A rr mutant protein, mutant gene, recombinant vector, engineered bacteria and construction method and application thereof

Abstract

The application discloses a kind of RR mutant protein, mutant gene, recombination carrier, engineering bacteria and its construction method and application, belong to molecular biological technique field.The amino acid sequence of RR mutant protein disclosed in the application is as shown in SEQ ID NO.1.The L-arginine yield is significantly improved after the RR protein is found to be mutated to RR mutant protein by carrying out ultraviolet mutagenesis treatment to corynebacterium glutamicum; RR The growth speed of the engineering bacteria obtained by using the mutant gene coding RR mutant protein is obviously accelerated, the L-arginine yield is obviously improved, the growth speed can also be improved, the fermentation time and cost are saved, the dominant bacteria can be formed compared with other bacteria, the growth of other bacteria is avoided, and the probability of contamination can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and particularly relates to an RR mutant protein, a mutant gene, a recombinant vector, an engineered bacterium, its construction method, and its application. Background Technology

[0002] L-arginine is an organic compound with the molecular formula C6H2O. 14 N4O2, with a density of 1.46 g / cm³ and a molecular weight of 174.201, is a white crystalline powder. It is abundant in protamine and other proteins, and is a basic component of various proteins, exhibiting a wide range of properties.

[0003] L-arginine is a basic amino acid, non-essential for adults but produced slowly in the body. It is an essential amino acid for infants and young children and has a certain detoxification effect. L-arginine has wide applications in the food, pharmaceutical, feed, and cosmetic industries. In the food industry, L-arginine is commonly used as a flavoring agent and beverage additive. In the pharmaceutical industry, it is used to produce various drugs for treating hyperammonemia, as an oral amino acid supplement, relieving fatigue, improving liver function, and regulating endocrine function. Furthermore, adding L-arginine to animal feed can promote animal growth and development, repair intestinal damage, enhance immunity, and prevent various diseases. As a basic amino acid, L-arginine is also widely used as a neutralizing agent and moisturizer in cosmetics.

[0004] Currently, L-arginine is mainly produced 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 L-arginine production, but there is still significant room for improvement in both yield and efficiency. Therefore, there is an urgent need to develop a method to increase the yield of L-arginine through microbial fermentation, providing technical support for large-scale, efficient fermentation production of L-arginine. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an RR mutant protein that can increase L-arginine production compared to the original protein.

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

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

[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 the RR mutant protein or the [other protein described herein]. RR The application of the mutant gene, the recombinant vector, or the engineered bacteria in the production of L-arginine.

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

[0012] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0013] The present invention provides an RR mutant protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0014] The present invention also provides RR Mutant 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.

[0015] The present invention also provides a recombinant vector, the recombinant vector containing the RR Mutant gene.

[0016] The present invention also provides an engineered bacterium, wherein the engineered bacterium contains the... RR Mutant gene or the recombinant vector.

[0017] Preferably, the originating bacteria of the engineered bacteria include Corynebacterium glutamicum.

[0018] Preferably, the engineered bacteria includes Corynebacterium glutamicum DXMC-DXA2, with accession number CCTCC NO: M2025350.

[0019] The present invention also provides a method for constructing the engineered bacteria, the method comprising: using the... RR The mutated gene replaces the starting strain RR Genes were used to obtain engineered bacteria.

[0020] The present invention also provides the RR mutant protein or the [other protein]. RR The application of the mutant gene, the recombinant vector, or the engineered bacteria in the production of L-arginine.

[0021] The present invention also provides a method for producing L-arginine, the method comprising: inoculating the engineered bacteria into a culture medium and fermenting.

[0022] Preferably, the fermentation conditions include: a fermentation temperature of 29~31℃ and a fermentation time of 12~96h.

[0023] The beneficial effects of this invention are:

[0024] This invention involves UV mutagenesis of Corynebacterium glutamicum, revealing that mutating the RR protein to the RR mutant protein with the amino acid sequence shown in SEQ ID NO.1 significantly increases L-arginine production. Homologous exchange is then used to transform the protein encoding the RR mutant protein... RR The mutated gene replaces the starting strain RR Following gene modification, the resulting engineered bacteria exhibited significantly accelerated growth and a marked increase in L-arginine production. This indicates that mutating the RR protein to an RR mutant protein can enhance L-arginine production. The present invention encodes the RR mutant protein... RR The engineered bacteria obtained by constructing mutant genes can not only increase the yield of L-arginine, but also increase the growth rate, which helps to save fermentation time and costs. Compared with other strains, they can form dominant strains, avoid the growth of other strains, and reduce the chance of contamination.

[0025] Biological Preservation Information

[0026] This invention relates to Corynebacterium glutamicum DXMC-DXA1, which is classified and named Corynebacterium glutamicum ( Corynebacterium glutamicum The sample is deposited at the China Center for Type Culture Collection (CCTCC) on March 3, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025349.

[0027] This invention relates to Corynebacterium glutamicum DXMC-DXA2, which is classified and named Corynebacterium glutamicum ( Corynebacterium glutamicum The sample is deposited at the China Center for Type Culture Collection (CCTCC) on March 3, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025350. Attached Figure Description

[0028] Figure 1 This is an agarose gel electrophoresis image from Example 5, where lanes 1 to 3 show positive colonies. RR Parallel experiments of amplified mutant gene fragments, with lane M serving as a marker;

[0029] Figure 2 The image shows the sequencing results from Example 5, with the red box indicating... RR The mutated gene contains bases 79-81. Detailed Implementation

[0030] The present invention provides an RR mutant protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0031] This invention utilizes ultraviolet irradiation to target Corynebacterium glutamicum (C. glutamicum). Corynebacterium glutamicum The *Corynebacterium glutamicum* strain DXMC-DXA1 (accession number CCTCC NO: M2025349) was mutagenized. 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 mutating the RR protein to the RR mutant protein can increase L-arginine production. In this invention, the RR mutant protein is formed by a mutation at amino acid position 27, from Leu to Pro.

[0032] The present invention also provides a protein encoding the RR mutant protein. RR Mutant gene; the RR The nucleotide sequence of the mutant gene is shown in SEQ ID NO.2. The RR protein is composed of... RR Gene encoding, the RR The nucleotide sequence of the gene is shown in SEQ ID NO.4. RR The mutated gene is formed when the 80th nucleotide is mutated from T to C.

[0033] The present invention also provides a recombinant vector, the recombinant vector containing the RR Mutant gene. In this invention, the starting vector of the recombinant vector can be conventionally selected according to actual needs. As one possible implementation, the starting vector preferably includes pK19mobsacB.

[0034] The present invention also provides an engineered bacterium, wherein the engineered bacterium contains the... RR The mutant gene or the recombinant vector. In this invention, the starting strain of the engineered bacteria preferably includes *Corynebacterium glutamicum*; as one possible implementation, the engineered bacteria preferably includes *Corynebacterium glutamicum* engineered strains. This invention does not specifically limit the type and source of the starting strain, and can conventionally select according to actual needs. As an optional implementation, the starting strain preferably includes *Corynebacterium glutamicum* ATCC 13032 or *Corynebacterium glutamicum* DXMC-DXA1. In some embodiments, the engineered bacteria preferably includes *Corynebacterium glutamicum* DXMC-DXA2, with accession number CCTCC NO: M2025350.

[0035] The present invention also provides a method for constructing the engineered bacteria, wherein the construction method preferably includes: using the... RR The mutated gene replaces the starting strain RR Genes were used to obtain engineered bacteria; the aforementioned RRThe preferred nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0036] In this invention, the starting strain preferably contains the... RR The strains containing the gene are preferably Corynebacterium glutamicum strains, more preferably Corynebacterium glutamicum ATCC 13032 or Corynebacterium glutamicum DXMC-DXA1.

[0037] In this invention, the replacement method can be conventionally selected according to actual needs. As an optional implementation method, it is achieved through same-source exchange. RR The mutated gene replaces the starting strain RR Gene.

[0038] In some embodiments of the present invention, the construction method preferably includes: taking the... RR The mutant gene is ligated to the upstream and downstream homologous arms and inserted into a linearized vector to obtain a recombinant plasmid; the recombinant plasmid is transformed into competent cells of the starting strain and screened to obtain engineered bacteria; the preferred nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO.5, and the preferred nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.6.

[0039] In this invention, the RR Mutant genes can be obtained through conventional methods, such as artificial in vitro synthesis or induction of mutant genes. RR Mutation of the gene in the strain to obtain a strain containing RR After obtaining the mutant gene strain, it is amplified by PCR. The type of linearization vector can be conventionally selected according to actual needs, with pK19mobsacB being a preferred option.

[0040] In this invention, conventional methods can be used to... RR One possible implementation involves linking the mutant gene to its upstream and downstream homologous arms and inserting it into a linearized vector, using a seamless cloning kit. RR The mutated gene is linked to upstream and downstream homologous arms and inserted into a linearized vector.

[0041] In this invention, the method of transfer 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.

[0042] The present invention also provides the RR mutant protein or the [other protein]. RR The application of the mutant gene, the recombinant vector, or the engineered bacteria in the production of L-arginine.

[0043] This invention, through ultraviolet irradiation to induce mutagenesis in Corynebacterium glutamicum DXMC-DXA1, found that mutating its RR protein to the RR mutant protein with the amino acid sequence shown in SEQ ID NO.1 increased L-arginine production; [the invention then describes a method for encoding the RR mutant protein.] RR The mutated gene replaces the starting strain RR The engineered bacteria obtained from the gene significantly increased the yield of L-arginine by 15.9% compared to the original strain, and also grew faster, which helped save fermentation time and costs. Compared with other strains, it could form a dominant strain, preventing the growth of other strains and reducing the chance of contamination.

[0044] The present invention also provides a method for producing L-arginine, the method comprising: inoculating the engineered bacteria into a culture medium and fermenting.

[0045] In this invention, the engineered bacteria is preferably Corynebacterium glutamicum, more preferably Corynebacterium glutamicum DXMC-DXA2, with accession number CCTCC NO: M2025350.

[0046] In this invention, the method preferably further includes: before inoculating the engineered bacteria, performing seed culture to obtain a seed solution. The culture medium for seed culture can be conventionally selected according to actual needs. In some embodiments, when the engineered bacteria is *Corynebacterium glutamicum*, the culture medium preferably includes the following components: 10g peptone, 5g yeast extract, 10g NaCl, and 5g glucose per 1L, with the remainder being water. The culture conditions for seed culture can be conventionally selected according to actual needs. In some embodiments, the seed culture temperature is preferably 25-37℃, more preferably 27-30℃ or 28-32℃; the seed culture rotation speed is preferably 100-400 rpm, more preferably 150-200 rpm, 180-220 rpm, or 210-300 rpm; and the seed culture time is preferably 8-24h, more preferably 12-16h, 14-18h, or 17-20h. The viable cell concentration of the seed solution is preferably 1×10⁻⁶. 7 ~1×10 9 CFU / mL, more preferably 3.6 × 10⁻⁶ 8 CFU / mL.

[0047] In this 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 culture medium volume percentage; more preferably, it is 2% to 5%, 4% to 6%, or 3% to 8%.

[0048] In this invention, the engineered bacteria are inoculated into a culture medium and fermented. The culture medium can be conventionally selected according to actual needs. In some embodiments, when the engineered bacteria is Corynebacterium glutamicum, the culture medium preferably includes the following components per 1L: 30g glucose, 40g corn steep liquor, 10g (NH4)2SO4, 15g CH3COONa, 8g urea, 1.67g L-alanine, 4g KH2PO4, 2.13g MgSO4·7H2O, 0.04g FeSO4·7H2O, 0.06g MnSO4·H2O, 0.002g biotin, 0.08g leucine, and the remainder water.

[0049] In this invention, the fermentation conditions can be conventionally selected according to actual needs. In some embodiments, the fermentation temperature is preferably 29~31℃, more preferably 30℃; the fermentation time is preferably 12~96h, more preferably 24~48h, 36~60h, or 50~72h.

[0050] The technical solutions provided by the present invention will be 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.

[0051] Unless otherwise specified, the following embodiments are all conventional methods.

[0052] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0053] In the following examples, Corynebacterium glutamicum ATCC 13032 was obtained from the U.S. Type Culture Collection, strain number ATCC 13032.

[0054] Example 1

[0055] Corynebacterium glutamicum DXMC-DXA1 was collected from Zhucheng City, Weifang City, Shandong Province, and deposited at the China Center for Type Culture Collection on March 3, 2025, with accession number CCTCC NO: M2025349.

[0056] Mutagenesis of Corynebacterium glutamicum DXMC-DXA1 was induced by ultraviolet irradiation: The activated Corynebacterium glutamicum DXMC-DXA1 bacterial culture was centrifuged, the supernatant was discarded, and the bacterial cells were diluted with physiological saline to 1×10⁻⁶. 8CFU / mL (10-fold dilution); preheat with UV lamp for 30 min, spread 3 mL of the diluted bacterial solution onto disposable petri dishes, and place under UV lamp for 0 s, 30 s, 60 s, 90 s, or 120 s of UV treatment, with 3 replicates for each gradient; after UV irradiation, spread on plates with appropriate dilution ratios for colony counting (incubate in the dark), and plot the lethality curve. The irradiation time with a lethality rate of 80%-90% was selected as the mutation irradiation time. Strains with increased L-arginine production under this irradiation time were screened. Before mutation, *Corynebacterium glutamicum* DXMC-DXA1 produced 1.21 g / L; the mutated *Corynebacterium glutamicum* DXMC-DXA1 produced 1.45 g / L. Genome sequencing revealed a mutation in its RR protein. The mutated amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0057] Example 2: Knockout Box Construction

[0058] 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 Mutant 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:

[0059] Upstream primer (RR-F): agggtatcgATGTTCCAACGCGTGGACG (SEQ ID NO.7);

[0060] Downstream primer (RR-R): ttcgccttaatgTCACGGAGCGGTCTCTCGC (SEQ ID NO.8);

[0061] The PCR amplification reaction system is as follows, with a total volume 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.

[0062] The PCR amplification program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 20 sec, 57℃ annealing for 15 sec, 72℃ extension for 1 min, 35 cycles, 72℃ extension for 5 min, and storage at 4℃.

[0063] 2. Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed to obtain the upstream homologous arm UP, the nucleotide sequence of which is shown in SEQ ID NO.5. The nucleotide sequences of the PCR amplification primers are as follows:

[0064] Upstream primer (UP-F): cccgggtaccgagctcgaatCGACAATGTCGCCCTTCAGG (SEQ ID NO.9);

[0065] Downstream primer (UP-R): cgttggaacatCGATACCCTTTTCGTCTTCCAG (SEQ ID NO.10);

[0066] The PCR amplification reaction system and PCR amplification procedure are the same as those described above. RR The same applies when the mutated gene is amplified.

[0067] 3. Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed to obtain the downstream homologous arm DOWN, the nucleotide sequence of which is shown in SEQ ID NO.6. The nucleotide sequences of the PCR amplification primers are as follows:

[0068] Upstream primer (DOWN-F): ctccgtgaCATTAAGGCGAATCGGCG (SEQ ID NO.11);

[0069] Downstream primer (DOWN-R): tgtaaaacgacggccagtgaTTCGAGTGTCACTGGCATATTCA (SEQ ID NO.12);

[0070] The PCR amplification reaction system and PCR amplification procedure are the same as those described above. RR The same applies when the mutated gene is amplified.

[0071] Example 3 Construction of recombinant plasmids

[0072] The amplification of the product obtained in Example 2 was performed using a seamless cloning kit. RR The mutant gene fragment was ligated to the upstream homologous arm UP and the downstream homologous arm DOWN, and then inserted into a linearized vector to obtain a recombinant plasmid.

[0073] The multi-fragment seamless cloning system is as follows, with a total volume of 10 μL: linearized vector pK19mobsacB 2 μL; upstream homologous arm UP 1 μL; downstream homologous arm DOWN 1 μL; RR 1 μL of the mutant gene fragment; 5 μL of 2×ClonExpress Mix.

[0074] The multi-fragment seamless cloning procedure is as follows: multi-fragment recombination reaction, 50℃, 15min; immediately place on ice to cool.

[0075] Example 4: Preparation and Transformation of Competent Cells

[0076] 1. Pick a single colony of Corynebacterium glutamicum ATCC 13032 and culture it in seed culture medium until the bacterial concentration reaches OD500. 600 The concentration was 0.9, and the cells were placed on ice to cool to 4°C. After cooling, the cells were centrifuged, washed four times with pre-cooled electroporation buffer, and the cells were resuspended in electroporation buffer to obtain Corynebacterium glutamicum ATCC 13032 competent cells.

[0077] The seed culture medium has the following components per liter: 10g peptone, 5g yeast extract, 10g sodium chloride, 5g glucose, and the remainder water;

[0078] The electroporation buffer solution has the following components per liter: 90g sorbitol, 90g mannitol, 100mL glycerol, and the remainder water.

[0079] 2. The recombinant plasmid obtained in Example 3 was transformed into the competent cells of Corynebacterium glutamicum ATCC13032 obtained in Step 1 by high voltage electroporation, transferred into liquid resuscitation medium, and cultured at 30°C for 14 h to screen transformants.

[0080] The liquid resuscitation medium has the following components per liter: 10g peptone, 5g yeast extract, 10g sodium chloride, 90g sorbitol, 70g mannitol, and the remainder water;

[0081] The conditions for high-voltage electric shock conversion are: 2500V electric shock for 5ms.

[0082] Example 5: Screening of positive colonies

[0083] Positive recombinant colonies from Example 4 (screening transformants) were selected and inoculated into solid LB medium containing 25 μg / mL kanamycin resistance and cultured overnight at 30°C. Single colonies growing on solid LB medium containing kanamycin resistance were identified as strains undergoing the first homologous recombination. Single colonies growing on solid LB medium containing kanamycin resistance were then inoculated into liquid LB medium containing 15 g / L sucrose and cultured overnight. The inoculated liquid LB medium was then spread onto solid LB medium containing 15 g / L sucrose and cultured overnight at 30°C. Single colonies growing on solid LB medium containing sucrose were identified as strains undergoing the second homologous recombination. Using single colonies growing on solid LB medium containing 15 g / L sucrose as templates, PCR amplification was performed using UP-F and DOWN-R primers. The amplification products were verified by agarose gel electrophoresis.

[0084] 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 remainder is water;

[0085] The nucleotide sequences of the PCR amplification primers are as follows:

[0086] UP-F: cccgggtaccgagctcgaatCGACAATGTCGCCCTTCAGG (SEQ ID NO.9);

[0087] DOWN-R: tgtaaaacgacggccagtgaTTCGAGTGTCACTGGCATATTCA (SEQ ID NO. 12);

[0088] The PCR amplification reaction system is as follows, with a total volume of 20 μL: 10 μL of 2×Phanta Max Master Mix; 1 μL of UP-F (10 μmol / L); 1 μL of DOWN-R (10 μmol / L); 1 μL of template; and 7 μL of ddH2O.

[0089] The PCR amplification program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 20 sec, 57℃ annealing for 15 sec, 72℃ extension for 1 min, 35 cycles, 72℃ extension for 5 min, and storage at 4℃.

[0090] Agarose gel electrophoresis was used to examine the PCR products, and the results were as follows: Figure 1 As shown in the figure, primers UP-F and DOWN-R can amplify a specific gene band, approximately 1300 bp in size, which is close to the theoretical value of 1368 bp. Sequencing was performed using two different primers, and the results are as follows... Figure 2 As shown, Figure 2 The bolded sequence in black is the correct sequence after the mutation. The results show that the sequencing results of both primers are consistent with the bolded sequence, indicating that the sequencing results of different primers all show the presence of the nucleotide sequence as shown in SEQ ID NO.2. RR Mutant genes indicate RR Gene replacement was successful, and Corynebacterium glutamicum DXMC-DXA2 was obtained. It was deposited at the China Center for Type Culture Collection on March 3, 2025, with accession number CCTCC NO: M2025350.

[0091] Example 6 L-Arginine Fermentation Test

[0092] 1. The *Corynebacterium glutamicum* DXMC-DXA2 and the original strain (*Corynebacterium glutamicum* ATCC13032) prepared in Example 5 were inoculated into 100 mL of LBG medium and cultured at 220 rpm and 30 °C for 16 h. The viable cell concentration of the resulting seed culture was 3.6 × 10⁻⁶. 8 CFU / mL;

[0093] 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 remainder is water.

[0094] 2. Subsequently, each culture medium was inoculated at a volume percentage of 5% into 100 mL of fermentation medium and fermented at 30°C for 50 h.

[0095] The fermentation medium has the following components: glucose 30 g / L, corn steep liquor 40 g / L, (NH4)2SO4 10 g / L, CH3COONa 15 g / L, urea 8 g / L, L-alanine 1.67 g / L, KH2PO4 4 g / L, MgSO4·7H2O 2.13 g / L, FeSO4·7H2O 0.04 g / L, MnSO4·H2O 0.06 g / L, biotin 0.002 g / L, leucine 0.08 g / L, with the remainder being water.

[0096] 3. Samples were taken at 12h, 24h, 36h, and 48h of fermentation, and the L-arginine content in the fermentation broth was determined by high-performance liquid chromatography (HPLC) using an Agilent Technologies instrument. The average value of the three fermentation measurements was taken, and the results are shown in Table 1. The OD was measured by ultraviolet spectrophotometer. 600 The values ​​are shown in Table 2.

[0097] Table 1. L-arginine production of Corynebacterium glutamicum DXMC-DXA2 and the original strain

[0098]

[0099] Table 2. OD values ​​of Corynebacterium glutamicum DXMC-DXA2 and the original strain 600 value

[0100]

[0101] As shown in Table 1, after 48 hours of fermentation, the L-arginine yield in the *Corynebacterium glutamicum* DXMC-DXA2 fermentation broth reached 1.24 g / L, while the original strain yielded only 1.07 g / L after 48 hours. The L-arginine yield of *Corynebacterium glutamicum* DXMC-DXA2 was 15.9% higher than that of the original strain. Furthermore, the fermentation time for *Corynebacterium glutamicum* DXMC-DXA2 to reach 1.01 g / L was 24 hours, while the original strain only achieved a yield of 0.99 g / L after 36 hours of fermentation. Compared to the original strain, this represents a saving of approximately 12 hours of fermentation time. This indicates that *Corynebacterium glutamicum* DXMC-DXA2 has a faster growth rate than the original strain, thus saving fermentation time and costs.

[0102] The data in Table 2 show that the OD values ​​of Corynebacterium glutamicum DXMC-DXA2 were [data missing] at 12h, 24h, 36h, and 48h of fermentation. 600 The values ​​were all greater than the OD of the original bacteria. 600 The value indicates that the biomass of Corynebacterium glutamicum DXMC-DXA2 also increased, with the largest increase of 14.5% observed after 12 hours of fermentation. Because Corynebacterium glutamicum DXMC-DXA2 grows rapidly, it can become the dominant bacterium compared to other strains, preventing the growth of other strains and thus reducing the chance of contamination.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of an RR mutant protein in the production of L-arginine, characterized in that, The amino acid sequence of the RR mutant protein is shown in SEQ ID NO.

1.

2. A kind RR The application of mutant genes in the production of L-arginine is characterized by, The RR The nucleotide sequence of the mutant gene is shown in SEQ ID NO.

2.

3. The application of a recombinant vector in the production of L-arginine, characterized in that, The recombinant vector contains RR Mutant gene, the RR The nucleotide sequence of the mutant gene is shown in SEQ ID NO.

2.

4. An engineered bacterium, characterized in that, The engineered bacteria contain RR Mutant gene or recombinant vector; the RR The nucleotide sequence of the mutant gene is shown in SEQ ID NO.2; the recombinant vector contains RR Mutant gene; the origin of the engineered bacteria is Corynebacterium glutamicum ATCC 13032.

5. The engineered bacteria according to claim 4, characterized in that, The engineered bacteria is Corynebacterium glutamicum DXMC-DXA2, with accession number CCTCC NO: M2025350.

6. The method for constructing the engineered bacteria according to any one of claims 4 to 5, characterized in that, The construction method includes: using RR The mutated gene replaces the starting strain RR Genes were used to obtain engineered bacteria; the aforementioned RR The nucleotide sequence of the mutant gene is shown in SEQ ID NO.

2.

7. The use of the engineered bacteria according to any one of claims 4 to 5 in the production of L-arginine.

8. A method for producing L-arginine, characterized in that, The method includes: inoculating the engineered bacteria according to any one of claims 4 to 5 into a culture medium and fermenting.

9. The method according to claim 8, characterized in that, The fermentation conditions include: fermentation temperature of 29~31℃ and fermentation time of 12~96h.

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