ATP-dependent CLP protease variant for improving fermentation level of L-arginine

Abstract

The invention discloses an ATP (adenosine triphosphate)-dependent CLP protease variant, which is a 307th mutant of ATP-dependent CLP protease with an amino acid sequence as shown in SEQ ID NO: 2, and after an encoding gene of the ATP-dependent CLP protease variant replaces an NCgl2585 / clpC gene in corynebacterium glutamicum for producing L-arginine, the fermentation level of the L-arginine can be effectively improved, and the L-arginine can be effectively degraded. The engineering strain with high yield and stable yield of the L-arginine is constructed.

Description

Technical Field

[0001] The invention belongs to the field of metabolic engineering, and in particular relates to an ATP-dependent CLP protease variant for improving the fermentation level of L-arginine and its use in constructing L-arginine producing bacteria. Background Art

[0002] L-arginine (L-Arg) is one of the semi-essential basic amino acids required by the human body. As a basic amino acid containing a guanidine group, it is an important intermediate metabolite of the urea cycle of organisms. It has a variety of unique physiological and pharmacological effects. It has good therapeutic effects on the treatment of physiological functions, cardiovascular diseases, stimulating the immune system, maintaining the nutritional balance of infants, and promoting human detoxification. For example, in clinical practice, in addition to being one of the main components of compound amino acid infusion, L-arginine and its salts are also widely used to treat various types of hepatic coma and those who are contraindicated from using sodium glutamate and those with abnormal alanine aminotransferase in viral livers, and have significant effects on viral hepatitis. It has therapeutic effects on intestinal ulcers, thrombosis, neurasthenia and other diseases. In addition, L-arginine is an important component of the formula of sports nutrition drinks, an important feed additive, and is also widely used in high-end breeding.

[0003] Since L-arginine has a large market demand, improving the production performance of strains can reduce production costs and expand downstream demand. The common method of producing L-arginine is through the use of microbial fermentation. L-Glutamic acid is the synthetic precursor of L-arginine. Corynebacterium glutamicum has a high natural glutamate flux, and using Corynebacterium glutamicum to produce L-arginine has a natural advantage.

[0004] For many years, our research group has conducted systematic research on the method of producing L-arginine by bacterial fermentation through metabolic engineering. See patent documents with publication numbers CN109957518A, CN110511899A, CN110511898A, CN110511952A, CN110564656A, CN110564758A, CN110564790A, CN114317582A, etc., which respectively report research work on the fermentation production of L-arginine using wild-type Corynebacterium glutamicum ATCC13032 as the starting strain. Summary of the invention

[0005] In order to further promote the industrial development of L-arginine production by microbial fermentation and meet market demand, we continue to genetically modify Corynebacterium glutamicum through metabolic engineering, mutagenesis / mutant strain screening and other technologies to cultivate an engineered strain that produces high and stable L-arginine. Specifically, the present invention includes the following technical solutions:

[0006] An ATP-dependent CLP protease variant, which is the following 307th position mutant of the ATP-dependent CLP protease with an amino acid sequence as shown in SEQ ID NO: 2, wherein the enzyme activity of the ATP-dependent CLP protease variant is lower than that of the wild-type ATP-dependent CLP protease with an amino acid sequence as shown in SEQ ID NO: 2, resulting in downregulation of ATP hydrolase activity.

[0007] The wild-type ATP-dependent CLP protease is encoded by the CGL_RS13350 / Cgl2678 / NCgl2585 / clpC gene (Genbank: NC_003450.3, Gene ID: 1020624).

[0008] In one embodiment, the mutant at position 307 of the above-mentioned ATP-dependent CLP protease is selected from the following group: E307K, E307G, E307D, E307Q, E307F, E307M, E307A.

[0009] Among them, the amino acid sequence of the E307K mutant is shown in SEQ ID NO:4, the amino acid sequence of the E307G mutant is shown in SEQ ID NO:6, the amino acid sequence of the E307D mutant is shown in SEQ ID NO:8, the amino acid sequence of the E307Q mutant is shown in SEQ ID NO:10, the amino acid sequence of the E307F mutant is shown in SEQ ID NO:12, the amino acid sequence of the E307M mutant is shown in SEQ ID NO:14, and the amino acid sequence of the E307A mutant is shown in SEQ ID NO:16.

[0010] A second aspect of the present invention provides a gene encoding the ATP-dependent CLP protease variant described above.

[0011] Preferably, the gene encoding the ATP-dependent CLP protease is a polynucleotide with a nucleotide sequence of SEQ ID NO: 1 (CGL_RS13350 / Cgl2678 / NCgl2585 / clpC, Genbank: NC_003450.3, Gene ID: 1020624), the gene encoding the E307K mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 3, the gene encoding the E307G mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 5, the gene encoding the E307D mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 7, the gene encoding the E307Q mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 9, the gene encoding the E307F mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 11, the gene encoding the E307M mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 13, and the gene encoding the E307A mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 15.

[0012] The third aspect of the present invention provides a DNA molecule comprising the above-mentioned gene, for example, an expression cassette / expression frame of mutants E307K, E307G, E307D, E307Q, E307F, E307M or E307A.

[0013] The fourth aspect of the present invention provides the use of the ATP-dependent CLP protease variant, the gene, or the DNA molecule described above in improving the fermentation level of L-arginine producing bacteria.

[0014] As a specific use, the L-arginine-producing bacteria are made to express the ATP-dependent ATP-dependent CLP protease variant as described above to replace the expression of the endogenous wild-type ATP-dependent CLP protease, that is, the encoding gene of the ATP-dependent CLP protease variant as described above is used to replace the original ATP-dependent CLP protease encoding gene in the genome of the L-arginine-producing bacteria.

[0015] In one embodiment, the use is a method for constructing an L-arginine engineered bacterium, comprising the following steps: using L-arginine producing bacteria as chassis cells, causing the original ATP-dependent CLP protease encoding gene in the genome to undergo E307K, E307G, E307D, E307Q, E307F, E307M or E307A mutations to obtain an engineered bacterium in which the ATP hydrolase activity is downregulated, resulting in an improved L-arginine fermentation level.

[0016] Optionally, the mutation step can be implemented by gene editing technology, antisense nucleic acid, and transcription regulation.

[0017] In one embodiment, the above-mentioned gene editing technology can be selected from the following groups: single-double exchange system, RAGATH-associated DNA nuclease (RAD) system, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system.

[0018] Preferably, the L-arginine producing bacteria, i.e., the chassis cells, are Corynebacterium glutamicum, for example, Corynebacterium glutamicum ATCC13032 is used as the chassis cells to knock out the genes NCgl2644 and argR, and then superimpose the mutation argB A26V M31V The resulting strain has a genotype of, for example, ATCC13032ΔNCgl2644ΔargRargB A26V M31V Alternatively, the glutamicum ATCC13032 cell was used as the chassis to mutate the gene NCgl2644, knock out argR, and then superimpose the mutation argB A26VM31V The resulting strain has a genotype of, for example, ATCC13032NCgl2644 A251V △argRargB A26V M31V .

[0019] The fifth aspect of the present invention provides a modified bacterium for producing L-arginine, which is constructed by the above method.

[0020] Preferably, the N-acetylglutamate synthase encoding gene NCgl2644 / cg3035 (Genbank: NC_003450.3, Gene ID: 1020685) in the genome of the modified bacteria is mutated or deleted, resulting in inactivation or weakened activity of N-acetylglutamate synthase; preferably, the mutation is A251V mutation.

[0021] Furthermore, the arginine operon regulatory gene argR (Genbank: AF041436.1) in the genome of the modified bacteria is down-regulated or knocked out, thereby relieving its repression of L-arginine synthesis.

[0022] Furthermore, the N-acetylglutamate kinase encoding gene argB (Genbank: BAB98789.1) in the genome of the modified bacteria is mutated to relieve the feedback inhibition of N-acetylglutamate kinase by L-arginine. Preferably, the anti-feedback inhibition mutations are A26V and M31V mutations.

[0023] The sixth aspect of the present invention provides the use of the modified bacteria described above in producing L-arginine by fermentation.

[0024] The genetically engineered bacteria can be directly used as a fermentation strain to produce L-arginine through fermentation, or can be used as a starting strain for further improvement to screen out production bacteria with further improved L-arginine production capacity and production performance.

[0025] When L-arginine is produced by fermenting the genetically engineered bacteria, the culture medium used in the fermentation can be any culture medium suitable for the growth and fermentation of Corynebacterium glutamicum.

[0026] According to a preferred embodiment of the present invention, the fermentation medium is composed of: 60g / L glucose, 5g / L corn steep liquor, 30g / L(NH4)2SO4, 8g / L KCl, 2g / L urea, 0.5g / L KH2PO4, 0.5g / L K2HPO4, 1g / L MgSO4·7H2O, 1g / LNaCl, 20mg / L FeSO4·7H2O, 10mg / L MnSO4·5H2O, 20mg / L niacin, 20mg / Lβ-alanine, 10mg / LVB1, 0.2mg / L biotin, 30g / L CaCO3, and KOH is adjusted to pH 7.7.

[0027] In a preferred embodiment, the L-arginine engineering bacteria fermentation includes a seed culture stage and a bacterial fermentation stage. The two stages use a seed culture medium and a fermentation medium respectively, and the fermentation medium may be the same as the seed culture medium or different from the seed culture medium.

[0028] Preferably, when the fermentation medium is different from the seed medium, the seed medium has the following composition: 3 g / L NaCl, 5 g / L yeast extract, 7 g / L beef extract, 10 g / L peptone, and 10 g / L glucose.

[0029] The invention newly discovers a mutation site capable of improving the fermentation level of L-arginine production bacteria, wherein the 307th position of the CGL_RS13350 / Cgl2678 / NCgl2585 / clpC gene (Genbank: NC_003450.3, Gene ID: 1020624) is partially mutated, and the mutation moderately downregulates the activity of ATP hydrolase, thereby effectively improving the fermentation level of L-arginine. The mutation site can be used to construct an engineering strain capable of producing L-arginine with high yield and stability, and has industrial application prospects. DETAILED DESCRIPTION

[0030] The present invention provides an ATP-dependent CLP protease variant with reduced ATP hydrolase activity, wherein the amino acid corresponding to position 307 of SEQ ID NO: 2 is replaced by another amino acid. The activity of the mutant ATP hydrolase is moderately reduced, thereby effectively improving the fermentation level of L-arginine producing bacteria.

[0031] The term gene CGL_RS13350 / Cgl2678 / NCgl2585 / clpC gene (Genbank: NC_003450.3, Gene ID: 1020624) encodes "ATP-dependent Clp protease ATP binding subunit", which can also be called "ATPase with chaperone activity, ATP binding subunit". The inventors' research found that after site-directed saturation mutagenesis of the 307th amino acid sequence of the gene CGL_RS13350 / Cgl2678 / NCgl2585 / ClpC, compared with other amino acid substitutions, mutation of E307 to K, G, D, Q, F, M and A can all increase the L-arginine production of Corynebacterium glutamicum.

[0032] As used herein, the term "(L-arginine production) increase" or "enhancement" can mean an increase of at least 10% compared to a reference level (such as a starting strain), for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase, or any increase between 10%-100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level.

[0033] Similarly, the terms "(ATP hydrolase activity) decreases", "decreases" or "downregulate" can mean a decrease of at least 10% compared to a reference level (such as a wild-type enzyme), for example, a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% decrease, or any decrease between 10%-100%, or a decrease of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level.

[0034] Taking into account the codon degeneracy or the preferred codons in the organism in which the mutant E307K, E307G, E307D, E307Q, E307F, E307M or E307A of the present invention is to be expressed, the polynucleotides of the present invention may be altered in the coding region without changing the amino acid sequence of the variant of the present invention. Therefore, it is clear that a polypeptide that can be translated into a polypeptide consisting of the amino acid sequence of the variant or a polypeptide that has homology or identity thereto due to codon degeneracy may also be included.

[0035] The gene encoding the ATP-dependent CLP protease may be derived from a microorganism of the genus Corynebacterium, specifically CGL_RS13350 / Cgl2678 / NCgl2585 / clpC from Corynebacterium glutamicum, but is not limited thereto.

[0036] Specifically, the ATP-dependent CLP protease may include, for example, the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 or a nucleotide sequence or amino acid sequence having more than 60% homology or identity thereto, but is not limited thereto. SEQ ID NO: 1 and SEQ ID NO: 2 can be obtained from NCBI, KEGG or Uiport, which are well-known databases. As an example, it may be derived from Corynebacterium or Corynebacterium glutamicum, and more specifically, it may be a polypeptide / protein containing the nucleotide sequence shown in SEQ ID NO: 1 or / and containing the amino acid sequence shown in SEQ ID NO: 2. However, it is not limited thereto. In addition, if it is an amino acid sequence having such homology or identity and exhibiting an efficacy corresponding to a protein, an auxiliary protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also within the scope.

[0037] The term "variant" refers to a variant in which one or more amino acids are conservatively substituted and / or modified so that the amino acid sequence of the variant is different from the amino acid sequence and function before the mutation. "Polypeptide" refers to a polypeptide whose characteristics are maintained. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant can be increased, unchanged or reduced compared to the polypeptide before the mutation. In addition, some variants may include variants in which one or more parts have been removed. Other variants may include variants in which parts of the mature protein are removed from the N-terminus and / or the C-terminus. The term "variant" means variant, variant polypeptide, mutant protein, mutation and variation.

[0038] The term "polynucleotide" refers to a DNA or RNA chain of a certain length or longer. A polynucleotide fragment encoding a variant. For example, a polynucleotide encoding an ATP-dependent CLP protease variant polypeptide of the present invention can be a polynucleotide sequence encoding an amino acid sequence of an ATP-dependent CLP protease variant polypeptide of the present invention, but is not limited thereto.

[0039] The gene NCgl2644 / cg3035 encodes N-acetylglutamate synthase, which can catalyze the synthesis of N-acetylglutamate from glutamate, and is the first step in the biosynthesis pathway from L-glutamate to L-arginine.

[0040] The gene argR (Genbank: AF041436.1) is a regulatory gene of the arginine operon that is ubiquitous in bacteria and has different functions in different bacteria. The inventors' research found that by knocking out this negative regulatory gene in the genome of Corynebacterium glutamicum, its repression of arginine synthesis can be relieved to a certain extent.

[0041] Gene argB (Genbank: BAB98789.1) encodes N-acetylglutamate kinase, which has the function of catalyzing acetylglutamate to generate acetylglutamate phosphate in Corynebacterium glutamicum, and is feedback inhibited by the end product L-arginine. The inventor's research found that by mutating N-acetylglutamate kinase, mutating the 26th alanine (A) to valine (V), and the 31st methionine (M) to valine (V), the feedback inhibition of L-arginine on it can be relieved to a certain extent, and the synthesis capacity of L-arginine in Corynebacterium glutamicum can be effectively improved. In this article, the mutant of the N-acetylglutamate kinase gene argB is abbreviated as argB A26V M31V .

[0042] It should be understood that technical scheme of the present invention is not only applicable to ATCC 13032, but also may be applicable to other Corynebacterium glutamicum (Corynebacterium glutamicum) that can ferment and produce L-arginine.These Corynebacterium glutamicum generally refer to a kind of Gram-positive rod-shaped bacteria, are used for fermentation and production of amino acid, particularly glutamic acid and lysine in industry.These Corynebacterium glutamicum also comprise ATCC13869, ATCC14067, ATCC13870, ATCC21831 etc.

[0043] In this article, the terms "Corynebacterium glutamicum ATCC13032 / ATCC13869 / ATCC14067 / ATCC13870 / ATCC21831", "strain ATCC13032 / ATCC13869 / ATCC14067 / ATCC13870 / ATCC21831", and "ATCC13032 / ATCC13869 / ATCC14067 / ATCC13870 / ATCC21831" have the same meaning and refer to the original strain ATCC13032 / ATCC13869 / ATCC14067 / ATCC13870 / ATCC21831 that is the object of genetic modification, which is the starting strain of L-arginine producing bacteria, i.e., the wild strain, and can all be purchased from strain collection institutions such as Shanghai Institute of Industrial Microbiology.

[0044] The present invention is described in further detail below in conjunction with specific embodiments. It should be understood that each description and embodiment disclosed herein can also be applied to each other description and embodiment. In other words, all combinations of elements such as various genes or proteins / polypeptides disclosed herein fall within the scope of the present invention. In addition, the scope of the present invention cannot be considered to be limited by the specific description described below.

[0045] Example

[0046] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned here refer to the mass percentages unless otherwise specified.

[0047] In the embodiments of the present invention, if there is no specific description on the experimental operation temperature, the temperature generally refers to room temperature (10-30° C.).

[0048] The term "solution" or "liquid" herein generally refers to an aqueous solution, in which water is usually the main solvent, which is easily understood by those skilled in the art.

[0049] Materials and methods

[0050] The whole gene synthesis, primer synthesis and sequencing in this article were all completed by Sangon Biotechnology (Shanghai) Co., Ltd.

[0051] The molecular biology experiments in this article, including plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., are mainly carried out with reference to "A Guide to Molecular Cloning Experiments" (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). For example, the competent cell transformation method and the competent cell preparation method are all carried out with reference to "A Guide to Molecular Cloning Experiments" (3rd edition), Chapter 1, page 96. If necessary, the specific experimental conditions can be determined by simple experiments.

[0052] Main culture media and buffers:

[0053] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0054] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder.

[0055] BHIS liquid medium: 37 g / L BHI, 91 g / L sorbitol.

[0056] BHIS solid medium: 37 g / L BHI, 91 g / L sorbitol, 20 g / L agar powder.

[0057] BHIS-suc solid medium: 37 g / L BHI, 91 g / L sorbitol, 200 g / L sucrose, 10 g / L glucose.

[0058] BYG medium: 3g / L NaCl, 5g / L yeast extract, 7g / L beef extract, 10g / L peptone, 10g / L glucose.

[0059] RG2 medium: 60g / L glucose, 5g / L corn steep liquor, 30g / L(NH4)2SO4, 8g / L KCl, 2g / L urea, 0.5g / L KH2PO4, 0.5g / L K2HPO4, 1g / L MgSO4·7H2O, 1g / L NaCl, 20mg / L FeSO4·7H2O, 10mg / LMnSO4·5H2O, 20mg / L niacin, 20mg / Lβ-alanine, 10mg / L VB1, 0.2mg / L biotin, 30g / LCaCO3, pH adjusted to 7.7 with KOH.

[0060] 20X electroporation master solution: 80 g / L glycine, 2% Tween 80.

[0061] In the following examples, when a culture medium containing kanamycin and spectinomycin was used, the final concentration of kanamycin in the culture medium was 25 μg / ml, and the final concentration of spectinomycin in the culture medium was 100 μg / ml.

[0062] Example 1: ATCC13032NCgl2644 A251V ΔargRargB A26V M31V Construction of strain (CIBT3568)

[0063] 1.1 Construction of related plasmids

[0064] The partial primer sequence information used in plasmid construction is shown in Table 1.

[0065] Table 1. List of primers used in plasmid construction

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] In Table 1, "-F" in the name stands for forward direction; "-R" stands for reverse direction.

[0073] 1.1.1pK18mobsacB-NCgl2644 A251V Plasmid construction

[0074] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and primer pairs 3 / 4 and 5 / 6 were used to amplify NCgl2644. A251V Upstream homology arm and downstream homology arm fragments; pK18mobsacB plasmid was used as a template, and primer pair 1 / 2 was used to amplify the pK18mobsacB backbone fragment. The primer sequences are shown in Table 1. The PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo): 2x PCR Buffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O to make up 50μl. The PCR program was 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragment was recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff Plus Multi OneStep Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α competent cells (commercial competent cells of Weidi Bio), and spread the recovery solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture at 37°C overnight, and obtain pK18mobsacB-NCgl2644 A251V Transformants of plasmids. Positive clones were identified by primers 12 / 13, and the plasmid was verified to be correct by sequencing with primers 12 / 13 / 14.

[0075] 1.1.2 Construction of pK18mobsacB-argR plasmid

[0076] Using the genome of Corynebacterium glutamicum ATCC13032 as a template, primer pairs 7 / 8 and 9 / 10 were used to amplify the upstream homology arm and downstream homology arm fragments of argR; using the pK18mobsacB plasmid as a template, primer pair 11 / 2 was used to amplify the pK18mobsacB backbone fragment, the primer sequences are shown in Table 1, and the PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo TOYOBO): 2x PCR Buffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, and ddH2O supplemented to 50μl. The PCR program was 99°C hot cover, 94°C pre-denaturation for 10 min; 98°C denaturation for 10 s, 60°C annealing for 30 s, 68°C extension for 1 min / kb; 32 cycles, and finally 68°C extension for 10 min; 16°C cooling for 10 min. The obtained fragments were recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff Plus Multi One Step Cloning Kit, Yisheng Biotech) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α transfection competent cells (commercial competent cells of Weidi Biotech), and spread the resuscitation solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture overnight at 37°C, and obtain transformants containing pK18mobsacB-argR plasmid. Positive clones were identified by 12 / 13 primers, and the plasmid was verified to be correct by sequencing with 12 / 13 / 1140 primers.

[0077] 1.1.3pK18mobsacB-argB A26V M31V Plasmid construction

[0078] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and primer pairs 15 / 16 and 17 / 18 were used to amplify argB A26V M31VUpstream homology arm and downstream homology arm fragments; primer pair 11 / 2 was used to amplify the pK18mobsacB backbone fragment. The primer sequences are shown in Table 1. The PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo): 2x PCR Buffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O to make up 50μl. The PCR program was 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragment was recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff Plus Multi One Step Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α competent cells (commercial competent cells of Weidi Bio), and spread the recovery solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture at 37°C overnight, and obtain pK18mobsacB-argB A26V M31V Transformants of plasmids. Positive clones were identified by primers 12 / 13, and the plasmid was verified to be correct by sequencing with primers 12 / 13 / 19.

[0079] 1.2 Strain construction

[0080] 1.2.1ATCC13032NCgl2644 A251V Strain construction

[0081] 1.2.1.1 Preparation of competent cells

[0082] Inoculate the glycerol strain of Corynebacterium glutamicum ATTC13032 into BHIS test tube medium and culture it overnight at 30°C and 220rpm. Inoculate it into a 50ml BHIS liquid medium shake flask at 2v / v%, add the electroporation mother solution to make the final concentration of 4g / L glycine and 0.1% Tween 80, and culture it on a constant temperature shaker at 30°C and 220rpm for 4-6h until the OD 600The value reaches about 1.0. On the clean bench, transfer all the bacterial liquid to a 50ml centrifuge tube, centrifuge at 4℃, 4500rpm for 10min, discard the supernatant, wash the bacteria with 10% glycerol, resuspend the bacteria, centrifuge at 4℃, 4500rpm for 10min, repeat the washing once, and discard the supernatant. Finally, add 350μl of 10% glycerol to suspend the bacteria, and divide it into 1.5ml centrifuge tubes. Each 100μl is prepared as a competent cell, and the competent cells can be stored in a -80℃ refrigerator.

[0083] 1.2.1.2pK18mobsacB-NCgl2644 A251V Electrotransformation of ATCC13032 competent cells

[0084] Select the plasmid with the correct sequencing, absorb 1μg into the competent cells of Corynebacterium glutamicum ATCC13032, mix well and transfer to the electroporation cup, perform electroporation under the conditions of 25uF, 2.5kV, 200Ω, and immediately transfer to 900μl 46℃ preheated BHIS liquid culture medium, and water bath in a 46℃ water bath for 6min, then place in a constant temperature shaker at 30℃, 220rpm for 1h to revive the bacteria. After revival, centrifuge the bacteria at 4500rpm for 3min, discard the supernatant, spread the bacteria on the BHIS plate containing kanamycin, and invert the plate in a 30℃ constant temperature incubator for 48 hours.

[0085] 1.2.1.3 SacB sucrose screen

[0086] Pick the transformants on the BHIS plate containing kanamycin, inoculate them into the non-resistant BHIS test tube culture medium, and culture them in a constant temperature shaker at 30°C, 220rpm for 24 hours to allow double exchange to occur. Dilute the bacteria 1000 times and spread them on a BHIS-suc plate containing 10% sucrose, invert and culture them in a constant temperature incubator at 30°C for 48 hours. Pick the transformants on the BHIS-suc plate, spot them on the BHIS plate and the BHIS plate containing kanamycin, invert and culture them in a constant temperature incubator at 30°C for 24 hours. Use primers 277 / 278 for PCR amplification to verify the transformants that grow on the BHIS plate but cannot grow on the BHIS plate containing kanamycin. The PCR amplification conditions are the same as step 1.1.1, and primers 277 / 278 / 14 are used for sequencing. The positive transformants with successful mutation of NCgl2644 gene were placed in 4 ml BHIS test tube medium, cultured on a constant temperature shaker at 30°C, 220 rpm for 24 hours, and maintained with 20% glycerol. The genotype of ATCC13032NCgl2644 was obtained. A251V strains.

[0087] 1.2.2ATCC13032NCgl2644 A251VConstruction of △argR strain

[0088] Prepare ATCC13032NCgl2644 according to the method in step 1.2.1 A251V Competent cells, pK18mobsacB-argR plasmid was transformed into ATCC13032NCgl2644 A251V In the BHIS medium containing 25 μg / ml kanamycin, the exchange recombinants were selected, and SacB sucrose counter-screening and spot plate verification were performed. Primers 1438 / 1439 were used for PCR amplification to verify the transformants that grew on BHIS plates but could not grow on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 1.1.1, and primers 1438 / 1439 / 1440 were used for sequencing. The positive transformants were transferred to 4 ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30°C, 220 rpm for 24 hours, using 20% ​​glycerol to preserve the bacteria. The genotype of ATCC13032NCgl2644 was obtained. A251V △argR strain.

[0089] 1.2.3ATCC13032NCgl2644 A251V △argRargB A26V M31V Strain construction

[0090] Prepare ATCC13032NCgl2644 according to the method in step 1.2.1 A251V △argR competent cells, pK18mobsacB-argB A26V M31V Plasmid transformation into ATCC13032NCgl2644 A251V In △argR, select the exchange recombinants on BHIS medium containing 25μg / ml kanamycin, and perform SacB sucrose counter-screening and spot plate verification. Use primers 385 / 386 for PCR amplification to verify the transformants that grow on BHIS plates but cannot grow on BHIS plates containing kanamycin. The PCR amplification conditions are the same as step 1.1.1, and primers 385 / 386 / 19 are used for sequencing. Positive transformants are transferred to 4ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30℃, 220rpm for 24 hours, using 20% ​​glycerol to preserve the bacteria. The genotype ATCC13032NCgl2644 was obtained. A251V △argRargB A26V M31V (CIBT3568) strain.

[0091] 1.3 Fermentation verification

[0092] 1.3.1 96-deep-well plate fermentation

[0093] Streak the strain from the glycerol tube onto the BHIS plate and culture at 30°C for about 48 hours. Pick a single clone and transfer it to a 96-deep-well plate containing 600 μl RG2 medium and culture it at 30°C and 240 rpm for 72 hours.

[0094] 1.3.2 Determination of arginine content by HPLC

[0095] Method: The OPA pre-column derivatization amino acid analysis method was used to determine the L-arginine content in the fermentation broth. Primary amino acids reacted with o-phthalaldehyde (OPA) in the presence of a sulfhydryl reagent to generate OPA-amino acids. The generated amino acid derivatives were separated by reversed-phase high performance liquid chromatography and detected by ultraviolet or fluorescence. Within a certain range, their absorbance was proportional to the amino acid concentration.

[0096] 1.3.3 Preparation of derivatization agent and mobile phase

[0097] Boric acid buffer: 0.4 M boric acid buffer, accurately weigh 6.183 g of boric acid, dissolve in ultrapure water, adjust to pH 10.2 with 10 N NaOH solution, and make up to 250 ml in a volumetric flask.

[0098] Derivatization agent: Accurately weigh 500 mg of solid o-phthalaldehyde (OPA), add 5 ml of anhydrous ethanol, add 500 μl of mercaptopropionic acid, and dilute to 50 ml with 0.4 M, pH 10.2 boric acid buffer.

[0099] Mobile phase A: 10mM Na2HPO4+10mM NaB4O7 solution, adjusted to pH 8.2, fixed to 1L, filtered through 0.22μm filter membrane and set aside;

[0100] Mobile phase B: ACN: MeOH: H2O = 45:45:10, constant volume 1L for standby use, reagent purity is HPLC grade;

[0101] 1.3.4 High performance liquid chromatography conditions:

[0102] Chromatographic column: Poroshell 120HPH-C18 4.6x 100mm, 4um (Cat. No.: 695970-702); Pre-column: Poroshell 120HPH-C18 4.6mm, 2.7um (Cat. No.: 820750-928) or Poroshell 120HPH-C184.6mm, 4.0um (Cat. No.: 820750-930); Flow rate: 1.0ml / min; Stop time: 28min; Column temperature: 40 degrees; DAD settings: UV on, visible light off, UV 338nm, 10nm (bandwidth), reference 390nm, 20nm.

[0103] Table 2. Gradient elution program

[0104]

[0105]

[0106] The automatic sampler takes 0.5 μl of sample, 2.5 μl of boric acid buffer, 0.5 μl of derivatization agent, and 32 μl of ultrapure water, respectively, and injects the mixture after derivatization. The L-arginine content in the sample can be obtained by comparing the peak area with the standard sample.

[0107] 1.3.5 L-arginine production by strain fermentation

[0108] According to the fermentation scheme and determination method in 1.3, the L-arginine fermentation yield of each strain was compared. Three parallel experiments were performed for each strain. The results are shown in Table 3.

[0109] Table 3. Comparison of L-arginine levels in fermented strains

[0110]

[0111] As shown in Table 3, the mutation NCgl2644 on the basis of ATCC13032 A251V , then superimposed knockout of argR and mutation of argB A26V M31V There is a clear positive effect on L-arginine production.

[0112] Example 2: Mutation of NCgl2585 (E307K, E484K, E645K, E484K E645K) in the strain of Example 1

[0113] 2.1 Plasmid construction

[0114] 2.1.1pK18mobsacB-NCgl2585 E307K Plasmid construction

[0115] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and primer pairs 509 / 510 and 508 / 706 were used to amplify NCgl2585. E307KUpstream homology arm and downstream homology arm fragments; use primer pair 1 / 2 to amplify the pK18mobsacB backbone fragment, the primer sequences are shown in Table 1, and the PCR system is as follows (the following PCR reagents are purchased from the KOD series of Toyobo): 2x PCRBuffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O to make up 50μl. The PCR program is 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragment was recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff Plus Multi One Step Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α competent cells (commercial competent cells of Weidi Bio), and spread the recovery solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture at 37°C overnight, and obtain the pK18mobsacB-NCgl2585 E307K Transformants of plasmids. Positive clones were identified by 12 / 13 primers, and the plasmid was verified to be correct by sequencing with 12 / 13 primers.

[0116] 2.1.2 pK18mobsacB-NCgl2585 E484K Plasmid construction

[0117] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and primer pairs 736 / 700 and 701 / 737 were used to amplify NCgl2585. E484K Upstream homology arm and downstream homology arm fragments; use primer pair 1 / 2 to amplify the pK18mobsacB backbone fragment, the primer sequences are shown in Table 1, and the PCR system is as follows (the following PCR reagents are purchased from the KOD series of Toyobo): 2x PCRBuffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O to make up 50μl. The PCR program is 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragment was recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff Plus Multi One Step Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α competent cells (commercial competent cells of Weidi Bio), and spread the recovery solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture at 37°C overnight, and obtain the pK18mobsacB-NCgl2585 E484K Transformants of plasmids. Positive clones were identified by 12 / 13 primers, and the plasmid was verified to be correct by sequencing with 12 / 13 primers.

[0118] 2.1.3pK18mobsacB-NCgl2585 E645K Plasmid construction

[0119] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and primer pairs 20 / 21 and 22 / 23 were used to amplify NCgl2585. E645K Upstream homology arm and downstream homology arm fragments; primer pair 2 / 11 was used to amplify the pK18mobsacB backbone fragment. The primer sequences are shown in Table 1. The PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo): 2x PCRBuffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O to make up 50μl. The PCR program was 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragment was recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff Plus Multi One Step Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α competent cells (commercial competent cells of Weidi Bio), and spread the recovery solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture at 37°C overnight, and obtain the pK18mobsacB-NCgl2585 E645K Transformants of plasmids. Positive clones were identified by 12 / 13 primers, and the plasmid was verified to be correct by sequencing with 12 / 13 primers.

[0120] 2.1.4pK18mobsacB-NCgl2585 E484K E645K Plasmid construction

[0121] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and primer pairs 933 / 702, 703 / 701, 700 / 934, NCgl2585 were used. E645K Recombinant fragment; primer pair 834 / 833 was used to amplify the pK18mobsacB backbone fragment. The primer sequences are shown in Table 1. The PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo): 2x PCR Buffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O to make up 50μl. The PCR program was 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragment was recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff Plus Multi One Step Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α competent cells (commercial competent cells of Weidi Bio), and spread the recovery solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture at 37°C overnight, and obtain the pK18mobsacB-NCgl2585 E484K E645K Transformants of plasmids were identified by using primers 1162 / 1163 to identify positive clones, and sequencing using primers 1162 / 1163 / 490 / 513 to verify that the plasmid was correct.

[0122] 2.2 Strain construction

[0123] 2.2.1 Strain CIBT3568NCgl2585 E307K Build

[0124] Prepare CIBT3568 competent cells according to the method in 1.2.1 and insert pK18mobsacB-NCgl2585 E307KThe plasmid was transferred into CIBT3568, and the exchange recombinants were selected on BHIS medium containing 25 μg / ml kanamycin, and SacB sucrose counter-screening and spot plate verification were performed. Primers 512 / 513 were used for PCR amplification to verify the transformants that grew on BHIS plates but could not grow on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 2.1.1, and primers 512 / 513 were used for sequencing. The positive transformants were transferred to 4 ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30°C, 220 rpm for 24 hours, using 20% ​​glycerol to preserve the bacteria. The genotype of CIBT3568NCgl2585 was obtained. E307K strains.

[0125] 2.2.2 Strain CIBT3568NCgl2585 E484K Build

[0126] Prepare CIBT3568 competent cells according to the method in 1.2.1 and insert pK18mobsacB-NCgl2585 E484K The plasmid was transferred into CIBT3568, and the exchange recombinants were selected on BHIS medium containing 25 μg / ml kanamycin, and SacB sucrose counter-screening and spot plate verification were performed. Primers 26 / 27 were used for PCR amplification to verify the transformants that grew on BHIS plates but could not grow on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 2.1.1, and primers 26 / 27 were used for sequencing. The positive transformants were transferred to 4 ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30°C, 220 rpm for 24 hours, using 20% ​​glycerol to preserve the bacteria. The genotype of CIBT3568NCgl2585 was obtained. E484K strains.

[0127] 2.2.3 Strain CIBT3568NCgl2585 E645K Build

[0128] Prepare CIBT3568 competent cells according to the method in 1.2.1 and insert pK18mobsacB-NCgl2585 E645K The plasmid was transferred into CIBT3568, and the exchange recombinants were selected on BHIS medium containing 25 μg / ml kanamycin, and SacB sucrose counter-screening and spot plate verification were performed. Primers 24 / 25 were used for PCR amplification to verify the transformants that grew on BHIS plates but could not grow on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 2.1.1, and primers 24 / 25 were used for sequencing. The positive transformants were transferred to 4 ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30°C, 220 rpm for 24 hours, using 20% ​​glycerol to preserve the bacteria. The genotype of CIBT3568NCgl2585 was obtained.E645K strains.

[0129] 2.2.4 Strain CIBT3568NCgl2585 E484K E645K Build

[0130] Prepare CIBT3568 competent cells according to the method in 1.2.1 and insert pK18mobsacB-NCgl2585 E484KE645K The plasmid was transferred into CIBT3568, and the exchange recombinants were selected on BHIS medium containing 25 μg / ml kanamycin, and SacB sucrose counter-screening and spot plate verification were performed. Primers 26 / 27 were used for PCR amplification to verify the transformants that grew on BHIS plates but could not grow on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 2.1.1, and primers 26 / 27 / 512 / 490 were used for sequencing. The positive transformants were transferred to 4 ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30°C, 220 rpm for 24 hours, using 20% ​​glycerol to preserve the bacteria. The genotype of CIBT3568NCgl2585 was obtained. E484K E645K strains.

[0131] 2.3 L-arginine production by strain fermentation

[0132] According to the fermentation scheme and determination method in step 1.3, the L-arginine fermentation yield of each strain was compared. Three parallel experiments were performed for each strain. The results are shown in Table 4.

[0133] Table 4. Comparison of L-arginine levels in fermented strains

[0134]

[0135] As shown in Table 4, the E307K mutant strain of NCgl2585 / clpC increased L-arginine by 139% compared with the wild-type strain; and the arginine production of the E307K mutant was significantly higher than that of E484K, E645K or the combination of the two. It was verified that the substitution of glutamic acid (E) at position 307 of the ATPase with chaperone activity by lysine (K) can further improve the arginine accumulation of L-arginine-producing bacteria. However, the substitution of glutamic acid (E) at position 484 by lysine (K) did not significantly improve the arginine accumulation.

[0136] Example 3: ATPase activity assay of NCgl2585 / ClpC and its mutants

[0137] 3.1 Plasmid construction

[0138] 3.1.1 Construction of pET28a-NCgl2585 plasmid

[0139] The NCgl2585 fragment was amplified using primer pair 696 / 697 with the genome of Corynebacterium glutamicum ATCC13032 as a template, and the pET28a plasmid backbone fragment was amplified using primer pair 698 / 699 with the pET28a plasmid as a template. The primer sequences are shown in Table 1. The PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo TOYOBO): 2x PCR Buffer for KODFX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O supplemented to 50μl. The PCR program is 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragments were recovered by Novozyme DNA purification kit and homologous recombination kit (Hieff Plus Multi One Step Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α transfection competent cells (commercial competent cells of Weidi Bio), and spread the resuscitation solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture overnight at 37°C, and obtain transformants containing pET28a-NCgl2585 plasmid. Positive clones were identified by 1148 / 1149 primers, and the plasmid was verified to be correct by sequencing with 1148 / 1149 / 513 / 490 primers.

[0140] 3.1.2 pET28a-NCgl2585 E307K Vector construction

[0141] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and primer pairs 696 / 960, 959 / 697, NCgl2585 were amplified. E307KThe upstream homology arm and the downstream homology arm were amplified with the pET28a plasmid as the template using primer pair 698 / 699. The primer sequences are shown in Table 1. The PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo): 2x PCR Buffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O to 50μl. The PCR program was 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragment was recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff Plus Multi OneStep Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α competent cells (commercial competent cells of Weidi Bio), and spread the recovery solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture at 37°C overnight, and obtain pET28a-NCgl2585 E307K Transformants of plasmids were identified by using primers 1148 / 1149 for positive clones and sequencing with primers 1148 / 1149 / 513 / 490 to verify that the plasmid was correct.

[0142] 3.1.3pET28a-NCgl2585 E484K Vector construction

[0143] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and primer pairs 696 / 700, 701 / 697 and NCgl2585 were amplified. E484K The upstream homology arm and the downstream homology arm were amplified with the pET28a plasmid as the template using primer pair 698 / 699. The primer sequences are shown in Table 1. The PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo): 2x PCR Buffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O to 50μl. The PCR program was 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragment was recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff Plus Multi OneStep Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α competent cells (commercial competent cells of Weidi Bio), and spread the recovery solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture at 37°C overnight, and obtain pET28a-NCgl2585 E484K Transformants of plasmids were identified by using primers 1148 / 1149 for positive clones and sequencing with primers 1148 / 1149 / 513 / 490 to verify that the plasmid was correct.

[0144] 3.1.4pET28a-NCgl2585 E645K Vector construction

[0145] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and primer pairs 696 / 703, 702 / 697 and NCgl2585 were amplified. E645K The upstream homology arm and the downstream homology arm were amplified with the pET28a plasmid as the template using primer pair 698 / 699. The primer sequences are shown in Table 1. The PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo): 2x PCR Buffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O to 50μl. The PCR program was 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragment was recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff Plus Multi OneStep Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α competent cells (commercial competent cells of Weidi Bio), and spread the recovery solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture at 37°C overnight, and obtain pET28a-NCgl2585 E645K Transformants of plasmids were identified by using primers 1148 / 1149 for positive clones and sequencing with primers 1148 / 1149 / 513 / 490 to verify that the plasmid was correct.

[0146] 3.1.5pET28a-NCgl2585 E484K E645K Vector construction

[0147] The genome of Corynebacterium glutamicum ATCC13032 was used as a template and primer pairs 696 / 700, 701 / 703, and 702 / 697 were used to amplify NCgl2585. E484KE645K The upstream homology arm and the downstream homology arm were amplified with the pET28a plasmid as the template using primer pair 698 / 699. The primer sequences are shown in Table 1. The PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo): 2x PCR Buffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O to 50μl. The PCR program was 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragment was recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff PlusMulti One Step Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α competent cells (commercial competent cells of Weidi Bio), and spread the recovery solution on kanamycin (final concentration 50 μg / mL) LB solid plate, culture at 37°C overnight, and obtain pET28a-NCgl2585 E484K E645K Transformants of plasmids were identified by using primers 1148 / 1149 for positive clones and sequencing with primers 1148 / 1149 / 513 / 490 to verify that the plasmid was correct.

[0148] 3.2 Protein expression and purification

[0149] Escherichia coli BL21 (DE3) △ clpAPX strain was used to express the recombinant NCgl2585 / ClpC and its mutant proteins from Corynebacterium glutamicum to prevent the unwanted co-purification of Escherichia coli Clp protein. Escherichia coli containing NCgl2585 and its mutant expression plasmids were cultured and grown to the mid-logarithmic growth phase (optical density at 600nm was 0.6-0.8) under the conditions of 37°C and 200rpm. After inducing expression by adding 1mM isopropyl-bD-thiogalactopyranoside (IPTG), the Escherichia coli culture was further shaken overnight under the conditions of 18°C ​​and 120rpm. After centrifugation, the cell pellet was resuspended in pre-cooled buffer A (100mM Tris / HCl, 150mM NaCl, pH8.0). The cells were crushed using a press and cell fragments were precipitated by centrifugation (12000rpm, 1h, 4°C). The following purification steps were carried out at 4°C. The lysate with His-tagged protein was loaded onto the HisTrap HP column, followed by washing with buffer B (20 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5 mM DTT, 10 mM imidazole) containing a gradient of imidazole from low to high, and finally eluted with buffer B containing 250 mM imidazole. Buffer exchange was performed using a centrifugal filter (Amicon Ultracel-30K, Merck) using buffer A supplemented with 30% glycerol and 5 mM DTT. The concentration and purity of the desired protein were determined by SDS-PAGE analysis and then Bradford assay using a bovine serum albumin (BSA) standard curve.

[0150] 3.3 ATPase activity assay

[0151] The ATPase activity of NCgl2585 and its mutants was determined using malachite green. The reaction was carried out in 30 μl buffer (20 mMTris / HCl, 100 mM NaCl, 10 mM MgCl2, 1 mM ATP) and 1 μM protein at 37°C for 30 minutes. Subsequently, 20 μl of the reaction solution was added to a solution containing 180 μl of ATPase activity (0.045% malachite green solution and 4.2% ammonium molybdate solution), mixed well, and then 25 μl of ATP stop solution (34% w / v sodium citrate aqueous solution) was added to terminate the reaction. The release of inorganic phosphate was measured by measuring the light absorption at 660 nm. The standard was prepared by dissolving a sodium phosphate solution in a buffer solution. The results of the comparison of the ATPase specific activity of NCgl2585 and its mutants are shown in Table 5.

[0152] Table 5. ATPase specific activity of NCgl2585 and its mutants

[0153]

[0154]

[0155] As shown in Table 5, the results of in vitro pure enzyme activity test showed that the ATPase specific activity of the E307K mutant of NCgl2585 was reduced by 86% compared with the wild type, which was significantly lower than that of E484K, E645K or the combination of the two. Combined with the arginine production in Table 4, we believe that the arginine production and the degree of reduction in ATPase activity are significantly correlated. The lower the ATP hydrolase activity, the higher the L-arginine production. It was verified that the replacement of glutamic acid (E) at position 307 of the NCgl2585 protein with lysine (K) would reduce the ATP hydrolase activity.

[0156] Example 4: Construction of NCgl2585 / ClpC 307th amino acid saturation mutant strain

[0157] 4.1 Construction of pJYS2-NCgl2585 plasmid

[0158] Plasmid pJYS2 was used as a template and primer pair 933 / 934 was used to amplify the pJYS2-NCgl2585 fragment. The primer sequences are shown in Table 1. The PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo): 2x PCR Buffer for KOD FX 25μl, dNTP 10μl, primer F 1.5μl, primer R 1.5μl, KOD FX 1μl, template 1μl, ddH2O to make up to 50μl. The PCR program was 99℃ hot cover, 94℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 60℃ annealing for 30s, 68℃ extension for 1min / kb; 32 cycles, and finally 68℃ extension for 10min; 16℃ cooling for 10min. The obtained fragment was recovered by the Novozyme DNA purification kit and purified by the homologous recombination kit (Hieff Plus Multi One Step Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α transfection competent cells (commercial competent cells of Weidi Bio), and spread the resuscitation solution on LB solid plates with kanamycin (final concentration 50 μg / mL), and culture overnight at 37°C to obtain transformants containing pJYS2-NCgl2585 plasmid. Positive clones were identified by 965 / 966 primers, and the plasmid was verified to be correct by 966 primer sequencing.

[0159] 4.2CIBT3568NCgl2585 E307 Saturation mutant strain construction

[0160] Prepare CIBT3568 competent cells according to the method in step 1.2.1, transfer the pJYS1 plasmid into CIBT3568, and culture for 48 hours in BHIS medium containing 25μg / ml kanamycin. Pick a single clone and inoculate it into BHIS liquid medium for overnight culture. Prepare CIBT3568::pJYS1 competent cells according to the method in step 2.1.2, transfer the pJYS2-NCgl2585 plasmid and ssDNA (primers 28 / 29 / 30 / 31 / 32 / 33 / 34 / 35 / 36 / 37 / 38 / 39 / 40 / 41 / 42 / 43 / 44 / 45 / 46) into CIBT3568::pJYS1, and culture for 48 hours in BHIS medium containing 25μg / ml kanamycin and 100μg / ml spectinomycin. Use primers 512 / 513 to perform PCR amplification on the clones that have grown, and use primer 512 to send for testing. Pick the transformants that mutate the amino acid at position 307 to different amino acids, inoculate them into non-antibiotic BHIS liquid medium and culture them at 37°C overnight, take part of the bacterial liquid and streak it on the non-antibiotic BHIS plate, and culture it at 37°C for 48h. Pick the BHIS plate clones, and spot them on BHIS plates, BHIS plates containing kanamycin, and BHIS plates containing spectinomycin, respectively, and culture them at 30°C for 24h. Use primers 512 / 513 to verify the clones that grow on BHIS plates but cannot grow on BHIS plates containing kanamycin and BHIS plates containing spectinomycin. The PCR amplification conditions are the same as step 2.1.1, and primer 512 is used for sequencing. The positive transformants are transferred to 4ml BHIS test tube culture medium, cultured on a constant temperature shaker at 30°C, 220rpm for 24 hours, and 20% glycerol is used to preserve the bacteria.

[0161] 4.3 L-arginine production by strain fermentation

[0162] According to the fermentation scheme and determination method in step 1.3, the L-arginine fermentation yield of each strain was compared. Three parallel experiments were performed for each strain. The results are shown in Table 6.

[0163] Table 6. Comparison of L-arginine levels in fermented strains

[0164] Fermentation strains Average L-arginine production (g / L) CIBT3568 4.58 <![CDATA[CIBT3568NCgl2585 E307K ]]> 11.18 <![CDATA[CIBT3568NCgl2585 E307* ]]> 0.45 <![CDATA[CIBT3568NCgl2585 E307G ]]> 8.72 <![CDATA[CIBT3568NCgl2585 E307D ]]> 8.59 <![CDATA[CIBT3568NCgl2585 E307P ]]> 0.22 <![CDATA[CIBT3568NCgl2585 E307Q ]]> 8.93 <![CDATA[CIBT3568NCgl2585 E307I ]]> 0.60 <![CDATA[CIBT3568NCgl2585 E307N ]]> 0.30 <![CDATA[CIBT3568NCgl2585 E307C ]]> 0.52 <![CDATA[CIBT3568NCgl2585 E307F ]]> 9.47 <![CDATA[CIBT3568NCgl2585 E307T ]]> 0.37 <![CDATA[CIBT3568NCgl2585 E307M ]]> 9.30 <![CDATA[CIBT3568NCgl2585 E307A ]]> 8.75 <![CDATA[CIBT3568NCgl2585 E307H ]]> 0.49 <![CDATA[CIBT3568NCgl2585 E307W ]]> 0.44 <![CDATA[CIBT3568NCgl2585 E307R ]]> 0.45 <![CDATA[CIBT3568NCgl2585 E307V ]]> 2.51 <![CDATA[CIBT3568NCgl2585 E307L ]]> 0.56 <![CDATA[CIBT3568NCgl2585 E307Y ]]> 0.47

[0165] As can be seen from Table 6, compared with the control strain CIBT33568, the 307th amino acid glutamic acid (E) of the NCgl2585 gene was replaced by glycine (G), which increased L-arginine by 90%; replaced by aspartic acid (D), which increased L-arginine by 88%; replaced by glutamine (Q), which increased L-arginine by 95%; replaced by phenylalanine (F), which increased L-arginine by 107%; replaced by methionine (M), which increased L-arginine by 103%; replaced by alanine (A), which increased L-arginine by 91%.

[0166] Example 5: ATCC13032ΔNCgl2644ΔargRargB A26V M31V Mutant strain construction

[0167] 5.1 Construction of pJYS3-ISClsp3-reRNA-NCgl2644 plasmid

[0168] Using pJYS3-ISClsp3-reRNA-crtYe as a template, 1911 / 1912 and 1913 / 1914 were used as primers to amplify the plasmid backbone; using Corynebacterium glutamicum ATCC 13032 as a template, 1915 / 1916 and 1917 / 1918 were used as primers to amplify the upstream and downstream homology arms of the NCgl2644 gene. The amplified fragments were recovered using a gel recovery kit and a homologous recombination kit (Hieff Plus Multi One Step Cloning Kit, Yisheng Bio) for recombination reaction. Take 10 μl of the reaction solution to transform DH5α competent cells, and spread the recovery solution on LB solid plates with kanamycin (final concentration 50 μg / mL), culture at 30°C overnight, obtain transformants containing pJYS3-ISClsp3-reRNA-NCgl2644 plasmid, and verify the plasmid is correct by sequencing.

[0169] 5.2 Construction of ATCC13032△NCgl2644 strain

[0170] 5.2.1 Transformation of pRecE564LT-spc Helper Plasmid

[0171] The glycerol bacteria of Corynebacterium glutamicum ATCC 13032 were inoculated into BHIS test tubes and cultured at 30°C overnight. The overnight cultured test tube culture solution was inoculated into 50 mL shake flask BHIS medium at a 2% v / v inoculum, and the electroporation mother solution was added to make the final concentration of glycine 4g / L and 0.1% Tween 80, and cultured at 30°C for 4-6h until the OD 600Reach about 1.0. On the clean bench, transfer all the bacterial solution to a 50ml centrifuge tube, and collect the bacteria by centrifugation at 4°C, 4500rpm for 10min. Centrifuge at 4°C, 4500rpm for 10min, repeat washing once, and discard the supernatant. Finally, add 350μl of 10% glycerol to suspend the bacteria, and divide them into 1.5ml centrifuge tubes. Prepare one competent cell for every 100μl. Transfer pRecE564LT-spc to Corynebacterium glutamicum ATCC 13032, collect the bacteria by centrifugation, and spread them on a BHIS plate containing spectinomycin (final concentration 50μg / mL), and culture at 30°C for 48 hours. Obtain Corynebacterium glutamicum ATCC 13032::pRecE564LT-spc strain.

[0172] 5.2.2 Transformation of pJYS3-ISClsp3-reRNA-NCgl2644 plasmid

[0173] Inoculate the Corynebacterium glutamicum ATCC 13032::pRecE564LT-spc strain into a BHIS test tube containing spectinomycin and culture at 30°C overnight; inoculate the overnight cultured test tube culture into a 50 mL shake flask BHIS medium at a 2% inoculum, add glycine at a final concentration of 4 g / L and 0.1% Tween 80, and add isopropyl-β-D-thiogalactoside at a final concentration of 2 mM to induce RecE*T expression. Culture at 30°C for 4-6 hours until OD 600 After reaching about 1.0, place in an ice bath for 20 minutes. Collect the bacteria by centrifugation at 4500rpm for 10 minutes at 4℃. Wash the bacteria twice with 10% glycerol, and prepare 5 100μl competent cells with 50ml culture medium. Add 1μg pJpJYS3-ISClsp3-reRNA-NCgl2644 plasmid to the competent cells, mix well and transfer to a 2mm electroporation cup, electroporate at 25μF, 2.5kV, and 200Ω, and immediately transfer to 900μl 46℃ preheated BHIS medium after electroporation. And incubate at 46℃ water bath for 6 minutes, and then incubate at 30℃ and 250rpm for 4 hours. Collect the bacteria by centrifugation and spread them on BHIS plates coated with kanamycin and spectinomycin, and culture at 30℃ for 48 hours. Use 1919 / 1920 primers for colony PCR verification.

[0174] 5.2.3 Plasmid solidification

[0175] The strain with the correct sequencing result was inoculated into a BHIS test tube, cultured overnight at 37°C, and part of the bacterial solution was streaked on a non-resistant BHIS plate, and cultured at 37°C for about 48 hours. Pick the plate transformants, spot them on BHIS plates, BHIS plates containing spectinomycin, and BHIS plates containing kanamycin, and invert them in a 30°C constant temperature incubator for 24 hours. Use primers 1919 / 1920 for PCR amplification to verify the transformants that grow on BHIS plates but cannot grow on BHIS plates containing kanamycin and BHIS plates containing spectinomycin, and then sequence them. The positive transformants with successful knockout of the NCgl2644 gene were placed in 4 ml of BHIS test tube culture medium, cultured on a constant temperature shaker at 30°C, 220rpm for 24 hours, and used 20% glycerol to preserve the bacteria. A strain with the genotype ATCC13032△NCgl2644 was obtained.

[0176] 5.3 Construction of ATCC13032△NCgl2644△argR strain

[0177] Prepare ATCC13032△NCgl2644 competent cells according to the method in step 2.1.2, transfer the pK18mobsacB-argR plasmid into ATCC13032△NCgl2644, select the exchange recombinant on BHIS medium containing 25μg / ml kanamycin, and perform SacB sucrose counter-screening and spot plate verification. Use primers 1438 / 1439 for PCR amplification to verify the transformants that grow on BHIS plates but cannot grow on BHIS plates containing kanamycin. The PCR amplification conditions are the same as those in step 2.1.1, and primers 1438 / 1439 / 1440 are used for sequencing. The positive transformants are transferred to 4ml BHIS test tube culture medium, cultured on a constant temperature shaker at 30℃, 220rpm for 24 hours, and 20% glycerol is used to maintain the bacteria. A strain with the genotype of ATCC13032△NCgl2644△argR was obtained.

[0178] 5.4ATCC13032△NCgl2644△argRargB A26V M31V Strain construction

[0179] Prepare ATCC13032△NCgl2644△argR competent cells according to the method in step 2.1.2, and insert pK18mobsacB-argB A26V M31VThe plasmid was transferred into ATCC13032△NCgl2644△argR, and the exchange recombinants were selected on BHIS medium containing 25μg / ml kanamycin, and SacB sucrose counter-screening and spot plate verification were performed. Primers 385 / 386 were used for PCR amplification to verify the transformants that grew on BHIS plates but could not grow on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 2.1.1, and primers 385 / 386 / 19 were used for sequencing. The positive transformants were transferred to 4ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30℃, 220rpm for 24 hours, using 20% ​​glycerol to preserve the bacteria. The genotype of ATCC13032△NCgl2644△argRargB was obtained. A26V M31V strains.

[0180] Comparison of L-arginine production by 5.5 strain fermentation

[0181] According to the fermentation scheme and determination method in step 1.3, the L-arginine fermentation yield of each strain was compared. Three parallel experiments were performed for each strain. The results are shown in Table 7.

[0182] Table 7. Comparison of L-arginine levels in fermented strains

[0183] Fermentation strains Average L-arginine production (g / L) ATCC13032 0 <![CDATA[ATCC13032△NCgl2644△argRargB A26V M31V ]]> 5.12

[0184] As shown in Table 7, NCgl2644 was knocked out based on ATCC13032, and argR and argB were knocked out. A26V M31V There is a clear positive effect on L-arginine production.

[0185] Example 6: Combining the effective mutations in Examples 2 and 4 onto the host of Example 5

[0186] 6.1 Strain construction

[0187] 6.1.1ATCC13032ΔNCgl2644ΔargRargB A26V M31V NCgl2585 E307K Strain construction

[0188] Prepare ATCC13032ΔNCgl2644ΔargRargB according to the method in step 1.2.1 A26V M31V Competent cells, pK18mobsacB-NCgl2585 E307KThe plasmid was transferred into competent cells, and the exchange recombinants were selected on BHIS medium containing 25 μg / ml kanamycin, and SacB sucrose counter-screening and spot plate verification were performed. Primers 512 / 513 were used for PCR amplification to verify the transformants that grew on BHIS plates but could not grow on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 2.1.1, and primers 512 / 513 were used for sequencing. The positive transformants were transferred to 4 ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30°C, 220 rpm for 24 hours, using 20% ​​glycerol to preserve the bacteria. The genotype of ATCC13032△NCgl2644△argRargB was obtained. A26V M31V NCgl2585 E307K strains.

[0189] 6.1.2ATCC13032ΔNCgl2644ΔargRargB A26V M31V NCgl2585 E484K Strain construction

[0190] Prepare ATCC13032ΔNCgl2644ΔargRargB according to the method in 1.2.1 A26V M31V Competent cells, pK18mobsacB-NCgl2585 E484K The plasmid was transferred into competent cells, and the exchange recombinants were selected on BHIS medium containing 25 μg / ml kanamycin, and SacB sucrose counter-screening and spot plate verification were performed. Primers 26 / 27 were used for PCR amplification to verify the transformants that grew on BHIS plates but could not grow on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 2.1.1, and primers 26 / 27 were used for sequencing. The positive transformants were transferred to 4 ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30°C, 220 rpm for 24 hours, using 20% ​​glycerol to preserve the bacteria. The genotype of ATCC13032△NCgl2644△argRargB was obtained. A26V M31V NCgl2585 E484K strains.

[0191] 6.1.3ATCC13032ΔNCgl2644ΔargRargB A26V M31V NCgl2585 E645K Strain construction

[0192] Prepare ATCC13032ΔNCgl2644ΔargRargB according to the method in 1.2.1 A26V M31VCompetent cells, pK18mobsacB-NCgl2585 E645K The plasmid was transferred into competent cells, and the exchange recombinants were selected on BHIS medium containing 25 μg / ml kanamycin, and SacB sucrose counter-screening and spot plate verification were performed. Primers 24 / 25 were used for PCR amplification to verify the transformants that grew on BHIS plates but could not grow on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 2.1.1, and primers 24 / 25 were used for sequencing. The positive transformants were transferred to 4 ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30°C, 220 rpm for 24 hours, using 20% ​​glycerol to preserve the bacteria. The genotype of ATCC13032△NCgl2644△argRargB was obtained. A26V M31V NCgl2585 E645K strains.

[0193] 6.1.4ATCC13032ΔNCgl2644ΔargRargB A26V M31V NCgl2585 E484K E645K Strain construction

[0194] Prepare ATCC13032ΔNCgl2644ΔargRargB according to the method in 1.2.1 A26V M31V Competent cells, pK18mobsacB-NCgl2585 E484K E645K The plasmid was transferred into competent cells, and the exchange recombinants were selected on BHIS medium containing 25 μg / ml kanamycin, and SacB sucrose counter-screening and spot plate verification were performed. Primers 26 / 27 were used for PCR amplification to verify the transformants that grew on BHIS plates but could not grow on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 2.1.1, and primers 26 / 27 / 512 / 490 were used for sequencing. The positive transformants were transferred to 4 ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30°C, 220 rpm for 24 hours, using 20% ​​glycerol to preserve the bacteria. The genotype of ATCC13032△NCgl2644△argRargB was obtained. A26VM31V NCgl2585 E484K E645K strains.

[0195] 6.1.5ATCC13032ΔNCgl2644ΔargRargB A26V M31V NCgl2585 E307

[0196] Prepare ATCC13032ΔNCgl2644ΔargRargB according to the method in step 1.2.1 A26V M31V Competent cells, transfer the pJYS1 plasmid into competent cells, and culture them in BHIS medium containing 25 μg / ml kanamycin for 48 h. Pick a single clone and inoculate it into BHIS liquid medium for overnight culture. Prepare ATCC13032△NCgl2644△argRargB according to the method in 2.1.2 A26V M31V ::pJYS1 competent cells, pJYS2-NCgl2585 plasmid and ssDNA (28 / 31 / 36 / 42 / 45 / 46) were transferred into competent cells, and cultured on BHIS medium containing 25μg / ml kanamycin and 100μg / ml spectinomycin for 48h. Use primers 512 / 513 to PCR amplify the grown clones, and use primer 512 for testing. Pick out the transformants of E307G, E307D, E307Q, E307F, E307M, and E307A, inoculate them into non-antibiotic BHIS liquid medium and culture them at 37℃ overnight, take part of the bacterial liquid and streak it on non-antibiotic BHIS plates, and culture them at 37℃ for 48h. Pick the BHIS plate clones, spot them on BHIS plates, BHIS plates containing kanamycin, and BHIS plates containing spectinomycin, and culture them at 30°C for 24h. Use primers 512 / 513 to verify the clones that grow on BHIS plates but cannot grow on BHIS plates containing kanamycin or BHIS plates containing spectinomycin. The PCR amplification conditions are the same as those in step 2.1.1, and primer 512 is used for sequencing. Positive transformants are transferred to 4ml BHIS test tube culture medium, cultured on a constant temperature shaker at 30°C, 220rpm for 24h, and maintained with 20% glycerol.

[0197] 6.2 L-arginine production by strain fermentation

[0198] According to the fermentation scheme and determination method in step 1.3, the L-arginine fermentation yield of each strain was compared. Three parallel experiments were performed for each strain. The results are shown in Table 8.

[0199] Table 8. Comparison of L-arginine levels in fermented strains

[0200]

[0201] As shown in Table 8, the mutants E645K, E484K, E645K, E307K, E307G, E307D, E307Q, E307F, E307M, and E307A of NCgl2585 can be used to increase the yield of L-arginine, and the yield is significantly higher than that of the mutations E484K, E645K, or a combination of the two. Combined with Tables 4, 5, and 6, it is suggested that reducing the activity of ATP hydrolase can effectively increase the yield of L-arginine.

[0202] The above-mentioned embodiments only represent several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An ATP-dependent CLP protease variant, which is the following 307th position mutant of the ATP-dependent CLP protease with an amino acid sequence as shown in SEQ ID NO: 2, wherein the ATP-dependent CLP protease variant has an ATP hydrolase activity lower than that of the wild-type ATP-dependent CLP protease with an amino acid sequence as shown in SEQ ID NO:

2.

2. The ATP-dependent CLP protease variant according to claim 1, characterized in that The mutant at position 307 is selected from the group consisting of E307K, E307G, E307D, E307Q, E307F, E307M, and E307A.

3. The ATP-dependent CLP protease variant according to claim 2, characterized in that The amino acid sequence of the E307K mutant is shown in SEQ ID NO:4, the amino acid sequence of the E307G mutant is shown in SEQ ID NO:6, the amino acid sequence of the E307D mutant is shown in SEQ ID NO:8, the amino acid sequence of the E307Q mutant is shown in SEQ ID NO:10, the amino acid sequence of the E307F mutant is shown in SEQ ID NO:12, the amino acid sequence of the E307M mutant is shown in SEQ ID NO:14, and the amino acid sequence of the E307A mutant is shown in SEQ ID NO:

16.

4. A gene encoding the ATP-dependent CLP protease variant according to any one of claims 1 to 3.

5. The gene according to claim 3, characterized in that The gene encoding the ATP-dependent CLP protease is a polynucleotide with a nucleotide sequence of SEQ ID NO: 1, the gene encoding the E307K mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 3, the gene encoding the E307G mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 5, the gene encoding the E307D mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 7, the gene encoding the E307Q mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 9, the gene encoding the E307F mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 11, the gene encoding the E307M mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO: 13, and the gene encoding the E307A mutant is a polynucleotide with a nucleotide sequence of SEQ ID NO:

15.

6. Use of the ATP-dependent CLP protease variant according to any one of claims 1 to 3, or the gene according to any one of claims 3 to 5, in improving the fermentation level of L-arginine producing bacteria.

7. The use according to claim 6, characterized in that The L-arginine-producing bacteria are made to express the ATP-dependent CLP protease variant as described in any one of claims 1 to 3 to replace the expression of the endogenous ATP-dependent CLP protease, that is, the original ATP-dependent CLP protease encoding gene in the genome of the L-arginine-producing bacteria is replaced with the encoding gene of the ATP-dependent CLP protease variant as described in any one of claims 3 to 5.

8. The use according to claim 7, characterized in that The invention discloses a method for constructing L-arginine engineering bacteria, comprising the following steps: using L-arginine producing bacteria as chassis cells, causing the original ATP-dependent CLP protease encoding gene in the genome to undergo E307K, E307G, E307D, E307Q, E307F, E307M or E307A mutations, thereby obtaining an engineering bacterium in which the activity of ATP hydrolase is downregulated, resulting in an improved L-arginine fermentation level.

9. The use according to claim 8, characterized in that The chassis cell is Corynebacterium glutamicum.

10. A modified bacterium for producing L-arginine, characterized in that The method is constructed by the method as claimed in claim 8 or 9.

11. The modified bacterium according to claim 10, wherein The N-acetylglutamate synthase encoding gene NCgl2644 / cg3035 (Genbank: NC_003450.3, Gene ID: 1020685) in the genome is mutated or deleted, resulting in the inactivation or weakening of the activity of N-acetylglutamate synthase; preferably, the mutation is an A251V mutation.

12. The modified bacterium according to claim 10 or 11, characterized in that The arginine operon regulatory gene argR (Genbank: AF041436.1) in the genome is down-regulated or knocked out, thereby relieving its repression of L-arginine synthesis.

13. The modified bacterium according to claim 10 or 11, characterized in that The N-acetylglutamate kinase encoding gene argB (Genbank: BAB98789.1) in the genome is mutated to relieve the feedback inhibition of N-acetylglutamate kinase by L-arginine. Preferably, the anti-feedback inhibition mutation is A26V and M31V mutation.

14. Use of the modified bacterium according to any one of claims 10 to 13 in the production of L-arginine by fermentation.

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