Recombinant bacteria with high yield of L-arginine and construction method thereof

Abstract

This invention provides a recombinant bacterium for high L-arginine production and its construction method, belonging to the field of bioengineering technology. The recombinant bacterium contains a yihU gene mutant or a yihU gene knockout. The nucleotide sequence of the yihU gene is shown in SEQ ID NO.2 of the sequence listing. The nucleotide sequence of the yihU gene mutant is shown in SEQ ID NO.1 of the sequence listing. Compared with the yihU gene with the nucleotide sequence of SEQ ID NO.2 in the sequence listing, the yihU gene mutant has a mutation of A to G at position 391. This invention constructs a recombinant bacterium by mutating or knocking out the yihU gene, and then applies the constructed recombinant bacterium to L-arginine fermentation production, which can significantly increase the yield of L-arginine.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant bacterium that produces high levels of L-arginine and its construction method. Background Technology

[0002] L-arginine is a conditionally essential amino acid, but it is an essential amino acid for individuals such as birds, carnivores, and young mammals whose ability to synthesize L-arginine is insufficient. L-arginine is widely involved in cellular metabolism and is closely related to bodily functions, such as hormone secretion, circulatory regulation, immune regulation, wound healing, and maintenance of the intestinal mucosal barrier.

[0003] The main methods for producing L-arginine are hydrolysis and microbial fermentation. Hydrolysis involves separating and purifying L-arginine-rich animal protein raw materials, such as hair, blood, and pigskin, after acid hydrolysis (usually hydrochloric acid). However, this method requires large amounts of hydrochloric acid, posing a serious pollution risk. Microbial fermentation is currently the primary method for L-arginine production, which can be divided into direct fermentation and precursor fermentation. Both methods utilize microorganisms to convert common, inexpensive industrial raw materials into the target product. Compared to hydrolysis, microbial fermentation is not only simpler in its production process and has less environmental impact, but the separation and purification of fermentation products are also easier. However, for L-arginine production via microbial fermentation, the low acid production level of the strains and unstable fermentation performance remain key research challenges. The yihU gene, a novel 4-hydroxybutyrate dehydrogenase, catalyzes the reduction of succinate semialdehyde to γ-hydroxybutyrate with the participation of NADH. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a recombinant strain that produces high levels of L-arginine and a method for constructing the strain. By mutating or knocking out the yihU gene, a recombinant strain is constructed, and then the constructed recombinant strain is applied to the fermentation production of L-arginine, which can significantly increase the yield of L-arginine.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] A recombinant bacterium that produces high levels of L-arginine, said recombinant bacterium containing a yihU gene mutant or a yihU gene knockout.

[0007] The recombinant bacteria originated from Escherichia coli.

[0008] Preferably, the starting strain of the recombinant bacteria is Escherichia coli CGMCC 1.366.

[0009] The recombinant bacteria do not express or attenuate the yiHU gene.

[0010] The nucleotide sequence of the yihU gene is shown in SEQ ID NO.2 of the sequence listing.

[0011] The nucleotide sequence of the yihU gene mutant is shown in SEQ ID NO.1 of the sequence listing. Compared with the yihU gene whose nucleotide sequence is SEQ ID NO.2 of the sequence listing, the A at position 391 of the yihU gene mutant is mutated to G.

[0012] The amino acid sequence of the protein encoded by the yihU gene mutant is shown in SEQ ID NO.13 of the sequence listing.

[0013] A method for constructing a recombinant bacterium that produces high levels of L-arginine, wherein when the recombinant bacterium contains a yihU gene mutant, the construction method involves amplifying the recombinant plasmid pTargetF-sgRNA-yihU as a template, introducing a mutation site, and obtaining the recombinant plasmid pTargetF-sgRNA-yihU. A391G Then, the recombinant plasmid pTargetF-sgRNA-yihU was used. A391G Import the starting strain;

[0014] Preferably, the primer used when introducing the mutation site is yihU. A391G -F and yihU A391G -R, the nucleotide sequences are shown in SEQ ID NO.11 and SEQ ID NO.12 in the sequence listing.

[0015] When the yihU gene is knocked out in the aforementioned recombinant bacteria, the construction method is to use the recombinant plasmid pTargetF-sgRNA-yihU as a template and KO-F and KO-R as amplification primers to amplify the recombinant plasmid pTargetF-sgRNA-yihU-KO. Then, the recombinant plasmid pTargetF-sgRNA-yihU-KO is introduced into the starting strain, and then the recombinant plasmid pTargetF-sgRNA-yihU-KO is knocked out.

[0016] Preferably, the nucleotide sequences of the amplification primers KO-F and KO-R are as shown in SEQ ID NO.14 and SEQ ID NO.15 of the sequence listing, respectively.

[0017] The recombinant plasmid pTargetF-sgRNA-yihU was obtained by ligating the yihU gene fragment with upstream and downstream homologous fragments using a seamless cloning kit and inserting it into a linearized vector.

[0018] The linearization carrier is pTargetF.

[0019] The target sequence of the sgRNA is shown in SEQ ID NO.16 of the sequence listing.

[0020] The yihU gene fragment was obtained by PCR amplification using genomic DNA of Escherichia coli CGMCC 1.366 as a template and yihU-F and yihU-R as primers; the nucleotide sequences of yihU-F and yihU-R are shown in SEQ ID NO.9 and SEQ ID NO.10 in the sequence listing.

[0021] The upstream homologous fragment was obtained by PCR amplification using genomic DNA of Escherichia coli CGMCC 1.366 as a template and UP-F and UP-R as primers; the nucleotide sequences of UP-F and UP-R are shown in SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing.

[0022] The downstream homologous fragment was obtained by PCR amplification using genomic DNA of Escherichia coli CGMCC 1.366 as a template and DOWN-F and DOWN-R as primers; the nucleotide sequences of DOWN-F and DOWN-R are shown in SEQ ID NO.7 and SEQ ID NO.8 in the sequence listing.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention involves site-directed mutagenesis of the yihU gene, mutating base A to G at position 391 of the nucleotide sequence to obtain the yihU gene mutant. Subsequently, engineered *E. coli* strains containing the yihU gene mutant and those without the yihU gene were constructed. Experimental results showed that, compared to the original *E. coli* CGMCC 1.366, both the engineered *E. coli* strains containing the yihU gene mutant and those without the yihU gene significantly increased L-arginine production while also reducing fermentation time. Attached Figure Description

[0025] Figure 1 This is an agarose gel electrophoresis image of positive bacteria.

[0026] Where M stands for Marker, and 1-2 are two parallel samples of the yihU gene amplification fragment.

[0027] Figure 2 This is a sequencing verification diagram of a positive clone.

[0028] Figure 3 This is an agarose gel electrophoresis image of positive bacteria.

[0029] Where M stands for Marker, and 1-6 are six parallel samples of the yihU gene knockout amplification fragment. Detailed Implementation

[0030] This invention provides a recombinant bacterium that produces high levels of L-arginine, wherein the recombinant bacterium contains a yihU gene mutant or a yihU gene knockout.

[0031] The recombinant bacteria described in this invention originated from Escherichia coli.

[0032] Preferably, the starting strain of the recombinant bacteria in this invention is Escherichia coli CGMCC 1.366.

[0033] The recombinant bacteria described in this invention do not express or attenuate the yiHU gene.

[0034] The nucleotide sequence of the yihU gene described in this invention is shown in SEQ ID NO.2 of the sequence listing.

[0035] The nucleotide sequence of the yihU gene mutant of the present invention is shown in SEQ ID NO.1 of the sequence listing. Compared with the yihU gene whose nucleotide sequence is SEQ ID NO.2 of the sequence listing, the A at position 391 of the yihU gene mutant is mutated to G.

[0036] In this invention, the protein encoded by the yihU gene mutant has the amino acid sequence shown in SEQ ID NO.13 of the sequence listing.

[0037] This invention also provides a method for constructing the aforementioned recombinant bacteria that produces high levels of L-arginine. When the recombinant bacteria contain a yihU gene mutant, the construction method involves amplifying the recombinant plasmid pTargetF-sgRNA-yihU as a template, introducing a mutation site, and obtaining the recombinant plasmid pTargetF-sgRNA-yihU. A391G Then, the recombinant plasmid pTargetF-sgRNA-yihU was used. A391G Import the starting strain;

[0038] Preferably, in this invention, the primer used to introduce the mutation site is yihU. A391G -F and yihU A391G -R, the nucleotide sequences are shown in SEQ ID NO.11 and SEQ ID NO.12 in the sequence listing.

[0039] In this invention, when the yihU gene is knocked out in the aforementioned recombinant bacteria, the construction method is to use the recombinant plasmid pTargetF-sgRNA-yihU as a template and KO-F and KO-R as amplification primers to amplify the recombinant plasmid pTargetF-sgRNA-yihU-KO. Then, the recombinant plasmid pTargetF-sgRNA-yihU-KO is introduced into the starting strain, and then the recombinant plasmid pTargetF-sgRNA-yihU-KO is knocked out.

[0040] Preferably, the nucleotide sequences of the amplification primers KO-F and KO-R of the present invention are shown in SEQ ID NO.14 and SEQ ID NO.15 in the sequence listing, respectively.

[0041] In this invention, the recombinant plasmid pTargetF-sgRNA-yihU is obtained by ligating the yihU gene fragment with upstream and downstream homologous fragments using a seamless cloning kit and inserting it into a linearized vector.

[0042] The linearization carrier described in this invention is pTargetF.

[0043] The target sequence of the sgRNA described in this invention is shown in SEQ ID NO.16 of the sequence listing.

[0044] The yihU gene fragment of the present invention was obtained by PCR amplification using genomic DNA of Escherichia coli CGMCC 1.366 as a template and yihU-F and yihU-R as primers; the nucleotide sequences of yihU-F and yihU-R are shown in SEQ ID NO. 9 and SEQ ID NO. 10 in the sequence listing.

[0045] The upstream homologous fragment of the present invention was obtained by PCR amplification using genomic DNA of Escherichia coli CGMCC 1.366 as a template and UP-F and UP-R as primers; the nucleotide sequences of UP-F and UP-R are shown in SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing.

[0046] The downstream homologous fragment of the present invention was obtained by PCR amplification using genomic DNA of Escherichia coli CGMCC 1.366 as a template and DOWN-F and DOWN-R as primers; the nucleotide sequences of DOWN-F and DOWN-R are shown in SEQ ID NO.7 and SEQ ID NO.8 in the sequence listing.

[0047] In this invention, Escherichia coli CGMCC 1.366 was purchased from the China General Microbiological Culture Collection Center.

[0048] In this invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0049] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0050] Example 1

[0051] 1. Upstream homologous arm (UP) amplification: Using genomic DNA of *E. coli* CGMCC 1.366 as a template, PCR amplification was performed using UP-F and UP-R primers to obtain the upstream homologous arm (UP) nucleotide sequence as shown in SEQ ID NO.3. The nucleotide sequences of the PCR amplification primers were: UP-F (as shown in SEQ ID NO.5 in the sequence listing); UP-R (as shown in SEQ ID NO.6 in the sequence listing). The PCR amplification reaction system was as follows: total volume 50 μL: 25 μL 2×PhantaMax Master Mix, 2 μL UP-F (10 μmol / L), 2 μL UP-R (10 μmol / L), 2 μL template, 19 μL ddH2O; the PCR amplification program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 56℃ annealing for 15 sec, 72℃ extension for 30 sec, 32 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0052] 2. Downstream homologous arm DOWN amplification: Using genomic DNA of *E. coli* CGMCC 1.366 as a template, and DOWN-F and DOWN-R as primers, PCR amplification was performed to obtain the downstream homologous arm DOWN nucleotide sequence as shown in SEQ ID NO.4. The nucleotide sequences of the PCR amplification primers were: DOWN-F (as shown in SEQ ID NO.7 in the sequence listing) and DOWN-R (as shown in SEQ ID NO.8 in the sequence listing). The PCR amplification system and procedure were the same as in step 1, except for the primers.

[0053] 3. Obtaining the yihU gene fragment: Using genomic DNA of *E. coli* CGMCC 1.366 as a template, and yihU-F and yihU-R as primers, PCR amplification was performed to obtain the yihU gene fragment. The nucleotide sequences of the PCR amplification primers are: yihU-F (as shown in SEQ ID NO. 9 in the sequence listing) and yihU-R (as shown in SEQ ID NO. 10 in the sequence listing). The PCR amplification system and procedure are the same as in step 1, except for the primers.

[0054] 4. Construction of recombinant plasmid pTargetF-sgRNA-yihU

[0055] The yihU gene fragment was inserted into a linearized vector using a seamless cloning kit to obtain the recombinant plasmid pTargetF-sgRNA-yihU.

[0056] The multi-fragment seamless cloning system consisted of a total volume of 10 μL: 2 μL of linearized vector pTargetF (Miaoling Biotechnology), 1 μL of upstream homologous arm UP, 1 μL of downstream homologous arm DOWN, 1 μL of yihU gene fragment, and 5 μL of 2×ClonExpress Mix. The multi-fragment seamless cloning procedure was as follows: multi-fragment recombination reaction, 50 °C, 15 min; immediately placed on ice for cooling.

[0057] The nucleotide sequence of the target sequence of the sgRNA is shown in SEQ ID NO.16 of the sequence listing.

[0058] 5. Recombinant vector pTargetF-sgRNA-yihU A391G The acquisition

[0059] Mutant primer design: Using pTargetF-sgRNA-yihU as the starting vector, and based on the principle of reverse overlap extension PCR, a pair of mutant primers yihU were designed to introduce the mutation site in a one-step process. A391G -F and yihU A391G -R, where the primer's nucleotide sequence is: yihU A391G -F (e.g., SEQ ID NO.11 in the sequence list), yihU A391G -R (e.g., SEQ ID NO. 12 in the sequence list).

[0060] The purpose of introducing mutation sites is to reduce the expression of the yiHU gene.

[0061] PCR amplification: PCR amplification was performed using the recombinant plasmid pTargetF-sgRNA-yihU constructed in step 4 as a template and the designed mutation site primers. The PCR amplification reaction system was as follows: total volume 50 μL: 25 μL 2×Phanta Max Master Mix, yihU A391G -F (10 μmol / L) 2 μL, yihU A391G2 μL of R (10 μmol / L), 2 μL of template, 1 μL of dNTP Mix (10 mM), 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and 17 μL of ddH2O; the PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 56℃ annealing for 15 sec, 72℃ extension for 3 min, 32 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0062] The amplification products were digested with Dpn I: the mixture consisted of 50 μL of the amplification product and 1 μL of Dpn I, which were gently mixed and incubated at 37°C for 1 h. The amplification products were then digested with Dpn I to remove the methylated template plasmid.

[0063] Recombination reaction: The amplification product after Dpn I digestion can be efficiently recombinated at the target mutation site under the catalysis of Exnase II to achieve pTargetF-sgRNA-yihU A391G The in vitro cyclization was performed on ice. Specific steps: The recombinant reaction system consisted of a total volume of 20 μL: 200 ng of Dpn I digest, 4 μL of 5×CE II Buffer, 2 μL of Exnase II, and 14 μL of ddH2O. The mixture was gently mixed using a pipette, and the reaction solution was collected to the bottom of the tube after a brief centrifugation. After incubating in a water bath at 37°C for 30 min, the solution was immediately placed on ice to cool.

[0064] 6. Recombinant product pTargetF-sgRNA-yihU A391G Transformation: Thaw cloned DH5α competent cells on ice, and take 10 μL of the recombinant product pTargetF-sgRNA-yihU. A391G Add to 100 μL of DH5α competent cells, gently tap the tube wall to mix, and incubate on ice for 30 min. Heat shock at 42°C for 45 sec, then immediately cool on ice for 2 min. Add 900 μL of LB liquid medium, and incubate at 37°C for 1 h with shaking at 220 rpm. Preheat LB solid medium plates in a 37°C incubator, centrifuge at 5000 rpm for 5 min, and discard 900 μL of supernatant. Resuspend the cells in the remaining medium and spread using a sterile spreader onto preheated LB solid medium plates at 37°C. Incubate upside down in a 37°C incubator for 12 h.

[0065] 7. Extract recombinant plasmid pTargetF-sgRNA-yihU A391G The recombinant product from step 6 was transformed into positive colonies grown on plates and subjected to pTargetF-sgRNA-yihU. A391G Plasmid extraction was performed using the Novizan plasmid extraction kit.

[0066] 8. Engineered Escherichia coli E. coli-yihU A391G The acquisition

[0067] (1) Preparation and transformation of competent cells

[0068] (i) Pick a single colony of Escherichia coli CGMCC 1.366, culture it in seed medium until the bacterial concentration OD600 is 0.6, place it on ice to cool to 4°C, centrifuge after cooling, wash the bacterial cells twice with pre-cooled buffer, resuspend the bacterial cells in buffer to obtain first competent cells;

[0069] (ii) Transform the plasmid pCas (knockout tool plasmid) into the first competent cells obtained in step (i), culture at 30°C for 12 h, and screen to obtain recombinant bacteria.

[0070] The above seed culture medium consists of the following components per liter: 8g peptone, 4g yeast extract, 8g sodium chloride, and the remainder water.

[0071] The above buffer solution comprises the following components per liter: 8g of anhydrous calcium chloride, with the remainder being water.

[0072] (iii) Prepare the recombinant bacteria obtained in step (ii) into second competent cells, and use the recombinant plasmid pTargetF-sgRNA-yihU obtained in step 5. A391G Transfected into second-competent cells, plated on solid LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar powder, and water balance) containing 50 μg / mL kanamycin resistance and 50 μg / mL spectinomycin resistance, and incubated overnight at 30°C. Positive colonies were screened to obtain *Escherichia coli* (E. coli-yihU). A391G -pCas-pTargertF.

[0073] (2) Validation of positive bacteria

[0074] Select positive colonies from step (1) (screen transformants), inoculate them into LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and the remainder water) containing 50 μg / mL kanamycin resistance and 50 μg / mL spectinomycin resistance, and incubate overnight at 30°C. After incubation, use the bacterial culture as a template and UP-F and DOWN-R as primers for PCR amplification. The PCR amplification system is 25 μL, 2 μL 2×Phanta Max Master Mix, 2 μL UP-F (10 μmol / L), 2 μL DOWN-R (10 μmol / L), 19 μL template, and dd H2O. The PCR amplification program is as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 sec, 55°C annealing for 15 sec, 72°C extension for 60 sec, 30 cycles; 72°C extension for 10 min, and storage at 4°C.

[0075] Agarose gel electrophoresis was used to examine the PCR products. The agarose gel electrophoresis image of the positive bacteria is shown below. Figure 1 . Figure 1 The results showed that using primers UP-F and DOWN-R amplified a specific gene band, close to the theoretical value of 1897 bp. Sequencing was then performed, and the sequencing verification image of the positive clone is shown below. Figure 2 . Figure 2 The results showed that the sequence was correct, indicating that yihU A391G Gene replacement was successful.

[0076] Example 2

[0077] 1. Obtaining the recombinant vector pTargetF-sgRNA-yihU-KO

[0078] Mutant primer design: Using the recombinant plasmid pTargetF-sgRNA-yihU constructed in step 4 of Example 1 as the starting vector, a pair of amplification primers KO-F and KO-R were designed based on the principle of reverse overlap extension PCR. The nucleotide sequences of the primers are: KO-F (as shown in SEQ ID NO.14 in the sequence listing) and KO-R (as shown in SEQ ID NO.15 in the sequence listing).

[0079] PCR amplification: Using the recombinant plasmid pTargetF-sgRNA-yihU constructed in step 4 of Example 1 as a template and amplification primers KO-F and KO-R, PCR amplification was performed. The PCR amplification reaction system was as follows: total system volume 50 μL: 25 μL 2×Phanta MaxMaster Mix, 2 μL KO-F (10 μmol / L), 2 μL KO-R (10 μmol / L), 2 μL template, 1 μL dNTP Mix (10 mM), 1 μL Phanta Max Super-Fidelity DNA Polymerase, and 17 μL ddH2O; the PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 56℃ annealing for 15 sec, 72℃ extension for 3 min, 32 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0080] The amplification products were digested with Dpn I: the mixture consisted of 50 μL of the amplification product and 1 μL of Dpn I, which were gently mixed and incubated at 37°C for 1 h. The amplification products were then digested with Dpn I to remove the methylated template plasmid.

[0081] Recombination reaction: The amplified product after Dpn I digestion was circularized in vitro with exnase II catalysis to achieve pTargetF-sgRNA-yihU-KO. The operation was performed on ice. Specific steps: The recombination reaction system consisted of a total volume of 20 μL: 200 ng of Dpn I digestion product, 4 μL of 5×CE II Buffer, 2 μL of Exnase II, and 14 μL of ddH2O. The mixture was gently mixed with a pipette, and the reaction solution was collected to the bottom of the tube after a brief centrifugation. The tube was incubated at 37°C for 30 min, and then immediately cooled on ice.

[0082] 2. Transformation with recombinant product pTargetF-sgRNA-yihU-KO: Thaw cloning DH5α competent cells on ice. Add 10 μL of recombinant product pTargetF-sgRNA-yihU-KO to 100 μL of DH5α competent cells, gently tap the tube wall to mix, and incubate on ice for 30 min. After heat shock in a 42℃ water bath for 45 sec, immediately cool on ice for 2 min. Add 900 μL of LB liquid medium and incubate at 37℃ for 1 h at 220 rpm. Preheat LB solid medium plates in a 37℃ incubator, centrifuge at 5000 rpm for 5 min, and discard 900 μL of supernatant. Resuspend the bacterial cells in the remaining medium and spread them onto preheated LB solid medium plates in a 37℃ incubator using a sterile spreader. Incubate upside down in a 37℃ incubator for 12 h.

[0083] 3. Extraction of recombinant plasmid pTargetF-sgRNA-yihU-KO: The recombinant product obtained in step 2 was transformed into positive colonies grown on plates and the recombinant plasmid pTargetF-sgRNA-yihU-KO was extracted using the Novizan plasmid extraction kit.

[0084] The recombinant plasmid pTargetF-sgRNA-yihU-KO is used to knock out the yihU gene in Escherichia coli.

[0085] 4. Obtaining the engineered E. coli strain E. coli-yihU-KO

[0086] (1) Preparation and transformation of competent cells

[0087] (i) Pick a single colony of Escherichia coli CGMCC 1.366, culture it in seed medium until the bacterial concentration OD600 is 0.6, place it on ice to cool to 4°C, centrifuge after cooling, wash the bacterial cells twice with pre-cooled buffer, resuspend the bacterial cells in buffer to obtain first competent cells;

[0088] (ii) Transform the plasmid pCasp (knockout tool plasmid) into the first competent cells obtained in step (i), culture at 30°C for 12 h, and screen to obtain recombinant bacteria.

[0089] The above seed culture medium consists of the following components per liter: 8g peptone, 4g yeast extract, 8g sodium chloride, and the remainder water.

[0090] The above buffer solution comprises the following components per liter: 8g of anhydrous calcium chloride, with the remainder being water.

[0091] (iii) The recombinant bacteria obtained in step (ii) were prepared as second competent cells. The recombinant plasmid pTargetF-sgRNA-yihU-KO obtained in step 3 of this embodiment was transformed into the second competent cells and plated on solid LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar powder, and water) containing 50 μg / mL kanamycin resistance and 50 μg / mL spectinomycin resistance. The medium was cultured overnight at 30 °C and positive colonies were screened to obtain Escherichia coli-yihU-KO-pCas-pTargertF.

[0092] (2) Elimination of plasmids pCas and pTargetF-sgRNA-yihU-KO

[0093] The *E. coli* obtained in step (1) was inoculated into LB medium containing 50 μg / mL kanamycin resistance and cultured at 30°C until the OD600 reached 1.0. IPTG was added to a final concentration of 0.5 mM and induced at 25°C for 24 h. The bacterial culture was then spread onto LB solid medium without resistance and cultured overnight at 37°C. Single colonies were picked and inoculated sequentially into LB solid medium without resistance, LB medium containing 50 μg / mL spectinomycin resistance, and LB medium containing 50 μg / mL kanamycin resistance. The strain that grew only on the resistance-free medium was selected as *E. coli* *yihU-KO* without plasmids pCas and pTargetF-sgRNA-yihU-KO.

[0094] (3) Verification

[0095] Pick the E. coli-yihU-KO from step (2) and inoculate it into LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and the remainder water) and incubate overnight at 30°C. After incubation, use the bacterial culture as a template and UP-F and DOWN-R as primers for PCR amplification. The PCR amplification system is 25 μL, 2× Phanta Max Master Mix 2 μL, UP-F (10 μmol / L) 2 μL, DOWN-R (10 μmol / L) 2 μL, template 19 μL, and dd H2O. The PCR amplification program is as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 sec, 55°C annealing for 15 sec, 72°C extension for 60 sec, 30 cycles; 72°C extension for 10 min, and storage at 4°C.

[0096] Agarose gel electrophoresis was used to examine the PCR products. The agarose gel electrophoresis image of the positive bacteria is shown below. Figure 3 . Figure 3 The results showed that a band close to the theoretical value of 1000bp was amplified using primers UP-F and DOWN-R, indicating that the yihU gene knockout was successful.

[0097] Example 3 L-arginine fermentation experiment

[0098] The *Escherichia coli* prepared in Example 1 was used. A391G-pCas-pTargertF, *E. coli*-yihU-KO prepared in Example 2, and the original strain *E. coli* CGMCC 1.366 were inoculated into 100 mL of LBG medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 5 g / L glucose, and the remainder water), and seed cultured at 220 rpm and 30°C for 16 h. Subsequently, at a 5% (v / v) inoculation rate, they were inoculated into 100 mL of fermentation medium (20 g / L glucose, 20 g / L (NH4)2SO4, 5 g / L urea, 2 g / L KH2PO4, 1.35 g / L MgSO4·7H2O, and 1.35 g / L FeSO4·7H2O). Fermentation was carried out for 50 h with 0.02 g / L of Bacillus subtilis, 0.04 g / L of MnSO4·H2O, 0.008 g / L of nicotinamide, 0.001 g / L of biotin, and the balance water. The fermentation broth was sampled and centrifuged immediately after sampling at 12000 rpm for 2 min. The supernatant of the centrifuged broth was collected, and the L-arginine content in the fermentation broth was determined by Agilent high-performance liquid chromatography. The average value of the three fermentation measurements was taken. The calculated L-arginine content is shown in Table 1.

[0099] Table 1. L-arginine production by engineered and original Escherichia coli strains

[0100]

[0101] The results showed that, compared with the original bacteria, after 48 hours of fermentation, *E. coli*-yihU... A391G The L-arginine content in the fermentation broths of *E. coli*-pCas-pTargertF and *E. coli*-yihU-KO reached 0.68 g / L and 0.77 g / L, respectively, while the L-arginine content in the fermentation broth of the original strain was 0.42 g / L after 48 h. The engineered *E. coli* strain *E. coli*-yihU... A391G The L-arginine production of E. coli-yihU-KO was increased by 61.9% and 83.3% respectively compared with the original strain; and compared with the original strain, the L-arginine production of the engineered strain reached 0.58 g / L after 24 hours, saving about 12 hours of fermentation time. This indicates that yihU A391G Replacing or knocking out the yihU gene in Escherichia coli with a gene mutant can increase the acid production level of L-arginine fermentation in Escherichia coli.

[0102] 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. A recombinant microorganism for high production of L-arginine, characterized by, The recombinant bacteria comprise a yihU gene mutant or a knockout yihU gene; The nucleotide sequence of the yihU gene is shown in SEQ ID NO. 2 in the sequence listing; The nucleotide sequence of the yihU gene mutant is shown in SEQ ID NO. 1 in the sequence listing, wherein A at position 391 of the yihU gene mutant is mutated to G, as compared with the yihU gene with the nucleotide sequence of SEQ ID NO. 2 in the sequence listing. The starting strain of the recombinant bacteria is Escherichia coli CGMCC 1.

366.

2. The recombinant microorganism of claim 1, wherein, The protein encoded by the yihU gene mutant has an amino acid sequence shown in SEQ ID NO. 13 in the sequence listing.

3. A method for constructing a recombinant microorganism producing L-arginine at a high yield according to any one of claims 1 to 2, characterized by, When the recombinant bacteria comprise a yihU gene mutant, the construction method is to amplify a recombinant plasmid pTargetF-sgRNA-yihU as a template, introduce a mutation site, and obtain a recombinant plasmid pTargetF-sgRNA-yihU A391G Then, the recombinant plasmid pTargetF-sgRNA-yihU A391G is introduced into the starting strain; When the recombinant bacteria knockout the yihU gene, the construction method is as follows: a recombinant plasmid pTargetF-sgRNA-yihU is used as a template, KO-F and KO-R are used as amplification primers to obtain a recombinant plasmid pTargetF-sgRNA-yihU-KO, then the recombinant plasmid pTargetF-sgRNA-yihU-KO is introduced into the starting strain, and the recombinant plasmid pTargetF-sgRNA-yihU-KO is knocked out.

4. The method for constructing a recombinant bacterium for high production of L-arginine according to claim 3, characterized by, The primers used to introduce the mutation site are yihU A391G -F and yihU A391G -R, the nucleotide sequences are SEQ ID NO. 11 and SEQ ID NO. 12 in the sequence listing, respectively; The nucleotide sequences of the amplification primers KO-F and KO-R are shown in SEQ ID NO. 14 and SEQ ID NO. 15 in the sequence listing, respectively.

5. The method for constructing a recombinant L-arginine high-producing bacterium according to claim 3, wherein The recombinant plasmid pTargetF-sgRNA-yihU is obtained by connecting a yihU gene fragment, an upstream homologous fragment and a downstream homologous fragment into a linearized vector through a seamless cloning kit; The yihU gene fragment is obtained by PCR amplification with the genomic DNA of Escherichia coli CGMCC 1.366 as a template and yihU-F and yihU-R as primers; the nucleotide sequences of yihU-F and yihU-R are shown in SEQ ID NO. 9 and SEQ ID NO. 10 in the sequence listing; The upstream homologous fragment is obtained by PCR amplification with the genomic DNA of Escherichia coli CGMCC 1.366 as a template and UP-F and UP-R as primers; the nucleotide sequences of UP-F and UP-R are shown in SEQ ID NO. 5 and SEQ ID NO. 6 in the sequence listing; The downstream homologous fragment is obtained by PCR amplification with the genomic DNA of Escherichia coli CGMCC 1.366 as a template and DOWN-F and DOWN-R as primers; the nucleotide sequences of DOWN-F and DOWN-R are shown in SEQ ID NO. 7 and SEQ ID NO. 8 in the sequence listing.

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