A method for improving the stability of the arginine-producing ability of a strain

Abstract

This invention discloses a method for improving the stability of arginine production capacity in bacterial strains, comprising the following steps: using *Corynebacterium glutamicum* producing L-arginine as the base strain, inactivating or attenuating the gene NCgl2644 / cg3035 in the genome. The method of this invention can effectively improve the genetic stability of L-arginine-producing strains.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and specifically, it relates to a method for improving the stability of arginine production capacity of a bacterial strain, and more particularly to a method for improving the genetic stability of arginine-producing bacteria through genetic engineering. Background Technology

[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 in the urea cycle and possesses a variety of unique physiological and pharmacological functions. It has good therapeutic effects on physiological functions, cardiovascular diseases, stimulating the immune system, maintaining nutritional balance in infants, and promoting detoxification. Experts consider it an important carrier for the transport and storage of amino acids in the body, playing a crucial role in intramuscular metabolism. It is an essential amino acid for the synthesis of cytoplasmic and nucleoproteins; it participates in creatine synthesis as the sole source of ammonia; as an important intermediate in the urea cycle, it plays a role in eliminating excess ammonia in the liver, preventing excessive ammonia accumulation and poisoning; it also has the function of regulating human immunity, inhibiting tumor growth, and promoting the healing of injured tissues. Furthermore, arginine is a direct precursor to nitric oxide, urea, ornithine, and creatine, and is an important element in the synthesis of inosine. It is also used in the synthesis of polyamines, citrulline, and glutamine. Therefore, L-arginine has important and wide-ranging applications in the pharmaceutical, food, and chemical industries. For example, in clinical practice, besides being a major component of compound amino acid infusions, L-arginine and its salts are widely used to treat various types of hepatic coma (contraindicated with monosodium glutamate) and viral hepatitis with abnormal alanine aminotransferase levels, showing significant efficacy in treating viral hepatitis. It also has therapeutic effects on intestinal ulcers, thrombosis, and neurasthenia. Furthermore, L-arginine is an important component of sports nutrition beverage formulations, a significant feed additive, and is widely used in high-end animal husbandry. Statistics show that the global demand for L-arginine currently exceeds 15,000 tons, and this demand is growing at a rate of 12%-15% annually.

[0003] There are two methods for producing L-arginine: protein hydrolysis extraction and microbial fermentation. Hydrolysis suffers from time-consuming operation, low yield and output, high cost, and serious pollution, making it unsuitable for large-scale production. Fermentation for L-arginine production is relatively simple and environmentally friendly, thus possessing great development potential and becoming an important trend in the amino acid industry both domestically and internationally. Internationally renowned amino acid companies such as Ajinomoto and Kyowa Hakko in Japan, and Deutsche Hakko in Germany primarily employ bio-fermentation and genetic engineering technologies for L-arginine production. However, domestic microbial fermentation production of L-arginine generally results in low acid production levels and high costs, with production levels and output far from meeting domestic demand. Therefore, research into improving L-arginine fermentation levels is of great significance.

[0004] The main fermentation strains for L-arginine that have been extensively studied include *Corynebacterium glutamicum*, *Brevibacterium flavum*, *Corynebacterium crenatum*, *Escherichia coli*, and *Bacillus subtilis*. However, the microbial strains currently used for L-arginine production are primarily *Corynebacterium glutamicum* and *Corynebacterium crenatum*. Genetic engineering technology plays a crucial role in the selection and breeding of high-yield L-arginine strains. Constructing high-yield L-arginine strains using genetic engineering is a highly efficient and rational breeding method.

[0005] However, many genetically engineered bacteria exhibit unstable genetic traits and inconsistent arginine production capabilities, leading to significant batch-to-batch variations in fermentation. They may also undergo degeneration and mutation after multiple generations, even losing their arginine production capacity after just a few passages, thus hindering industrial-scale production. For example, the inventors used wild-type Corynebacterium glutamicum ATCC13032 as the starting strain, knocked out argR, and introduced the (A26V M31V) mutation of argB to remove argB feedback inhibition, obtaining genetically engineered strain number 1441 (genotype ATCC13032ΔargRargB). A26V M31V This significantly improved the arginine production capacity of Corynebacterium glutamicum, achieving an arginine yield of 6.231 ± 0.023 g / L. However, subsequent experiments revealed that this genetically engineered strain was highly unstable, exhibiting significant batch-to-batch variability, and lost its arginine production capacity after subculturing (strain number 1442 was obtained one generation after subculturing strain number 1441). Therefore, improving the genetic stability of arginine-producing engineered strains remains an urgent need. Summary of the Invention

[0006] To overcome the shortcomings of existing L-arginine-producing strains, such as unstable genetic traits and low fermentation levels, this invention utilizes genetic engineering technology to modify the genome of Corynebacterium glutamicum, including engineered Corynebacterium glutamicum strains. By enhancing genes related to L-arginine production and weakening branched metabolic pathways, the genetic stability and L-arginine production capacity of the strains can be significantly improved.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for improving the stability of a strain's ability to produce arginine includes the following steps: using a Corynebacterium glutamicum that produces L-arginine as the base strain, inactivating or attenuating the gene NCgl2644 / cg3035 in the genome.

[0009] The inactivation or attenuation of the above-mentioned gene NCgl2644 / cg3035 can be selected from the following methods: knocking out the open reading frame of gene NCgl2644 / cg3035; mutating any amino acid in the open reading frame of gene NCgl2644 / cg3035 to a stop codon; mutating amino acid A at position 251 in the open reading frame of gene NCgl2644 / cg3035.

[0010] The mutation of amino acid A at position 251 of the gene NCgl2644 / cg3035 can be selected from the following groups: mutation to the stop codon TGA or TAA; mutation to amino acid V, D, E, F, G, K, L, M, N, P, R, S, T, W or Y.

[0011] The aforementioned basic strains can be selected from ATCC13032, ATCC13870, ATCC21831, etc., preferably from the genome (ΔargR,argBmut(A26V M31V)) engineered strains of the aforementioned strains, namely ATCC13032(ΔargR,argBmut(A26VM31V)), ATCC13870(ΔargR,argBmut(A26V M31V)), ATCC21831(ΔargR,argBmut(A26VM31V)), etc., more preferably Corynebacterium glutamicum ATCC13032(ΔargR,argBmut(A26V M31V)) or its derived mutant strains, wherein

[0012] The *Corynebacterium glutamicum* ATCC13032(ΔargR,argBmut(A26V M31V)) was obtained by knocking out the argR gene of *Corynebacterium glutamicum* ATCC13032 and introducing the (A26V M31V) mutation of argB.

[0013] The derived mutant strains were selected from strain CCTCC NO: M2017760 reported in patent application CN201711430988.5, strain ATCC13032(ΔargR,argBmut(A26V M31V),NCgl0083mut2) reported in patent application CN201810493046.X, strain ATCC13032(ΔargR,argBmut(A26V M31V),NCgl0742mut3) reported in patent application CN201810492999.4, strain ATCC13032(ΔargR,argBmut(A26V M31V),NCgl2374mut4) reported in patent application CN201810492998.X, and strain ATCC13032(ΔargR,argBmut(A26V M31V),NCgl2374mut4) reported in patent application CN201810569947.2. The strains ATCC13032 (ΔargR,argBmut(A26V M31V),NCgl2585mut51) reported in patent application CN201810569343.8 and ATCC13032 (ΔargR,argBmut(A26V M31V),NCgl2585mut52) reported in patent application CN201810569948.7.

[0014] In a preferred embodiment, the basic strain is Corynebacterium glutamicum ATCC13032 (ΔargR,argBmut(A26V M31V)), and the method includes the following steps:

[0015] A. The gene NCgl2644 / cg3035 in the genome was inactivated or weakened to obtain the mutant strain ATCC13032 (cg3035mut) of gene NCgl2644 / cg3035.

[0016] B. Knock out the argR gene in the genome of the mutant strain ATCC13032(cg3035mut) described in step A to obtain the gene knockout strain ATCC13032((cg3035mut,ΔargR).

[0017] C. Mutate the argB gene in the genome of the gene knockout strain ATCC13032(cg3035mut,ΔargR) described in step B with A26V and M31V mutations to obtain the genetically engineered strain ATCC13032((cg3035mut,ΔargR,argBmut(A26V M31V)).

[0018] Step A above can be implemented using gene editing technology, which can employ the CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas related transposon system INTEGRATE system, or CAST system.

[0019] The INTEGRATE system mentioned above refers to the gene editing tool (Insertion of transposable elements by guide RNA-assisted targeting) developed by Sam Sternberg's research group; the CAST system refers to the gene editing tool (CRISPR-associated transposase) developed by Zhang Feng's research group.

[0020] In one embodiment, the gene knockout strain ATCC13032 (cg3035mut, ΔargR) described in step B above can be prepared by the following method:

[0021] B1. Using the ATCC13032 genome as a template, PCR amplification was performed using primer argR-aL-F with sequence SEQ ID NO:15 and primer argR-aL-R with sequence SEQ ID NO:16 to obtain an argR-aL fragment of approximately 1 kb.

[0022] B2. Using the ATCC13032 genome as a template, PCR amplification was performed using primer argR-aR-F with sequence SEQ ID NO:17 and primer argR-aR-R with sequence SEQ ID NO:18 to obtain an argR-aR fragment of approximately 1 kb.

[0023] B3. Plasmid pK18mobsacB with GenBank accession number FJ437239.1 was digested with HindIII and EcoRI, and a 5.7kb vector fragment was obtained by gel recovery.

[0024] B4. Gibson was used to connect the above-mentioned argR-aL fragment, argR-aR fragment and vector fragment, transformed into DH5α competent cells, plated on kanamycin LB plates and cultured overnight;

[0025] B5. Transformants were verified by PCR amplification using primers argR-aL-F and argR-aR-R, yielding plasmid pK18mobsacB-argR;

[0026] B6. Preparation of competent cells of Corynebacterium glutamicum ATCC13032 (cg3035mut);

[0027] B7. Transform the plasmid pK18mobsacB-argR into ATCC13032 (cg3035mut) competent cells;

[0028] B8. Perform SacB sucrose reverse screening, and use primers argR-aL-F and argR-aR-R for PCR amplification to verify the transformants that grow on BHIS plates but cannot grow on BHIS plates containing kanamycin, and obtain strain ATCC13032 (cg3035mut, ΔargR).

[0029] The genetically engineered strain described in step C above can be prepared by the following method:

[0030] C1. Using the ATCC13032 genome as a template, PCR amplification was performed using primer argB-aL-F with sequence SEQ ID NO:19 and primer argB-aL-R with sequence SEQ ID NO:20 to obtain an argB-aL fragment of approximately 1 kb.

[0031] C2. Using the ATCC13032 genome as a template, PCR amplification was performed using primer argB-aR-F with sequence SEQ ID NO:21 and primer argB-aR-R with sequence SEQ ID NO:22 to obtain an argB-aR fragment of about 1kb.

[0032] C3. Plasmid pK18mobsacB with GenBank accession number FJ437239.1 was digested with HindIII and EcoRI, and a 5.7kb vector fragment was obtained by gel recovery.

[0033] C4. Gibson was used to connect the above-mentioned argB-aL fragment, argB-aR fragment and vector fragment, transformed into DH5α competent cells, plated on kanamycin LB plates and cultured overnight;

[0034] C5. Transformants were verified by PCR amplification using primers argB-aL-F and argB-aR-R, resulting in plasmid pK18mobsacB-argBmut.

[0035] C6. Preparation of competent cells of Corynebacterium glutamicum ATCC13032 (cg3035mut, ΔargR);

[0036] C7. Transform the plasmid pK18mobsacB-argBmut into ATCC13032 (cg3035mut, ΔargR) competent cells;

[0037] C8. Perform SacB sucrose reverse screening, and use primers argB-aL-F and argB-aR-R for PCR amplification to verify the transformants that grow on BHIS plates but cannot grow on BHIS plates containing kanamycin, and obtain strain ATCC13032 (cg3035mut,ΔargR,argBmut(A26V M31V)).

[0038] The conversion described in steps B7 and C7 above can be either calcium chloride conversion or electroconversion, with electroconversion being preferred.

[0039] Steps A, B, and C can be performed in any way they are combined or reversed, as long as each step can achieve its respective function.

[0040] According to a second aspect of the present invention, a genetically engineered bacterium is provided, which is constructed according to the method described above.

[0041] According to a third aspect of the present invention, the application of the above-described genetically engineered bacteria in the production of L-arginine is provided.

[0042] This genetically engineered bacterium can be used directly as a fermentation strain to produce L-arginine through fermentation, or it can be used as a starting strain for further improvement in order to screen out new production strains with further enhanced L-arginine production capacity.

[0043] When L-arginine is produced by fermenting the above-mentioned genetically engineered bacteria, the culture medium used for fermentation can be any culture medium suitable for the growth and fermentation of Corynebacterium glutamicum.

[0044] According to a preferred embodiment of the present invention, the fermentation medium is composed of the following: 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 / L MnSO4·5H2O, 20 mg / L nicotinic acid, 20 mg / L β-alanine, 10 mg / L LVB1, 0.2 mg / L biotin, 30 g / L CaCO3, and KOH adjusted to pH 7.7.

[0045] In a preferred embodiment, the fermentation process of the L-arginine-producing bacteria includes a seed culture stage and a cell fermentation stage. These two stages utilize a seed culture medium and a fermentation culture medium, respectively. The fermentation culture medium may be the same as or different from the seed culture medium.

[0046] Preferably, when the fermentation medium is different from the seed medium, the seed medium composition 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.

[0047] This invention, by inactivating or weakening the gene NCgl2644 / cg3035 in the genome of Corynebacterium glutamicum ATCC13032, can greatly improve the genetic stability, i.e., the transmission stability, of the L-arginine-producing genetically engineered strain Corynebacterium glutamicum ATCC13032 (ΔargR,argBmut(A26V M31V)) and its derived mutant strains, and has the value of popularization and promotion. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of plasmid pK18mobsacB, donated by Shuangjiang Liu of the Institute of Microbiology, Chinese Academy of Sciences. GenBank: FJ437239.1. For more information, please see https: / / www.ncbi.nlm.nih.gov / nuccore / 215434894.

[0049] Figure 2 This is a schematic diagram of the recombinant plasmid pK18mobsacB-argR constructed in this invention.

[0050] Figure 3 This is a schematic diagram of the recombinant plasmid pK18mobsacB-argBmut constructed in this invention.

[0051] Figure 4 This is a schematic diagram of the recombinant plasmid pJYS3_Δcg3035 constructed in this invention.

[0052] Figure 5 This is a schematic diagram of the recombinant plasmid pCgsgRNA_cg3035 constructed in this invention. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0054] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage.

[0055] Currently, the original Corynebacterium glutamicum strains that can be used or have the potential to be used for L-arginine production include ATCC13032, ATCC13870, and ATCC21831. After genetic engineering of their genomes (ΔargR, argBmut(A26V M31V)), these original strains have been transformed into engineered strains such as ATCC13032 (ΔargR, argBmut(A26VM31V)), ATCC13870 (ΔargR, argBmut(A26V M31V)), and ATCC21831 (ΔargR, argBmut(A26VM31V)). These engineered strains can be transformed from being unable to produce L-arginine to being able to produce it, or from having a weak ability to produce L-arginine to having a strong ability to produce it. Many genetically engineered strains suffer from low passage stability; therefore, improving their passage stability remains an urgent problem to be solved.

[0056] In this article, the terms "Corynebacterium glutamicum ATCC13032 / ATCC13870 / ATCC21831", "strain ATCC13032 / ATCC13870 / ATCC21831", and "ATCC13032 / ATCC13870 / ATCC21831" have the same meaning. They all refer to the original strain ATCC13032 / ATCC13870 / ATCC21831, which is the L-arginine-producing bacterium originating strain, i.e., the wild-type strain, and was purchased from the Shanghai Institute of Industrial Microbiology.

[0057] This invention improves the genetic stability (generational stability) of Corynebacterium glutamicum, including strains Corynebacterium glutamicum ATCC13032 / 13870 / 21831 (ΔargR,argBmut(A26V M31V)) and its derived mutant strains, primarily by inactivating or attenuating the gene NCgl2644 / cg3035 in the genome of Corynebacterium glutamicum ATCC13032 / ATCC13870 / ATCC21831 in any modified manner. Inactivation or attenuation includes knocking out the open reading frame (OPF) of the NCgl2644 / cg3035 gene; or mutating amino acid A at position 251 of the OPF to the stop codon TGA or TAA, or mutating it to other amino acids V, D, E, F, G, K, L, M, N, P, R, S, T, W, or Y. Subsequently, argR is knocked out in the obtained mutant strains, and argB is mutated to an L-arginine-resistant feedback repression genotype.

[0058] The gene NCgl2644 / cg3035 encodes N-acetylglutamate synthase, which catalyzes the synthesis of N-acetylglutamate from glutamate. This is the first step in the biosynthetic pathway from glutamate to arginine. The inventors' research has shown that inactivation or attenuation of this gene can improve the genetic stability of L-arginine-producing Corynebacterium glutamicum ATCC13032 / 13870 / 21831 (ΔargR,argBmut(A26VM31V)) and its derived mutant strains.

[0059] The gene argR is a gene that regulates the arginine operon, which is ubiquitous in bacteria and has different functions in different bacteria. The inventors found that by knocking out this negative regulatory gene in the genome of Corynebacterium glutamicum, its inhibition on arginine synthesis can be relieved to some extent.

[0060] N-acetylglutamate kinase catalyzes the synthesis of N-acetylglutamate from N-acetylglutamate, which is subject to feedback inhibition by the final product L-arginine. The inventors discovered that by mutating N-acetylglutamate kinase, specifically changing alanine (A) at position 26 to valine (V) and methionine (M) at position 31 to valine (V), the synthesis capacity of L-arginine in Corynebacterium glutamicum ATCC13032 / ATCC13870 / ATCC21831 can be effectively enhanced. In this paper, the mutated gene of this N-acetylglutamate kinase gene argB is abbreviated as argBmut or argBmut(A26VM31V), which is an L-arginine-resistant feedback inhibition genotype.

[0061] In the examples, through the above-described genetic engineering operations, a series of L-arginine-producing Corynebacterium glutamicum were obtained, such as the genetically engineered strain ATCC13032 cg3035mutΔargRargB. A26V M31V Including ATCC13032Δcg3035ΔargRargB A26V M31V and ATCC13032 cg3035 A251X ΔargRargB A26V M31V (where X is a stop codon such as TGA, TAA, amino acid V, D, E, F, G, K, L, M, N, P, R, S, T, W, or Y).

[0062] For ease of description, this article refers to the mutations Δcg3035 (knockout) and cg3035 in the gene NCgl2644 / cg3035. A251X (Mutation at amino acid A at position 251) is uniformly identified as cg3035mut.

[0063] Furthermore, among the *Corynebacterium glutamicum* strains with enhanced genetic stability mentioned above, the genetically engineered strain ATCC13032Δcg3035ΔargRargB... A26V M31V and ATCC13032 cg3035 A251X ΔargRargB A26V M31V Based on this, to further increase the L-arginine production of the strain, the NCgl2585 E484K mutation (see patent documents CN110564790A, i.e., CN201810569948.7), or the NCgl2585 E645K mutation (see patent documents CN110564758A, i.e., CN201810569343.8), or both NCgl2585 E484K and E645K mutations can be added simultaneously, which can increase the arginine production by more than 0.5 times. In other words, the technical solution of this invention for improving the genetic stability of L-arginine-producing bacteria is also applicable to derivative strains of engineered bacteria ATCC13032 (ΔargR,argBmut(A26V M31V)), thereby simultaneously improving the genetic stability and production capacity of L-arginine-producing bacteria.

[0064] It should be understood that in constructing the genetically engineered bacterium ATCC13032Δcg3035ΔargRargB of the present invention... A26V M31V and ATCC13032 cg3035 A251X ΔargRargB A26V M31V (Where X is the stop codon TGA, TAA, amino acid V, D, E, F, G, K, L, M, N, P, R, S, T, W, or Y) In the specific operation, the order of steps A, B, and C is not fixed according to the English alphabetical order from front to back. They can be operated in a cross or reversed manner, as long as each step can achieve its own function and complete the directional change of the host cell genotype.

[0065] The technical solution of the present invention will be specifically described below using the example of improving the passage stability of engineered strains of Corynebacterium glutamicum that produce L-arginine, including ATCC13032 / 13870 / 21831 (ΔargR,argBmut(A26V M31V)).

[0066] Example

[0067] Materials and methods

[0068] The whole genome synthesis, primer synthesis, and sequencing in this article were all completed by Sangon Biotech (Shanghai) Co., Ltd.

[0069] The molecular biology experiments in this article, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were mainly conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the methods for competent cell transformation and competent cell preparation were based on Chapter 1, page 96 of *Molecular Cloning: A Laboratory Manual* (3rd Edition). Specific experimental conditions could be determined through simple experiments when necessary.

[0070] Main culture media and buffer solutions:

[0071] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0072] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder.

[0073] BHIS liquid medium: 37 g / L BHI, 91 g / L sorbitol.

[0074] BHIS solid medium: 37 g / L BHI, 91 g / L sorbitol, 20 g / L agar powder.

[0075] BHIS-suc solid medium: 37 g / L BHI, 91 g / L sorbitol, 200 g / L sucrose, 10 g / L glucose.

[0076] BYG medium: 3 g / L NaCl, 5 g / L yeast extract, 7 g / L beef extract, 10 g / L peptone, 10 g / L glucose.

[0077] 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 / L MnSO4·5H2O, 20 mg / L nicotinic acid, 20 mg / L β-alanine, 10 mg / L VB1, 0.2 mg / L biotin, 30 g / L CaCO3, KOH adjusted to pH 7.7.

[0078] 20X electroporation mother liquor: 80 g / L glycine, 2% Tween 80.

[0079] In the following examples, when using a culture medium containing kanamycin, the final concentration of kanamycin in the culture medium is 50 μg / ml.

[0080] The primer sequence information used in the following examples is shown in Table 1.

[0081] Table 1. List of primers used in the examples

[0082]

[0083]

[0084] In Table 1, "-F" in the name represents positive; "-R" represents negative.

[0085] Example 1: Knocking out the open reading frame of the NCgl2644 / cg3035 gene using the CRISPR-Cpf1 single plasmid system

[0086] 1.1 Genome extraction from ATCC13032:

[0087] A small amount of ATCC13032 glycerol bacteria (purchased from Shanghai Institute of Industrial Microbiology) was transferred to a BHIS test tube and cultured on a constant temperature shaker at 30°C and 220 rpm for 18 hours. The bacterial cells were collected by centrifugation at 12,000 rpm, and the ATCC13032 genome was extracted using the Axygen bacterial genome mini-extraction kit.

[0088] 1.2 Construction of knockout plasmids

[0089] Using pJYS3_crtYf plasmid (Nat Commun. 2017 May 4; 8: 15179.) as a template, a 4268bp fragment 1 was obtained using F1 / R1 primers, a 5584bp fragment 2 was obtained using F2 / R2 primers, and a 1026bp fragment 3 was obtained using F3 / R3 primers and a 965bp fragment 4 was obtained using F4 / R4 primers, using the ATCC13032 genome extracted in step 1.1 as a template. The fragments were then transformed into DH5α competent cells (commercially available competent cells purchased from Nanjing Novizan Biotechnology Co., Ltd.), plated on kanamycin LB plates, and cultured overnight.

[0090] Positive transformants were inoculated into LB liquid test tubes, and plasmids were extracted and sequenced using an Axygen plasmid extraction kit to obtain the correctly knocked-out plasmid pJYS3_Δcg3035, the structure of which is shown below. Figure 4 As shown.

[0091] The PCR system was as follows (the PCR reagents were purchased from TOYOBO's KOD series): KOD Buffer 5 μl, dNTP 5 μl, MgSO4 4 μl, primer argR-aL-F 0.5 μl, primer argR-aL-R 0.5 μl, KOD Plus Neo 1 μl, template 0.4 μl, and ddH2O to make up to 50 μl. The PCR program was: 99℃ hot cap, 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 68℃ extension for 60 s; 35 cycles, with a final extension at 68℃ for 10 min; cooling at 16℃ for 10 min to amplify the target band, and gel recovery of the target fragment.

[0092] 1.3 Preparation of Corynebacterium glutamicum ATCC13032 competent cells:

[0093] Streaking *Corynebacterium glutamicum* ATTC13032 onto a BHIS plate and incubating overnight at 30°C. Single colonies were then picked and incubated overnight at 30°C and 220 rpm in BHIS test tubes. 1 ml of the bacterial culture was inoculated into a 100 ml shake flask containing BHIS liquid medium and incubated on a shaker at 30°C and 220 rpm for 4-6 hours until the OD600 value reached approximately 1.0. On a clean bench, the entire bacterial culture was transferred to 50 ml centrifuge tubes and centrifuged at 4500 × g at 4°C. The supernatant was discarded, and the cells were washed with 10% glycerol to resuspend them. The cells were centrifuged again at 4500 × g at 4°C, and the washing was repeated once, discarding the supernatant. Finally, 600 μl of 10% glycerol was added to resuspend the cells, which were then aliquoted into 1.5 ml centrifuge tubes. Each 90 μl tube prepared a competent cell line. Competent cells can be stored at -80°C.

[0094] 1.4pJYS3_Δcg3035 electroporated ATCC13032 competent cells:

[0095] Select the correctly sequenced pJYS3_Δcg3035 plasmid, and add 3 μl (more than 1 μg) to *Corynebacterium glutamicum* ATCC13032 competent cells. After mixing, transfer to an electroporation cuvette and electroporate at 25 uF, 2.5 kV, and 200 Ω for 5.6 ms. Immediately after electroporation, transfer to 900 μl of preheated BHIS liquid medium at 46 °C and incubate in a 46 °C water bath for 6 min. Then, incubate in a constant temperature shaker at 30 °C and 220 rpm for 1 h to allow the cells to recover. After recovery, spread 50 μl of the cells onto a BHIS plate containing kanamycin and incubate upside down in a 30 °C incubator for 48 h.

[0096] 1.5 Identification of positive strains

[0097] PCR amplification was performed to verify single colonies growing on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 1.2. Primers F5 / R5 were used for identification. After successful knockout of cg3035, the positive band size was 2335bp, and the negative band size was 3200bp.

[0098] 1.6 Positive transformants lost plasmid

[0099] Positive transformants were picked and transferred to 4 ml of BHIS medium in test tubes. After incubation at 37°C and 220 rpm for 12 hours on a shaker, the culture was streaked onto antibiotic-free plates and incubated for another 24 hours at 37°C. Single colonies were then streaked onto both kanamycin-containing and antibiotic-free BHIS plates. Transformants that grew on antibiotic-free BHIS plates but not on kanamycin-containing BHIS plates were selected and preserved using 20% ​​glycerol. A strain with the genotype ATCC13032Δcg3035, containing the knockout of the NCgl2644 / cg3035 gene open reading frame, was obtained.

[0100] Example 2: Using the CRISPR-Cas9 dual plasmid system to mutate the NCgl2644 / cg3035 gene at position 251 (A mutation).

[0101] 2.1 Construction of mutant plasmids

[0102] Using pCgsgRNA_crtYf plasmid (ACS Synth Biol. 2020 Jul 17; 9(7): 1897-1906.) as a template, a 4401bp fragment was amplified using F6 / R6 primers. The fragment was then transformed into Escherichia coli DH5α competent cells, plated on spectinomycin LB plates, and cultured overnight.

[0103] Positive transformants were inoculated into LB liquid test tubes, and plasmids were extracted and sequenced using an Axygen plasmid extraction kit to obtain the correct plasmid pCgsgRNA_cg3035, the structure of which is shown below. Figure 5 As shown.

[0104] The PCR system is the same as in 1.2.

[0105] 2.2 Design of Single-Link Repair Template

[0106] To mutate the A at position 251 of the NCgl2644 / cg3035 gene to the stop codon TGA, TAA, or other amino acids V, D, E, F, G, K, L, M, N, P, R, S, T, W, or Y, a single-strand repair template was designed, as shown in Table 2. The sequences in Table 2 were synthesized as primers and dissolved to a concentration of 2 μg / μL.

[0107] Table 2. List of Single-Link Repair Templates

[0108] Single-strand repair template Template sequence (5’→3’) TGA (stop) cctgaccacgccggcggggcaaacAgtGTGAatcacgcgcgccaccatcacggctgcgg TAA (stop) cctgaccacgccggcggggcaaacAgtGTAAatcacgcgcgccaccatcacggctgcgg GTG (V) cctgaccacgccggcggggcaaacAgtGGTGatcacgcgcgccaccatcacggctgcgg GAT (D) cctgaccacgccggcggggcaaacAgtGGATatcacgcgcgccaccatcacggctgcgg GAA (E) cctgaccacgccggcggggcaaacAgtGGAAatcacgcgcgccaccatcacggctgcgg TTC (F) cctgaccacgccggcggggcaaacAgtGTTCatcacgcgcgccaccatcacggctgcgg GGC (G) cctgaccacgccggcggggcaaacAgtGGGCatcacgcgcgccaccatcacggctgcgg AAG (K) cctgaccacgccggcggggcaaacAgtGAAGatcacgcgcgccaccatcacggctgcgg CTG (L) cctgaccacgccggcggggcaaacAgtGCTGatcacgcgcgccaccatcacggctgcgg ATG (M) cctgaccacgccggcggggcaaacAgtGATGatcacgcgcgccaccatcacggctgcgg AAC (N) cctgaccacgccggcggggcaaacAgtGAACatcacgcgcgccaccatcacggctgcgg CCA (P) cctgaccacgccggcggggcaaacAgtGCCAatcacgcgcgccaccatcacggctgcgg CGC (R) cctgaccacgccggcggggcaaacAgtGCGCatcacgcgcgccaccatcacggctgcgg TCC (S) cctgaccacgccggcggggcaaacAgtGTCCatcacgcgcgccaccatcacggctgcgg ACC (T) cctgaccacgccggcggggcaaacAgtGACCatcacgcgcgccaccatcacggctgcgg TGG(W) cctgaccacgccggcggggcaaacAgtGTGGatcacgcgcgccaccatcacggctgcgg TAC(Y) cctgaccacgccggcggggcaaacAgtGTACatcacgcgcgccaccatcacggctgcgg

[0109] 2.3 Transform the pCgCas9_recT plasmid into Corynebacterium glutamicum ATCC13032 competent cells.

[0110] First, the pCgCas9_recT plasmid (ACS Synth Biol. 2020 Jul 17; 9(7): 1897-1906.) was transformed into prepared Corynebacterium glutamicum ATCC13032 competent cells. The preparation method of competent cells was the same as that of electroporation in steps 1.3 and 1.4. ATCC13032 strain containing the pCgCas9_recT plasmid was obtained.

[0111] 2.4 The pCgsgRNA_cg3035 plasmid was transformed into competent cells of ATCC13032 strain containing the pCgCas9_recT plasmid.

[0112] Competent cells of strain ATCC13032 containing pCgCas9_recT plasmid were prepared according to the competent cell preparation method in step 1.3. 500 ng of pCgsgRNA_cg3035 plasmid and 1-10 ng of single-stranded repair template were transferred into the prepared competent cells. After revival, the bacterial cells were plated on BHIS plates containing kanamycin and spectinomycin and incubated upside down in a 30°C incubator for 48 hours.

[0113] 2.5 Identification of positive transformants

[0114] PCR amplification was performed using primers F-cg3035 and R-cg3035 to verify transformants on BHIS plates containing kanamycin and spectinomycin. PCR amplification conditions were the same as in step 1.2, yielding a band of approximately 1 kb. Sequencing of the PCR product yielded successfully mutated transformants. Positive transformants were picked into 4 ml of BHIS culture medium, and plasmids were discarded as described in step 1.6. The positive transformants were then cultured on a shaker at 30°C and 220 rpm for 24 hours, using 20% ​​glycerol for preservation. The genotype of the gene NCgl2644 / cg3035 with an A mutation at position 251 was determined to be ATCC13032 cg3035. A251X The strains include ATCC13032 cg3035 A251TGA ATCC13032 cg3035 A251TAA ATCC13032 cg3035 A251V ATCC13032cg3035 A251D ATCC13032 cg3035 A251E ATCC13032 cg3035A251F ATCC13032 cg3035 A251G ATCC13032 cg3035 A251K ATCC13032 cg3035 A251L ATCC13032 cg3035 A251M ATCC13032cg3035 A251N ATCC13032 cg3035 A251P ATCC13032 cg3035 A251R ATCC13032 cg3035 A251S ATCC13032 cg3035 A251T ATCC13032 cg3035 A251W ATCC13032 cg3035 A251Y .

[0115] Example 3: Preparation of strain ATCC13032 cg3035mutΔargR with argR gene knocked out

[0116] 3.1 Construction of the knockout plasmid pK18mobsacB-argR

[0117] 3.1.1 Using the ATCC13032 genome extracted in step 1.1 as a template, the argR-aL fragment was amplified by PCR using primers argR-aL-F and argR-aL-R. The PCR system was as follows (the following PCR reagents were purchased from TOYOBO's KOD series): KOD Buffer 5 μl, dNTP 5 μl, MgSO4 4 μl, primer argR-aL-F 0.5 μl, primer argR-aL-R 0.5 μl, KOD Plus Neo 1 μl, template 0.4 μl, and ddH2O to make up to 50 μl. The PCR program was: 99℃ hot-lid, 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 68℃ extension for 60 s; 35 cycles, with a final extension at 68℃ for 10 min; and cooling at 16℃ for 10 min. A fragment of approximately 1 kb was amplified, and the target fragment was recovered by gel electrophoresis.

[0118] 3.1.2 Using the ATCC13032 genome extracted in step 1.1 as a template, the argR-aR fragment (approximately 1 kb) was amplified under the same PCR conditions as in step 3.1.1 using primers argR-aR-F and argR-aR-R. The target fragment was then recovered from the gel.

[0119] 3.1.3 Plasmid pK18mobsacB (a gift from Liu Shuangjiang of the Institute of Microbiology, Chinese Academy of Sciences, whose structure is shown in Figure 1) was extracted using Axygen's plasmid extraction kit. Figure 1 As shown in the figure, the plasmid was digested with HindIII and EcoRI, and the fragment was recovered by gel to obtain a 5.7kb vector fragment.

[0120] 3.1.4 Gibson ligation of the above fragments was used to transform DH5α competent cells, which were then plated on kanamycin LB plates and cultured overnight.

[0121] 3.1.5 Transformants were verified by PCR amplification using primers argR-aL-F and argR-aR-R. The PCR system was as follows (PCR reagents were purchased from TOYOBO's KOD series): KOD Buffer 2 μl, dNTP 2 μl, MgSO4 1.6 μl, primers argR-aL-F 0.4 μl, primers argR-aR-R 0.4 μl, KOD Plus Neo 0.4 μl, bacterial cells as template, and ddH2O to a final volume of 20 μl. The PCR program was: 99℃ heat treatment, 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 68℃ extension for 120 s, 35 cycles; final extension at 68℃ for 10 min; and cooling at 16℃ for 10 min. The positive band was approximately 2 kb. Positive transformants were inoculated into LB liquid test tubes, and plasmids were extracted and sequenced using an Axygen plasmid extraction kit to obtain the correct plasmid pK18mobsacB-argR, the structure of which is shown below. Figure 2 As shown.

[0122] 3.2 Preparation of Corynebacterium glutamicum ATCC13032Δcg3035 or ATCC13032 cg3035 A251X competent cells:

[0123] Pick Corynebacterium glutamicum ATCC13032Δcg3035 or ATCC13032cg3035 A251X Streak the bacteria on BHIS plates and incubate overnight at 30°C. Pick a single colony and transfer it to a BHIS test tube, incubating overnight at 30°C and 220 rpm. Inoculate 1 ml of the bacterial culture into a 100 ml shake flask of BHIS liquid medium and incubate on a shaker at 30°C and 220 rpm for 4-6 hours until OD (dose retardation). 600The value reaches approximately 1.0. On a clean bench, transfer the entire bacterial culture to a 50ml centrifuge tube, centrifuge at 4500×g at 4°C, discard the supernatant, wash the cells with 10% glycerol to resuspend them, centrifuge again at 4500×g at 4°C, repeat the washing once, and discard the supernatant. Finally, add 600μl of 10% glycerol to resuspend the cells, aliquot into 1.5ml centrifuge tubes, preparing one competent cell line of 90μl each. Competent cells can be stored at -80°C.

[0124] 3.3pK18mobsacB-argR electroconversion ATCC13032Δcg3035 or ATCC13032 cg3035 A251X competent cells:

[0125] Select the plasmid that was correctly sequenced, and aspirate 3 μl (or more than 1 μg) into *Corynebacterium glutamicum* ATCC13032Δcg3035 or ATCC13032 cg3035. A251X After mixing, the competent cells were transferred to an electroporation cuvette and electroporated at 25 μF, 2.5 kV, and 200 Ω for 5.6 ms. Immediately after electroporation, the cells were transferred to 900 μl of preheated BHIS liquid medium at 46 °C and incubated in a 46 °C water bath for 6 min. Then, the cells were incubated in a shaker at 30 °C and 220 rpm for 1 h to allow for cell resuscitation. After resuscitation, 50 μl of the cells were spread onto a BHIS plate containing kanamycin and incubated upside down in a 30 °C incubator for 48 h.

[0126] 3.4SacB sucrose reverse screening:

[0127] Transformants from BHIS plates containing kanamycin were picked and inoculated into antibiotic-free BHIS culture tubes. The tubes were incubated at 30°C and 220 rpm for 24 hours in a shaker to allow for double crossover. The bacterial cells were diluted 1000-fold and spread onto BHIS-suc plates containing 20% ​​sucrose. These plates were then incubated upside down at 30°C for 48 hours. Transformants from the BHIS-suc plates were picked and spotted onto both BHIS plates and BHIS plates containing kanamycin, and incubated upside down at 30°C for 24 hours. PCR amplification was performed using primers argR-aL-F and argR-aR-R to verify transformants that grew on BHIS plates but not on BHIS plates containing kanamycin. PCR amplification conditions were the same as in steps 1.2.5. A positive band indicating successful argR gene knockout was approximately 2 kb, and a negative band was approximately 2.5 kb. Positive transformants were picked and transferred to 4 ml of BHIS medium in test tubes, and cultured on a shaker at 30°C and 220 rpm for 24 hours. The culture was maintained using 20% ​​glycerol. Genotypes of ATCC13032 cg3035mutΔargR were obtained, including (ATCC13032Δcg3035ΔargR and ATCC13032 cg3035). A251X ΔargR) strain.

[0128] Example 4: Strains with mutated argB gene ATCC13032 cg3035mutargB mut(A26V M31V) Preparation

[0129] 4.1 Construction of the mutant plasmid pK18mobsacB-argBmut:

[0130] 4.1.1 Using the ATCC13032 genome extracted in step 1.1 as a template, the argB-aL fragment (approximately 1 kb) was amplified using primers argB-aL-F and argB-aL-R, and the target fragment was recovered by gel electrophoresis.

[0131] 4.1.2 Using the ATCC13032 genome extracted in step 1.1 as a template, the argB-aR fragment (approximately 1 kb) was amplified using primers argB-aR-F and argB-aR-R, and the target fragment was recovered by gel electrophoresis.

[0132] 4.1.3 Plasmid pK18mobsacB (a gift from Liu Shuangjiang of the Institute of Microbiology, Chinese Academy of Sciences, whose structure is shown in Figure 1) was extracted using Axygen's plasmid extraction kit. Figure 1 As shown in the figure, the plasmid was digested with HindIII and EcoRI, and the fragment was recovered by gel to obtain a 5.7kb vector fragment.

[0133] 4.1.4 Gibson ligation of the above fragments was used to transform DH5α competent cells (commercially available competent cells purchased from Nanjing Novizan Biotechnology Co., Ltd.), which were then plated on kanamycin LB plates and cultured overnight.

[0134] 4.1.5 Using primers argB-aL-F and argB-aR-R, the transformants were amplified and verified under the same PCR conditions as in step 1.2.5. The positive band was approximately 2kb. The positive transformants were inoculated into LB liquid tubes, and plasmids were extracted and sequenced to obtain the correct plasmid pK18mobsacB-argBmut, the structure of which is shown below. Figure 3 As shown.

[0135] 4.2 Preparation of Corynebacterium glutamicum ATCC13032 Δcg3035 ΔargR or ATCC13032 cg3035 A251X ΔargR competent cells:

[0136] Pick Corynebacterium glutamicum ATCC13032Δcg3035ΔargR or ATCC13032 cg3035 A251X Streak ΔargR on BHIS plates and incubate overnight at 30°C. Pick single colonies and transfer them to BHIS test tubes, incubating overnight at 30°C and 220 rpm. Inoculate 1 ml of the bacterial culture into a 100 ml shake flask of BHIS liquid medium and incubate on a shaker at 30°C and 220 rpm for 4-6 hours until OD (dose retardation). 600 The value reaches approximately 1.0. On a clean bench, transfer the entire bacterial culture to a 50ml centrifuge tube, centrifuge at 4500×g at 4°C, discard the supernatant, wash the cells with 10% glycerol to resuspend them, centrifuge again at 4500×g at 4°C, repeat the washing once, and discard the supernatant. Finally, add 600μl of 10% glycerol to resuspend the cells, aliquot into 1.5ml centrifuge tubes, preparing one competent cell line of 90μl each. Competent cells can be stored at -80°C.

[0137] 4.3pK18mobsacB-argBmut electroconversion ATCC13032Δcg3035ΔargR or ATCC13032cg3035 A251X ΔargR competent cells:

[0138] Select the plasmid that was correctly sequenced, and aspirate 3 μl (or more than 1 μg) into *Corynebacterium glutamicum* ATCC13032Δcg3035ΔargR or ATCC13032 cg3035. A251XIn ΔargR competent cells, after mixing, the mixture was transferred to an electroporation cuvette and electroporated at 25 μF, 2.5 kV, and 200 Ω for 5.6 ms. Immediately after electroporation, the cells were transferred to 900 μl of preheated BHIS liquid medium at 46 °C and incubated in a 46 °C water bath for 6 min. Then, the cells were incubated in a shaker at 30 °C and 220 rpm for 1 h to allow for cell resuscitation. After resuscitation, 50 μl of the cells were spread onto a BHIS plate containing kanamycin and incubated upside down in a 30 °C incubator for 48 h.

[0139] 4.4SacB sucrose reverse screening:

[0140] Transformants from BHIS plates containing kanamycin were picked and inoculated into antibiotic-free BHIS culture tubes. The tubes were incubated at 30°C and 220 rpm for 24 hours in a shaker to allow for double crossover. The bacterial cells were diluted 1000-fold and spread onto BHIS-suc plates containing 20% ​​sucrose. These plates were then incubated upside down at 30°C for 48 hours. Transformants from the BHIS-suc plates were picked and spotted onto both BHIS plates and BHIS plates containing kanamycin, and incubated upside down at 30°C for 24 hours. PCR amplification was performed using primers argB-aL-F and argB-aR-R to verify transformants that grew on BHIS plates but not on BHIS plates containing kanamycin. The PCR amplification conditions were the same as in step 3.1.5. A band of approximately 2 kb was obtained. Sequencing of the PCR product revealed transformants with successful argB mutations. Positive transformants were picked and transferred to 4 ml of BHIS medium in test tubes, and cultured on a shaker at 30°C and 220 rpm for 24 hours. The culture was maintained using 20% ​​glycerol. The resulting genotype was ATCC13032 cg3035mutargB. mut(A26V M31V) (including ATCC13032Δcg3035ΔargR argB) mut(A26V M31V) and ATCC13032 cg3035 A251X ΔargRargB mut(A26V M31V) ) strains.

[0141] Example 5: ATCC13032 cg3035mutΔargR argB mut(A26V M31V) Preparation of Derivative Strains

[0142] To improve the genetic stability of strain ATCC13032 cg3035mutargB mut(A26V M31V) (including ATCC13032Δcg3035ΔargR argB)mut(A26V M31V) Or ATCC13032 cg3035 A251X ΔargRargB mut(A26V M31V) The arginine production capacity of [a specific strain] was investigated, and its derivative strains were constructed.

[0143] 5.1 Referring to patent CN110564790A, the strain ATCC13032Δcg3035ΔargRargB obtained in mutation example 4 was modified. mut(A26V M31V) Or ATCC13032 cg3035 A251X ΔargRargB mut(A26V M31V) The ClpC gene is ClpC E484K The strain ATCC13032Δcg3035ΔargR argB was obtained. mut(A26V M31V) ClpC E484K Or ATCC13032cg3035 A251X ΔargRargB mut(A26V M31V) ClpC E484K .

[0144] 5.2 Referring to patent CN110564758A, the strain ATCC13032Δcg3035ΔargRargB obtained in Example 4 was mutated. mut(A26V M31V) Or ATCC13032 cg3035 A251X ΔargRargB mut(A26V M31V) The ClpC gene is ClpC E645K The strain ATCC13032Δcg3035ΔargR argB was obtained. mut(A26V M31V) ClpC E645K Or ATCC13032cg3035 A251X ΔargRargB mut(A26V M31V) ClpC E645K .

[0145] 5.3 Referring to patents CN110564790A and CN110564758A, the strain ATCC13032Δcg3035ΔargR argB obtained in Example 4 was mutated. mut(A26V M31V) Or ATCC13032 cg3035 A251X ΔargRargB mut(A26V M31V)The ClpC gene is ClpC E484KE645K The strain ATCC13032Δcg3035ΔargR argB was obtained. mut(A26V M31V) ClpC E484KE645K Or ATCC13032 cg3035 A251X ΔargRargB mut(A26V M31V) ClpC E484KE645K .

[0146] The arginine production of these derived strains was generally more than 0.5 times higher than that of the original strains before the gene NCgl2644 / cg3035 was inactivated or weakened.

[0147] Example 6: Fermentation test of arginine-producing strains

[0148] For the original strain ATCC13032, the arginine-producing strain ATCC13032ΔargRargB mut(A26V M31V) (Including strain 1441 and its next-generation strain 1442), ATCC13032 cg3035mutΔargR argB modified by inactivating or attenuating the gene NCgl2644 / cg3035. mut(A26V M31V) The strain and its derivatives were used to test arginine production capacity through fermentation. The steps are as follows:

[0149] 6.1 Shake-flask fermentation of strain 6.1

[0150] 6.1.1 Activation: Streak the bacterial culture from a glycerol tube onto a BHIG plate and incubate at 30°C for about 2 days.

[0151] 6.1.2 Seed culture: After mixing the BYG medium, dispense 10 ml / bottle into 250 ml shake flasks. Use an inoculation loop to scoop out single colonies obtained from subculture and inoculate them into the seed bottle. Incubate at 30℃ and 220 rpm for 24 h.

[0152] 6.1.3 Shake Flask Fermentation: Prepare RG2 medium, mix thoroughly, and dispense 45 ml / flask into 500 ml single-baffle shake flasks (pre-filled with 1.5 g calcium carbonate). Inoculate at least 5 ml of seed culture into each RG2 shake flask. Incubate at 30°C and 220 rpm for approximately 48-72 hours, until glucose is depleted. Centrifuge the fermentation broth at high speed, collect the supernatant, dilute 50 times, and determine the arginine yield by HPLC.

[0153] 6.2 Determination of Arginine Content by High Performance Liquid Chromatography (HPLC)

[0154] 6.2.1 Methods: The L-arginine content in the fermentation broth was determined using the pre-column derivatization amino acid analysis method with OPA. Primary amino acids react with o-phthalaldehyde (OPA) in the presence of a thiol reagent to form OPA-amino acids. The resulting amino acid derivatives were separated by reversed-phase high-performance liquid chromatography and detected by ultraviolet or fluorescence. Within a certain range, the absorbance value was directly proportional to the amino acid concentration.

[0155] 6.2.2 Preparation of Derivatizing Agents and Mobile Phase

[0156] Borate buffer: Accurately weigh 6.183g of boric acid into 0.4M borate buffer, dissolve it in ultrapure water, adjust the pH to 10.2 with 10N NaOH solution, and make up to 250ml in a volumetric flask.

[0157] Derivatizing agent: Accurately weigh 500 mg of o-phthalaldehyde (OPA) solid, add 5 ml of anhydrous ethanol, add 500 μl of mercaptopropionic acid, and bring the volume up to 50 ml with 0.4 M, pH 10.2 borate buffer.

[0158] Mobile phase A: 40mM NaH2PO4 solution. Accurately weigh 5.5g NaH2PO4·H2O, dissolve it in ultrapure water, adjust the pH to 7.8 with 10NNaOH solution, bring the volume to 1L, filter through a 0.22μm filter membrane, and use for later use.

[0159] Mobile phase B: ACN:MeOH:H2O = 45:45:10, bring to a final volume of 1L for later use. The reagent purity is HPLC grade.

[0160] 6.2.3 High Performance Liquid Chromatography (HPLC) Determination Conditions:

[0161] Column: ZORBAX Eclipse-AAA 4.6 x 75 mm, 3.5 μm; Flow rate: 1.2 ml / min; Stop time: 14 min; Column temperature: 40 degrees; DAD settings: UV 338 nm, 10 nm (bandwidth), reference 390 nm, 20 nm (bandwidth).

[0162] The elution procedure is shown in Table 3.

[0163] Table 3. HPLC elution program

[0164]

[0165]

[0166] The autosampler dispensed 0.5 μl of sample, 2.5 μl of borate buffer, 0.5 μl of derivatizing agent, and 32 μl of ultrapure water, respectively. After mixing and derivatizing, the sample was injected. The arginine peak elution time was 7.9 minutes. The L-arginine content in the sample could be obtained by comparing the peak area with the standard sample.

[0167] 6.3 L-arginine yield from strain fermentation

[0168] Based on the fermentation protocol in step 6.1 and the determination method in step 6.2, the L-arginine fermentation yield of each strain was compared. Three parallel experiments were performed for each strain, and the results are shown in the table below:

[0169] Table 4. Comparison of L-arginine levels in fermentation by different strains

[0170]

[0171]

[0172] As can be seen from Table 4, strain ATCC13032ΔargRargB A26V M31V After subculturing, it loses its ability to produce arginine and lacks genetic stability. This is observed in the L-arginine-producing strain ATCC13032ΔargRargB. mut(A26V M31V) After inactivating or attenuating the gene NCgl2644 / cg3035, the resulting ATCC13032 cg3035mutΔargR argB mut(A26V M31V) The strain and its derivatives still maintain L-arginine production capacity.

[0173] The following is a comparison of ATCC13032 cg3035mutΔargR argB. mut(A26V M31V) The genetic stability of the strain was investigated.

[0174] Example 7: Stability of arginine production capacity of strains analyzed by subculturing

[0175] Select the gene NCgl2644 / cg3035 knockout strain ATCC13032Δcg3035ΔargR argB mut(A26V M31V) The mutant strain ATCC13032 cg3035, containing amino acid A at position 251. A251TGA ΔargRargB A26V M31V and ATCC13032cg3035 A251V ΔargRargB A26V M31V Genetic stability was investigated through passage and fermentation.

[0176] 7.1 Passage for Stability Analysis

[0177] 7.1.1 Prepare and mix the BYG medium, then dispense 10 ml / bottle into 250 ml shake flasks. Inoculate the glycerol strain of the strain to be investigated into three separate bottles of BYG medium. Incubate at 30℃ and 220 rpm for 24 h.

[0178] 7.1.2 Inoculate the above culture medium at 2% v / v into fresh BYG medium and incubate at 30°C and 220 rpm for 24 h.

[0179] 7.1.3 Repeat the steps in 7.1.2 to transfer the data and complete the generation three times.

[0180] 7.1.4 After three subcultures using BYG, transfer 2% v / v into 50 ml of RG2 medium. Incubate at 30℃ and 220 rpm for 48 h.

[0181] 7.1.5 Take the 48-hour endpoint fermentation broth and dilute it 10... 5 -10 6 After multiplication, 100 μl of the solution was spread onto antibiotic-free BHIS plates and incubated at 30°C for 48 h. After single colonies grew, fermentation was performed to verify that the strain could maintain the percentage of arginine-producing colonies after the above three subcultures.

[0182] 7.2 Shake-flask fermentation of strains

[0183] 7.2.1 Seed culture: After mixing the BYG medium, dispense 10 ml / bottle into 250 ml shake flasks. Use an inoculation loop to scoop out single colonies obtained from subculturing in step 7.1 and inoculate them into the seed bottle. Incubate at 30℃ and 220 rpm for 24 h.

[0184] 7.2.2 Shake Flask Fermentation: Prepare RG2 medium, mix thoroughly, and dispense 45 ml / flask into 500 ml single-baffle shake flasks (pre-filled with 1.5 g calcium carbonate). Inoculate at least 5 ml of the seed culture from step 7.2.1 into the RG2 shake flasks. Incubate at 30°C and 220 rpm for approximately 48-72 h, until glucose is depleted. Centrifuge the fermentation broth at high speed, collect the supernatant, dilute 50 times, and determine the arginine yield by HPLC.

[0185] 7.3 Stability analysis of the arginine production capacity of the strain:

[0186] According to the subculturing scheme in step 7.1, each strain underwent three parallel subculturing experiments, resulting in six plates. Ten single colonies were selected from each plate for fermentation, and the L-arginine fermentation yield of the strains was summarized in Table 5.

[0187] Table 5. L-arginine yield and percentage of strains producing arginine after three generations of subculturing.

[0188]

[0189]

[0190] As shown in Table 5, strain ATCC13032ΔargRargB A26V M31V After subculturing, it loses its ability to produce arginine and lacks genetic stability; strain ATCC13032Δcg3035ΔargR argB mut(A26V M31V) and ATCC13032 cg3035 A251V ΔargRargB mut(A26V M31V) After three generations, the mother strain maintained more than 70% of its L-arginine production capacity.

[0191] Regarding the strains with amino acid A at position 251 mutated to the stop codon TAA, and those mutated to amino acids D, E, F, G, K, L, M, N, P, R, S, T, W, and Y respectively, the results of genetic stability studies after subculturing were compared with those of ATCC13032Δcg3035ΔargR argB. mut(A26V M31V) ATCC13032 cg3035 A251TGA ΔargRargB A26V M31V and ATCC13032cg3035 A251V ΔargRargB mut(A26V M31V) Similarly, even after three generations, the mother strain can maintain more than 70% of its L-arginine production capacity.

[0192] Example 8: Fermentation test of strain ATCC13870 / ATCC21831 after gene NCgl2644 / cg3035 modification.

[0193] To verify whether the modification scheme of inactivating or weakening the NCgl2644 / cg3035 gene is applicable to other L-arginine-producing strains, Corynebacterium glutamicum ATCC13870 and Corynebacterium glutamicum ATCC21831 were used as examples for corresponding modifications, and fermentation tests were conducted, as follows:

[0194] 8.1 Referring to Examples 1 and 2, ATCC13870Δcg3035 and ATCC13870cg3035 were constructed respectively. A251V , ATCC21831Δcg3035, ATCC21831 cg3035 A251V strains.

[0195] 8.2 Referring to Example 3, construct ATCC13870Δcg3035ΔargR and ATCC13870 cg3035 respectively. A251V ΔargR,ATCC21831Δcg3035ΔargR,ATCC21831 cg3035 A251V ΔargR strain.

[0196] 8.3 Referring to Example 4, construct ATCC13870Δcg3035ΔargR argB respectively. mut(A26V M31V) ATCC13870 cg3035 A251V ΔargR argB mut(A26V M31V) , ATCC21831Δcg3035ΔargR argB mut(A26V M31V) ATCC21831 cg3035 A251V ΔargR argB mut(A26V M31V) strains.

[0197] 8.4 Referring to Example 6, for strains ATCC13870, ATCC21831, and ATCC13870Δcg3035ΔargRargB mut(A26V M31V) ATCC13870 cg3035 A251V ΔargR argB mut(A26V M31V) , ATCC21831Δcg3035ΔargR argB mut(A26V M31V) ATCC21831 cg3035 A251V ΔargR argB mut(A26V M31V) Fermentation was used to test arginine production capacity.

[0198] The L-arginine fermentation yield of each strain was compared. Three parallel experiments were conducted for each strain, and the results are shown in Table 6 below.

[0199] Table 6. Comparison of L-arginine levels in fermentation by strains

[0200] strain number genotype Arginine production (g / L) ATCC13870 wild type 0 ATCC21831 wild type 0 13870Δcg3035 <![CDATA[ATCC13870Δcg3035ΔargR argB mut(A26V M31V) ]]> 6.486±0.117 <![CDATA[13870cg3035 A251V ]]> <![CDATA[ATCC13870cg3035 A251V ΔargR argB mut(A26V M31V) ]]> 6.794±0.146 21831Δcg3035 <![CDATA[ATCC21831Δcg3035ΔargR argB mut(A26V M31V) ]]> 5.986±0.213 <![CDATA[21831cg3035 A251V ]]> <![CDATA[ATCC21831cg3035 A251V ΔargR argB mut(A26V M31V) ]]> 6.725±0.124

[0201] Example 9: Stability of arginine production capacity of strain ATCC13870 / ATCC21831 by subculturing analysis

[0202] Selected strains: ATCC13870Δcg3035ΔargR argBmut(A26V M31V) ATCC13870 cg3035 A251V ΔargR argB mut(A26V M31V) , ATCC21831Δcg3035ΔargR argB mut(A26V M31V) ATCC21831cg3035 A251V ΔargR argB mut(A26V M31V) Genetic stability was investigated through subculturing and fermentation, and the results are shown in Table 7 below.

[0203] Table 7. L-arginine yield and percentage of strains producing arginine after three generations of subculturing.

[0204]

[0205]

[0206] The above experimental results show that the modification scheme of the present invention, which inactivates or weakens the NCgl2644 / cg3035 gene of the arginine-producing strain, can greatly improve the stability of the strain's arginine production capacity, making the genetically engineered strain possible for industrial application. sequence list <110> Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences <120> A method to improve the stability of arginine production capacity of bacterial strains <130> SHPI2010515 <160> twenty two <170> SIPOSequenceListing 1.0 <210> 1 <211> 42 <212> DNA <213> Artificial sequence() <400> 1 gacgtcaggg gctgggcacc gatttaaata aaacgaaagg ct 42 <210> 2 <211> 30 <212> DNA <213> Artificial sequence() <400> 2 gcgggactct ggggttcgcg gaatcatgac 30 <210> 3 <211> 42 <212> DNA <213> Container() <400> 3 acgcaccagt catcgttgat tcagaagac tcgtcaaga gg <210> 4 <211> 45 <212> DNA <213> Container() <400> 4 cggtgcccag cccctgacgt catctacaac lives cggat <210> 5 <211> 40 <212> DNA <213> Container() <400> 5 cgcgaacccc agagtcccgc gtcgtctttg ccctgcattg <210> 6 <211> 40 <212> DNA <213> Container() <400> 6 agatatgcct cctgtgcgtg cggaaacggg gaagactagg <210> 7 <211> 46 <212> DNA <213> Container() <400> 7 cctagtcttc cccgtttccg cacgcacagg aggcatatct ccaggt <210> 8 <211> 48 <212> DNA <213> Container() <400> 8 ccttcttgac gagttcttct gaatcaacga tgactggtgc gtggtcag <210> 9 <211> 20 <212> DNA <213> Container() <400> 9 cggcggttga gtttgatgac <210> 10 <211> 20 <212> DNA <213> Container() <400> 10 cggaatccga agagccaga <210> 11 <211> 48 <212> DNA <213> Container() <400> 11 ccccgccggc gtggtcaggc throw aaacgaagg ctcagtcg <210> 12 <211> 49 <212> DNA <213> Container() <400> 12 cgcctgacca cgccggcggg gatctacaac agtagaatt cggatccat <210> 13 <211> 20 <212> DNA <213> Container() <400> 13 ttcgcggttt tatgcacgtc <210> 14 <211> 20 <212> DNA <213> Container() <400> 14 tggtttcctg caccgctaat <210> 15 <211> 59 <212> DNA <213> Artificial sequence () <400> 15 acaatttcac acaggaaaca gctatgacat gattacggac ttcacaacca caggttcgc 59 <210> 16 <211> 54 <212> DNA <213> Artificial sequence () [[ID=X]]X<21]]<400> 16 gcgattaaca agccttgaac taggggcgct gtcttacctc ggctggttgg ccag 54 <210> 17 <211> 53 <212> DNA <213> Artificial sequence () <400> 17 gtggctgctg gccaaccagc cgaggtaaga cagcgcccct agttcaaggc ttg 53 <210> 18 <211> 59 <212> DNA <213> Artificial sequence () <400> 18 agtgccaagc ttgcatgcct gcaggtcgac tctagagcac gagacggtcc tcaaccatg 59 <210> 19 <211> 59 <212> DNA <213> Artificial sequence () <400> 19 acaatttcac acaggaaaca gctatgacat gattacggtg acaatggtgc agctgctgc 59 <210> 20 <211> 57 <212> DNA Note: There seems to be an incorrect tag "X<21]]" in the original text which might be a formatting error. I've translated it as is while pointing it out. <213> Container() <400> 20 57. cacggtgcgc aagagacca cgtcggcagc aaaaacagcc ttgagatcat catccac <210> 21 <211> 57 <212> DNA <213> Container() <400> 21 57. gatgatgatc tcaaggctgt ttttgctgcc gacgtggtct tcttgcgcac cgtgggc <210> 22 <211> 59 <212> DNA <213> Container() <400> 22 agtgccaagc ttgcatgcct gcaggtcgac tctagaggtg gcccaacgcg ttgactgcg

Claims

1. A method for improving the stability of arginine production capacity of a bacterial strain, comprising the following steps: Using *Corynebacterium glutamicum* producing L-arginine as the basic strain, the gene NCgl2644 / cg3035 in the genome was inactivated or attenuated. The inactivation or attenuation of NCgl2644 / cg3035 was selected from the following methods: knocking out the open reading frame (OPF) of NCgl2644 / cg3035; or mutating amino acid A at position 251 of the ORF of NCgl2644 / cg3035. The mutation at position 251 of amino acid A in NCgl2644 / cg3035 was selected from the following groups: a mutation to the stop codon TGA or TAA; or a mutation to amino acid V, D, E, F, G, K, L, M, N, P, R, S, T, W, or Y. The basic strain is Corynebacterium glutamicum ATCC13032∆argR, argBmutA26V M31V.

2. The method as described in claim 1, characterized in that, The basic strain is *Corynebacterium glutamicum* ATCC13032∆argR, argBmutA26V M31V, and the following steps are included: A. Inactivate or attenuate the gene NCgl2644 / cg3035 in the genome to obtain the mutant strain ATCC13032cg3035mut; B. Knock out the argR gene in the genome of the NCgl2644 / cg3035 mutant strain ATCC13032cg3035mut described in step A to obtain the gene knockout strain ATCC13032cg3035mut, ΔargR; C. Mutate the argB gene in the genome of the gene knockout strain ATCC13032cg3035mut, ΔargR described in step B with A26V and M31V mutations to obtain the genetically engineered strain ATCC13032cg3035mut, ΔargR, argBmutA26VM31V.

3. The method as described in claim 2, characterized in that, Step A is implemented using gene editing technology, wherein the gene editing employs the CRISPR-Cas9 system, the CRISPR-Cpf1 system, the CRISPR-Cas-related transposon system INTEGRATE system, or the CAST system.

4. The method as described in claim 2, characterized in that, The gene knockout strain ATCC13032cg3035mut, ΔargR described in step B was prepared using the following method: B1. Using the ATCC13032 genome as a template, PCR amplification was performed using primer argR-aL-F with sequence SEQ ID NO: 15 and primer argR-aL-R with sequence SEQ ID NO: 16 to obtain a 1kb argR-aL fragment; B2. Using the ATCC13032 genome as a template, PCR amplification was performed using primer argR-aR-F with sequence SEQ ID NO: 17 and primer argR-aR-R with sequence SEQ ID NO: 18 to obtain a 1kb argR-aR fragment; B3. The plasmid pK18mobsacB with GenBank accession number FJ437239.1 was digested with HindIII and EcoRI, and a 5.7kb vector fragment was obtained by gel recovery. B4. Gibson was used to connect the above argR-aL fragment, argR-aR fragment and vector fragment, transformed into DH5α competent cells, plated on kanamycin LB plates and cultured overnight; B5. Transformants were verified by PCR amplification using primers argR-aL-F and argR-aR-R, yielding plasmid pK18mobsacB-argR; B6. Preparation of Corynebacterium glutamicum ATCC13032cg3035mut competent cells; B7. Transform plasmid pK18mobsacB-argR into ATCC13032cg3035mut competent cells; B8. Perform SacB sucrose reverse screening, and use primers argR-aL-F and argR-aR-R for PCR amplification to verify transformants that grow on BHIS plates but cannot grow on BHIS plates containing kanamycin, and obtain strain ATCC13032cg3035mut, ΔargR.

5. The method as described in claim 4, characterized in that, The genetically engineered strain described in step C is prepared by the following method: C1. Using the ATCC13032 genome as a template, PCR amplification was performed using primer argB-aL-F with sequence SEQ ID NO: 19 and primer argB-aL-R with sequence SEQ ID NO: 20 to obtain a 1kb argB-aL fragment; C2. Using the ATCC13032 genome as a template, PCR amplification was performed using primer argB-aR-F with sequence SEQ ID NO: 21 and primer argB-aR-R with sequence SEQ ID NO: 22 to obtain a 1kb argB-aR fragment; C3. Plasmid pK18mobsacB with GenBank accession number FJ437239.1 was digested with HindIII and EcoRI, and a 5.7kb vector fragment was obtained by gel recovery. C4. Gibson was used to connect the above argB-aL fragment, argB-aR fragment and vector fragment, transformed into DH5α competent cells, plated on kanamycin LB plates and cultured overnight; C5. Transformants were verified by PCR amplification using primers argB-aL-F and argB-aR-R, yielding plasmid pK18mobsacB-argBmut; C6. Preparation of Corynebacterium glutamicum ATCC13032cg3035mut, ΔargR competent cells; C7. Transform plasmid pK18mobsacB-argBmut into ATCC13032cg3035mut, ΔargR competent cells; C8. Perform SacB sucrose reverse screening, and use primers argB-aL-F and argB-aR-R for PCR amplification to verify transformants that grow on BHIS plates but not on BHIS plates containing kanamycin, obtaining strains ATCC13032cg3035mut, ∆argR, argBmutA26V M31V.

6. A genetically engineered bacterium ATCC13032cg3035mut, ∆argR, argBmutA26V M31V, characterized in that, Constructed according to the method described in any one of claims 1-5.

7. The application of the genetically engineered bacteria as described in claim 6 for the production of L-arginine.

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