RR mutant protein, mutant gene, recombinant vector, engineering bacterium and construction method and application of RR mutant protein, mutant gene, recombinant vector and engineering bacterium

Abstract

The invention discloses an RR mutant protein, a mutant gene, a recombinant vector, an engineering bacterium and a construction method and application of the RR mutant protein, the mutant gene, the recombinant vector and the engineering bacterium, and belongs to the technical field of molecular biology. The amino acid sequence of the RR mutant protein disclosed by the invention is as shown in SEQ ID NO. 1. Corynebacterium glutamicum is subjected to ultraviolet mutagenesis treatment, and it is found that after RR protein is mutated into RR mutant protein, the yield of L-arginine is remarkably increased; according to the present invention, the growth speed of the engineering bacteria constructed by using the RR mutant gene encoding the RR mutant protein is significantly accelerated, the L-arginine yield is significantly improved, the growth speed can be improved, the fermentation time and the fermentation cost can be easily saved, the dominant bacteria can be formed compared to other strains, the growth of other strains can be avoided, and the bacterial contamination probability can be reduced.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and in particular relates to an RR mutant protein, a mutant gene, a recombinant vector, an engineering bacterium, and a construction method and application thereof. Background Art

[0002] L-arginine is an organic compound with the molecular formula C6H 14 N₄O₂, with a density of 1.46 g / cm³ and a molecular weight of 174.201, is a white crystalline powder. It is found in large quantities in protamine and other proteins, and is a basic component of various proteins, making it a very common substance.

[0003] L-arginine is a basic amino acid. It's non-essential for adults, but its production in the body is slow. It's essential for infants and young children, and has a certain detoxifying effect. L-arginine has a wide range of applications in the food, pharmaceutical, feed, and cosmetics industries. In the food industry, L-arginine is often used as a food flavoring and beverage additive. In the pharmaceutical industry, L-arginine is used in the production of various medications for the treatment of hyperammonemia, oral amino acid supplements, fatigue relief, liver function improvement, and endocrine regulation. Furthermore, adding L-arginine to animal feed can promote growth and development, repair intestinal damage, enhance immunity, and prevent the occurrence of various diseases. As an alkaline amino acid, L-arginine is also widely used as a neutralizer and moisturizer in cosmetics.

[0004] Currently, L-arginine can be produced primarily through protein hydrolysis and microbial fermentation. However, protein hydrolysis suffers from drawbacks such as cumbersome operation, high purification costs, low recovery efficiency, and environmental pollution. In recent years, microbial fermentation has become the mainstream method for producing L-arginine, but the yield and production efficiency of L-arginine still have significant room for improvement. Therefore, there is an urgent need to develop a method for increasing L-arginine yield via microbial fermentation to provide technical support for large-scale, efficient fermentation production of L-arginine. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a RR mutant protein that can increase L-arginine production compared to before mutation.

[0006] Another object of the present invention is to provide a RR Mutated gene.

[0007] Another object of the present invention is to provide a recombinant vector.

[0008] Another object of the present invention is to provide an engineered bacterium.

[0009] Another object of the present invention is to provide a method for constructing the engineered bacteria.

[0010] Another object of the present invention is to provide a RR mutant protein or the RR Application of the mutant gene, the recombinant vector or the engineered bacteria in producing L-arginine.

[0011] Another object of the present invention is to provide a method for producing L-arginine.

[0012] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a RR mutant protein, the amino acid sequence of the RR mutant protein is shown in SEQ ID NO.1.

[0013] The present invention also provides a RR Mutated gene, the RR The mutant gene encodes the RR mutant protein; RR The nucleotide sequence of the mutant gene is shown in SEQ ID NO.2.

[0014] The present invention also provides a recombinant vector, which contains the RR Mutated gene.

[0015] The present invention also provides an engineered bacterium, wherein the engineered bacterium contains the RR Mutant gene or the recombinant vector.

[0016] Preferably, the starting bacteria of the engineered bacteria include Corynebacterium glutamicum.

[0017] Preferably, the engineered bacteria include Corynebacterium glutamicum DXMC-DXA2, with a deposit number of CCTCC NO: M2025350.

[0018] The present invention also provides a method for constructing the engineered bacteria, the method comprising: RR The mutant gene replaces the original strain RR Gene, get engineered bacteria.

[0019] The present invention also provides a RR mutant protein or the RR Application of the mutant gene, the recombinant vector or the engineered bacteria in producing L-arginine.

[0020] The present invention also provides a method for producing L-arginine, which comprises: inoculating the engineered bacteria into a culture medium and fermenting.

[0021] Preferably, the fermentation conditions include: fermentation temperature 29-31° C., and fermentation time 12-96 h.

[0022] Beneficial effects of the present invention: The present invention subjected Corynebacterium glutamicum to ultraviolet mutagenesis treatment and found that after the RR protein was mutated into the RR mutant protein with the amino acid sequence shown in SEQ ID NO.1, the L-arginine production was significantly improved. RR The mutant gene replaces the original strain RR After the gene was added, the growth rate of the engineered bacteria was significantly accelerated and the production of L-arginine was significantly improved. This shows that after the RR protein was mutated into the RR mutant protein, the L-arginine production could be increased. RR The engineered bacteria obtained by constructing mutant genes can not only increase the production of L-arginine, but also increase the growth rate, which is beneficial to saving fermentation time and cost. Compared with other bacterial species, they can form dominant bacteria, avoid the growth of other bacterial species, and reduce the chance of contamination.

[0023] Biological deposit information The present invention Corynebacterium glutamicum DXMC-DXA1 is classified and named Corynebacterium glutamicum ( Corynebacterium glutamicum ), deposited in China Center for Type Culture Collection (CCTCC), the deposit date is March 3, 2025, the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2025349.

[0024] The present invention Corynebacterium glutamicum DXMC-DXA2 is classified and named Corynebacterium glutamicum ( Corynebacterium glutamicum ), deposited in China Center for Type Culture Collection (CCTCC), the deposit date is March 3, 2025, the deposit address is Wuhan University, Wuhan, China, the deposit number is CCTCC NO: M2025350. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the agarose gel electrophoresis test in Example 5, where lanes 1 to 3 are positive colonies. RR Parallel experiments of mutant gene amplified fragments, lane M is the marker; Figure 2 The sequencing result diagram in Example 5, the red box indicates RR The mutated gene bases 79-81. DETAILED DESCRIPTION

[0026] The present invention provides a RR mutant protein, the amino acid sequence of the RR mutant protein is shown in SEQ ID NO.1.

[0027] The present invention treats Corynebacterium glutamicum ( Corynebacterium glutamicum) DXMC-DXA1 was mutagenized. The Corynebacterium glutamicum DXMC-DXA1 is deposited as CCTCC NO: M2025349. After mutagenesis, it was found that the L-arginine production of the strain increased after the RR protein was mutated to the RR mutant protein with the amino acid sequence shown in SEQ ID NO. 1; the amino acid sequence of the RR protein is shown in SEQ ID NO. 3. This indicates that the RR mutant protein can increase L-arginine production. In the present invention, the RR mutant protein has a mutation at amino acid position 27 of the RR protein, from Leu to Pro.

[0028] The present invention also provides a method for encoding the RR mutant protein. RR mutant gene; RR The nucleotide sequence of the mutant gene is shown in SEQ ID NO. 2. RR Gene encoding, the RR The nucleotide sequence of the gene is shown in SEQ ID NO.4. RR The mutant gene has a mutation at the 80th nucleotide, from T to C.

[0029] The present invention also provides a recombinant vector, which contains the RR Mutant gene. In the present invention, the starting vector of the recombinant vector can be conventionally selected according to actual needs. As an embodiment, the starting vector preferably includes pK19mobsacB.

[0030] The present invention also provides an engineered bacterium, wherein the engineered bacterium contains the RR Mutant gene or the recombinant vector. In the present invention, the starting bacteria of the engineered bacteria preferably include Corynebacterium glutamicum; as an optional embodiment, the engineered bacteria preferably include Corynebacterium glutamicum engineered bacteria. The present invention has no special restrictions on the strain type and source of the starting bacteria, and can be conventionally selected according to actual needs. As an optional embodiment, the strain of the starting bacteria preferably includes Corynebacterium glutamicum ATCC 13032 or Corynebacterium glutamicum DXMC-DXA1. In some embodiments, the engineered bacteria preferably include Corynebacterium glutamicum DXMC-DXA2, with a deposit number of CCTCC NO: M2025350.

[0031] The present invention also provides a method for constructing the engineered bacteria, which preferably comprises: RR The mutant gene replaces the original strain RR Gene, to obtain an engineered bacterium; RR The nucleotide sequence of the gene is preferably shown as SEQ ID NO.4.

[0032] In the present invention, the starting strain preferably contains RR The strain containing the gene preferably includes a Corynebacterium glutamicum strain, more preferably includes Corynebacterium glutamicum ATCC 13032 or Corynebacterium glutamicum DXMC-DXA1.

[0033] In the present invention, the replacement method can be conventionally selected according to actual needs. As an optional embodiment, the replacement method can be selected by homologous exchange with the RR The mutant gene replaces the original strain RR Gene.

[0034] In some embodiments of the present invention, the construction method preferably includes: RR The mutant gene is connected to the upstream and downstream homology arms and inserted into the linearized vector to obtain a recombinant plasmid; the recombinant plasmid is transformed into competent cells of the starting strain and screened to obtain an engineered bacterium; the nucleotide sequence of the upstream homology arm is preferably as shown in SEQ ID NO.5, and the nucleotide sequence of the downstream homology arm is preferably as shown in SEQ ID NO.6.

[0035] In the present invention, the RR The mutant gene can be obtained by conventional means, for example, by artificial in vitro synthesis, or by inducing RR The strain containing the gene was mutated to obtain RR After the mutant gene strain is obtained by PCR amplification, the linearized vector type can be selected according to actual needs, preferably including pK19mobsacB.

[0036] In the present invention, conventional methods can be used to RR The mutant gene is connected to the upstream and downstream homology arms and inserted into the linearized vector. As an embodiment, the seamless cloning kit is used to clone the mutant gene into the linearized vector. RR The mutant gene was ligated with the upstream and downstream homology arms and inserted into the linearized vector.

[0037] In the present invention, the method of conversion can be conventionally selected according to actual needs, preferably including high-voltage electric shock conversion; the voltage of the high-voltage electric shock conversion is preferably 1800~2500V, and the electric shock time of the high-voltage electric shock conversion is preferably 3~7ms, more preferably 5ms.

[0038] The present invention also provides a RR mutant protein or the RR Application of the mutant gene, the recombinant vector or the engineered bacteria in producing L-arginine.

[0039] The present invention mutagenesis treatment is performed on Corynebacterium glutamicum DXMC-DXA1 by ultraviolet irradiation, and it is found that after the RR protein thereof is mutated into the RR mutant protein with the amino acid sequence as shown in SEQ ID NO.1, the L-arginine production is increased; RR The mutant gene replaces the original strain RR Compared with the starting strain, the genetically engineered bacteria can significantly increase the production of L-arginine, with the yield increased by 15.9%, and grow faster, which helps save fermentation time and cost. Compared with other strains, it can form a dominant bacteria, avoid the growth of other strains, and reduce the chance of contamination.

[0040] The present invention also provides a method for producing L-arginine, which comprises: inoculating the engineered bacteria into a culture medium and fermenting.

[0041] In the present invention, the engineered bacteria is preferably an engineered bacterium of Corynebacterium glutamicum, more preferably Corynebacterium glutamicum DXMC-DXA2, with a deposit number of CCTCC NO: M2025350.

[0042] In the present invention, the method preferably further comprises: before inoculating the engineered bacteria, seed culture is first performed to obtain seed liquid. The culture medium for the seed culture can be conventionally selected according to actual needs. In some embodiments, when the engineered bacteria is the engineered bacteria of Corynebacterium glutamicum, the culture medium for the seed culture preferably comprises the following components: 10 g of peptone, 5 g of yeast extract, 10 g of NaCl and 5 g of glucose per 1 L, and the balance is water. The culture conditions for the seed culture can be conventionally selected according to actual needs. In some embodiments, the temperature for the seed culture is preferably 25-37°C, more preferably 27-30°C or 28-32°C; the rotation speed for the seed culture is preferably 100-400 rpm, more preferably 150-200 rpm or 180-220 rpm or 210-300 rpm; the time for the seed culture is preferably 8-24 h, more preferably 12-16 h or 14-18 h or 17-20 h. The viable bacteria concentration of the seed liquid is preferably 1×10 7 ~1×10 9 CFU / mL, more preferably 3.6×10 8 CFU / mL.

[0043] In the present invention, the inoculation amount of the engineered bacteria can be conventionally selected according to actual needs. In some embodiments, the seed liquid of the engineered bacteria is preferably inoculated into the culture medium at an inoculation amount of 1% to 10% of the volume percentage of the culture medium; more preferably 2% to 5% or 4% to 6% or 3% to 8%.

[0044] In the present invention, the engineered bacteria are inoculated into a culture medium for fermentation; the culture medium can be conventionally selected according to actual needs. In some embodiments, when the engineered bacteria is an engineered bacterium of Corynebacterium glutamicum, the culture medium preferably includes the following components: per liter, 30 g of glucose, 40 g of corn steep liquor, 10 g of (NH4)2SO4, 15 g of CH3COONa, 8 g of urea, 1.67 g of L-alanine, 4 g of KH2PO4, 2.13 g of MgSO4·7H2O, 0.04 g of FeSO4·7H2O, 0.06 g of MnSO4·H2O, 0.002 g of biotin, 0.08 g of leucine, and the balance of water.

[0045] In the present invention, the fermentation conditions can be conventionally selected according to actual needs. In some embodiments, the fermentation temperature is preferably 29-31°C, more preferably 30°C; the fermentation time is preferably 12-96h, more preferably 24-48h or 36-60h or 50-72h.

[0046] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] In the following examples, unless otherwise specified, all methods are conventional.

[0048] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0049] In the following examples, Corynebacterium glutamicum ATCC 13032 was obtained from the American Type Culture Collection, with the strain number ATCC 13032.

[0050] Example 1 Corynebacterium glutamicum DXMC-DXA1 was collected from Zhucheng City, Weifang City, Shandong Province, and deposited in the China Center for Type Culture Collection on March 3, 2025, with the deposit number CCTCC NO: M2025349.

[0051] The activated Corynebacterium glutamicum DXMC-DXA1 bacterial suspension was centrifuged, the supernatant was discarded, and the bacterial cells were diluted with physiological saline to 1×10 8CFU / mL (10-fold dilution); preheat the UV lamp for 30 minutes. Spread 3 mL of the diluted bacterial solution onto a disposable Petri dish and place under UV light for 0, 30, 60, 90, or 120 seconds, with three replicates for each gradient. After UV irradiation, plate the appropriate dilution ratios for colony count (incubated in the dark) and plot a lethality curve. The irradiation time with a lethality of 80%-90% was selected as the irradiation time for mutation. Strains were screened for increased L-arginine production at this irradiation time. The yield of Corynebacterium glutamicum DXMC-DXA1 before mutation was 1.21 g / L, while the mutant strain, DXMC-DXA1, yielded 1.45 g / L. Genome sequencing revealed a mutation in its RR protein. The amino acid sequence of the mutated strain is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2.

[0052] Example 2 Knockout Frame Construction 1. Using the genomic DNA of the mutant Corynebacterium glutamicum DXMC-DXA1 obtained in Example 1 as a template, PCR amplification was performed to obtain RR Mutated gene fragments, RR The nucleotide sequence of the mutant gene is shown in SEQ ID NO. 2, and the nucleotide sequences of the PCR amplification primers are as follows: Upstream primer (RR-F): aggtatcgATGTTCCAACGCGTGGACG (SEQ ID NO. 7); Downstream primer (RR-R): ttcgccttaatgTCACGGAGCGGTCTCTCGC (SEQ ID NO. 8); The PCR amplification reaction system was as follows, with a total system of 50 μL: 2×Phanta Max Master Mix 25 μL; upstream primer (10 μmol / L) 2 μL; downstream primer (10 μmol / L) 2 μL; template 2 μL; ddH2O 19 μL.

[0053] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 20 sec, annealing at 57°C for 15 sec, extension at 72°C for 1 min, extension at 72°C for 5 min, and storage at 4°C.

[0054] 2. PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC 13032 as a template to obtain the upstream homology arm UP. The nucleotide sequence is shown in SEQ ID NO. 5. The nucleotide sequences of the PCR amplification primers are as follows: Upstream primer (UP-F): cccgggtaccgagctcgaatCGACAATGTCGCCCTTCAGG (SEQ ID NO. 9); Downstream primer (UP-R): cgttggaacatCGATACCCTTTTCGTCTTCCAG (SEQ ID NO. 10); The reaction system and PCR amplification procedure were the same as those mentioned above. RR The same is true when the mutant gene is amplified.

[0055] 3. PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC 13032 as a template to obtain the downstream homology arm DOWN. The nucleotide sequence is shown in SEQ ID NO. 6. The nucleotide sequences of the PCR amplification primers are as follows: Upstream primer (DOWN-F): ctccgtgaCATTAAGGCGAATCGGCG (SEQ ID NO. 11); Downstream primer (DOWN-R): tgtaaaacgacggccagtgaTTCGAGTGTCACTGGCATATTCA (SEQ ID NO. 12); The reaction system and PCR amplification procedure were the same as those mentioned above. RR The same is true when the mutant gene is amplified.

[0056] Example 3 Recombinant plasmid construction The amplified product from Example 2 was cloned using a seamless cloning kit. RR The mutant gene fragment was connected to the upstream homology arm UP and the downstream homology arm DOWN, and inserted into the linearized vector to prepare a recombinant plasmid; The multi-fragment seamless cloning system is as follows, with a total volume of 10 μL: 2 μL of linearized vector pK19mobsacB; 1 μL of upstream homology arm UP; 1 μL of downstream homology arm DOWN; RR 1 μL of mutant gene fragment; 5 μL of 2×ClonExpress Mix.

[0057] The multi-fragment seamless cloning procedure was as follows: multi-fragment recombination reaction, 50°C, 15 min; immediately placed on ice to cool.

[0058] Example 4 Preparation and transformation of competent cells 1. Pick a single colony of Corynebacterium glutamicum ATCC 13032 and culture it in seed culture medium until the bacterial concentration OD 600The concentration of the purified protein was 0.9, and the cells were placed on ice and cooled to 4°C. After cooling, the cells were centrifuged and washed four times with pre-cooled electroporation buffer. The cells were resuspended in electroporation buffer to prepare competent cells of Corynebacterium glutamicum ATCC 13032. The seed culture medium has the following components per liter: 10g peptone, 5g yeast powder, 10g sodium chloride, 5g glucose, and the balance water; The electrotransfer buffer solution has the following components per liter: 90 g of sorbitol, 90 g of mannitol, 100 mL of glycerol, and the balance of water.

[0059] 2. The recombinant plasmid prepared in Example 3 was transformed into the competent cells of Corynebacterium glutamicum ATCC13032 prepared in step 1 by high-voltage electroporation, and the cells were transferred into liquid recovery medium. The cells were cultured at 30° C. for 14 h, and transformants were screened. The liquid recovery medium has the following components per liter: 10g peptone, 5g yeast powder, 10g sodium chloride, 90g sorbitol, 70g mannitol, and the balance water; The conditions for high-voltage electric shock conversion are: 2500V electric shock for 5ms.

[0060] Example 5 Positive colony screening The positive recombinant colonies (transformants screened) from Example 4 were selected and inoculated into solid LB medium containing 25 μg / mL kanamycin resistance and cultured overnight at 30°C. The single colony growing on the solid LB medium containing kanamycin resistance was the strain that underwent the first homologous recombination. The single colony growing on the solid LB medium containing kanamycin resistance was selected and inoculated into liquid LB medium containing 15 g / L sucrose and cultured overnight. The colony was then plated into solid LB medium containing 15 g / L sucrose and cultured overnight at 30°C. The single colony growing on the solid LB medium containing sucrose was the strain that underwent the second homologous recombination. The single colony growing on the solid LB medium containing 15 g / L sucrose was selected as the template, and PCR amplification was performed using primers UP-F and DOWN-R. The amplified product was verified by agarose gel electrophoresis. The solid LB medium has the following components: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L sucrose, 15 g / L agar powder, and the balance is water; The nucleotide sequences of the PCR amplification primers are as follows: UP-F: cccgggtaccgagctcgaatCGACAATGTCGCCCTTCAGG (SEQ ID NO.9); DOWN-R: tgtaaaacgacggccagtgaTTCGAGTGTCACTGGCATATTCA (SEQ ID NO. 12); The PCR amplification reaction system was as follows, with a total volume of 20 μL: 2× Phanta Max Master Mix 10 μL; UP-F (10 μmol / L) 1 μL; DOWN-R (10 μmol / L) 1 μL; template 1 μL; ddH2O 7 μL; The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 20 sec, annealing at 57°C for 15 sec, extension at 72°C for 1 min, extension at 72°C for 5 min, and storage at 4°C; Agarose gel electrophoresis was used to test the PCR products. Figure 1 As shown in the figure, it can be seen that the use of primers UP-F and DOWN-R can amplify a specific gene band with a size of about 1300bp, which is close to the theoretical value of 1368bp. The results are shown in the figure. Figure 2 As shown, Figure 2 The black bold sequence is the correct sequence after mutation. The results show that the sequencing results of the two primers are consistent with the black bold sequence, indicating that the sequencing results of different primers all contain the nucleotide sequence shown in SEQ ID NO.2 RR Mutated genes, indicating RR The gene replacement was successful, and Corynebacterium glutamicum DXMC-DXA2 was obtained, which was deposited in the China Center for Type Culture Collection on March 3, 2025, with the deposit number CCTCC NO: M2025350.

[0061] Example 6 L-arginine fermentation test 1. Corynebacterium glutamicum DXMC-DXA2 prepared in Example 5 and the original bacteria (Corynebacterium glutamicum ATCC13032) were inoculated into 100 mL of LBG medium, respectively, and seed culture was carried out at 220 rpm and 30°C for 16 h. The viable bacterial concentration of the obtained seed solution was 3.6×10 8 CFU / mL; The LBG medium has the following components: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 5 g / L glucose, and the balance is water.

[0062] 2. Then, inoculate 5% by volume into 100 mL of fermentation medium and ferment at 30°C for 50 h. The fermentation medium has the following components: 30 g / L glucose, 40 g / L corn steep liquor, 10 g / L (NH4)2SO4, 15 g / L CH3COONa, 8 g / L urea, 1.67 g / L L-alanine, 4 g / L KH2PO4, 2.13 g / L MgSO4·7H2O, 0.04 g / L FeSO4·7H2O, 0.06 g / L MnSO4·H2O, 0.002 g / L biotin, 0.08 g / L leucine, and the balance is water.

[0063] 3. Samples were taken at 12 h, 24 h, 36 h and 48 h of fermentation, and the L-arginine content in the fermentation broth was determined by high performance liquid chromatography (Agilent Technologies). The average value of the three fermentation measurements was taken, and the results are shown in Table 1. The OD was measured by UV spectrophotometer. 600 The results are shown in Table 2.

[0064] Table 1 L-arginine production of Corynebacterium glutamicum DXMC-DXA2 and original bacteria

[0065] Table 2 OD of Corynebacterium glutamicum DXMC-DXA2 and original bacteria 600 value

[0066] It can be seen from the data in Table 1 that the production of L-arginine in the fermentation broth of Corynebacterium glutamicum DXMC-DXA2 can reach 1.24 g / L after 48 hours of fermentation, while the production of L-arginine in the fermentation broth of the original strain is 1.07 g / L after 48 hours of fermentation. The L-arginine production of Corynebacterium glutamicum DXMC-DXA2 is 15.9% higher than that of the original strain; when the L-arginine production of Corynebacterium glutamicum DXMC-DXA2 reaches 1.01 g / L, the fermentation time is 24 hours, while the L-arginine production of the original strain reaches 0.99 g / L after 36 hours of fermentation. Compared with the original strain, the fermentation time is saved by about 12 hours, indicating that Corynebacterium glutamicum DXMC-DXA2 grows faster than the original strain, which can save fermentation time and cost.

[0067] The data in Table 2 show that the OD values ​​of Corynebacterium glutamicum DXMC-DXA2 were 0.01, 0.06, 0.13, and 0.17, respectively, at 12 h, 24 h, 36 h, and 48 h of fermentation. 600 The values ​​were greater than the OD of the original bacteria 600The values ​​indicate that the biomass of C. glutamicum DXMC-DXA2 also increased, with the largest increase reaching 14.5% at 12 hours of fermentation. Because C. glutamicum DXMC-DXA2 grows faster, it can become dominant over other strains, preventing their growth and thus reducing the risk of contamination.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An RR mutant protein, characterized in that, The amino acid sequence of the RR mutant protein is shown in SEQ ID NO.

1.

2. A RR mutated gene, characterized in that The RR mutant gene encodes the RR mutant protein as claimed in claim 1; the RR nucleotide sequence of the mutant gene is as shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, The recombinant vector contains the RR mutant gene as claimed in claim 2.

4. An engineered bacterium, characterized in that, The engineered bacteria contain the RR mutant gene described in claim 2 or the recombinant vector described in claim 3.

5. The engineered bacterium according to claim 4, characterized in that, The starting strain of the engineered bacterium includes Corynebacterium glutamicum.

6. The engineered bacterium according to claim 5, wherein The engineered bacterium includes Corynebacterium glutamicum DXMC-DXA2, with the deposit number of CCTCC NO: M2025350.

7. The construction method of the engineering bacteria according to any one of claims 5 to 6, characterized in that The construction method includes: replacing the RR gene in the starting strain with the RR mutant gene to obtain the engineered strain.

8. Use of the RR mutant protein according to claim 1, or the mutant gene according to claim 2, or the recombinant vector according to claim 3, or the engineered bacterium according to any one of claims 4 to 6 in the production of L-arginine. RR ​ 9. A method for producing L-arginine, characterized in that, The method includes: inoculating the engineered bacterium according to any one of claims 4 to 6 into a culture medium and performing fermentation.

10. The method according to claim 9, wherein The conditions for the fermentation include: a fermentation temperature of 29 to 31 °C and a fermentation time of 12 to 96 h.

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