Murj protein mutants and uses thereof

Abstract

The application provides a kind of MurJ protein mutant and its application.The mutant includes at least one of the following ①-③ mutations: ① the mutation of the 373th amino acid of MurJ protein from P to S;② the mutation of the 422th amino acid of MurJ protein from A to T;③ the mutation of the 882th amino acid of MurJ protein from T to I.The MurJ protein variant provided by the application containing P373S, A422T, T882I mutation site can improve the yield and conversion rate of amino acid, and also can ensure the growth performance of strain.Using the recombinant microorganism expressing the above-mentioned MurJ protein variant for amino acid fermentation production, the amino acid fermentation production performance is significantly improved, especially glutamic acid, lysine, proline, with high yield and sugar acid conversion rate, providing a powerful tool for large-scale industrial production of amino acid.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular, to a mutant of MurJ protein and application thereof. BACKGROUND

[0002] L-glutamic acid, chemical name α-amino glutaric acid, molecular formula C5H9NO4, molecular weight 147.13076 Da, contains two carboxyl groups in the molecule, is an acidic amino acid. Glutamic acid is one of the most abundant amino acids, which plays an important role in nutrition and signal transduction in addition to participating in protein synthesis. Glutamic acid or its monosodium salt (MSG) can be used as a food additive to enhance the umami of food, so it is widely produced industrially. At present, the most commonly used method for producing glutamic acid is fermentation method, compared with the complexity of enzymatic method and chemical synthesis process and the use of toxic raw materials, fermentation method has the advantages of safe raw material source, low production cost, single product, etc.

[0003] Although the research on the production of glutamic acid by fermentation method has developed for several decades, the production performance of glutamic acid strains still needs to be further improved, which also restricts the large-scale industrial production of glutamic acid and is not conducive to saving production cost.

[0004] Similar to glutamic acid, other amino acid production also faces the problem of high cost due to low fermentation production index, so it is necessary to improve the performance of amino acid production strains. SUMMARY

[0005] The purpose of the present application is to provide a mutant of MurJ protein and application thereof.

[0006] Another purpose of the present application is to provide a genetically engineered strain for producing amino acid (L-amino acid) and a construction method and application thereof.

[0007] In order to achieve the purpose of the present application, in the first aspect, the present application provides a mutant of MurJ protein, which comprises at least one of the following ①-③ mutations:

[0008] ① the mutation of the 373th amino acid of MurJ protein from P to S;

[0009] ② the mutation of the 422th amino acid of MurJ protein from A to T;

[0010] ③ the mutation of the 882th amino acid of MurJ protein from T to I.

[0011] In the present application, the MurJ protein (murein biosynthesis protein) is from Corynebacterium glutamicum, and its reference sequence number in NCBI is WP_216312835.1. The protein is encoded by the murj gene (i.e. the BBD29_15285 gene).

[0012] Preferably, the mutant comprises a mutation of the amino acid at position 373 from P to S, a mutation of the amino acid at position 422 from A to T, and a mutation of the amino acid at position 882 from T to I (SEQ ID NO: 2).

[0013] In a second aspect, the present application provides a nucleic acid molecule encoding the mutant of the MurJ protein.

[0014] In a third aspect, the present application provides a biological material containing the nucleic acid molecule, including but not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or transgenic cell line.

[0015] In a fourth aspect, the present application provides any of the following applications of the nucleic acid molecule or the biological material containing the nucleic acid molecule:

[0016] (1) for the fermentative production of amino acids (L-amino acids);

[0017] (2) for improving the fermentative yield of amino acids (L-amino acids);

[0018] (3) for constructing genetically engineered bacteria producing amino acids (L-amino acids).

[0019] In the present application, the amino acids include but are not limited to glutamic acid, lysine, proline.

[0020] In a fifth aspect, the present application provides a method for constructing genetically engineered bacteria producing amino acids, which utilizes genetic engineering means to introduce mutations in the genome of bacteria having the ability to produce amino acids, so that the encoded MurJ protein comprises at least one of the following ①-③ mutations:

[0021] ① a mutation of the amino acid at position 373 of the MurJ protein from P to S;

[0022] ② a mutation of the amino acid at position 422 of the MurJ protein from A to T;

[0023] ③ a mutation of the amino acid at position 882 of the MurJ protein from T to I.

[0024] Preferably, the bacteria are of the genus Escherichia, Corynebacterium, or Bacillus.

[0025] More preferably, the bacteria is Corynebacterium glutamicum.

[0026] In one embodiment of the present application, the Corynebacterium glutamicum is strain MHZ-0112-8 (CGMCC No. 11941), see ZL201610119402.2.

[0027] In another embodiment of the present application, the Corynebacterium glutamicum is strain MHZ-0701 (CGMCC No. 13757), see ZL201710385500.5.

[0028] In yet another embodiment of the present application, the Corynebacterium glutamicum is strain MHZ-0912-6 (CGMCC No. 11942), see ZL201610119394.1.

[0029] In a sixth aspect, the present application provides an amino acid-producing genetically engineered bacterium constructed according to the above method.

[0030] In a seventh aspect, the present application provides the use of the genetically engineered bacterium constructed according to the above method in the fermentation production of amino acids (including glutamic acid, lysine, proline) or in the improvement of the fermentation yield of amino acids.

[0031] By means of the above technical solution, the present application has at least the following advantages and beneficial effects:

[0032] The MurJ protein variant provided by the present application containing the mutation sites P373S, A422T and T882I can improve the yield and conversion rate of amino acids, while also ensuring the growth performance of the strain. The use of recombinant microorganisms expressing the above-mentioned MurJ protein variant for amino acid fermentation production can significantly improve the performance of amino acid fermentation production, especially glutamic acid, lysine and proline, with high yield and sugar acid conversion rate. DETAILED DESCRIPTION

[0033] The present application provides a method for improving the fermentation production capacity of L-amino acids, particularly glutamic acid, lysine and proline.

[0034] The present application adopts the following technical solution:

[0035] The application provides a mutant BBD29_15285 protein (i.e. a mutant of a MurJ protein), wherein the proline at position 373 of the protein encoded by the BBD29_15285 gene (i.e. a murj gene) is mutated into serine, the alanine at position 422 is mutated into threonine, and the threonine at position 882 is mutated into isoleucine. Specifically, the amino acid sequence of the protein is shown in SEQ ID NO: 2.

[0036] The application also provides a DNA molecule encoding the above-mentioned BBD29_15285 protein variant, wherein the DNA molecule is obtained by mutating the base at position 1117 of the BBD29_15285 gene from C to T, the base at position 1264 from G to A, and the base at position 2645 from C to T. Specifically, the nucleotide sequence of the DNA molecule is shown in SEQ ID NO: 1.

[0037] The application also provides a recombinant strain of Corynebacterium glutamicum, which is obtained by expressing the above-mentioned DNA molecule in a Corynebacterium glutamicum strain producing glutamic acid, proline or lysine, wherein the Corynebacterium glutamicum strain producing glutamic acid is MHZ-0112-8, the Corynebacterium glutamicum strain producing proline is MHZ-0701, and the Corynebacterium glutamicum strain producing lysine is MHZ-0912-6.

[0038] The application also provides a method for constructing the above-mentioned recombinant strain of Corynebacterium glutamicum, which comprises the following steps:

[0039] (1) constructing a recombinant plasmid in which the proline at position 373 of the protein encoded by the BBD29_15285 gene is mutated into serine, a recombinant plasmid in which the alanine at position 422 of the protein encoded by the BBD29_15285 gene is mutated into threonine, a recombinant plasmid in which the threonine at position 882 of the protein encoded by the BBD29_15285 gene is mutated into isoleucine, and a recombinant plasmid in which the three mutations are combined;

[0040] (2) transforming the recombinant plasmid into Corynebacterium glutamicum, screening the transformants with a selective medium containing kanamycin, and culturing the screened transformants in a liquid brain heart infusion medium;

[0041] (3) diluting the culture and spreading it on a solid brain heart infusion medium containing 10% sucrose, and culturing to obtain the recombinant strain.

[0042] Specifically, the method for constructing the recombinant strain comprises:

[0043] (1) Construct the recombinant plasmid pK18-BBD29_15285(P373S) in which the proline at the 373rd position of the protein encoded by the BBD29_15285 gene is mutated into serine, the recombinant plasmid pK18-BBD29_15285(A422T) in which the alanine at the 422nd position of the protein encoded by the BBD29_15285 gene is mutated into threonine, the recombinant plasmid pK18-BBD29_15285(T882I) in which the threonine at the 882nd position of the protein encoded by the BBD29_15285 gene is mutated into isoleucine, and the recombinant plasmid pK18-BBD29_15285(P373S+A422T+T882I) in which the three mutations are combined.

[0044] (2) Transform the recombinant plasmid into coryneform bacteria, screen the transformants with a selection medium containing 15 mg / L kanamycin, and culture the screened transformants in a liquid brain heart infusion medium at 31.5°C with 220 rpm shaking overnight.

[0045] (3) Dilute the culture and spread on a solid brain heart infusion medium containing 10% sucrose, and incubate at 31.5°C for 36 h to obtain the recombinant strain.

[0046] In the specific embodiments provided in the present application, the starting strain is coryneform bacteria MHZ-0112-8 with the preservation number of CGMCC No.11941, coryneform bacteria MHZ-0701 with the preservation number of CGMCC No.13757, or coryneform bacteria MHZ-0912-6 with the preservation number of CGMCC No.11942.

[0047] The pure culture of coryneform bacteria MHZ-0112-8 has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 25, 2015, and the preservation number is CGMCC No.11941. Coryneform bacteria MHZ-0112-8 (CGMCC No.11941) has been disclosed in ZL201610119402.2.

[0048] The starting strain MHZ-0701 is Corynebacterium glutamicum, and a pure culture of Corynebacterium glutamicum MHZ-0701 has been deposited with the China General Microbiological Culture Collection Center (CGMCC, located at No. 1, Huixiyuan, Beichen West Road, Chaoyang District, Beijing, China, and managed by the Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101) on March 15, 2017, and the deposit number is CGMCC No. 13757. The Corynebacterium glutamicum MHZ-0701 (CGMCC No. 13757) mentioned in the present application has been disclosed in ZL201710385500.5.

[0049] The starting strain MHZ-0912-6 is Corynebacterium glutamicum, and a pure culture of Corynebacterium glutamicum MHZ-0912-6 has been deposited with the China General Microbiological Culture Collection Center (CGMCC, located at No. 1, Huixiyuan, Beichen West Road, Chaoyang District, Beijing, China, and managed by the Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101) on December 25, 2015, and the deposit number is CGMCC No. 11942. The Corynebacterium glutamicum MHZ-0912-6 (CGMCC No. 11942) mentioned in the present application has been disclosed in ZL201610119394.1.

[0050] The present application also provides the use of the above-mentioned recombinant Corynebacterium glutamicum strain in the fermentation production of glutamic acid, lysine or proline.

[0051] Specifically, the present application provides a method for producing an amino acid, comprising:

[0052] The above-mentioned recombinant Corynebacterium glutamicum strain is inoculated into a slant medium for slant culture, and the bacterial lawn on the slant culture medium is picked and inoculated into a seed culture medium for seed culture, and the seed culture is transferred into a fermentation medium for fermentation.

[0053] In the method for producing glutamic acid by fermentation provided by the present application, the slant culture medium used is: yeast powder 5 g / L, beef extract 10 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar powder 2.5 g / L, pH 7.0-7.2.

[0054] The seed culture medium used is: glucose 25 g / L, urea 3.0 g / L, K2HPO4·3H2O 2.2 g / L, MgSO4·7H2O 0.9 g / L, corn syrup 33 mL / L, soybean cake hydrolysate 22 mL / L, pH 7.0-7.2.

[0055] The fermentation medium used is: glucose 60 g / L, ammonium sulfate 15 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.4 g / L, FeSO4·7H2O 1.0 mg / L, MnSO4·5H2O 1.0 mg / L, VB1 200 μg / L, biotin 300 μg / L, soybean hydrolysate 0.48 g / L, pH 7.2-7.5.

[0056] The seed culture method comprises: under the condition of 31.5℃ and 220 rpm, the seed culture is cultured to the middle and later logarithmic growth phase, and the culture time is 10-14 h.

[0057] The fermentation culture method comprises: under the condition of 31.5℃ and 220 rpm, the seed culture is cultured for 12-20 h.

[0058] In the proline fermentation production method provided by the application, the seed activation culture medium used is: yeast extract 1%, peptone 1%, sodium chloride 0.5%, glucose 0.5%, agar 2%, pH 7.2.

[0059] The seed culture medium used is: corn syrup 2.5%, glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.1%, urea 0.1%, CaCO3 0.5%, pH 7.2.

[0060] The fermentation medium used is: corn syrup 0.6%, glucose 12.0%, ammonium sulfate 3.7%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.1%, CaCO3 4%, VH 70 μg / L, VB1 80 μg / L, pH 7.2.

[0061] The fermentation culture method comprises: 33℃, 220r / min oscillation culture for 72 h.

[0062] In the lysine fermentation production method provided by the application, the slant culture medium used is: yeast powder 5.0 g / L, beef extract 10 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar powder 2.5 g / L, pH 7.0-7.2. The prepared culture medium is sterilized at 121℃ and 0.1 MPa for 30 min.

[0063] The seed culture medium used is: glucose 25 g / L, urea 3.0 g / L, K2HPO4·3H2O 2.2 g / L, MgSO4·7H2O 0.9 g / L, corn syrup 33 mL / L, soybean cake hydrolysate 22 mL / L, pH 7.0-7.2. The prepared culture medium is sterilized at 121℃ and 0.1 MPa for 15 min.

[0064] The fermentation medium used was: glucose 60 g / L, (NH4)2SO4 25 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O 1.0 g / L, yeast powder 10 g / L, CaCO3 30 g / L, NaOH to adjust pH 7.0.

[0065] The fermentation culture method included: 31.5℃, 220 r / min shaking culture for 16 h.

[0066] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If not specifically indicated, the examples are all according to the conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or the conditions suggested by the manufacturer's instructions.

[0067] The primer sequences used in the following examples are shown in Table 1.

[0068] Table 1 Primer sequences

[0069]

[0070]

[0071] Example 1 Construction of recombinant plasmid carrying single-point mutation and combined mutation of P373S, A422T and T882I

[0072] A. Construction of pK18-BBD29_15285 (P373S) recombinant plasmid

[0073] The primers were designed according to the sequence of BBD29_15285 gene in NCBI database, and the sequences of the primers are shown in Table 1. Using Phusion ultra-fidelity polymerase (New England BioLabs), P373S-UP-1F / P373S-UP-1R, P373S-DN-2F / P373S-DN-2R as primer pairs, the genome of C. glutamicum MHZ-0112-8 as a template, a recombinant fragment was prepared, and the PCR program was as follows: denaturation at 98℃ for 10 s, annealing at 50℃ for 20 s, extension at 72℃ for 15 s, for 30 cycles, and complete extension at 72℃ for 10 min. The obtained fragment was purified by agarose gel recovery kit (Tiangen), and then P373S-UP-1F / P373S-DN-2R was used as a primer pair, and the above and below homologous arms were used as templates to prepare a recombinant fragment, and the PCR program was as follows: denaturation at 98℃ for 10 s, annealing at 50℃ for 20 s, extension at 72℃ for 30 s, for 30 cycles, and complete extension at 72℃ for 10 min. The obtained recombinant fragment was purified by agarose gel recovery kit (Tiangen), and then digested by XbaI / PstI, and pK18-mobsacB was digested by XbaI / PstI at the same time, and the fragment and the vector were ligated by T4 DNA ligase (TransGen Biotech), and Trans1T1 competent cells (TransGen Biotech) were transformed, kanamycin-resistant clones were picked, and XbaI / PstI enzyme digestion was used for identification to obtain positive clones of the insertion of the fragment into pK18mobsacB, and further identification of the correct inserted fragment was performed by sequencing (Invitrogen). The obtained plasmid was named as pK18-BBD29_15285(P373S).

[0074] B, construction of pK18-BBD29_15285(A422T) recombinant plasmid

[0075] The primers were designed according to the sequence of BBD29_15285 gene in NCBI database, and the sequences of the primers are shown in Table 1. Using Phusion ultra-fidelity polymerase (New England BioLabs), A422T-UP-1F / A422T-UP-1R, A422T-DN-2F / A422T-DN-2R as primer pairs, the genome of C. glutamicum MHZ-0112-8 as a template, a recombinant fragment was prepared, and the PCR program was as follows: denaturation at 98℃ for 10 s, annealing at 50℃ for 20 s, extension at 72℃ for 15 s, for 30 cycles, and complete extension at 72℃ for 10 min. The obtained fragment was purified by agarose gel recovery kit (Tiangen), and then A422T-UP-1F / A422T-DN-2R was used as a primer pair, and the above and below homologous arms were used as templates to prepare a recombinant fragment, and the PCR program was as follows: denaturation at 98℃ for 10 s, annealing at 50℃ for 20 s, extension at 72℃ for 30 s, for 30 cycles, and complete extension at 72℃ for 10 min. The obtained recombinant fragment was purified by agarose gel recovery kit (Tiangen), and then digested by XbaI / PstI, and pK18-mobsacB was digested by XbaI / PstI at the same time, and the fragment and the vector were ligated by T4 DNA ligase (TransGen Biotech), and Trans1T1 competent cells (TransGen Biotech) were transformed, kanamycin-resistant clones were picked, and positive clones of the fragment inserted into pK18mobsacB were identified by XbaI / PstI digestion, and the inserted fragment was further identified to be correct by sequencing (Invitrogen). The obtained plasmid was named as pK18-BBD29_15285(A422T).

[0076] C, construction of pK18-BBD29_15285(T882I) recombinant plasmid

[0077] The primers were designed according to the sequence of BBD29_15285 gene in NCBI database, and the sequences of the primers are shown in Table 1. Using Phusion ultra-fidelity polymerase (New England BioLabs), T882I-UP-1F / T882I-UP-1R, T882I-DN-2F / T882I-DN-2R as a primer pair, the genome of C. glutamicum MHZ-0112-8 as a template, a recombinant fragment was prepared, and the PCR program was as follows: denaturation at 98℃ for 10 s, annealing at 50℃ for 20 s, extension at 72℃ for 15 s, for 30 cycles, and complete extension at 72℃ for 10 min. The obtained fragment was purified by agarose gel recovery kit (Tiangen), and then T882I-UP-1F / T882I-DN-2R was used as a primer pair, and the above and below homologous arms were used as templates to prepare a recombinant fragment, and the PCR program was as follows: denaturation at 98℃ for 10 s, annealing at 50℃ for 20 s, extension at 72℃ for 30 s, for 30 cycles, and complete extension at 72℃ for 10 min. The obtained recombinant fragment was purified by agarose gel recovery kit (Tiangen), and then digested by XbaI / PstI, and pK18-mobsacB was digested by XbaI / PstI at the same time, and the fragment and the vector were connected by T4 DNA ligase (TransGen Biotech), and Trans1T1 competent cells (TransGen Biotech) were transformed, kanamycin-resistant clones were picked, and XbaI / PstI enzyme digestion was used for identification to obtain positive clones of the insertion of the fragment into pK18mobsacB, and further identification of the correct inserted fragment was performed by sequencing (Invitrogen). The obtained plasmid was named as pK18-BBD29_15285(T882I).

[0078] D, construction of pK18-BBD29_15285(P373S+A422T+T882I) recombinant plasmid

[0079] The primers were designed according to the sequence of BBD29_15285 gene in NCBI database, and the sequences of the primers are shown in Table 1. Using Phusion ultra-fidelity polymerase (New England BioLabs), P373S-UP-1F / P373S-UP-1R, P373S-DN-2F / A422T-UP-1R, A422T-DN-2F / T882I-UP-1R, T882I-DN-2F / T882I-DN-2R as primer pairs, the genome of C. glutamicum MHZ-0112-8 as a template, recombinant fragments were prepared, and the PCR program was as follows: denaturation at 98℃ for 10 s, annealing at 50℃ for 20 s, extension at 72℃ for 15 s, for 30 cycles, and complete extension at 72℃ for 10 min. The obtained fragments were purified by agarose gel recovery kit (Tiangen), and then P373S-UP-1F / T882I-DN-2R was used as a primer pair, and the four single fragments were used as templates to prepare a fusion fragment, and the PCR program was as follows: denaturation at 98℃ for 10 s, annealing at 50℃ for 20 s, extension at 72℃ for 30 s, for 30 cycles, and complete extension at 72℃ for 10 min. The obtained fusion fragment was purified by agarose gel recovery kit (Tiangen), and then digested by XbaI / PstI, and pK18-mobsacB was digested by XbaI / PstI at the same time, and the fragment and the vector were ligated by T4 DNA ligase (TransGen Biotech), and Trans1T1 competent cells (TransGen Biotech) were transformed, kanamycin-resistant clones were picked, and positive clones in which the fragment was inserted into pK18mobsacB were identified by XbaI / PstI digestion, and the inserted fragment was further identified to be correct by sequencing (Invitrogen). The obtained plasmid was named as pK18-BBD29_15285 (P373S+A422T+T882I).

[0080] Example 2 Mutation introduction in the starting strain MHZ-0112-8

[0081] The pK18-BBD29_15285 (P373S) was transformed into C. glutamicum MHZ-0112-8, and the exchange recombinants were selected on the selection medium containing 15 mg / L of kanamycin. The Fast Taq DNA polymerase (TransGen Biotech) was used for colony PCR identification of KanR clones with P373S-UP-1F / P85, P82 / P373S-DN-2R primer pairs. The PCR program was 30 s at 94℃, 30 s at 50℃, 45 s at 72℃, for a total of 30 cycles, and 10 min at 72℃ for complete extension. The clones that amplified 1.2 kb and 1.1 kb fragments with the two primer pairs were positive clones. The selected positive clones were inoculated into an antibiotic-free BHI medium and cultured for 12-14 h. The bacterial solution was diluted by 100-1000 times and then spread on a solid BHI medium containing 10% sucrose and cultured for 36 h. The selected strains were further verified for kanamycin resistance phenotype. The positive recombinants obtained were amplified and sequenced with ID-F / ID-R, and the strain with correct sequencing was named MHZ-0112-16.

[0082] The pK18-BBD29_15285 (P373S) was transformed into C. glutamicum MHZ-0112-8, and the exchange recombinants were selected on the selection medium containing 15 mg / L of kanamycin. The Fast Taq DNA polymerase (TransGen Biotech) was used for colony PCR identification of KanR clones with P373S-UP-1F / P85, P82 / P373S-DN-2R primer pairs. The PCR program was 30 s at 94℃, 30 s at 50℃, 45 s at 72℃, for a total of 30 cycles, and 10 min at 72℃ for complete extension. The clones that amplified 1.2 kb and 1.1 kb fragments with the two primer pairs were positive clones. The selected positive clones were inoculated into an antibiotic-free BHI medium and cultured for 12-14 h. The bacterial solution was diluted by 100-1000 times and then spread on a solid BHI medium containing 10% sucrose and cultured for 36 h. The selected strains were further verified for kanamycin resistance phenotype. The positive recombinants obtained were amplified and sequenced with ID-F / ID-R, and the strain with correct sequencing was named MHZ-0112-16.

[0083] The pK18-BBD29_15285 (T882I) was transformed into C. glutamicum MHZ-0112-8, and the exchange recombinants were selected on the selective medium containing 15 mg / L of kanamycin. Colony PCR was performed on the KanR clones using Fast Taq DNA polymerase (TransGen Biotech) with T882I-UP-1F / P85, P82 / T882I-DN-2R primer pairs, and the PCR program was 30 s at 94℃, 30 s at 50℃, 45 s at 72℃, for a total of 30 cycles, and 10 min at 72℃ for complete extension. The clones that amplified 1.2 kb and 1.1 kb fragments with the two primer pairs were positive clones. The selected positive clones were inoculated into an antibiotic-free BHI medium and cultured for 12-14 h, and then diluted 100-1000 times and spread on a solid BHI medium containing 10% sucrose and cultured for 36 h. The selected strains were further verified for kanamycin resistance phenotype, and the positive recombinants obtained were amplified and sequenced with ID-F / ID-R. The strain with correct sequencing was named MHZ-0112-18.

[0084] The pK18-BBD29_15285 (T882I) was transformed into C. glutamicum MHZ-0112-8, and the exchange recombinants were selected on the selective medium containing 15 mg / L of kanamycin. Colony PCR was performed on the KanR clones using Fast Taq DNA polymerase (TransGen Biotech) with T882I-UP-1F / P85, P82 / T882I-DN-2R primer pairs, and the PCR program was 30 s at 94℃, 30 s at 50℃, 45 s at 72℃, for a total of 30 cycles, and 10 min at 72℃ for complete extension. The clones that amplified 1.2 kb and 1.1 kb fragments with the two primer pairs were positive clones. The selected positive clones were inoculated into an antibiotic-free BHI medium and cultured for 12-14 h, and then diluted 100-1000 times and spread on a solid BHI medium containing 10% sucrose and cultured for 36 h. The selected strains were further verified for kanamycin resistance phenotype, and the positive recombinants obtained were amplified and sequenced with ID-F / ID-R. The strain with correct sequencing was named MHZ-0112-18.

[0085] Example 3 Shake flask verification of mutant strain glutamic acid production

[0086] The recombinant C. glutamicum constructed in Example 2 was fermented to verify its glutamic acid production performance, as follows:

[0087] The strain frozen in a glycerol tube at -80°C was inoculated in a slant medium for activation, and after 24 h of incubation at 31.5°C, a bacterial lawn was grown. A bacterial lawn was picked from the freshly activated slant and inoculated in the seed culture medium described above, and after incubation at 31.5°C, 220 rpm to the mid-logarithmic growth phase for 12 h, a seed solution was prepared. The seed solution described above was inoculated into a 500 mL flask containing 20 mL of fermentation medium at a 10% inoculation amount, and incubated at 31.5°C, 220 rpm for 16 h. After the sugar was completely consumed, the concentration of L-glutamic acid accumulated in the medium was determined.

[0088] The medium formula is as follows:

[0089] Slant medium: yeast powder 5 g / L, beef extract 10 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar powder 2.5 g / L, pH 7.0-7.2. The prepared culture medium was sterilized at 121°C, 0.1 MPa for 30 min.

[0090] Seed medium: glucose 25 g / L, urea 3 g / L, K2HPO4·3H2O 2.2 g / L, MgSO4·7H2O 0.9 g / L, corn syrup 33 mL / L, soybean cake hydrolysate 22 mL / L, pH 7.0-7.2. The prepared culture medium was sterilized at 121°C, 0.1 MPa for 15 min.

[0091] Fermentation medium: glucose 60 g / L, ammonium sulfate 15 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.4 g / L, FeSO4·7H2O 1.0 mg / L, MnSO4·5H2O 1 mg / L, VB1 200 μg / L, biotin 300 μg / L, soybean hydrolysate 0.48 g / L, adjusted to pH 7.2-7.5 with NaOH. The prepared culture medium was sterilized at 121°C, 0.1 MPa for 15 min, and then 1.0 g of hot-sterilized calcium carbonate was added.

[0092] The fermentation results are shown in Table 2. After the P373S, A422T, and T882I point mutations were made to BBD29_15285 based on the starting strain MHZ-0112-8, the glutamic acid conversion rate was improved. In particular, after the combined mutation, the sugar acid conversion rate was increased from 51.8% to 61.0%, which was 9.2 percentage points higher than that of the starting strain.

[0093] Table 2 Detection of glutamic acid content of mutant strains

[0094] Strain OD 600 (×100)]]> Glu (g / L) Sugar acid conversion % MHZ-0112-8 0.413 31.1 51.8 MHZ-0112-16 0.398 33.4 55.7 MHZ-0112-17 0.405 32.9 54.8 MHZ-0112-18 0.412 33.7 56.2 MHZ-0112-19 0.391 36.6 61.0

[0095] Example 4 Mutant introduction and shake flask verification of recombinant plasmid pK18-BBD29_15285 (P373S+A422T+T882I) in the starting strain MHZ-0701

[0096] A. Mutant strain construction

[0097] The pK18-BBD29_15285 (P373S+A422T+T882I) was transformed into Corynebacterium glutamicum MHZ-0701, and the exchange recombinants were selected on a selective medium containing 15 mg / L of kanamycin. Colony PCR was performed on the KanR clones using Fast Taq DNA polymerase (TransGen Biotech) with P373S-UP-1F / P85, P82 / T882I-DN-2R primer pairs, and the PCR program was 30 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 45 s, and 72°C for 10 min. Clones that amplified 2.95 kb and 2.8 kb fragments with the two primer pairs, respectively, were positive clones. The selected positive clones were inoculated into an antibiotic-free BHI medium and cultured for 12-14 h, and then diluted 100-1000 times and plated on a solid BHI medium containing 10% sucrose and cultured for 36 h. The selected strains were further verified for kanamycin resistance phenotype, and the positive recombinants obtained were amplified and sequenced with ID-F / ID-R. The strain with correct sequencing was named MHZ-0703.

[0098] B. Shake flask verification of mutant strain for proline production

[0099] 1. Culture medium

[0100] Seed activation medium: yeast extract 1%, peptone 1%, sodium chloride 0.5%, glucose 0.5%, agar 2%, pH 7.2.

[0101] Seed medium: corn steep liquor 2.5%, glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.1%, urea 0.1%, CaCO3 0.5%, pH 7.2.

[0102] Fermentation medium: corn steep liquor 0.6%, glucose 12.0%, ammonium sulfate 3.7%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.1%, CaCO3 4%, VH 70 μg / L, VB1-80 μg / L, pH 7.2.

[0103] 2. Shake flask fermentation

[0104] (1) Seed culture: picking 1 ring of MHZ-0701, MHZ-0703 slant seed to 500 mL flask containing 20 mL seed culture medium, 33°C, 220 r / min shaking culture for 16-22 h.

[0105] (2) Fermentation culture: inoculating 2 mL seed liquid into 500 mL flask containing 20 mL fermentation culture medium, 33°C, 220 r / min shaking culture for 72 h.

[0106] (3) Taking 1 mL fermentation liquid to centrifuge (12000 rpm, 2 min), collecting supernatant, using HPLC to detect L-proline content in fermentation liquid of engineering bacteria and control bacteria, and the results are shown in Table 3.

[0107] Table 3 L-proline content detection

[0108] Strain MHZ-0701 (starting strain) MHZ-0703 (engineered strain) OD 562 ]]> 43.2 40.1 L-proline concentration g / L 35.9 43.1 Sugar acid conversion % 29.9 35.9

[0109] It can be seen that the conversion rate of L-proline of the starting strain MHZ-0701 is 29.9%, and the conversion rate of L-proline of the engineering bacteria MHZ-0703 is 35.9%. The conversion rate is relatively increased by 6 percentage points.

[0110] Example 5 Mutant introduction of recombinant plasmid pK18-BBD29_15285 (P373S+A422T+T882I) in starting strain MHZ-0912-6 and shake flask verification

[0111] A, mutant strain construction

[0112] pK18-BBD29_15285 (P373S+A422T+T882I) was transformed into Corynebacterium glutamicum MHZ-0912-6, and the exchange recombinants were selected on a selection medium containing 15 mg / L kanamycin. Fast Taq DNA polymerase (TransGen Biotech) was used for colony PCR identification of KanR clones with P373S-UP-1F / P85, P82 / T882I-DN-2R primer pairs. The PCR program was: 94°C for 30 s, 50°C for 30 s, 72°C for 45 s, a total of 30 cycles, and 72°C for 10 min. Two pairs of primers amplified 2.95 kb and 2.8 kb fragments, respectively. The positive clones were selected and inoculated into an antibiotic-free BHI medium and cultured for 12-14 h. The bacterial liquid was diluted by 100-1000 times and then spread on a solid BHI medium containing 10% sucrose and cultured for 36 h. The selected strains were further subjected to kanamycin resistance phenotype verification. The positive recombinants obtained were amplified and sequenced with ID-F / ID-R, and the strain with correct sequencing was named MHZ-0912-7.

[0113] B. Lysine production of mutant strains in shake flask

[0114] 1. Medium:

[0115] Slope medium: yeast powder 5.0 g / L, beef extract 10 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar powder 2.5 g / L, pH 7.0-7.2. The prepared medium was sterilized at 121 °C 0.1 MPa for 30 min.

[0116] Seed medium: glucose 25 g / L, urea 3.0 g / L, K2HPO4·3H2O 2.2 g / L, MgSO4·7H2O 0.9 g / L, corn syrup 33 mL / L, soybean cake hydrolysate 22 mL / L, pH 7.0-7.2. The prepared medium was sterilized at 121 °C 0.1 MPa for 15 min.

[0117] Fermentation medium: glucose 60 g / L, (NH4)2SO4 25 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O 1.0 g / L, yeast powder 10 g / L, CaCO3 30 g / L, NaOH to adjust pH 7.0.

[0118] 2. Shake flask fermentation

[0119] The strain stored in a glycerol tube at -80 °C was inoculated in the above-mentioned slope medium for activation, and after 24 h of culture at 31.5 °C, a bacterial lawn was grown. A bacterial lawn was picked from the freshly activated slope and inoculated in the above-mentioned seed medium, which was cultured at 31.5 °C, 220 rpm for 12 h until the mid-logarithmic growth phase. The seed liquid was inoculated into a 500 mL shake flask containing 20 mL of fermentation medium at a 10% inoculation amount, and cultured at 31.5 °C, 220 rpm for 16 h. After the glucose was completely consumed, the concentration of accumulated lysine in the medium was determined by HPLC method.

[0120] The results are shown in Table 4 (OD 562 is the turbidity of the diluted 100 times culture solution at 562 nm and represents the cell amount, Lys (g / L) represents the amount of accumulated L-lysine. After introducing the P373S+A422T+T882I mutation in MHZ-0912-6, the lysine increased from 37.5 g / L to 40.4 g / L, and the conversion rate increased from 62.5% to 67.3%, an increase of 4.8% percentage points.

[0121] Table 4 Detection of lysine content of mutant strains

[0122] Strain MHZ-0912-6 (starting strain) MHZ-0912-7 (engineered strain) OD 600 ]] 39.7 38.6 L-lysine concentration g / L 37.5 40.4 Sugar acid conversion % 62.5 67.3

[0123] While the application has been described in detail with particular references to specific embodiments thereof, it will be understood by those skilled in the art that various modifications or changes in form and details can be made therein without departing from the spirit and scope thereof. It is therefore intended that the application be construed as including all such modifications and changes as fall within the scope of the appended claims. SEQUENCE LISTING <110> Li Yan <120> Mutants of MurJ protein and uses thereof <130> KHP211117877.5 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 3345 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 1 atgaatggtc aacaagtgag ttcttcgctt tcgaataatt cggagcagtc cggcctgcgt 60 ggcaggatcg ttgctccagc accgccggcg cctgtgcccg aggcgcgcaa gaaggctgtc 120 gcacgcacgg atggtgatcg ctcgagtttg aaaaactcgc ctacggcatc cgccacccag 180 gcagcccaga cgcgtctggc agaaccggaa ccagaaaagc acacctccga ttctgatgtg 240 gtgcgctcga ctggctccat ggcaatagcc acgctgctga gtcgtatcac cggtttcctg 300 cgcaccgtga tgattggtgc ggcgctgtcg ccggctatcg cgtcggcgtt caacactgcc 360 aacacgctgc ccaacctgat cactgagatc gtgttgggtg cggtgctgac atcgctggtt 420 attccggtgc ttacccgcgc ggaaaaagaa gacgccgacg gcggttccgg gttcttcagg 480 cggctgctca ccctgtcggt gacgctgctg ggcggtgtca ccatcctgtc gattatcggc 540 gcgccgctgc tgacacggat gatgctgtcc tctgagggac aagtcaacgt ggtcatgtcc 600 acggcctttg cgtattggct gctgccacag attttcttct acggcctgtt tgccctgttc 660 atggctgtgt tgaacacccg tgaagtgttc aaacccggcg cgtgggcacc tgttgtcaac 720 aatgtgatca ccttgaccgt gctgggcgtg tacatggtgc tgcctgcgcg tttgcacccg 780 catgagcagg tgggcatttt tgatccgcag atcattttcc tcggcgtggg caccaccctt 840 ggtgtggttg cacagtgtct aatcatgatt ccgtacctgc gtcgcgcggg cattgatatg 900 cgccctctgt ggggtatcga tgcgcgtttg aagcagttcg gtggcatggc catggcgatc 960 atcgtgtacg tggcgatctc ccagttcggt tacatcatca ccactcgcat tgcgtcgatt 1020 gcagacgatg ctgcgccgtt tatttatcag cagcactgga tgttgctgca agttccttat 1080 ggcatcattg gcgtcacctt gctcaccgcg attatgtcgc gactgtcccg caacgcggca 1140 gacggcgacg atagggcagt agtctctgac cttcagttgg gttccaagct gaccttcatc 1200 gcactgatcc ccatcgtggt gttcttcacc gccttcggtg tccctattgc caatggcctt 1260 tttacctacg gccaattcga tgccaacgcc gccaacatcc ttggttggac tctgagcttc 1320 tctgctttca cgctgattcc ttacgctttg gtgctgctac atctgcgtgt gttttatgcg 1380 cgtgaagagg tctggacccc aaccttcatc atcgccggca tcaccgccac caaggtcgtg 1440 ctgtccctgt tggcaccgct gctgtcgagc tccccggagc gtgtggtggt gcttcttggt 1500 gcggccaacg gtttcagttt catcaccggc gcggtcatcg gcgcgtatct gttgcgcaag 1560 aaactcggcc tgttgggtat gcgctctttg gctaaaacct ccctgtgggc gttgggctct 1620 gcggcggttg gtgcagcagc agcatgggcg ttggggtggc tgattcaagc cgtcgtgggc 1680 gatttcttgc tgggcactct aagctccgta ggctacttgt tgtacctggc tgtgttgggt 1740 gtcttcttca tcatcatcac cggcatcgtg ctgtcacgtt ctggtctgcc agaggtccaa 1800 aacttaggcc aggcactgac ccgcatcccg ggtatgagtc gctttattcg cccgaacacc 1860 aagatctctt tggatgtcgg cgaagtctcc cagcaagatt tctccaccca gctggtcgcg 1920 ccaagcgagt tctccgcaac ccctgttccg ccaccaatgt ccgccggtat tgtccgcgga 1980 cctcgcctgg ttcccggcgc cccagtcggc gacggtcgct tccgcttgct tgccgatcac 2040 ggcggcgtcc agggtgcacg tttctggcag gcccgcgaga tcgccaccgg caaggaagtc 2100 gcgctgatct tcgtggatac ttccggcaac gccccatttg cgccactgtc ttcggcagcc 2160 gcagcgggca tcgcctacga ggtgcagcgc cgcaccaaga agctggccag cttgggcagc 2220 ttggcggtgg cccccaatat ctactccgag gcgtaccgca acggttgcct cattgtggcc 2280 gattgggtgc ctggctccag cttgagcgcc gtcgcggaat ccggtgccga tccccgcgcc 2340 gccgcgttcg cgctcgcgga actaactgaa accatcggcg aggcccacga gatgggtatc 2400 ccggccggct tggacaacaa gtgccgcatc cgcatcaaca ccgacggcca tgccgtcctc 2460 gccttcccgg cgattttgcc cgatgcctca gagctccgcg acgccaagtc cctggcctcg 2520 gccgccgaga tgcttatcga cgcgaccctc gctcccagcg acgtcaaggc aatggtcact 2580 GAAGCCCAGG GGCTAGCTAC AGAAGACAA TCCCATTACG CATCATTTCG CATGGCGATG 2640 CGCATCTGCG GACTGTTCCA CCAGAACAAC CCACCTTGCG TGGTGAAGAA GGAAAAGACA 2700 CCAAAGCCTG CGACACGTGA TGTTTCGGTG CCTCCGACTA CACCCTCAAG GGTCATGGC 2760 GCCATCGCCG CTGTGGTGAT CATCTTGGT TTCCCTGGTG GCTGCCGGTA CCACGTTCCC 2820 ACCAGCTTCT TCAGCAGCAC CAACGAAACA TCCCCATTGG CTTCTGTTGA AGCCACCTTC 2880 ACTTCTGCAA CACCAGAACT GGTGGGCCAC CGGTCTACCT GGATCTGGAT CAAGCCCAGC 2940 ACGTGGGATG ACGGTGCAGA ACAGATGTCA CCGACGTACA CCGACGGCAA CACCTCCACC 3000 GCGTGGACCT CCACCAGCGG CGACGGCCTC CTAGTTGACC TGTCCTCGCC TGCCCAGCTC 3060 GACCAGTACA TCTTGACCAC CAGCACCAGC TCTGACAGCA ACAGTGAACC TGACCCTGAA 3120 ATCTACGCAT TCAACGACGC CTCACCACAC TCCCTGTCGG AAGGCATCGA GATCGGCACT 3180 GTGGATTATT CCAGCCGCAG CCTCAGCCAC AGCATCCGCG ATCCTCCCAA GCTTCCGGGT 3240 CAGGTGGAAT CCATGATGAT TCTGATCGAT GAGGTTCATT CCTCACAAAC CTCAGACACC 3300 aatccacaga tgcagatcgc tgaagtacaa ctcgttggtt ggtaa 3345 <210> 2 <211> 1114 <212> PRT <213> Artificial Sequence <400> 2 Met Asn Gly Gln Gln Val Ser Ser Ser Leu Ser Asn Asn Ser Glu Gln 1 5 10 15 Ser Gly Leu Arg Gly Arg Ile Val Ala Pro Ala Pro Pro Ala Pro Val 20 25 30 Pro Glu Ala Arg Lys Lys Ala Val Ala Arg Thr Asp Gly Asp Arg Ser 35 40 45 Ser Leu Lys Asn Ser Pro Thr Ala Ser Ala Thr Gln Ala Ala Gln Thr 50 55 60 Arg Leu Ala Glu Pro Glu Pro Glu Lys His Thr Ser Asp Ser Asp Val 65 70 75 80 Val Arg Ser Thr Gly Ser Met Ala Ile Ala Thr Leu Leu Ser Arg Ile 85 90 95 Thr Gly Phe Leu Arg Thr Val Met Ile Gly Ala Ala Leu Ser Pro Ala 100 105 110 Ile Ala Ser Ala Phe Asn Thr Ala Asn Thr Leu Pro Asn Leu Ile Thr 115 120 125 Glu Ile Val Leu Gly Ala Val Leu Thr Ser Leu Val Ile Pro Val Leu 130 135 140 Thr Arg Ala Glu Lys Glu Asp Ala Asp Gly Gly Ser Gly Phe Phe Arg 145 150 155 160 Arg Leu Leu Thr Leu Ser Val Thr Leu Leu Gly Gly Val Thr Ile Leu 165 170 175 Ser Ile Ile Gly Ala Pro Leu Leu Thr Arg Met Met Leu Ser Ser Glu 180 185 190 Gly Gln Val Asn Val Val Met Ser Thr Ala Phe Ala Tyr Trp Leu Leu 195 200 205 Pro Gln Ile Phe Phe Tyr Gly Leu Phe Ala Leu Phe Met Ala Val Leu 210 215 220 Asn Thr Arg Glu Val Phe Lys Pro Gly Ala Trp Ala Pro Val Val Asn 225 230 235 240 Asn Val Ile Thr Leu Thr Val Leu Gly Val Tyr Met Val Leu Pro Ala 245 250 255 Arg Leu His Pro His Glu Gln Val Gly Ile Phe Asp Pro Gln Ile Ile 260 265 270 Phe Leu Gly Val Gly Thr Thr Leu Gly Val Val Ala Gln Cys Leu Ile 275 280 285 Met Ile Pro Tyr Leu Arg Arg Ala Gly Ile Asp Met Arg Pro Leu Trp 290 295 300 Gly Ile Asp Ala Arg Leu Lys Gln Phe Gly Gly Met Ala Met Ala Ile 305 310 315 320 Ile Val Tyr Val Ala Ile Ser Gln Phe Gly Tyr Ile Ile Thr Thr Arg 325 330 335 Ile Ala Ser Ile Ala Asp Asp Ala Ala Pro Phe Ile Tyr Gln Gln His 340 345 350 Trp Met Leu Leu Gln Val Pro Tyr Gly Ile Ile Gly Val Thr Leu Leu 355 360 365 Thr Ala Ile Met Ser Arg Leu Ser Arg Asn Ala Ala Asp Gly Asp Asp 370 375 380 Arg Ala Val Val Ser Asp Leu Gln Leu Gly Ser Lys Leu Thr Phe Ile 385 390 395 400 Ala Leu Ile Pro Ile Val Val Phe Phe Thr Ala Phe Gly Val Pro Ile 405 410 415 Ala Asn Gly Leu Phe Thr Tyr Gly Gln Phe Asp Ala Asn Ala Ala Asn 420 425 430 Ile Leu Gly Trp Thr Leu Ser Phe Ser Ala Phe Thr Leu Ile Pro Tyr 435 440 445 Ala Leu Val Leu Leu His Leu Arg Val Phe Tyr Ala Arg Glu Glu Val 450 455 460 Trp Thr Pro Thr Phe Ile Ile Ala Gly Ile Thr Ala Thr Lys Val Val 465 470 475 480 Leu Ser Leu Leu Ala Pro Leu Leu Ser Ser Ser Pro Glu Arg Val Val 485 490 495 Val Leu Leu Gly Ala Ala Asn Gly Phe Ser Phe Ile Thr Gly Ala Val 500 505 510 Ile Gly Ala Tyr Leu Leu Arg Lys Lys Leu Gly Leu Leu Gly Met Arg 515 520 525 Ser Leu Ala Lys Thr Ser Leu Trp Ala Leu Gly Ser Ala Ala Val Gly 530 535 540 Ala Ala Ala Ala Trp Ala Leu Gly Trp Leu Ile Gln Ala Val Val Gly 545 550 555 560 Asp Phe Leu Leu Gly Thr Leu Ser Ser Val Gly Tyr Leu Leu Tyr Leu 565 570 575 Ala Val Leu Gly Val Phe Phe Ile Ile Ile Thr Gly Ile Val Leu Ser 580 585 590 Arg Ser Gly Leu Pro Glu Val Gln Asn Leu Gly Gln Ala Leu Thr Arg 595 600 605 Ile Pro Gly Met Ser Arg Phe Ile Arg Pro Asn Thr Lys Ile Ser Leu 610 615 620 Asp Val Gly Glu Val Ser Gln Gln Asp Phe Ser Thr Gln Leu Val Ala 625 630 635 640 Pro Ser Glu Phe Ser Ala Thr Pro Val Pro Pro Pro Met Ser Ala Gly 645 650 655 Ile Val Arg Gly Pro Arg Leu Val Pro Gly Ala Pro Val Gly Asp Gly 660 665 670 Arg Phe Arg Leu Leu Ala Asp His Gly Gly Val Gln Gly Ala Arg Phe 675 680 685 Trp Gln Ala Arg Glu Ile Ala Thr Gly Lys Glu Val Ala Leu Ile Phe 690 695 700 Val Asp Thr Ser Gly Asn Ala Pro Phe Ala Pro Leu Ser Ser Ala Ala 705 710 715 720 Ala Ala Gly Ile Ala Tyr Glu Val Gln Arg Arg Thr Lys Lys Leu Ala 725 730 735 Ser Leu Gly Ser Leu Ala Val Ala Pro Asn Ile Tyr Ser Glu Ala Tyr 740 745 750 Arg Asn Gly Cys Leu Ile Val Ala Asp Trp Val Pro Gly Ser Ser Leu 755 760 765 Ser Ala Val Ala Glu Ser Gly Ala Asp Pro Arg Ala Ala Ala Phe Ala 770 775 780 Leu Ala Glu Leu Thr Glu Thr Ile Gly Glu Ala His Glu Met Gly Ile 785 790 795 800 Pro Ala Gly Leu Asp Asn Lys Cys Arg Ile Arg Ile Asn Thr Asp Gly 805 810 815 His Ala Val Leu Ala Phe Pro Ala Ile Leu Pro Asp Ala Ser Glu Leu 820 825 830 Arg Asp Ala Lys Ser Leu Ala Ser Ala Ala Glu Met Leu Ile Asp Ala 835 840 845 Thr Leu Ala Pro Ser Asp Val Lys Ala Met Val Thr Glu Ala Gln Gly 850 855 860 Leu Ala Thr Glu Asp Asn Pro Asp Tyr Ala Ser Leu Ala Met Ala Met 865 870 875 880 Arg Ile Cys Gly Leu Phe Thr Glu Glu Pro Thr His Leu Val Val Lys 885 890 895 Lys Glu Lys Thr Pro Lys Pro Ala Thr Arg Asp Gly Phe Gly Ala Ser 900 905 910 Asp Tyr Thr Val Lys Gly Met Ala Ala Ile Ala Ala Val Val Ile Ile 915 920 925 Leu Val Ser Leu Val Ala Ala Gly Thr Ala Phe Leu Thr Ser Phe Phe 930 935 940 Gly Ser Ser Thr Asn Glu Gin Ser Pro Leu Ala Ser Val Glu Ala Thr 945 950 955 960 Thr Ser Ala Thr Pro Glu Pro Val Gly Pro Pro Val Tyr Leu Asp Leu 965 970 975 Asp Gin Ala Arg Thr Trp Asp Asp Gly Ala Gly Thr Asp Val Thr Asp 980 985 990 Val Thr Asp Gly Asn Thr Ser Thr Ala Trp Thr Ser Thr Gly Gly Asp 995 1000 1005 Gly Leu Leu Val Asp Leu Ser Thr Pro Ala Arg Leu Asp Arg Val Ile 1010 1015 1020 Leu Thr Thr Gly Thr Gly Ser Asp Ser Asn Val Thr Ser Thr Val Lys 1025 1030 1035 1040 Ile Tyr Ala Phe Asn Asp Ala Ser Pro His Ser Leu Ser Glu Gly Ile 1045 1050 1055 Glu Ile Gly Thr Val Asp Tyr Ser Gly Arg Ser Leu Ser His Ser Ile 1060 1065 1070 Arg Asp Ser Ser Lys Leu Pro Gly Gin Val Glu Ser Met Val He Leu 1075 1080 1085 Val Asp Glu Val His Ser Ser Gin Thr Ser Asp Thr Asn Pro Gin Met 1090 1095 1100 Gln He Ala Glu Val Gin Leu Val Gly Trp 1105 1110

Claims

1. Mutant of the MurJ protein, characterized in that, the mutation of the 373th amino acid from P to S, the mutation of the 422th amino acid from A to T and the mutation of the 882th amino acid from T to I; The reference sequence number of the MurJ protein on NCBI is WP_216312835.

1.

2. A nucleic acid molecule encoding the mutant of the MurJ protein according to claim 1.

3. A biological material containing the nucleic acid molecule according to claim 2, wherein the biological material is a recombinant DNA molecule, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.

4. The nucleic acid molecule according to claim 2 or the biological material according to claim 3 for any of the following uses: (1) for the fermentation production of amino acids; (2) for improving the fermentation yield of amino acids; (3) for constructing genetically engineered bacteria producing amino acids; wherein The amino acids include glutamic acid, lysine and proline.

5. A method for constructing genetically engineered bacteria producing amino acids, characterized by, The mutation of the 373th amino acid from P to S, the mutation of the 422th amino acid from A to T and the mutation of the 882th amino acid from T to I are introduced into the genome of bacteria with the ability of producing amino acids by genetic engineering, so that the encoded MurJ protein contains the mutations; The reference sequence number of the MurJ protein on NCBI is WP_216312835.

1. The amino acids include glutamic acid, lysine and proline.

6. The method of claim 5, wherein, The bacteria are Escherichia (E. coli) Escherichia ), Corynebacterium (C. glutamicum) Corynebacterium ), Bacillus (B. subtilis) Bacillus ) species.

7. The method of claim 6, wherein, The bacteria are Corynebacterium glutamicum Corynebacterium glutamicum ).

8. The method of claim 7, wherein, The Corynebacterium glutamicum is strain MHZ-0112-8, MHZ-0701 or MHZ-0912-6.

9. The genetically engineered bacteria producing amino acids obtained by the method according to any one of claims 5-8.

10. The genetically engineered bacteria according to claim 9 for the fermentation production of amino acids or for improving the fermentation yield of amino acids; wherein The amino acids include glutamic acid, lysine and proline.

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