times. Among them, YPV-pET28(a)- yjiG is a strain containing the pET28(a) expression vector, and IPTG induction is required during the fermentation process. The specific induction method is: add IPTG with a final concentration of 0.1 mM after inoculation in fermentation culture to induce yjiG the overexpression of the gene. After the fermentation is completed, the content of L-valine is detected by high performance liquid chromatography (HPLC), and the results are the average values of three repetitions, as shown in Table 9. The experimental result P<0.05 (*) indicates a significant difference, and P<0.01 (**) indicates a highly significant difference.

[0204] L-valine fermentation medium: The solvent is water, and the solutes and their concentrations are yeast extract powder 4 g / L, corn steep liquor dry powder 2 g / L, peptone 4 g / L, methionine 2 g / L, KH2PO4•3H2O 7 g / L, MgSO4•7H2O 2 g / L, CoCl2 20 mg / L, (NH4)2SO4 3 g / L, citric acid 2 g / L, FeSO4•7H2O 50 mg / L, MnSO4•7H2O 30 mg / L, VH 20 mg / L, VB1 1.5 mg / L, VB3 1.5 mg / L VB 12 1.5 g / L, antifoaming agent 0.3 mL / L, (NH4)2SO4 3 g / L, pH value 7.0.

[0205] L-valine fermentation culture conditions:

[0206] Fermentation control temperature: 35 °C;

[0207] Fermentation cycle: 30 h;

[0208] Dissolved oxygen electrode calibration method: Calibrate the zero point in a saturated sodium sulfite solution and calibrate the full scale in air;

[0209] L-valine fermentation involves two-stage aerobic-oxygen-limited fermentation. The cells are first cultured under aerobic fermentation. In the early stage, the air volume, rotation speed, and sugar feeding rate are adjusted to control the dissolved oxygen at about 25%. When the OD 600 value reaches 50 - 60, the rotation speed is reduced to 400 rpm and the air volume is reduced to 2 L / min; and the aerobic fermentation is converted to oxygen-limited fermentation.

[0210]

[0211] Note: ns represents YPV-pET28(a)- yjiG Compared with CGMCC No.22721, there is no significant difference in the yield of L-valine (P > 0.05).

[0212] As shown by the above fermentation results, whether for the L-valine-producing strain CGMCC No.22721 or the model strain W3110, yjiG the knockout of the yjiG gene is helpful for the improvement of L-valine yield, while the overexpression of the

[0213] gene has no improvement effect on the production of L-valine. yjiG As can be seen from all the above fermentation results, whether for the strains producing the corresponding L-amino acids with high yields or the model strain W3110, yjiG the knockout of the

[0214] gene is helpful for the improvement of the yields of L-threonine, L-tryptophan, L-arginine, and L-valine, while the overexpression of the gene has no improvement effect on the production of L-threonine, L-tryptophan, L-arginine, and L-valine.

[0214] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Use of recombinant Escherichia coli in the fermentative production of L-amino acids; The recombinant Escherichia coli is obtained by inhibiting the expression of SPMB family inner membrane proteins in recipient Escherichia coli; Inhibition of the expression of the SPMB family inner membrane proteins in the recipient Escherichia coli is achieved by the following method: knocking out the expression of the coding gene of the SPMB family inner membrane proteins in the genome of the recipient Escherichia coli; The SPMB family inner membrane protein is the protein with the amino acid sequence shown in SEQ ID No. 6; The L-amino acid is L-threonine, L-tryptophan, L-arginine, and / or L-valine.

2. Use of inhibition of the expression of SPMB family inner membrane proteins in increasing the yield of L-amino acids in Escherichia coli; Inhibition of the expression of the SPMB family inner membrane proteins is achieved by the following method: knocking out the expression of the coding gene of the SPMB family inner membrane proteins in the genome of recipient Escherichia coli; The SPMB family inner membrane protein is the protein with the amino acid sequence shown in SEQ ID No. 6; The L-amino acid is L-threonine, L-tryptophan, L-arginine, and / or L-valine.

3. Use of a substance for inhibiting the expression of SPMB family inner membrane proteins in increasing the yield of L-amino acids in Escherichia coli; The substance is a gene editing tool or siRNA or shRNA targeting the coding gene of the SPMB family inner membrane proteins; The SPMB family inner membrane protein is the protein with the amino acid sequence shown in SEQ ID No. 6; The L-amino acid is L-threonine, L-tryptophan, L-arginine, and / or L-valine.

4. A method for producing L-amino acids, comprising the following steps: culturing recombinant Escherichia coli and obtaining L-amino acids from the fermentation product; The recombinant Escherichia coli is obtained by inhibiting the expression of SPMB family inner membrane proteins in recipient Escherichia coli; Inhibition of the expression of the SPMB family inner membrane proteins in the recipient Escherichia coli is achieved by the following method: knocking out the expression of the coding gene of the SPMB family inner membrane proteins in the genome of the recipient Escherichia coli; The SPMB family inner membrane protein is the protein with the amino acid sequence shown in SEQ ID No. 6; The L-amino acid is L-threonine, L-tryptophan, L-arginine, and / or L-valine.

5. A method for constructing an engineered bacterium for producing L-amino acids, comprising the following steps: inhibiting the expression of SPMB family inner membrane proteins in Escherichia coli; Inhibition of the expression of the SPMB family inner membrane proteins is achieved by the following method: knocking out the expression of the coding gene of the SPMB family inner membrane proteins in the genome of recipient Escherichia coli; The SPMB family inner membrane protein is the protein with the amino acid sequence shown in SEQ ID No. 6; The L-amino acid is L-threonine, L-tryptophan, L-arginine, and / or L-valine.

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