CN114317582A - Method for improving stability of arginine producing capacity of strain - Google Patents

Abstract

The invention discloses a method for improving the stability of arginine producing capacity of a strain, which comprises the following steps: the gene NCgl2644/cg3035 in the genome is inactivated or attenuated by using Corynebacterium glutamicum producing L-arginine as a basic strain. The method can effectively improve the genetic stability of the L-arginine producing strain.

Description

Method for improving stability of arginine producing capacity of strain

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a method for improving the stability of arginine producing capacity of a strain, in particular to a method for improving the genetic stability of an arginine producing strain through genetic engineering.

Background

L-arginine (L-argnine, L-Arg for short) is one of semi-essential basic amino acids needed in human body, is an important intermediate metabolite of organism urea circulation as a basic amino acid containing guanidyl, has various unique physiological and pharmacological effects, has good curative effects on treating physiological functions, cardiovascular diseases, stimulating immune system, maintaining nutrition balance of infants, promoting detoxification of human body and the like, is called as an important carrier for transporting and storing amino acid in the body by experts, and is extremely important in intramuscular metabolism. It is an essential amino acid for the synthesis of cytoplasmic and nuclear proteins; participate in creatine synthesis as the only ammonia source; as an important intermediate of the urea cycle, the urea cycle plays a role in removing excessive ammonia in the liver and prevents poisoning caused by excessive accumulation of ammonia; it also has effects in regulating immunity, inhibiting tumor growth, and promoting wound tissue healing. Arginine is a direct precursor of nitric oxide, urea, ornithine and myostatin, an important element in the synthesis of myostatin, and is used as a synthesis of polyamines, citrulline and glutamine. Therefore, the L-arginine has important and wide application in the fields of medicine, food and chemical industry. For example, in clinic, besides being one of the main components of compound amino acid infusion, L-arginine and its salts are widely used for treating various hepatic coma patients who are forbidden to use sodium glutamate and viral hepatic glutamic pyruvic transaminase abnormal patients, and have obvious curative effect on viral hepatitis. It can be used for treating intestinal ulcer, thrombosis, and neurasthenia. In addition, the L-arginine is an important component of a sports nutritional beverage formula, is also an important feed additive, and is widely applied to high-end breeding industry. Statistically, the worldwide demand for L-arginine is currently over 15000 tons, and the demand increases at a rate of 12% -15% per year.

There are two methods for producing L-arginine: the first is protein hydrolysis extraction method, and the second is microorganism fermentation method. The hydrolysis method has the problems of time-consuming operation, low yield and output, high cost and the like, and has serious pollution, so the hydrolysis method is not suitable for large-scale production. The fermentation method for producing the L-arginine has relatively simple process and is environment-friendly, so the method has great development potential and becomes an important development trend of the domestic and foreign amino acid industry. International famous amino acid companies such as Japanese monosodium glutamate, Synergestin and Degaosha in Germany mainly use biological fermentation and genetic engineering techniques for L-arginine production. However, the acid production level of L-arginine produced by domestic microbial fermentation is generally low, the cost is high, and the production level and the yield can not meet the domestic requirements far, so the research for improving the L-arginine fermentation level has important significance.

Most of L-arginine fermentation strains are mainly Corynebacterium glutamicum (Corynebacterium glutamicum), Brevibacterium flavum (Brevibacterium flavum), Corynebacterium crenatum (Corynebacterium crenatum), Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis) and the like, but currently, microorganism strains for producing L-arginine are mainly Corynebacterium glutamicum and Corynebacterium crenatum. The genetic engineering technology has an important promoting effect on the breeding of the arginine high-yielding strain, and the construction of the L-arginine high-yielding strain by utilizing the genetic engineering is a high-efficiency and rational breeding means.

However, many genetically engineered bacteria have unstable genetic characters and unstable arginine secretion capacity, which results in large differences among fermentation batches, and can cause degeneration and variation phenomena after passage, and even lose the arginine production capacity after several passages, thus causing difficulty in industrial scale production and application. For example, the inventors obtained a genetically engineered strain number 1441 (genotype ATCC 13032. delta. argRogB) by knocking out argR and introducing an argB mutation (A26V M31V) by releasing feedback inhibition of argB using a wild type Corynebacterium glutamicum ATCC13032 as a starting strain^{A26V M31V}) The arginine producing capacity of corynebacterium glutamicum is greatly improved, the arginine yield is 6.231 +/-0.023 g/L, but subsequent experiments show that the genetic engineering strain is quite unstable, the fermentation batch difference is large, and the arginine producing capacity is lost after passage (the number 1442 is the strain obtained after the first generation of the number 1441 strain). Therefore, improvement of genetic stability of arginine engineered strains is always an urgent need.

Disclosure of Invention

In order to overcome the defects of unstable genetic character and low fermentation level of the existing L-arginine production strain, the invention utilizes a genetic engineering technology to modify the genome of corynebacterium glutamicum including corynebacterium glutamicum engineering bacteria, and can greatly improve the genetic stability and the L-arginine production capacity of the strain by enhancing genes related to L-arginine production and weakening branch metabolic pathways.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for improving the stability of arginine producing ability of a strain comprises the following steps: the gene NCgl2644/cg3035 in the genome is inactivated or attenuated by using Corynebacterium glutamicum producing L-arginine as a basic strain.

The inactivation or attenuation of the above-mentioned gene NCgl2644/cg3035 may be selected from the following modes: knocking out an open reading frame of a gene NCgl2644/cg 3035; mutating any amino acid in the open reading frame of the gene NCgl2644/cg3035 to a stop codon; the 251 st amino acid A of the open reading frame of the gene NCgl2644/cg3035 is mutated.

The 251 st amino acid A mutation of the gene NCgl2644/cg3035 may be selected from the group consisting of: mutation is stop codon TGA or TAA; the mutation is amino acid V, D, E, F, G, K, L, M, N, P, R, S, T, W or Y.

The above-mentioned basic strain may be selected from ATCC13032, ATCC13870, ATCC21831 and the like, preferably from the genome of the above-mentioned strain (Δ argR, argBmut (a26V M31V)) engineering bacteria, namely ATCC13032(Δ argR, argBmut (a26V M31V)), ATCC13870(Δ argR, argBmut (a26V M31V)), ATCC21831(Δ argR, argBmut (a26V M31V)) and the like, more preferably corynebacterium glutamicum ATCC13032(Δ argR, argBmut (a26V M31V)) or a mutant strain derived therefrom, wherein Δ argR, argBmut (a26V M31V)) are preferably used

The Corynebacterium glutamicum ATCC13032 (. DELTA.argR, argBmut (A26V M31V)) was obtained by knocking out the argR gene of Corynebacterium glutamicum ATCC13032 and introducing a mutation (A26V M31V) of argB,

the derivative mutant strain is selected from the strains CCTCC NO: M2017760 reported in patent application CN201711430988.5, ATCC13032(Δ argR, argBmut (A26V M31V), NCgl0083mut2) reported in patent application CN 2015826, ATCC13032(Δ argR, argBmut (A26V M31V), NCgl0742mut3) reported in patent application CN201810492999.4, ATCC13032(Δ argR, argBmut (A26V M31V), NCgl2374mut4) reported in patent application CN201810569947.2, ATCC13032(Δ argR, argBmut (A26M V M31V), NCgl2620mut6), CN 3748 (ATCC13032, Δ arg 2526A 2526, Δ arg 5926A 5926), ATCC13032(Δ argR 5926A 5926) reported in patent application CN 5926, NCgl 13032(Δ argBmut 5926M 5926), and NCgl 5926 Bmut 13032(Δ arg 5926A 5926).

In a preferred embodiment, the above base strain is Corynebacterium glutamicum ATCC13032(Δ argR, argBmut (A26V M31V)), comprising the following steps:

A. inactivating or weakening the gene NCgl2644/cg3035 in the genome to obtain a mutant strain ATCC13032(cg3035mut) of the gene NCgl2644/cg 3035;

B. knocking out argR gene in the genome of NCgl2644/cg3035 mutant strain ATCC13032(cg3035mut) in the step A to obtain gene knocked-out strain ATCC13032((cg3035mut, delta argR);

C. A26V and M31V mutations were made in the argB gene in the genome of the knockout strain ATCC13032(cg3035mut, Δ argR) described in step B to obtain the genetically engineered strain ATCC13032((cg3035mut, Δ argR, argBmut (A26V M31V)).

The step A can be implemented by a gene editing technology, and the gene editing can adopt a CRISPR-Cas9 system, a CRISPR-Cpf1 system, a CRISPR-Cas related transposition system INTEGRATE system or a CAST system.

The INTEGRATE system refers to the gene editing tool (Insertion of transposable element for guiding RNA assisted targeting) developed by Sam Sternberg research group; the CAST system is a gene editing tool (CRISPR-associated transposase) developed by the tensor research group.

In one embodiment, the knockout strain ATCC13032(cg3035mut, Δ argR) described in step B above can be prepared by the following method:

B1. using ATCC13032 genome as a template, and using a primer argR-aL-F with a sequence of SEQ ID NO. 15 and a primer argR-aL-R with a sequence of SEQ ID NO. 16 to carry out PCR amplification to obtain an argR-aL fragment with about 1 kb;

B2. carrying out PCR amplification by using an ATCC13032 genome as a template and a primer argR-aR-F with a sequence of SEQ ID NO. 17 and a primer argR-aR-R with a sequence of SEQ ID NO. 18 to obtain an argR-aR fragment with about 1 kb;

B3. the plasmid pK18mobsacB with GenBank accession number FJ437239.1 is digested with HindIII and EcoRI, and the 5.7kb vector fragment is obtained by gel recovery;

gibson ligated argR-aL, argR-aR and vector fragments as described above, transformed DH 5. alpha. competent cells, plated on kanamycin LB plates, and cultured overnight;

B5. carrying out PCR amplification by using primers argR-aL-F and argR-aR-R to verify a transformant, and obtaining a plasmid pK18 mobsacB-argR;

B6. preparing competent cells of Corynebacterium glutamicum ATCC13032(cg3035 mut);

B7. plasmid pK18mobsacB-argR was transformed into ATCC13032(cg3035mut) competent cells;

B8. SacB sucrose counter-screening was performed, PCR amplification was performed using primers argR-aL-F and argR-aR-R, and transformants that grew on BHIS plates but could not grow on kanamycin-containing BHIS plates were verified to obtain strain ATCC13032(cg3035mut,. DELTA.argR).

The genetically engineered strain in the step C can be prepared by the following method:

C1. using ATCC13032 genome as template, using primer argB-aL-F with sequence SEQ ID NO. 19 and primer argB-aL-R with sequence SEQ ID NO. 20 to carry out PCR amplification, obtaining about 1kb argB-aL fragment;

C2. carrying out PCR amplification by using ATCC13032 genome as a template and a primer argB-aR-F with a sequence of SEQ ID NO. 21 and a primer argB-aR-R with a sequence of SEQ ID NO. 22 to obtain an argB-aR fragment with about 1 kb;

C3. the plasmid pK18mobsacB with GenBank accession number FJ437239.1 is digested with HindIII and EcoRI, and the 5.7kb vector fragment is obtained by gel recovery;

gibson ligated the argB-aL, argB-aR and vector fragments described above, transformed DH 5. alpha. competent cells, plated on kanamycin LB plates, and cultured overnight;

C5. carrying out PCR amplification by using primers argB-aL-F and argB-aR-R to verify a transformant, and obtaining a plasmid pK18 mobsacB-argBmut;

C6. preparing competent cells of Corynebacterium glutamicum ATCC13032(cg3035mut, Δ argR);

C7. plasmid pK18mobsacB-argBmut was transformed into ATCC13032(cg3035mut, Δ argR) competent cells;

C8. SacB sucrose counter-screening was performed, PCR amplification was performed using primers argB-aL-F and argB-aR-R, and transformants that grew on BHIS plates but could not grow on kanamycin-containing BHIS plates were verified, yielding strain ATCC13032(cg3035mut,. DELTA.argR, argBmut (A26V M31V)).

The transformation in the above steps B7 and C7 may be calcium chloride transformation or electro-transformation, preferably electro-transformation.

The steps a, B and C may be arbitrarily crossed and reversed, as long as each step can realize its own function.

According to a second aspect of the present invention, there is provided a genetically engineered bacterium constructed according to the above-described method.

According to a third aspect of the present invention, there is provided the use of the above genetically engineered bacterium in the production of L-arginine.

The genetically engineered bacterium can be directly used as a fermentation strain to produce the L-arginine through fermentation, and can also be used as an original strain to be further improved so as to screen out a new production strain with further improved L-arginine production capacity.

When L-arginine is produced by fermentation of the above genetically engineered bacterium, the medium used in the fermentation may be any medium suitable for growth and fermentation of Corynebacterium glutamicum.

According to a preferred embodiment of the invention, the fermentation medium consists of: 60g/L glucose, 5g/L corn steep liquor, 30g/L (NH)₄)₂SO₄8g/L KCl, 2g/L urea, 0.5g/L KH₂PO₄，0.5g/L K₂HPO₄，1g/L MgSO₄·7H₂O，1g/L NaCl，20mg/L FeSO₄·7H₂O，10mg/L MnSO₄·5H₂O, 20mg/L nicotinic acid, 20mg/L beta-alanine10mg/L VB1, 0.2mg/L biotin, 30g/L CaCO₃KOH was adjusted to pH 7.7.

In a preferred embodiment, the fermentation of the L-arginine producing bacteria comprises a seed culture stage and a bacterial fermentation stage. The two stages use seed culture medium and fermentation culture medium, which may be the same as or different from the seed culture medium.

Preferably, when the fermentation medium is not the same as the seed medium, the seed medium consists of: 3g/L NaCl, 5g/L yeast extract, 7g/L beef extract, 10g/L peptone and 10g/L glucose.

The gene NCgl2644/cg3035 in the genome of the Corynebacterium glutamicum ATCC13032 is inactivated or weakened, so that the genetic stability, namely the passage stability of the genetic engineering strain Corynebacterium glutamicum ATCC13032 (delta argR, argBmut (A26V M31V)) producing L-arginine and derivative mutant strains thereof can be greatly improved, and the method has popularization and popularization values.

Drawings

FIG. 1 is a schematic representation of plasmid pK18mobsacB, which is presented by Liu Shuangjiang, institute of microbiology, academy of sciences of China. GenBank: FJ 437239.1. See https:// www.ncbi.nlm.nih.gov/nuccore/215434894 for specific information.

FIG. 2 is a schematic diagram of a recombinant plasmid pK18mobsacB-argR constructed according to the present invention.

FIG. 3 is a schematic diagram of recombinant plasmid pK18mobsacB-argBmut constructed according to the present invention.

FIG. 4 is a schematic diagram of recombinant plasmid pJYS3_ Δ cg3035 constructed in the present invention.

FIG. 5 is a schematic diagram of a recombinant plasmid pCgsgRNA _ cg3035 constructed according to the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.

The original Corynebacterium glutamicum which can be currently used for the production, or which has the potential to be used for the production of L-arginine, includes ATCC13032, ATCC13870, ATCC21831 and the like. These original strains were genetically engineered to have genomes of (Δ argR, argBmut (A26V M31V)) and then engineered to have ATCC13032(Δ argR, argBmut (A26V M31V)), ATCC13870(Δ argR, argBmut (A26V M31V)), ATCC21831(Δ argR, argBmut (A26V M31V)), and the like, which are engineering bacteria, which could be changed from being incapable of producing L-arginine to being capable of producing L-arginine or from being weak in L-arginine production to being strong in L-arginine production. Many genetically engineered bacteria have the defect of low passage stability, so how to improve the passage stability of the genetically engineered bacteria is an urgent problem to be solved.

In this context, the terms "Corynebacterium glutamicum ATCC13032/ATCC13870/ATCC 21831", "strain ATCC13032/ATCC13870/ATCC 21831", "ATCC 13032/ATCC13870/ATCC 21831" mean the same meanings, and all refer to the original strain ATCC13032/ATCC13870/ATCC21831 which is the subject of genetic modification, and is a wild strain which is the L-arginine producing strain, and is purchased from the institute for Industrial microbiology, Shanghai province.

The invention improves the genetic stability (passage stability) of Corynebacterium glutamicum including strain Corynebacterium glutamicum ATCC13032/13870/21831 (delta argR, argBmut (A26V M31V)) and derivative mutant strains thereof, mainly by inactivating or weakening the gene NCgl2644/cg3035 in Corynebacterium glutamicum ATCC13032/ATCC13870/ATCC21831 genome in any modification mode, wherein the inactivation or weakening comprises knocking out the NCgl2644/cg3035 gene open reading frame; or the 251 th amino acid A of the open reading frame is mutated into a stop codon TGA or TAA, or into other amino acids V, D, E, F, G, K, L, M, N, P, R, S, T, W or Y. Then, the argR is knocked out on the basis of the obtained mutant strain, and argB is mutated into an anti-L-arginine feedback inhibition genotype.

The gene NCgl2644/cg3035 encodes N-acetyl glutamic acid synthase which can catalyze glutamic acid to synthesize N-acetyl glutamic acid, and is the first reaction of a biosynthetic pathway from glutamic acid to arginine, and the research of the inventor proves that the gene is inactivated or weakened to improve the genetic stability of Corynebacterium glutamicum ATCC13032/13870/21831 (delta argR, argBmut (A26V M31V)) and mutant strains derived from the Corynebacterium glutamicum.

The gene argR is an arginine operon regulating gene which is ubiquitous in bacteria and has different functions in different bacteria.

N-acetylglutamate kinase (N-acetylglutamate kinase) catalyzes the synthesis of N-acetylglutamyl phosphate from N-acetylglutamate and is feedback-inhibited by the final product, L-arginine. The inventors have found that L-arginine synthesizing ability of Corynebacterium glutamicum ATCC13032/ATCC13870/ATCC21831 can be effectively improved by mutating N-acetylglutamate kinase to thereby change alanine (A) at position 26 to valine (V) and methionine (M) at position 31 to valine (V). Herein, the mutant gene of the N-acetylglutamate kinase gene argB is abbreviated as argBmut or argBmut (A26V M31V), which is an anti-L-arginine feedback inhibition genotype.

In the examples, a series of L-arginine-producing Corynebacterium glutamicum strains with significantly improved genetic stability, such as the genetically engineered strain ATCC13032cg3035 mut Δ argRargB, were obtained by the above genetic engineering procedures^{A26V M31V}Including ATCC 13032. delta. cg 3035. delta. argRogB^{A26V M31V}And ATCC13032cg3035^A251XΔargRargB^{A26V M31V}(wherein X is a stop codon TGA, TAA, amino acid V, D, E, F, G, K, L, M, N, P, R, S, T, W or Y), and the like.

For convenience of description, the mutations Δ cg3035 (knock-outs) and cg3035 of the gene NCgl2644/cg3035 are described herein^A251X(amino acid A mutation at position 251) is collectively designated cg3035 mut.

Further, the Corynebacterium glutamicum having improved genetic stability described above includes the genetically engineered strain ATCC 13032. delta. cg 3035. delta. argRgB^{A26V M31V}And ATCC13032cg3035^A251XΔargRargB^{A26V M31V}On the basis of (A), in order to increase the L-arginine production of the strainIn addition, the amount of arginine produced can be increased by 0.5-fold or more by adding the NCgl 2585E 484K mutation (see CN110564790A, CN201810569948.7), the NCgl 2585E 645K mutation (see CN110564758A, CN201810569343.8), or the NCgl 2585E 484K E645K mutation simultaneously. In other words, the technical scheme of the invention for improving the genetic stability of the L-arginine producing strain is also applicable to the derivative strain of the engineering strain ATCC13032 (delta argR, argBmut (A26V M31V)), thereby simultaneously improving the genetic stability and the production capacity of the L-arginine producing strain.

It is understood that the genetically engineered bacterium ATCC13032 delta cg3035 delta argRorgB of the invention is constructed^{A26V M31V}And ATCC13032cg3035^A251XΔargRargB^{A26V M31V}(wherein X is a stop codon TGA, TAA, amino acid V, D, E, F, G, K, L, M, N, P, R, S, T, W or Y), the sequence of step A, step B and step C is not fixed from the front to the back according to the English letter sequence, and they can be operated alternately or reversely, as long as each step can realize the respective function and accomplish the oriented change of host cell genotype.

The technical scheme of the invention is specifically explained below by taking the example of improving the generation stability of L-arginine-producing engineered Corynebacterium glutamicum including ATCC13032/13870/21831 (delta argR, argBmut (A26V M31V)).

Examples

Materials and methods

The whole gene synthesis, primer synthesis and sequencing herein were performed by Biotechnology engineering (Shanghai) Inc.

The molecular biological experiments herein include plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., which are mainly performed with reference to molecular cloning, a guide to experiments (third edition), J. SammBruk, D.W. Lassel (America), Huangpetang, et al, scientific Press, Beijing, 2002). For example, the methods for competent cell transformation and competent cell preparation are described in Chapter 1, 96 of molecular cloning, A laboratory Manual (third edition). The specific experimental conditions can be determined by simple experiments if necessary.

Main medium and buffer:

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

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

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

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

BHIS-suc solid Medium: 37g/LBHI, 91g/L sorbitol, 200g/L sucrose, 10g/L glucose.

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

RG2 medium: 60g/L glucose, 5g/L corn steep liquor, 30g/L (NH)₄)₂SO₄8g/L KCl, 2g/L urea, 0.5g/L KH₂PO₄，0.5g/L K₂HPO₄，1g/L MgSO₄·7H₂O，1g/L NaCl，20mg/L FeSO₄·7H₂O，10mg/L MnSO₄·5H₂O, 20mg/L nicotinic acid, 20mg/L beta-alanine, 10mg/L VB1, 0.2mg/L biotin, 30g/L CaCO₃KOH was adjusted to pH 7.7.

20X electrotransfer mother liquor: 80g/L glycine, 2% Tween 80.

In the following examples, when a kanamycin-containing medium was used, the final concentration of kanamycin in the medium was 50. mu.g/ml.

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

TABLE 1 primer List used in the examples

In Table 1, "-F" in the name represents the forward direction; "-R" represents reverse.

Example 1: knocking out NCgl2644/cg3035 gene open reading frame by CRISPR-Cpf1 single plasmid system

1.1ATCC13032 genome extraction:

a small amount of ATCC13032 glycerol strain (purchased from Shanghai institute of Industrial microorganisms) was inoculated into a BHIS test tube, cultured at 30 ℃ for 18 hours at 220rpm on a constant temperature shaker, centrifuged at 12000rpm to collect the strain, and the ATCC13032 genome was extracted using the Axygen bacterial genome miniprep kit.

1.2 knockout plasmid construction

Using pJYS3_ crtYf plasmid (Nat Commun.2017May 4; 8:15179.) as a template, 4268bp fragment 1 obtained using F1/R1 as primers, 5584bp fragment 2 obtained using F2/R2 as primers, ATCC13032 genome extracted in step 1.1 as a template, 1026bp fragment 3 obtained using F3/R3 as primers, 965bp fragment 4 obtained using F4/R4 as primers, Gibson ligation of the above 4 fragments, DH 5. alpha. competent cells (commercially available competent cells from Nanjing nuo-Town Biotech Co., Ltd.) were transformed, kanamycin LB plate was coated, and overnight culture was carried out.

The positive transformant is inoculated to an LB liquid test tube, and plasmid extraction and sequencing are carried out by using a plasmid extraction kit of Axygen, so as to obtain a correct knockout plasmid pJYS3_ delta cg3035, wherein the structure of the plasmid is shown in FIG. 4.

The PCR system was as follows (the following PCR reagents were purchased from the KOD series of TOYOBO Toyo): KOD Buffer 5. mu.l, dNTP 5. mu.l, MgSO₄4 μ l, 0.5 μ l of primer argR-aL-F, 0.5 μ l of primer argR-aL-R, 1 μ l of KOD Plus Neo, 0.4 μ l of template, ddH₂O make up to 50. mu.l. The PCR procedure was 99 ℃ hot-capping, 95 ℃ pre-denaturation for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 68 ℃ for 60 s; 35 cycles, final extension at 68 ℃ for 10 min; cooling at 16 deg.c for 10min to amplify target band and recovering target segment.

1.3 preparation of Corynebacterium glutamicum ATCC13032 competent cells:

corynebacterium glutamicum ATTC13032 was picked and streaked onto BHIS plates, incubated overnight in a 30 ℃ incubator, and a single colony was picked into BHIS tube medium and incubated overnight at 30 ℃ and 220 rpm. Inoculating 1ml of the bacterial liquid into a 100ml of BHIS liquid culture medium shake flask, and culturing at 30 ℃ and 220rpm for 4-6h on a constant temperature shaking bed until the OD600 value reaches about 1.0. The whole amount of the suspension was transferred to a 50ml centrifuge tube on a clean bench, centrifuged at 4500 Xg at 4 ℃ and the supernatant was discarded, the cells were washed with 10% glycerol and resuspended, centrifuged at 4500 Xg at 4 ℃ and the supernatant was discarded after 1-time washing. Finally, 600. mu.l of 10% glycerol was added to suspend the cells, and the suspension was dispensed into 1.5ml centrifuge tubes, and each 90. mu.l of the suspension was prepared into a competent cell, which was stored in a freezer at-80 ℃.

1.4pJYS3_ Δ cg3035 electroporation of ATCC13032 competent cells:

selecting pJYS3_ delta cg3035 plasmid with correct sequencing, sucking 3mu l (more than 1 mu g) of the plasmid into corynebacterium glutamicum ATCC13032 competent cells, uniformly mixing, transferring the plasmid into an electric rotating cup, carrying out electric shock under the conditions of 25uF,2.5kV and 200 omega, wherein the electric shock time is 5.6ms, immediately transferring the plasmid into 900 mu l of BHIS liquid culture medium preheated at 46 ℃, carrying out water bath in a water bath kettle at 46 ℃ for 6min, then placing the medium in a constant temperature shaking table at 30 ℃ and 220rpm for culturing for 1h, and recovering the thalli. After recovery, 50. mu.l of the cells were spread on a BHIS plate containing kanamycin, and the plate was inverted and cultured in a 30 ℃ incubator for 48 hours.

1.5 Positive Strain identification

And (3) verifying a single colony growing on the BHIS plate containing kanamycin by PCR amplification, wherein the PCR amplification condition is the same as that in step 1.2, and the single colony is identified by using a primer F5/R5, and the size of a positive band after cg3035 successfully knocks out is 2335bp, and the size of an unbknocked negative band is 3200 bp.

1.6 Positive transformant plasmids

Positive transformants were picked up in 4ml of BHIS test tube medium, and after 12 hours of incubation at 37 ℃ and 220rpm on a constant temperature shaker, the non-resistant plates were streaked, and further incubation at 37 ℃ in a constant temperature incubator for 24 hours, single colonies were streaked respectively on kanamycin-containing BHIS plates and non-anti-BHIS plates, and transformants which grew on the non-anti-BHIS plates but could not grow on the kanamycin-containing BHIS plates were picked up and kept sterile with 20% glycerol. A strain with the genotype of ATCC 13032. delta. cg3035 was obtained in which the open reading frame of the NCgl2644/cg3035 gene was knocked out.

Example 2: mutation of 251-site A of NCgl2644/cg3035 gene by using CRISPR-Cas9 two-plasmid system

2.1 mutant plasmid construction

The 4401bp fragment was amplified using pCgsgRNA _ crTYf plasmid (ACS Synth biol.2020Jul 17; 9(7):1897-1906.) as a template and F6/R6 as primers, and the fragment was transferred into E.coli DH 5. alpha. competent cells, spread on spectinomycin LB plate, and cultured overnight.

The positive transformant is connected with an LB liquid test tube, and plasmid extraction and sequencing are carried out by using a plasmid extraction kit of Axygen, so as to obtain the correct plasmid pCgsgRNA _ cg3035, wherein the structure is shown in figure 5.

The PCR system was the same as 1.2.

2.2 design of Single-stranded repair templates

To mutate the 251 nd A of the NCgl2644/cg3035 gene to the stop codon TGA, TAA, or other amino acid V, D, E, F, G, K, L, M, N, P, R, S, T, W, Y, a single-stranded repair template was designed, see Table 2. The sequences in Table 2 were synthesized as primers and dissolved to a concentration of 2. mu.g/. mu.L.

TABLE 2 list of single-stranded repair templates

Single-chain 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

2.3 transfer of the pCgCas9_ recT plasmid into competent cells of Corynebacterium glutamicum ATCC13032

Firstly, pCgCas9_ recT plasmid (ACS Synth biol.2020Jul 17; 9(7):1897-1906.) was transferred into prepared Corynebacterium glutamicum ATCC13032 competent cells, and the competent preparation method and the electrotransformation method were performed in the same steps 1.3 and 1.4. ATCC13032 strain containing the pCgCas9_ recT plasmid was obtained.

2.4 transfer of pCgsgRNA _ cg3035 plasmid into competent cells of ATCC13032 Strain containing pCgcAS9_ recT plasmid

Preparing a competent cell of an ATCC13032 strain containing a pCgCas9_ recT plasmid according to the competent preparation method of the step 1.3, transferring 500ng of pCgSgRNA _ cg3035 plasmid and 1-10 ng of single-chain repair template into the prepared competent cell, coating thalli on a BHIS plate containing kanamycin and spectinomycin after recovery, and inverting the plate to culture in a constant temperature incubator at 30 ℃ for 48 hours.

2.5 identification of Positive transformants

PCR amplification is carried out by using primers F-cg3035 and R-cg3035, transformants on BHIS plates containing kanamycin and spectinomycin are verified, the PCR amplification conditions are the same as the step 1.2, a band of about 1Kb is obtained, and sequencing is carried out on a PCR product to obtain the transformants with successful mutation. Positive transformants were picked into 4ml of BHIS tube medium, plasmid was removed as described in step 1.6, and the positive transformants were cultured on a constant temperature shaker at 30 ℃ for 24 hours at 220rpm, using 20% glycerol for conservation. The 251 th A mutation of the gene NCgl2644/cg3035 was obtained as ATCC13032cg3035^A251XThe strain of (1), comprising ATCC13032cg3035^A251TGA、ATCC13032 cg3035^A251TAA、ATCC13032 cg3035^A251V、ATCC13032 cg3035^A251D、ATCC13032 cg3035^A251E、ATCC13032 cg3035^A251F、ATCC13032 cg3035^A251G、ATCC13032 cg3035^A251K、ATCC13032 cg3035^A251L、ATCC13032 cg3035^A251M、ATCC13032 cg3035^A251N、ATCC13032 cg3035^A251P、ATCC13032 cg3035^A251R、ATCC13032 cg3035^A251S、ATCC13032 cg3035^A251T、ATCC13032 cg3035^A251W、ATCC13032 cg3035^A251Y。

Example 3: preparation of argR gene-knocked-out strain ATCC13032cg3035 mut delta argR

3.1 construction of knock-out plasmid pK18mobsacB-argR

3.1.1 PCR amplification of argR-aL fragment using primers argR-aL-F and argR-aL-R using ATCC13032 genome extracted in step 1.1 as template, the PCR system was as follows (the following PCR reagents were purchased from KOD series of TOYOBO, Toyo): KOD Buffer 5. mu.l, dNTP 5. mu.l, MgSO₄4 μ l, 0.5 μ l of primer argR-aL-F, 0.5 μ l of primer argR-aL-R, 1 μ l of KOD Plus Neo, 0.4 μ l of template, ddH₂O make up to 50. mu.l. The PCR procedure was 99 ℃ hot-capping, 95 ℃ pre-denaturation for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 68 ℃ for 60 s; 35 cycles, final extension at 68 ℃ for 10 min; cooling at 16 deg.C for 10 min. Amplification ofThe fragment of about 1kb was obtained, and the desired fragment was recovered by gel electrophoresis.

3.1.2 ArgR-aR fragment was amplified using the primers argR-aR-F and argR-aR-R under the same PCR conditions as in step 3.1.1 using the ATCC13032 genome extracted in step 1.1 as a template, about 1kb, and the desired fragment was recovered by gel.

3.1.3 plasmid pK18mobsacB (presented by Liu Shuangjiang, institute of microbiology, China academy of sciences) was extracted using the plasmid extraction kit from Axygen, the structure of which is shown in FIG. 1, the plasmid was digested with HindIII and EcoRI, and the gel was recovered to obtain a 5.7kb vector fragment.

3.1.4Gibson ligated the above fragments, transformed DH 5. alpha. competent cells, plated on kanamycin LB plates and cultured overnight.

3.1.5 validation of transformants by PCR amplification Using primers argR-aL-F and argR-aR-R, the PCR system was as follows (the following PCR reagents were purchased from KOD series of TOYOBO Toyo): KOD Buffer 2. mu.l, dNTP 2. mu.l, MgSO₄1.6. mu.l, 0.4. mu.l of primer argR-aL-F, 0.4. mu.l of primer argR-aR-R, 0.4. mu.l of KOD Plus Neo, as a template, as a cell, ddH₂O make up to 20. mu.l. The PCR procedure was 99 ℃ hot-capping, 95 ℃ pre-denaturation for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 68 ℃ for 120s for 35 cycles; finally, extending for 10min at 68 ℃; cooling at 16 deg.C for 10 min. The positive band was about 2 kb. The positive transformant is connected with an LB liquid test tube, and plasmid extraction kit of Axygen is used for extracting plasmid sequencing to obtain the correct plasmid pK18mobsacB-argR, and the structure of the plasmid is shown in figure 2.

3.2 preparation of Corynebacterium glutamicum ATCC 13032. delta. cg3035 or ATCC13032cg3035^A251XCompetent cells:

picking Corynebacterium glutamicum ATCC13032 delta cg3035 or ATCC13032cg3035^A251XStreaking on BHIS plate, culturing overnight in 30 deg.C incubator, picking single colony to BHIS test tube culture medium, and culturing overnight at 30 deg.C and 220 rpm. Inoculating 1ml of the bacterial liquid into a 100ml BHIS liquid culture medium shake flask, culturing at 30 ℃ and 220rpm on a constant temperature shaking bed for 4-6h to OD₆₀₀The value reaches about 1.0. Transferring the bacterial liquid into a 50ml centrifuge tube on a clean bench, centrifuging at 4 deg.C and 4500 Xg, discarding supernatant, washing the thallus with 10% glycerol, resuspending the thallus, centrifuging at 4 deg.C and 4500 Xg,the washing was repeated 1 time and the supernatant was discarded. Finally, 600. mu.l of 10% glycerol was added to suspend the cells, and the suspension was dispensed into 1.5ml centrifuge tubes, and each 90. mu.l of the suspension was prepared into a competent cell, which was stored in a freezer at-80 ℃.

3.3pK18mobsacB-argR electrotransformation of ATCC 13032. delta. cg3035 or ATCC13032cg3035^A251XCompetent cells:

the correctly sequenced plasmids were selected and 3. mu.l (more than 1. mu.g) were pipetted into Corynebacterium glutamicum ATCC 13032. delta. cg3035 or ATCC 13032. cg3035^A251XAnd uniformly mixing the competent cells, transferring the mixture into an electric rotating cup, carrying out electric shock under the conditions of 25uF,2.5kV and 200 omega, wherein the electric shock time is 5.6ms, immediately transferring the mixture into 900 mu l of BHIS liquid culture medium preheated at 46 ℃, carrying out water bath in a water bath kettle at 46 ℃ for 6min, and then placing the mixture in a constant-temperature shaking table at 30 ℃ and 220rpm for culturing for 1h to recover the thalli. After recovery, 50. mu.l of the cells were spread on a BHIS plate containing kanamycin, and the plate was inverted and cultured in a 30 ℃ incubator for 48 hours.

3.4SacB sucrose reverse sieving:

transformants on the kanamycin-containing BHIS plates were picked, inoculated into non-resistant BHIS tube medium, and cultured in a constant temperature shaker at 30 ℃ and 220rpm for 24 hours to allow double crossover. The cells were diluted 1000 times and spread on BHIS-suc plates containing 20% sucrose, and the plates were inverted and cultured in a 30 ℃ incubator for 48 hours. BHIS-suc plate transformants were picked, spotted on a plate BHIS plate and a BHIS plate containing kanamycin, respectively, and inverted and cultured in a 30 ℃ incubator for 24 hours. The transformants that grew on the BHIS plate but failed to grow on the BHIS plate containing kanamycin were verified by PCR amplification using the primers argR-aL-F and argR-aR-R under the conditions of 1.2.5 steps in PCR amplification, and the positive band of successful argR gene knockout was about 2kb and the negative band was 2.5K. Positive transformants were picked into 4ml of BHIS tube medium, cultured at 30 ℃ for 24 hours at 220rpm on a constant temperature shaker, and then sterilized with 20% glycerol. The genotypes of ATCC13032cg3035 mut Δ argR were obtained (ATCC13032 Δ cg3035 Δ argR and ATCC13032cg3035^A251XΔ argR).

Example 4: strain ATCC13032cg3035 mutargB mutated in the argB gene^{mut(A26V M31V)}Preparation of

4.1 construction of the mutant plasmid pK18 mobsacB-argBmut:

4.1.1 amplification of argB-aL fragment, about 1kb, using primers argB-aL-F and argB-aL-R with the ATCC13032 genome extracted in step 1.1 as template, and recovering the desired fragment.

4.1.2 ArgB-aR fragments were amplified using primers argB-aR-F and argB-aR-R, about 1kb, using the ATCC13032 genome extracted in step 1.1 as template, and the fragment of interest was recovered in gel.

4.1.3 plasmid pK18mobsacB (presented by Liu Shuangjiang, institute of microbiology, China academy of sciences) was extracted using the plasmid extraction kit from Axygen, the structure of which is shown in FIG. 1, and the plasmid was digested with HindIII and EcoRI, and the gel was recovered to obtain a 5.7kb vector fragment.

4.1.4Gibson ligated the above fragments, DH 5. alpha. competent cells (commercially available from Biotech, Inc., N.K.) were transformed, plated on kanamycin LB plates, and cultured overnight.

4.1.5 amplification of the verified transformants using primers argB-aL-F and argB-aR-R following the same PCR conditions as in step 1.2.5, the positive band was approximately 2 kb. The positive transformant is connected with an LB liquid test tube, and the extracted plasmid is sequenced to obtain the correct plasmid pK18mobsacB-argBmut, and the structure of the plasmid is shown in figure 3.

4.2 preparation of Corynebacterium glutamicum ATCC 13032. delta. cg 3035. delta. argR or ATCC13032cg3035^A251XΔ argR competent cells:

picking Corynebacterium glutamicum ATCC13032 delta cg3035 delta argR or ATCC13032cg3035^A251XDelta argR was streaked on a BHIS plate, cultured overnight in a 30 ℃ incubator, and a single colony was picked up in a BHIS test tube medium and cultured overnight at 30 ℃ and 220 rpm. Inoculating 1ml of the bacterial liquid into a 100ml BHIS liquid culture medium shake flask, culturing at 30 ℃ and 220rpm on a constant temperature shaking bed for 4-6h to OD₆₀₀The value reaches about 1.0. The whole amount of the suspension was transferred to a 50ml centrifuge tube on a clean bench, centrifuged at 4500 Xg at 4 ℃ and the supernatant was discarded, the cells were washed with 10% glycerol and resuspended, centrifuged at 4500 Xg at 4 ℃ and the supernatant was discarded after 1-time washing. Finally, 600. mu.l of 10% glycerol is added to suspend the thalli, and the thalli are subpackagedInto a 1.5ml centrifuge tube, one competent cell was prepared per 90. mu.l, and the competent cell was stored in a-80 ℃ freezer.

4.3pK18mobsacB-argBmut electrotransformation of ATCC 13032. delta. cg 3035. delta. argR or ATCC 13032. cg3035^A251XΔ argR competent cells:

the correctly sequenced plasmid was selected and 3. mu.l (more than 1. mu.g) was pipetted into Corynebacterium glutamicum ATCC 13032. delta. cg 3035. delta. argR or ATCC 13032. cg3035^A251XDelta argR competent cells are uniformly mixed and then transferred into an electric rotating cup, electric shock is carried out under the conditions of 25uF,2.5kV and 200 omega, the electric shock time is 5.6ms, the cells are immediately transferred into 900 mul of BHIS liquid culture medium preheated at 46 ℃ after electric shock, water bath is carried out in a water bath kettle at 46 ℃ for 6min, and then the cells are cultured for 1h at 220rpm in a constant temperature shaking table at 30 ℃ so as to enable the cells to be recovered. After recovery, 50. mu.l of the cells were spread on a BHIS plate containing kanamycin, and the plate was inverted and cultured in a 30 ℃ incubator for 48 hours.

4.4SacB sucrose reverse sieving:

transformants on the kanamycin-containing BHIS plates were picked, inoculated into non-resistant BHIS tube medium, and cultured in a constant temperature shaker at 30 ℃ and 220rpm for 24 hours to allow double crossover. The cells were diluted 1000 times and spread on BHIS-suc plates containing 20% sucrose, and the plates were inverted and cultured in a 30 ℃ incubator for 48 hours. BHIS-suc plate transformants were picked, spotted on a plate BHIS plate and a BHIS plate containing kanamycin, respectively, and inverted and cultured in a 30 ℃ incubator 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 failed to grow on kanamycin-containing BHIS plates, PCR amplification conditions were synchronized at step 3.1.5 to obtain a band of about 2kb, and PCR products were sequenced to obtain transformants in which argB successfully mutated. Positive transformants were picked into 4ml of BHIS tube medium, cultured at 30 ℃ for 24 hours at 220rpm on a constant temperature shaker, and then sterilized with 20% glycerol. The genotype was obtained as ATCC13032cg3035 mutargB^{mut(A26V M31V)}(including ATCC 13032. delta. cg 3035. delta. argR argB^{mut(A26V M31V)}And ATCC13032cg3035^A251XΔargRargB^{mut(A26V M31V)}) The strain of (1).

Example 5:ATCC13032 cg3035mutΔargR argB^{mut(A26V M31V)}preparation of derivative strains

In order to increase the genetic stability of the strain ATCC13032cg3035 mutargB^{mut(A26V M31V)}(including ATCC 13032. delta. cg 3035. delta. argR argB^{mut(A26V M31V)}Or ATCC13032cg3035^A251XΔargRargB^{mut(A26V M31V)}) The arginine producing ability of (1), and constructing a derivative strain thereof.

5.1 mutation of the Strain ATCC 13032. delta. cg 3035. delta. argR argB obtained in example 4, see patent CN110564790A^{mut(A26V M31V)}Or ATCC13032cg3035^A251XΔargRargB^{mut(A26V M31V)}The ClpC gene of (a) is ClpC^E484KThe strain ATCC 13032. delta. cg 3035. delta. argR argB was obtained^{mut(A26V M31V)}ClpC^E484KOr ATCC13032cg3035^A251XΔargRargB^{mut(A26V M31V)}ClpC^E484K。

5.2 mutation of the Strain ATCC 13032. delta. cg 3035. delta. argR argB obtained in example 4, see patent CN110564758A^{mut(A26V M31V)}Or ATCC13032cg3035^A251XΔargRargB^{mut(A26V M31V)}The ClpC gene of (a) is ClpC^E645KThe strain ATCC 13032. delta. cg 3035. delta. argR argB was obtained^{mut(A26V M31V)}ClpC^E645KOr ATCC13032cg3035^A251XΔargRargB^{mut(A26V M31V)}ClpC^E645K。

5.3 with reference to patents CN110564790A and CN110564758A, the strain ATCC 13032. delta. cg 3035. delta. argR argB obtained in example 4 was mutated^{mut(A26V M31V)}Or ATCC13032cg3035^A251XΔargRargB^{mut(A26V M31V)}The ClpC gene of (a) is ClpC^E484KE645KThe strain ATCC 13032. delta. cg 3035. delta. argR argB was obtained^{mut(A26V M31V)}ClpC^E484KE645KOr ATCC13032cg3035^A251XΔargRargB^{mut(A26V M31V)}ClpC^E484KE645K。

The arginine production of the derivative strains is generally improved by more than 0.5 times compared with the original strain before inactivation or attenuation modification of the gene NCgl2644/cg 3035.

Example 6: fermentation test of arginine producing strains

For the original strain ATCC13032 and the arginine producing strain ATCC13032 delta argRoggB^{mut(A26V M31V)}(including 1441 strain, and 1442 strain, the next generation), ATCC13032cg3035 mut Δ argR argB inactivated or attenuated by NCgl2644/cg3035^{mut(A26V M31V)}Bacterial strains and derivatives thereof, tested for arginine production capacity by fermentation. The method comprises the following steps:

6.1 fermentation of strains in shake flasks

6.1.1 activation: streaking the strain from the glycerin tube to the flat plate BHIG, and culturing at 30 ℃ for about 2 days.

6.1.2 seed culture: the BYG culture medium is prepared, mixed uniformly and then distributed into 10 ml/bottle to 250ml shake bottles, a single colony obtained by passage is dug by an inoculating ring and inoculated into a seed bottle, and the single colony is cultured for 24 hours at 30 ℃ and 220 rpm.

6.1.3 Shake flask fermentation: RG2 medium is prepared, mixed uniformly, dispensed into 45 ml/bottle to 500ml single baffle shake flask (pre-filled with 1.5g calcium carbonate), inoculated with more than 5ml seed solution into RG2 shake flask, cultured at 30 deg.C and 220rpm for about 48-72 h, until glucose is exhausted. And centrifuging the fermentation liquor at a high speed, taking the supernatant, diluting by 50 times, and detecting the yield of arginine by HPLC.

6.2 high Performance liquid chromatography HPLC determination of arginine content

6.2.1 method: the content of L-arginine in the fermentation broth was determined using OPA pre-column derivatized amino acid analysis. The first-order amino acid reacts with o-phthalaldehyde (OPA) in the presence of a sulfhydryl reagent to generate OPA-amino acid, the generated amino acid derivative is separated by reversed-phase high performance liquid chromatography and then is detected by ultraviolet or fluorescence, and the light absorption value of the amino acid derivative is in direct proportion to the concentration of the amino acid in a certain range.

6.2.2 derivatization Agents and flow phase formulation

Boric acid buffer: 6.183g of boric acid is accurately weighed by 0.4M boric acid buffer solution, dissolved in ultrapure water, adjusted to pH 10.2 by 10N NaOH solution and added into a volumetric flask with the volume of 250 ml.

A derivatizing agent: 500mg of an o-phthalaldehyde (OPA) solid was weighed out accurately, 5ml of absolute ethanol was added, 500. mu.l of mercaptopropionic acid was added, and the volume was adjusted to 50ml with 0.4M, pH 10.2.2 of boric acid buffer.

Mobile phase A: 40mM NaH₂PO₄Solution, accurately weighing 5.5g NaH₂PO₄·H₂Dissolving O in ultrapure water, adjusting the pH value to 7.8 by using a 10N NaOH solution, metering the volume to 1L, and filtering by using a 0.22 mu m filter membrane for later use.

Mobile phase B: ACN MeOH H₂O45: 45:10, constant volume of 1L for standby, and the purity of the reagent is HPLC grade.

6.2.3 high performance liquid chromatography assay conditions:

a chromatographic column: ZORBAX Eclipse-AAA 4.6x 75mm, 3.5 μm; flow rate: 1.2 ml/min; stopping time: 14 min; column temperature: 40 degrees; setting the DAD: UV 338nm, 10nm (bandwidth), referenced 390nm, 20nm (bandwidth).

The elution procedure is shown in table 3.

TABLE 3 HPLC elution procedure

An automatic sample injector respectively takes 0.5 mu l of sample, 2.5 mu l of boric acid buffer solution, 0.5 mu l of derivative agent and 32 mu l of ultrapure water, and the sample injection is carried out after the mixture and the derivative, and the peak time of arginine is 7.9 minutes. And comparing the standard sample according to the peak area to obtain the content of the L-arginine in the sample.

6.3L-arginine production by fermentation with the Strain

The L-arginine fermentation yields of the strains were compared according to the fermentation protocol in step 6.1 and the assay in step 6.2, with 3 replicates per strain, with the results shown in the following table:

TABLE 4 comparison of L-arginine levels in fermentation of strains

As can be seen from Table 4, the strain ATCC 13032. delta. argRogB^{A26V M31V}The arginine producing ability is lost after passage, and the genetic stability is not good. For the L-arginine producing strain ATCC13032 delta argRogB^{mut(A26V M31V)}ATCC13032cg3035 mut delta argR argB obtained after inactivation or attenuation modification of the gene NCgl2644/cg3035^{mut(A26V M31V)}The strain and the derivative strain thereof still keep the L-arginine production capacity.

ATCC13032cg3035 mut Δ argR argB^{mut(A26V M31V)}Investigation of genetic stability of strains

Example 7: stability of arginine producing ability of strain by subculture analysis

The NCgl2644/cg3035 knockout strain ATCC13032 delta cg3035 delta argR argB is selected^{mut(A26V M31V)}251 amino acid A mutant strain ATCC13032cg3035^A251TGAΔargRargB^{A26V M31V}And ATCC13032cg3035^A251VΔargRargB^{A26V M31V}And (4) carrying out genetic stability investigation through passage and fermentation.

7.1 passages for stability analysis

7.1.1 preparing BYG culture medium, mixing uniformly, subpackaging 10 ml/bottle to 250ml shake bottles, and respectively inoculating the glycerol strain of the strain to be tested into three bottles of BYG culture medium. Incubated at 30 ℃ and 220rpm for 24 h.

7.1.2 the above culture broth was inoculated at 2% v/v into fresh BYG medium and cultured at 30 ℃ and 220rpm for 24 hours.

7.1.3 repeat the step of 7.1.2 and complete the passage three times.

7.1.4 after three passages of BYG above, 2% v/v was transferred into 50ml of RG2 medium. Incubated at 30 ℃ and 220rpm for 48 h.

7.1.5 taking 48h end-point fermentation liquid, diluting 10%⁵-10⁶After doubling, 100. mu.l of the plate was plated with non-anti-BHIS and incubated at 30 ℃ for 48 hours. After the single fungus grows out, the method is carried outAnd fermentation verification shows that the bacterial strain can keep the proportion of bacterial colonies capable of producing arginine after three passages.

7.2 fermentation of strains in shake flasks

7.2.1 seed culture: the BYG culture medium is prepared, mixed evenly and then distributed into 10 ml/bottle to 250ml shake bottles, a single colony obtained by passage in the step 7.1 is dug by an inoculating ring and inoculated into a seed bottle, and the single colony is cultured for 24 hours at 30 ℃ and 220 rpm.

7.2.2 Shake flask fermentation: RG2 medium was prepared, mixed well and dispensed into 45 ml/flask to 500ml single baffle shake flask (pre-filled with 1.5g calcium carbonate), inoculated with more than 5ml of the seed solution from step 7.2.1 into RG2 shake flask, incubated at 30 ℃ and 220rpm for about 48-72 h until glucose was consumed. And centrifuging the fermentation liquor at a high speed, taking the supernatant, diluting by 50 times, and detecting the yield of arginine by HPLC.

7.3 stability analysis of arginine producing ability of the Strain:

according to the passage scheme in step 7.1, each strain was subjected to 3 parallel passage experiments to obtain 6 plates. 10 single colonies were picked for fermentation on each plate, and the measured L-arginine fermentation yields of the strains are summarized in Table 5.

TABLE 5L-arginine production by fermentation of the third generation strains and the ratio of the produced strains

As can be seen from Table 5, the strain ATCC 13032. delta. argRoggB^{A26V M31V}The arginine producing ability is lost after passage, and the genetic stability is not existed; strain ATCC 13032. delta. cg 3035. delta. argR argB^{mut(A26V M31V)}And ATCC13032cg3035^A251VΔargRargB^{mut(A26V M31V)}After three passages, the production capacity of the mother bacteria L-arginine can be maintained by more than 70 percent.

As for the 251 th amino acid A, the mutation is the stop codon TAA and the mutation is the amino acidD. E, F, G, K, L, M, N, P, R, S, T, W and Y, and ATCC 13032. delta. cg 3035. delta. argR argB^{mut(A26V M31V)}、ATCC13032 cg3035^A251TGAΔargRargB^{A26V M31V}And ATCC13032cg3035^A251VΔargRargB^{mut(A26V M31V)}Similarly, the L-arginine producing ability of the mother bacteria can be maintained above 70% after three passages.

Example 8: strain ATCC13870/ATCC21831 gene NCgl2644/cg3035 post-transformation fermentation test

To verify whether the modification scheme for inactivation or attenuation of the gene NCgl2644/cg3035 is applicable to other L-arginine-producing strains, the corresponding modifications were continued, for example, in Corynebacterium glutamicum ATCC13870 and Corynebacterium glutamicum ATCC21831, and fermentation tests were performed, as follows:

8.1 with reference to example 1 and example 2, ATCC 13870. delta. cg3035 and ATCC13870 cg3035 were constructed respectively^A251V，ATCC21831Δcg3035，ATCC21831 cg3035^A251VAnd (3) strain.

8.2 with reference to example 3, ATCC 13870. delta. cg 3035. delta. argR, ATCC13870 cg3035, respectively, were constructed^A251VΔargR，ATCC21831Δcg3035ΔargR，ATCC21831 cg3035^A251VA Δ argR strain.

8.3 with reference to example 4, ATCC 13870. delta. cg 3035. delta. argR argB was constructed^{mut(A26V M31V)}，ATCC13870 cg3035^A251VΔargR argB^{mut(A26V M31V)}，ATCC21831Δcg3035ΔargR argB^{mut(A26V M31V)}，ATCC21831 cg3035^A251VΔargR argB^{mut(A26V M31V)}And (3) strain.

8.4 with reference to example 6, for the strains ATCC13870, ATCC21831, ATCC13870 Δ cg3035 Δ argR argB^{mut(A26V M31V)}，ATCC13870 cg3035^A251VΔargR argB^{mut(A26V M31V)}，ATCC21831Δcg3035ΔargR argB^{mut(A26V M31V)}，ATCC21831 cg3035^A251VΔargR argB^{mut(A26V M31V)}Arginine production capacity was tested by fermentation.

The L-arginine fermentation yields of the respective strains were compared, and 3 parallel experiments were performed for each strain, and the results are shown in Table 6 below.

TABLE 6 comparison of L-arginine levels in fermentation of strains

Strain numbering	Genotype(s)	Arginine yield (g/L)
			ATCC13870	wild type	0
ATCC21831	wild type	0
			13870Δcg3035	ATCC13870Δcg3035ΔargR argBmut(A26V M31V)	6.486±0.117
13870cg3035A251V	ATCC13870cg3035A251VΔargR argBmut(A26V M31V)	6.794±0.146
			21831Δcg3035	ATCC21831Δcg3035ΔargR argBmut(A26V M31V)	5.986±0.213
21831cg3035A251V	ATCC21831cg3035A251VΔargR argBmut(A26V M31V)	6.725±0.124

Example 9: stability of arginine producing ability of strain ATCC13870/ATCC21831 by subculture

Selecting a strain ATCC13870 delta cg3035 delta argR argB^{mut(A26V M31V)}，ATCC13870 cg3035^A251VΔargR argB^{mut(A26V M31V)}，ATCC21831Δcg3035ΔargR argB^{mut(A26V M31V)}，ATCC21831 cg3035^A251VΔargR argB^{mut(A26V M31V)}Genetic stability studies by passage and fermentation were performed and the results are shown in table 7 below.

Table 7L-arginine production by fermentation of the third Generation later and the ratio of the produced strains

The experimental results show that the modification scheme of inactivating or weakening the gene NCgl2644/cg3035 of the arginine producing strain can greatly improve the stability of the arginine producing capacity of the strain, so that the genetic engineering strain has the possibility of industrial application.

Sequence listing

<110> China academy of sciences molecular plant science remarkable innovation center

<120> a method for improving the stability of arginine producing ability of a strain

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gacgtcaggg gctgggcacc gatttaaata aaacgaaagg ct 42

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gcgggactct ggggttcgcg gaatcatgac 30

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acgcaccagt catcgttgat tcagaagaac tcgtcaagaa gg 42

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cggtgcccag cccctgacgt catctacaac agtagaaatt cggat 45

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cgcgaacccc agagtcccgc gtcgtctttg ccctgcattg 40

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agatatgcct cctgtgcgtg cggaaacggg gaagactagg 40

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cctagtcttc cccgtttccg cacgcacagg aggcatatct ccaggt 46

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ccttcttgac gagttcttct gaatcaacga tgactggtgc gtggtcag 48

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cggcggttga gtttgatgac 20

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cggaatccga agagccagaa 20

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ccccgccggc gtggtcaggc gatttaaata aaacgaaagg ctcagtcg 48

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cgcctgacca cgccggcggg gatctacaac agtagaaatt cggatccat 49

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ttcgcggttt tatgcacgtc 20

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tggtttcctg caccgctaat 20

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acaatttcac acaggaaaca gctatgacat gattacggac ttcacaacca caggttcgc 59

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gtggctgctg gccaaccagc cgaggtaaga cagcgcccct agttcaaggc ttg 53

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gatgatgatc tcaaggctgt ttttgctgcc gacgtggtct tcttgcgcac cgtgggc 57

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agtgccaagc ttgcatgcct gcaggtcgac tctagaggtg gcccaacgcg ttgactgcg 59

Claims (10)

1. A method for improving the stability of arginine producing ability of a strain comprises the following steps:

the gene NCgl2644/cg3035 in the genome is inactivated or attenuated by using Corynebacterium glutamicum producing L-arginine as a basic strain.

2. The method of claim 1, wherein the inactivation or attenuation of the gene NCgl2644/cg3035 is selected from the group consisting of: knocking out an open reading frame of a gene NCgl2644/cg 3035; mutating any amino acid in the open reading frame of the gene NCgl2644/cg3035 to a stop codon; the 251 st amino acid A of the open reading frame of the gene NCgl2644/cg3035 is mutated.

3. The method of claim 2, wherein the mutation at amino acid a at position 251 of the gene NCgl2644/cg3035 is selected from the group consisting of: mutation is stop codon TGA or TAA; the mutation is amino acid V, D, E, F, G, K, L, M, N, P, R, S, T, W or Y.

4. The method according to claim 1, wherein the base strain is Corynebacterium glutamicum ATCC13032(Δ argR, argBmut (A26V M31V)) or a mutant strain derived therefrom, wherein

The Corynebacterium glutamicum ATCC13032 (. DELTA.argR, argBmut (A26V M31V)) was obtained by knocking out the argR gene of Corynebacterium glutamicum ATCC13032 and introducing a mutation (A26V M31V) of argB,

the derivative mutant strain is selected from the strains CCTCC NO: M2017760 reported in patent application CN201711430988.5, ATCC13032(Δ argR, argBmut (A26V M31V), NCgl0083mut2) reported in patent application CN 2015826, ATCC13032(Δ argR, argBmut (A26V M31V), NCgl0742mut3) reported in patent application CN201810492999.4, ATCC13032(Δ argR, argBmut (A26V M31V), NCgl2374mut4) reported in patent application CN201810569947.2, ATCC13032(Δ argR, argBmut (A26M V M31V), NCgl2620mut6), CN 3748 (ATCC13032, Δ arg 2526A 2526, Δ arg 5926A 5926), ATCC13032(Δ argR 5926A 5926) reported in patent application CN 5926, NCgl 13032(Δ argBmut 5926M 5926), and NCgl 5926 Bmut 13032(Δ arg 5926A 5926).

5. The method according to claim 4, wherein the base strain is Corynebacterium glutamicum ATCC13032(Δ argR, argBmut (A26V M31V)), comprising the following steps:

A. inactivating or weakening the gene NCgl2644/cg3035 in the genome to obtain a mutant strain ATCC13032(cg3035mut) of the gene NCgl2644/cg 3035;

B. knocking out argR gene in the genome of NCgl2644/cg3035 mutant strain ATCC13032(cg3035mut) in the step A to obtain gene knocked-out strain ATCC13032((cg3035mut, delta argR);

C. A26V and M31V mutations were made in the argB gene in the genome of the knockout strain ATCC13032(cg3035mut, Δ argR) described in step B to obtain the genetically engineered strain ATCC13032((cg3035mut, Δ argR, argBmut (A26V M31V)).

6. The method of claim 5, wherein step a is performed by a gene editing technique employing a CRISPR-Cas9 system, a CRISPR-Cpf1 system, a CRISPR-Cas related transposition system INTEGRATE system, or a CAST system.

7. The method of claim 5, wherein the knockout strain ATCC13032(cg3035mut, Δ argR) in step B is prepared by the following method:

B1. using ATCC13032 genome as a template, and using a primer argR-aL-F with a sequence of SEQ ID NO. 15 and a primer argR-aL-R with a sequence of SEQ ID NO. 16 to carry out PCR amplification to obtain an argR-aL fragment with about 1 kb;

B2. carrying out PCR amplification by using an ATCC13032 genome as a template and a primer argR-aR-F with a sequence of SEQ ID NO. 17 and a primer argR-aR-R with a sequence of SEQ ID NO. 18 to obtain an argR-aR fragment with about 1 kb;

B3. the plasmid pK18mobsacB with GenBank accession number FJ437239.1 is digested with HindIII and EcoRI, and the 5.7kb vector fragment is obtained by gel recovery;

gibson ligated argR-aL, argR-aR and vector fragments as described above, transformed DH 5. alpha. competent cells, plated on kanamycin LB plates, and cultured overnight;

B5. carrying out PCR amplification by using primers argR-aL-F and argR-aR-R to verify a transformant, and obtaining a plasmid pK18 mobsacB-argR;

B6. preparing competent cells of Corynebacterium glutamicum ATCC13032(cg3035 mut);

B7. plasmid pK18mobsacB-argR was transformed into ATCC13032(cg3035mut) competent cells;

B8. SacB sucrose counter-screening was performed, PCR amplification was performed using primers argR-aL-F and argR-aR-R, and transformants that grew on BHIS plates but could not grow on kanamycin-containing BHIS plates were verified to obtain strain ATCC13032(cg3035mut,. DELTA.argR).

8. The method of claim 7, wherein the genetically engineered strain in step C is prepared by:

C1. using ATCC13032 genome as template, using primer argB-aL-F with sequence SEQ ID NO. 19 and primer argB-aL-R with sequence SEQ ID NO. 20 to carry out PCR amplification, obtaining about 1kb argB-aL fragment;

C2. carrying out PCR amplification by using ATCC13032 genome as a template and a primer argB-aR-F with a sequence of SEQ ID NO. 21 and a primer argB-aR-R with a sequence of SEQ ID NO. 22 to obtain an argB-aR fragment with about 1 kb;

C3. the plasmid pK18mobsacB with GenBank accession number FJ437239.1 is digested with HindIII and EcoRI, and the 5.7kb vector fragment is obtained by gel recovery;

gibson ligated the argB-aL, argB-aR and vector fragments described above, transformed DH 5. alpha. competent cells, plated on kanamycin LB plates, and cultured overnight;

C5. carrying out PCR amplification by using primers argB-aL-F and argB-aR-R to verify a transformant, and obtaining a plasmid pK18 mobsacB-argBmut;

C6. preparing competent cells of Corynebacterium glutamicum ATCC13032(cg3035mut, Δ argR);

C7. plasmid pK18mobsacB-argBmut was transformed into ATCC13032(cg3035mut, Δ argR) competent cells;

C8. SacB sucrose counter-screening was performed, PCR amplification was performed using primers argB-aL-F and argB-aR-R, and transformants that grew on BHIS plates but could not grow on kanamycin-containing BHIS plates were verified, yielding strain ATCC13032(cg3035mut,. DELTA.argR, argBmut (A26V M31V)).

9. A genetically engineered bacterium ATCC13032(cg3035mut, Δ argR, argBmut (a26V M31V)) or a mutant strain derived therefrom, constructed according to the method of any one of claims 1 to 7.

10. The use of the genetically engineered bacterium of claim 9 for the production of L-arginine.

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