An ATP-dependent CLP protease variant that improves L-arginine fermentation

Abstract

The present invention discloses an ATP-dependent CLP protease variant, which is a 307th mutant of the ATP-dependent CLP protease with an amino acid sequence as shown in SEQ ID NO: 2. After the encoding gene of the ATP-dependent CLP protease variant replaces the NCgl2585 / clpC gene in L-arginine-producing Corynebacterium glutamicum, the fermentation level of L-arginine can be effectively improved, and an engineered strain with high and stable L-arginine production can be constructed.

Description

Technical Field

[0001] The present 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-arginine, abbreviated as 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 in the urea cycle of organisms. It has many unique physiological and pharmacological effects and has good therapeutic effects in treating physiological functions, cardiovascular diseases, stimulating the immune system, maintaining nutritional balance in infants, and promoting human detoxification. For example, in clinical practice, in addition to being one of the main components of compound amino acid infusions, L-arginine and its salts are also widely used to treat various types of hepatic coma who are contraindicated with monosodium glutamate and those with abnormal alanine aminotransferase in viral liver diseases, and have significant therapeutic effects on viral hepatitis. It has therapeutic effects on intestinal ulcers, thrombosis, and neurasthenia. In addition, L-arginine is an important component of sports nutrition drink formulas, an important feed additive, and is widely used in high-end breeding.

[0003] Since L-arginine has a large market demand, improving strain production performance can reduce production costs and expand downstream demand. A common method for producing L-arginine is through microbial fermentation. L-glutamate is the synthetic precursor of L-arginine. Corynebacterium glutamicum has a high natural glutamate flux, which gives it a natural advantage in producing L-arginine using Corynebacterium glutamicum.

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

[0005] 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 enzymatic 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 ATP-dependent CLP protease is selected from the group consisting of E307K, E307G, E307D, E307Q, E307F, E307M, and 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 above-mentioned ATP-dependent CLP protease variant, or the above-mentioned gene, or the above-mentioned DNA molecule in improving the fermentation level of L-arginine-producing bacteria.

[0014] As a specific application, 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 an L-arginine-producing bacterium as a chassis cell, 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 transcriptional regulation.

[0017] In one embodiment, the above-mentioned gene editing technology can be selected from the following groups: single-double crossover system, RAGATH-associated DNAnuclease (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 NCgl2644 gene is mutated using Corynebacterium glutamicum ATCC13032 as the base cell, argR is knocked out, and argB is superimposed. 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 weakening of the 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 releasing its repressive inhibition on 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] A sixth aspect of the present invention provides use of the modified bacteria described above in producing L-arginine by fermentation.

[0024] The genetically engineered bacteria can be directly used as fermentation strains to produce L-arginine through fermentation, or can be used as starting strains 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 fermentation of the L-arginine engineered bacteria comprises a seed culture stage and a bacterial fermentation stage, wherein the two stages use a seed culture medium and a fermentation medium, respectively, and the fermentation medium may be the same as or different from the seed culture medium.

[0028] Preferably, when the fermentation medium is different from the seed medium, the composition of the seed medium is as follows: 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 present invention newly discovered a mutation site that can improve the fermentation level of L-arginine-producing bacteria. The partial mutation at position 307 of the CGL_RS13350 / Cgl2678 / NCgl2585 / clpC gene (Genbank: NC_003450.3, Gene ID: 1020624) moderately downregulates the activity of ATP hydrolase, thereby effectively improving the fermentation level of L-arginine. Utilizing this mutation site, an engineered strain capable of high and stable L-arginine production can be constructed, showing promising prospects for industrial application. 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 with another amino acid. This mutant moderately reduces ATP hydrolase activity, thereby effectively improving the fermentation level of L-arginine-producing bacteria.

[0031] The gene CGL_RS13350 / Cgl2678 / NCgl2585 / clpC (Genbank: NC_003450.3, Gene ID: 1020624) encodes the "ATP-dependent Clp protease ATP-binding subunit," also known as the "ATPase with chaperone activity, ATP-binding subunit." The inventors' research has found that site-directed saturation mutagenesis at amino acid position 307 of the CGL_RS13350 / Cgl2678 / NCgl2585 / ClpC gene revealed that mutating E307 to K, G, D, Q, F, M, or A increased L-arginine production in Corynebacterium glutamicum compared to other amino acid substitutions.

[0032] As used herein, the term "(L-arginine production) increase" or "elevate" 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% and 100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold compared to a reference level.

[0033] Similarly, the terms "(ATP hydrolase activity) decreases," "decreases," or "downregulates" 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% and 100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold compared to a reference level.

[0034] Taking into account codon degeneracy or codon preference in the organism in which the mutants E307K, E307G, E307D, E307Q, E307F, E307M, or E307A of the present invention are to be expressed, the polynucleotides of the present invention may be altered in the coding region without altering the amino acid sequence of the variants of the present invention. Thus, it is apparent that polypeptides that can be translated into a polypeptide consisting of the amino acid sequence of the variant or having homology or identity thereto due to codon degeneracy are also encompassed.

[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 the genus 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 efficacy corresponding to that of the 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. A "polypeptide" refers to a polypeptide whose properties 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 decreased 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 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. It also refers to a polynucleotide fragment encoding a variant. For example, a polynucleotide encoding an ATP-dependent CLP protease variant polypeptide of the present invention may be a polynucleotide sequence encoding the 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 catalyzes 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 has 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 subject to feedback inhibition 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 to it can be relieved to a certain extent, effectively improving the synthetic capacity of L-arginine in Corynebacterium glutamicum. In this article, the mutant of this N-acetylglutamate kinase gene argB is abbreviated as argB A26V M31V .

[0042] Should be understood that technical scheme of the present invention is not only applicable to ATCC 13032, also may be applicable to other can ferment and produce L-arginine Corynebacterium glutamicum (Corynebacterium glutamicum).These Corynebacterium glutamicum generally refer to a kind of Gram-positive rod-shaped bacteria, are used for fermentation and produce amino acid, particularly L-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, all referring 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., a wild strain, and can all be purchased from culture collection institutions such as the Shanghai Institute of Industrial Microbiology.

[0044] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that each description and embodiment disclosed herein is also applicable to each other description and embodiment. In other words, all combinations of the various genes or proteins / polypeptides disclosed herein fall within the scope of the present invention. In addition, the scope of the present invention should not 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 therein, unless otherwise specified, refer to the percentage by mass.

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

[0048] When referred to herein, "solution" or "liquid" 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 Biotech (Shanghai) Co., Ltd.

[0051] The molecular biology experiments herein, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were primarily performed with reference to Molecular Cloning: A Laboratory Manual (3rd edition), edited by J. Sambrook and D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the competent cell transformation method and competent cell preparation method were performed with reference to Chapter 1, page 96 of Molecular Cloning: A Laboratory Manual (3rd edition). Specific experimental conditions can be determined through simple experiments if necessary.

[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, and 10 g / L glucose.

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

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

[0060] 20X electroporation masterbatch: 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 sequence information of some primers 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; Using the pK18mobsacB plasmid as a template, 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 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 a final extension of 68℃ for 10min; cooling at 16℃ for 10min. The resulting fragment was recovered using the Novozymes DNA purification kit and purified using the Homologous Recombination Kit (Hieff The recombination reaction was performed using the Plus Multi OneStep Cloning Kit (Yisheng Bio). 10 μl of the reaction solution was transformed into DH5α competent cells (commercial competent cells from Weidi Bio). The resuscitation solution was spread on LB solid plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C to obtain a cell containing pK18mobsacB-NCgl2644. A251V Plasmid transformants were identified by using primers 12 / 13 for positive clones, and the plasmid was verified to be correct by sequencing using 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 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. The PCR system is as follows (the following PCR reagents were purchased from the KOD series of Toyobo): 2x PCR Buffer for KOD FX 25 μl, dNTP 10 μl, primer F 1.5 μl, primer R 1.5 μl, KOD FX 1 μl, template 1 μl, and ddH2O supplemented to 50 μl. The PCR program was as follows: 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, with a final extension at 68°C for 10 min; and cooling at 16°C for 10 min. The resulting fragments were recovered using the Novozymes DNA purification kit and cloned using the homologous recombination kit (Hieff Recombination was performed using the Plus Multi One-Step Cloning Kit (Yisheng Bio). 10 μl of the reaction solution was transformed into DH5α competent cells (commercially available from Weidi Bio). The resuscitated solution was spread onto LB plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C to obtain transformants containing the pK18mobsacB-argR plasmid. Positive clones were identified using primers 12 / 13, and plasmid identity was verified using sequencing using primers 12 / 13 / 1140.

[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 TOYOBO's KOD series): 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 a final extension of 68℃ for 10min; cooling at 16℃ for 10min. The resulting fragment was recovered using the Novozymes DNA purification kit and purified using the Homologous Recombination Kit (Hieff The recombination reaction was carried out using the Plus Multi One Step Cloning Kit (Yisheng Bio). 10 μl of the reaction solution was transformed into DH5α competent cells (commercial competent cells from Weidi Bio). The resuscitation solution was spread on LB solid plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C to obtain a cell containing pK18mobsacB-argB. A26V M31V Plasmid transformants were identified by using primers 12 / 13 for positive clones and sequencing with primers 12 / 13 / 19 for plasmid verification.

[0079] 1.2 Strain construction

[0080] 1.2.1ATCC13032NCgl2644 A251V Strain construction

[0081] 1.2.1.1 Preparation of competent cells

[0082] Inoculate glycerol strains of Corynebacterium glutamicum ATTC13032 into BHIS test tube culture medium and culture overnight at 30°C, 220 rpm. Inoculate into 50 ml BHIS liquid medium shake flasks at a 2 v / v% inoculum, add electroporation stock solution to a final concentration of 4 g / L glycine and 0.1% Tween 80, and culture on a constant temperature shaker at 30°C, 220 rpm for 4-6 hours until the OD reaches 0. 600The value reaches approximately 1.0. On a clean bench, transfer the entire bacterial suspension to a 50ml centrifuge tube and centrifuge at 4500 rpm for 10 minutes at 4°C. Discard the supernatant. Wash the cells with 10% glycerol and resuspend them. Centrifuge at 4500 rpm for 10 minutes at 4°C. Repeat the wash cycle once and discard the supernatant. Finally, add 350 μl of 10% glycerol to resuspend the cells. Aliquot the cells into 1.5ml centrifuge tubes. Prepare one aliquot of competent cells per 100 μl. Competent cells can be stored in a -80°C freezer.

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

[0084] Select the correctly sequenced plasmid and pipette 1 μg into competent cells of Corynebacterium glutamicum ATCC13032. Mix thoroughly and transfer to an electroporation cuvette. Electroporate at 25 μF, 2.5 kV, and 200 Ω. Immediately after electroporation, transfer to 900 μl of BHIS liquid medium preheated at 46°C and incubate in a 46°C water bath for 6 minutes. Then, incubate in a thermostatic shaker at 30°C and 220 rpm for 1 hour to allow the cells to recover. After recovery, centrifuge the cells at 4500 rpm for 3 minutes, discard the supernatant, and spread the cells onto BHIS plates containing kanamycin. The plates are then inverted and incubated in a 30°C incubator for 48 hours.

[0085] 1.2.1.3 SacB sucrose reverse screening

[0086] Pick the transformants on the BHIS plate containing kanamycin, inoculate them into the BHIS test tube culture medium without resistance, and culture them in a constant temperature shaker at 30°C and 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 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 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 in step 1.1.1, and primers 277 / 278 / 14 are used for sequencing. The positive transformants with successful mutation of NCgl2644 gene were transferred into 4 ml BHIS test tube culture medium and cultured on a constant temperature shaker at 30°C and 220 rpm for 24 hours. The bacteria were maintained with 20% glycerol. 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 process, 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 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 and 220 rpm for 24 hours. 20% glycerol was used to maintain 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 transformed into ATCC13032NCgl2644 A251V In △argR, 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 1.1.1, and primers 385 / 386 / 19 were used for sequencing. Positive transformants were transferred to 4ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30℃ and 220rpm for 24 hours. 20% glycerol was used to maintain the bacteria. The genotype ATCC13032NCgl2644 was obtained. A251V △argRargB A26V M31V strain (CIBT3568).

[0091] 1.3 Fermentation Verification

[0092] 1.3.1 96-deep-well plate fermentation

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

[0094] 1.3.2 Determination of arginine content by HPLC

[0095] Methods: L-arginine content in the fermentation broth was determined using OPA pre-column derivatization amino acid analysis. Primary amino acids react with o-phthalaldehyde (OPA) in the presence of a sulfhydryl reagent to form OPA-amino acids. The resulting amino acid derivatives are separated by reversed-phase high-performance liquid chromatography and detected by ultraviolet or fluorescence. Within a certain range, their absorbance is 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 the volume in a 250 ml volumetric flask.

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

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

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

[0101] 1.3.4 High performance liquid chromatography conditions:

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

[0103] Table 2. Gradient elution program

[0104]

[0105]

[0106] The autosampler dispenses 0.5 μl of sample, 2.5 μl of borate buffer, 0.5 μl of derivatizing agent, and 32 μl of ultrapure water, mixing and derivatizing before injection. The L-arginine content in the sample is determined by comparing the peak area with the standard.

[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 superimpose the knockout of argR and the 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; 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 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 a final extension of 68℃ for 10min; cooling at 16℃ for 10min. The resulting fragment was recovered using the Novozymes DNA purification kit and purified using the Homologous Recombination Kit (Hieff The recombination reaction was performed using the Plus Multi One Step Cloning Kit (Yisheng Bio). 10 μl of the reaction solution was transformed into DH5α competent cells (commercial competent cells from Weidi Bio). The resuscitation solution was spread on LB solid plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C to obtain a cell containing pK18mobsacB-NCgl2585. E307K Plasmid transformants were identified by using primers 12 / 13 to identify positive clones, and the plasmid was verified to be correct by sequencing using primers 12 / 13.

[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; 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 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 a final extension of 68℃ for 10min; cooling at 16℃ for 10min. The resulting fragment was recovered using the Novozymes DNA purification kit and purified using the Homologous Recombination Kit (Hieff The recombination reaction was performed using the Plus Multi One Step Cloning Kit (Yisheng Bio). 10 μl of the reaction solution was transformed into DH5α competent cells (commercial competent cells from Weidi Bio). The resuscitation solution was spread on LB solid plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C to obtain a cell containing pK18mobsacB-NCgl2585. E484K Plasmid transformants were identified by using primers 12 / 13 to identify positive clones, and the plasmid was verified to be correct by sequencing using primers 12 / 13.

[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 TOYOBO's KOD series): 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 a final extension of 68℃ for 10min; cooling at 16℃ for 10min. The resulting fragment was recovered using the Novozymes DNA purification kit and purified using the Homologous Recombination Kit (Hieff The recombination reaction was performed using the Plus Multi One Step Cloning Kit (Yisheng Bio). 10 μl of the reaction solution was transformed into DH5α competent cells (commercial competent cells from Weidi Bio). The resuscitation solution was spread on LB solid plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C to obtain a cell containing pK18mobsacB-NCgl2585. E645K Plasmid transformants were identified by using primers 12 / 13 to identify positive clones, and the plasmid was verified to be correct by sequencing using primers 12 / 13.

[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 was as follows (the following PCR reagents were purchased from TOYOBO's KOD series): 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, with a final extension of 68℃ for 10min; and cooling at 16℃ for 10min. The resulting fragment was recovered using the Novozymes DNA purification kit and purified using the Homologous Recombination Kit (Hieff The recombination reaction was performed using the Plus Multi One Step Cloning Kit (Yisheng Bio). 10 μl of the reaction solution was transformed into DH5α competent cells (commercial competent cells from Weidi Bio). The resuscitation solution was spread on LB solid plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C to obtain a cell containing pK18mobsacB-NCgl2585. E484K E645K Plasmid transformants were identified by using primers 1162 / 1163 to identify positive clones, and the plasmid was verified to be correct by sequencing using primers 1162 / 1163 / 490 / 513.

[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 and 220 rpm for 24 hours. 20% glycerol was used to maintain 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 and 220 rpm for 24 hours. 20% glycerol was used to maintain 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 and 220 rpm for 24 hours. 20% glycerol was used to maintain 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 and 220 rpm for 24 hours. 20% glycerol was used to maintain 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 of NCgl2585 / clpC increased L-arginine production by 139% compared to the wild-type strain. Furthermore, the E307K mutant significantly increased arginine production compared to E484K, E645K, or a combination of the two. This demonstrates that substitution of glutamic acid (E) at position 307 with lysine (K) in the ATPase with chaperone activity further enhances arginine accumulation in L-arginine-producing strains. However, substitution of glutamic acid (E) at position 484 with lysine (K) had no significant effect.

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

[0137] 3.1 Construction of plasmids used

[0138] 3.1.1 Construction of pET28a-NCgl2585 plasmid

[0139] The NCgl2585 fragment was amplified using the genome of Corynebacterium glutamicum ATCC13032 as a template using primer pair 696 / 697. The pET28a plasmid backbone fragment was amplified using primer pair 698 / 699. The primer sequences are shown in Table 1. The PCR system was as follows (the following PCR reagents were purchased from Toyobo's KOD series): 25 μl of 2x PCR Buffer for KODFX, 10 μl of dNTPs, 1.5 μl of Primer F, 1.5 μl of Primer R, 1 μl of KODFX, 1 μl of template, and ddH2O to 50 μl. The PCR program was: 99°C heated lid, initial denaturation at 94°C for 10 min; denaturation at 98°C for 10 s, annealing at 60°C for 30 s, and extension at 68°C for 1 min / kb; 32 cycles, with a final extension at 68°C for 10 min; followed by a 10-min cooling at 16°C. The obtained fragments were recovered by Novozyme DNA purification kit and homologous recombination kit (Hieff Recombination was performed using the Plus Multi One-Step Cloning Kit (Yisen Biotech). 10 μl of the reaction solution was transformed into DH5α competent cells (commercially available from Weidi Biotech). The resuscitation solution was plated onto LB plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C to obtain transformants containing the pET28a-NCgl2585 plasmid. Positive clones were identified using primers 1148 / 1149, and plasmid identity was verified by sequencing using primers 1148 / 1149 / 513 / 490.

[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 and downstream homology arms were amplified using the pET28a plasmid as a template using primer pair 698 / 699. The primer sequences are shown in Table 1. The PCR system was 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, and 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 a final extension of 68℃ for 10min; cooling at 16℃ for 10min. The resulting fragment was recovered using the Novozymes DNA purification kit and purified using the Homologous Recombination Kit (Hieff The recombination reaction was carried out using the Plus Multi OneStep Cloning Kit (Yisheng Bio). 10 μl of the reaction solution was transformed into DH5α competent cells (commercial competent cells from Weidi Bio). The resuscitation solution was spread on LB solid plates containing kanamycin (final concentration 50 μg / mL) and cultured at 37°C overnight to obtain a culture containing pET28a-NCgl2585. E307K Plasmid transformants were identified by using primers 1148 / 1149 for positive clones, and sequencing was performed using primers 1148 / 1149 / 513 / 490 to verify 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, NCgl2585 were amplified. E484K The upstream and downstream homology arms were amplified using the pET28a plasmid as a template using primer pair 698 / 699. The primer sequences are shown in Table 1. The PCR system was 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, and 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 a final extension of 68℃ for 10min; cooling at 16℃ for 10min. The resulting fragment was recovered using the Novozymes DNA purification kit and purified using the Homologous Recombination Kit (Hieff The recombination reaction was carried out using the Plus Multi OneStep Cloning Kit (Yisheng Bio). 10 μl of the reaction solution was transformed into DH5α competent cells (commercial competent cells from Weidi Bio). The resuscitation solution was spread on LB solid plates containing kanamycin (final concentration 50 μg / mL) and cultured at 37°C overnight to obtain a culture containing pET28a-NCgl2585. E484K Plasmid transformants were identified by using primers 1148 / 1149 for positive clones, and sequencing was performed using primers 1148 / 1149 / 513 / 490 to verify 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, NCgl2585 were amplified. E645K The upstream and downstream homology arms were amplified using the pET28a plasmid as a template using primer pair 698 / 699. The primer sequences are shown in Table 1. The PCR system was 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, and 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 a final extension of 68℃ for 10min; cooling at 16℃ for 10min. The resulting fragment was recovered using the Novozymes DNA purification kit and purified using the Homologous Recombination Kit (Hieff The recombination reaction was carried out using the Plus Multi OneStep Cloning Kit (Yisheng Bio). 10 μl of the reaction solution was transformed into DH5α competent cells (commercial competent cells from Weidi Bio). The resuscitation solution was spread on LB solid plates containing kanamycin (final concentration 50 μg / mL) and cultured at 37°C overnight to obtain a culture containing pET28a-NCgl2585. E645K Plasmid transformants were identified by using primers 1148 / 1149 for positive clones, and sequencing was performed using primers 1148 / 1149 / 513 / 490 to verify the plasmid was correct.

[0146] 3.1.5 pET28a-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 and downstream homology arms were amplified using the pET28a plasmid as a template using primer pair 698 / 699. The primer sequences are shown in Table 1. The PCR system was 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, and 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 a final extension of 68℃ for 10min; cooling at 16℃ for 10min. The resulting fragment was recovered using the Novozymes DNA purification kit and purified using 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). The resuscitation solution is spread on LB solid plates containing kanamycin (final concentration 50 μg / mL) and cultured at 37°C overnight to obtain pET28a-NCgl2585. E484K E645K Plasmid transformants were identified by using primers 1148 / 1149 for positive clones, and sequencing was performed using primers 1148 / 1149 / 513 / 490 to verify the plasmid was correct.

[0148] 3.2 Protein expression and purification

[0149] Escherichia coli BL21 (DE3) △ clpAPX strain was used to express recombinant NCgl2585 / ClpC and its mutant proteins from Corynebacterium glutamicum to prevent unwanted co-purification of Escherichia coli Clp protein. Escherichia coli containing NCgl2585 and its mutant expression plasmids were cultured and grown to mid-logarithmic growth (optical density at 600nm was 0.6-0.8) at 37°C and 200rpm. After inducing expression by adding 1mM isopropyl-bD-thiogalactopyranoside (IPTG), the Escherichia coli culture was further shaken overnight at 18°C ​​and 120rpm. After centrifugation, the cell pellet was resuspended in pre-cooled buffer A (100mM Tris / HCl, 150mM NaCl, pH 8.0). The cells were broken 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 containing the His-tagged protein was loaded onto a HisTrap HP column, followed by washing with a gradient of buffer B containing imidazole from low to high (20 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5 mM DTT, 10 mM imidazole), and finally eluting the protein with buffer B containing 250 mM imidazole. The buffer was exchanged with buffer A supplemented with 30% glycerol and 5 mM DTT using a centrifugal filter (Amicon Ultracel-30K, Merck). The concentration and purity of the desired protein were determined by SDS-PAGE analysis followed by 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 at 37°C for 30 minutes in 30 μl of buffer (20 mM Tris / HCl, 100 mM NaCl, 10 mM MgCl2, 1 mM ATP) and 1 μM protein. Subsequently, 20 μl of the reaction solution was added to 180 μl of ATPase activity reaction solution (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 absorbance at 660 nm. The standard was prepared by dissolving sodium phosphate solution in buffer. 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, in vitro pure enzyme activity assays indicate that the ATPase specific activity of the E307K mutant of NCgl2585 was 86% lower than that of the wild-type, significantly lower than that of E484K, E645K, or the combination of the two. Combined with the arginine production data in Table 4, we believe that there is a significant correlation between arginine production and the degree of ATPase activity reduction. Lower ATP hydrolase activity is associated with higher L-arginine production. This confirms that the substitution of glutamic acid (E) at position 307 of the NCgl2585 protein with lysine (K) reduces ATP hydrolase activity.

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

[0157] 4.1 Construction of pJYS2-NCgl2585 plasmid

[0158] Using plasmid pJYS2 as a template, primer pair 933 / 934 was used to amplify the pJYS2-NCgl2585 fragment. The primer sequences are shown in Table 1. The PCR system was as follows (the following PCR reagents were purchased from Toyobo's KOD series): 2x PCR Buffer for KOD FX 25μl, dNTPs 10μl, Primer F 1.5μl, Primer R 1.5μl, KOD FX 1μl, template 1μl, and ddH2O to 50μl. The PCR program was: 99°C heated lid, 94°C initial denaturation for 10min; 98°C denaturation for 10s, 60°C annealing for 30s, 68°C extension for 1min / kb; 32 cycles, with a final extension at 68°C for 10min; and cooling at 16°C for 10min. The resulting fragment was recovered using a Novozymes DNA purification kit and purified using a homologous recombination kit (Hieff Recombination was performed using the Plus Multi One-Step Cloning Kit (Yisheng Bio). 10 μl of the reaction solution was transformed into DH5α competent cells (commercially available from Weidi Bio). The resuscitation solution was spread onto LB plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C to obtain transformants containing the pJYS2-NCgl2585 plasmid. Positive clones were identified using primers 965 / 966, and plasmid identity was verified using sequencing with the 966 primer.

[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 in BHIS medium containing 25 μg / ml kanamycin for 48 hours. Pick a single colony 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 in BHIS medium containing 25 μg / ml kanamycin and 100 μg / ml spectinomycin for 48 hours. Use primers 512 / 513 to PCR amplify the clones that have grown, and use primer 512 for testing. Pick out transformants that have mutated amino acid position 307 to different amino acids, inoculate them into antibiotic-free BHIS liquid medium, and culture them at 37°C overnight. Take a portion of the bacterial liquid and streak it onto antibiotic-free BHIS plates and culture them at 37°C for 48 hours. Pick BHIS plate clones and spot them onto BHIS plates, BHIS plates containing kanamycin, and BHIS plates containing spectinomycin, respectively, and culture them at 30°C for 24 hours. Use primers 512 / 513 to verify clones that grow on BHIS plates but cannot grow on BHIS plates containing kanamycin or BHIS plates containing spectinomycin. PCR amplification conditions are the same as in step 2.1.1, and primer 512 is used for sequencing. Positive transformants are 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. Use 20% glycerol to maintain 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, primers 1911 / 1912 and 1913 / 1914 were used to amplify the plasmid backbone; using Corynebacterium glutamicum ATCC 13032 as a template, primers 1915 / 1916 and 1917 / 1918 were used to amplify the upstream and downstream homology arms of the NCgl2644 gene. The amplified fragments were recovered using a gel recovery kit and cloned using a homologous recombination kit (Hieff Recombination was performed using the Plus Multi One Step Cloning Kit (Yisun Bio). 10 μl of the reaction solution was transformed into DH5α competent cells. The resuscitation solution was spread onto LB plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 30°C to obtain transformants containing the pJYS3-ISClsp3-reRNA-NCgl2644 plasmid. The plasmid was verified by sequencing.

[0169] 5.2 Construction of ATCC13032ΔNCgl2644 strain

[0170] 5.2.1 Transformation of pRecE564LT-spc Helper Plasmid

[0171] The glycerol culture of Corynebacterium glutamicum ATCC 13032 was inoculated into a BHIS test tube and cultured overnight at 30°C. The overnight cultured test tube culture solution was inoculated into a 50 mL shake flask BHIS medium at a 2% v / v inoculum volume, and the electroporated mother solution was added to a final concentration of 4 g / L glycine and 0.1% Tween 80. The culture was then cultured at 30°C for 4-6 h until the OD 600Reach about 1.0. On the clean bench, transfer all the bacterial liquid to a 50ml centrifuge tube and collect the bacteria by centrifugation at 4°C and 4500rpm for 10 minutes. Centrifuge at 4°C and 4500rpm for 10 minutes, repeat washing once, and discard the supernatant. Finally, add 350μl of 10% glycerol to suspend the bacteria, divide it into 1.5ml centrifuge tubes, and 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 coated with 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 Corynebacterium glutamicum ATCC 13032::pRecE564LT-spc into a BHIS tube containing spectinomycin and incubate overnight at 30°C. Inoculate a 2% inoculum of the overnight incubation tube into a 50 mL shake flask of BHIS medium. Add glycine to a final concentration of 4 g / L and 0.1% Tween 80. Induce RecE*T expression by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 2 mM. Incubate at 30°C for 4-6 hours until the OD reaches 0. 600 After reaching about 1.0, place in an ice bath for 20 minutes. Collect the cells by centrifugation at 4500 rpm for 10 minutes at 4°C. Wash the cells 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 cuvette. Electroporate at 25μF, 2.5kV, and 200Ω. Immediately after electroporation, transfer to 900μl of 46°C preheated BHIS medium. Incubate in a 46°C water bath for 6 minutes, then incubate at 30°C and 250rpm for 4 hours. Collect the cells by centrifugation and spread them on BHIS plates coated with kanamycin and spectinomycin, and culture at 30°C for 48 hours. Colony PCR verification is performed using primers 1919 / 1920.

[0174] 5.2.3 Plasmid curing

[0175] The strain with the correct sequencing result was inoculated into a BHIS test tube and cultured at 37°C overnight. A portion of the bacterial liquid was streaked onto an antibiotic-free BHIS plate and cultured at 37°C for about 48 hours. Pick the transformants on the plate and spot them on BHIS plates, BHIS plates containing spectinomycin, and BHIS plates containing kanamycin, respectively. They were inverted and cultured in a 30°C constant temperature incubator for 24 hours. PCR amplification was performed using primers 1919 / 1920 to verify the transformants that grew on the BHIS plate but could not grow on the BHIS plate containing kanamycin or the BHIS plate containing spectinomycin, and then sequenced. The positive transformants with successful knockout of the NCgl2644 gene were placed in 4 ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30°C and 220 rpm for 24 hours. 20% glycerol was used to maintain the bacteria. A strain with the genotype ATCC13032△NCgl2644 was obtained.

[0176] 5.3 Construction of ATCC13032ΔNCgl2644ΔargR strain

[0177] Prepare ATCC13032ΔNCgl2644 competent cells according to the method in step 2.1.2. Transform the pK18mobsacB-argR plasmid into ATCC13032ΔNCgl2644. Select for exchange recombinants on BHIS medium supplemented with 25 μg / ml kanamycin, perform SacB sucrose counterselection, and perform spot plating verification. Transformants that grow on BHIS plates but not on BHIS plates supplemented with kanamycin were verified by PCR amplification using primers 1438 / 1439. PCR amplification conditions were the same as in step 2.1.1, and sequencing was performed using primers 1438 / 1439 / 1440. Positive transformants were transferred to 4 ml of BHIS tubes and cultured on a shaker at 30°C, 220 rpm for 24 hours. The strain was maintained in 20% glycerol. This resulted in the strain with the genotype ATCC13032ΔNCgl2644ΔargR.

[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℃ and 220rpm for 24 hours, using 20% ​​glycerol to maintain the bacteria. The genotype of ATCC13032△NCgl2644△argRargB was obtained. A26V M31V strains.

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

[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 then 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 transformed 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 and 220 rpm for 24 hours, using 20% ​​glycerol to maintain 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 transformed into competent cells, 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 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 4ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30℃ and 220rpm for 24 hours, using 20% ​​glycerol to maintain 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 transformed 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 and 220 rpm for 24 hours, using 20% ​​glycerol to maintain 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 transformed into competent cells, 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 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 4ml of BHIS test tube culture medium and cultured on a constant temperature shaker at 30℃ and 220rpm for 24 hours, using 20% ​​glycerol to maintain 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, pJYS1 plasmid was transferred into competent cells, cultured in BHIS medium containing 25 μg / ml kanamycin for 48 h. Single colonies were picked and inoculated into BHIS liquid medium for overnight culture. ATCC13032△NCgl2644△argRargB was prepared according to the method in 2.1.2. A26V M31V ::pJYS1 competent cells were transformed with the pJYS2-NCgl2585 plasmid and ssDNA (28 / 31 / 36 / 42 / 45 / 46) and cultured for 48 h in BHIS medium supplemented with 25 μg / ml kanamycin and 100 μg / ml spectinomycin. The resulting clones were amplified by PCR using primers 512 / 513 and tested using primer 512. Transformants of E307G, E307D, E307Q, E307F, E307M, and E307A were selected and inoculated into antibiotic-free BHIS liquid medium and cultured overnight at 37°C. A portion of the bacterial suspension was streaked onto antibiotic-free BHIS plates and cultured at 37°C for 48 h. Select colonies from the BHIS plates and spot-plate them onto BHIS plates, BHIS plates containing kanamycin, and BHIS plates containing spectinomycin. Incubate at 30°C for 24 hours. Use primers 512 / 513 to verify colonies that grow on BHIS plates but not on BHIS plates containing kanamycin or spectinomycin. PCR amplification conditions are the same as in step 2.1.1, and sequencing is performed using primer 512. Positive transformants are transferred to 4 ml of BHIS tube culture medium and cultured on a shaker at 30°C, 220 rpm, for 24 hours. Use 20% glycerol for maintenance.

[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, NCgl2585 mutants E645K, E484K, E645K, E307K, E307G, E307D, E307Q, E307F, E307M, and E307A can be used to increase L-arginine production, with yields significantly higher than those obtained with mutations E484K, E645K, or a combination of the two. Combined with Tables 4, 5, and 6, this suggests that reducing ATP hydrolase activity can effectively increase L-arginine production.

[0202] The above-described embodiments represent only a few implementation methods of the present invention. Although the descriptions thereof are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and such modifications and improvements are all within the scope of protection of the present invention.

Claims

1. An ATP-dependent CLP protease variant, which is a mutant of the ATP-dependent CLP protease with an amino acid sequence as shown in SEQ ID NO: 2, wherein only the 307th position is mutated, and the mutant is selected from the following group: an E307K mutant with an amino acid sequence as shown in SEQ ID NO: 4, an E307G mutant with an amino acid sequence as shown in SEQ ID NO: 6, an E307D mutant with an amino acid sequence as shown in SEQ ID NO: 8, an E307Q mutant with an amino acid sequence as shown in SEQ ID NO: 10, an E307F mutant with an amino acid sequence as shown in SEQ ID NO: 12, an E307M mutant with an amino acid sequence as shown in SEQ ID NO: 14, and an E307A mutant with an amino acid sequence as shown in SEQ ID NO:

16.

2. A gene encoding the ATP-dependent CLP protease variant according to claim 1.

3. The gene according to claim 2, wherein 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.

4. Use of the ATP-dependent CLP protease variant according to claim 1, or the gene according to claim 2 or 3, in improving the fermentation level of L-arginine-producing bacteria.

5. The use according to claim 4, characterized in that The L-arginine-producing bacteria are made to express the ATP-dependent CLP protease variant as claimed in claim 1 to replace the endogenous ATP-dependent CLP protease, that is, the encoding gene of the ATP-dependent CLP protease variant as claimed in claim 2 or 3 is used to replace the original ATP-dependent CLP protease encoding gene in the genome of the L-arginine-producing bacteria.

6. A method for constructing L-arginine engineering bacteria, characterized in that: The method comprises the following steps: causing the L-arginine-producing bacteria to express the ATP-dependent CLP protease variant as claimed in claim 1 to replace the endogenous ATP-dependent CLP protease, that is, replacing the original ATP-dependent CLP protease encoding gene in the genome of the L-arginine-producing bacteria with the encoding gene of the ATP-dependent CLP protease variant as claimed in claim 2 or 3.

7. The method according to claim 6, wherein The L-arginine producing bacterium is Corynebacterium glutamicum.

8. A modified bacterium for producing L-arginine, characterized in that It is constructed by the method according to claim 6 or 7.

9. The modified bacterium according to claim 8, wherein N-acetylglutamate synthase encoding gene in the genome NCgl2644 / cg3035 Mutated or deleted, resulting in inactivation or weakening of N-acetylglutamate synthase activity, where the gene NCgl2644 / cg3035 As shown in Genbank: NC_003450.3, Gene ID: 1020685.

10. The modified bacterium according to claim 9, wherein The mutation is a gene NCgl2644 / cg3035 A251V mutation.

11. The modified bacterium according to any one of claims 8 to 10, wherein Arginine operon regulated genes in the genome argR The gene is down-regulated or knocked out, thereby relieving its inhibition on L-arginine synthesis. argR As shown in Genbank: AF041436.

1.

12. The modified bacterium according to any one of claims 8 to 10, wherein N-acetylglutamate kinase encoding gene in the genome argB Mutated to relieve feedback inhibition of N-acetylglutamate kinase by L-arginine, where the gene argB As shown in Genbank: BAB98789.

1.

13. The modified bacterium according to claim 12, wherein Genes that resist feedback inhibition argB The mutations are A26V and M31V mutations.

14. Use of the modified bacterium according to any one of claims 8 to 13 in the fermentative production of L-arginine.

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