Escherichia coli genetically engineered bacteria for fermentative production of l-citrulline, construction method and application

Abstract

This invention relates to a genetically engineered *E. coli* strain for the fermentation production of L-citrulline, its construction method, and its application, belonging to the field of genetic engineering technology. It focuses on the encoding of glutamate dehydrogenase in *E. coli* W3110. gdh Gene A, glutamate-pyruvate aminotransferase ala A and the ATP-binding subunit of the L-arginine ABC transporter. art The P gene was overexpressed; simultaneously, the enzyme encoding arginine succinate synthase was activated. arg G gene and encoding argininosuccinate lyase arg The H gene was knocked out, and the resulting recombinant strain was used to ferment and produce L-citrulline. The acid production was high and it can be applied to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the fermentation production of L-citrulline, specifically to the Escherichia coli genetically engineered strain for fermenting L-citrulline, its construction method, and its application. Background Technology

[0002] Citrulline is a neutral, non-essential, non-protein amino acid and an intermediate in the urea cycle, playing a vital role in maintaining blood ammonia balance. It can be used to treat rheumatoid arthritis; prevent cardiovascular disease; has excellent antioxidant properties, scavenging free radicals in the body and, to some extent, improving immunity and delaying aging; it can also increase intracellular cyclic guanosine monophosphate levels, potentially preventing male diseases and improving sexual function.

[0003] Currently, there are four main methods for producing citrulline:

[0004] First, it can be extracted from plants rich in citrulline, but the yield and extraction rate are low.

[0005] Secondly, chemical organic synthesis generates a large amount of industrial wastewater and waste gas during the synthesis process, which is very harmful to the environment and is prone to producing D-type enantiomers, which is not conducive to subsequent separation and purification.

[0006] Third, enzyme-catalyzed production, using fecal streptococci (… Streptococcus faecalis Listeria monocytogenes (); Listeria monocytogenes ); Lactococcus lactis ( Lactococcus lactis Arginine deiminoase produced by microorganisms such as arginine catalyzes the production of citrulline from arginine;

[0007] Fourth, microbial fermentation is a production method. Direct fermentation yields high output and high product purity. No harmful substances are generated during the production process, extraction and purification are convenient, and costs are reduced, making it suitable for industrial production.

[0008] L-citrulline is an amino acid in the glutamate family. It is synthesized from glutamate as a precursor through the catalysis of 8-9 enzymes. However, L-citrulline does not accumulate in large quantities in microorganisms as an intermediate metabolite. Instead, it is catalyzed by arginine succinate synthase to produce arginine, resulting in a low citrulline content. Summary of the Invention

[0009] To address the shortcomings of low acid production during fermentation in existing citrulline production processes, this invention aims to: 1) provide a genetically engineered *E. coli* strain for the fermentation of L-citrulline; 2) provide a method for constructing the genetically engineered *E. coli* strain; 3) provide the application of the genetically engineered *E. coli* strain in the fermentation production of L-citrulline; and 4) provide a method for the fermentation production of L-citrulline. This invention utilizes genetic engineering techniques, starting with *E. coli* W3110 as the starting strain, for genetic modification, and optimizes the corresponding fermentation control scheme, resulting in a significant increase in the fermentation yield of L-citrulline.

[0010] The specific solution adopted in this invention is as follows:

[0011] A genetically engineered *Escherichia coli* strain for the fermentation production of L-citrulline, wherein the genetically engineered strain is *Escherichia coli* W3110 as the host strain and is genetically modified to encode arginine succinate synthase. arg G gene and encoding argininosuccinate lyase arg The H gene was knocked out; simultaneously, the enzyme encoding glutamate dehydrogenase was inhibited. good Gene A, glutamate-pyruvate aminotransferase good A and the ATP-binding subunit of the L-arginine ABC transporter. art The P gene is overexpressed.

[0012] The above-mentioned method for constructing the genetically engineered *Escherichia coli* strain for fermentation to produce L-citrulline involves using CRISPR-Cas9 gene editing technology to modify the host bacterial genome. arg G and arg The H gene is knocked out and then integrated. good A, good A and art Plasmid containing the P gene fragment.

[0013] The construction method specifically includes the following steps:

[0014] Step 1: Construction of pGBR plasmid: Using primer Sg- arg GF, Sg- arg GR and Sg- arg HF, Sg- arg HR, constructing PCR-containing target sequences respectively arg G-pGRB and arg H-pGRB plasmid; wherein, primer Sg- arg GF, Sg- arg GR and Sg- arg HF, Sg- arg The nucleotide sequences of HR are shown in SEQ ID NO: 04~07;

[0015] Step 2: Preparation of recombinant DNA fragments: The recombinant DNA fragments consist of the upstream and downstream homologous arms of the knockout gene; the knockout gene is amplified separately using PCR. arg G gene and knockout arg The upstream and downstream homologous arms of the H gene were extracted, and then the upstream and downstream homologous fragments were ligated to prepare knockout samples. arg G recombinant DNA fragment and knockout arg H recombinant DNA fragment;

[0016] Step 3: Transform the pCas9 plasmid into Escherichia coli W3110 to prepare competent cells of W3110 strain containing the pCas9 plasmid;

[0017] Step 4: Simultaneously transform the pGBR plasmid constructed in Step 1 and the recombinant DNA fragment constructed in Step 2 into competent cells of strain W3110 containing the pCas9 plasmid obtained in Step 3. Verify and screen for positive recombinants, eliminate the tool plasmid, and obtain W3110-Δ. arg G-△ arg H mutant strain;

[0018] Step 5: Amplify separately good A, good A, art Using the P gene fragment and the PBR332 plasmid fragment, a gene recombination method was employed to construct a structure containing... good A, good A and art The recombinant plasmid of the P gene was then transferred into the W3110-△ gene obtained in step four. arg G-△ arg The genetically engineered Escherichia coli strain that produces L-citrulline through fermentation was obtained from competent cells of the H mutant strain through positive verification.

[0019] The application of the above-mentioned genetically engineered Escherichia coli in the fermentation production of L-citrulline.

[0020] A method for producing L-citrulline by fermentation, using the above-mentioned Escherichia coli genetically engineered bacteria for fermentation production.

[0021] As a further optimization of the above-mentioned method for producing L-citrulline by fermentation, the fermentation medium formula used for fermentation is as follows: glucose: 10-30 g / L, yeast powder 2-8 g / L, peptone 2-8 g / L, magnesium sulfate 1-5 g / L, ammonium sulfate 1-5 g / L, dipotassium hydrogen phosphate 3-7 g / L, ferrous sulfate and manganese sulfate 15-30 mg / L, arginine 0.1-0.3 g / L; the remainder is water, pH 6.8-7.2.

[0022] As a further optimization of the above-mentioned method for producing L-citrulline by fermentation, the fermentation process conditions are as follows: inoculum size of fermentation broth 10%-15%, pH 6.5-7.5, culture temperature 30-40℃, and aeration rate of 0.2-1.0 m³ / h. 3 The stirring speed is 100-600 r / min, and the fermentation time is 30-65 h.

[0023] Beneficial effects: This study used Escherichia coli W3110 as the starting strain to overexpress the gene for glutamate dehydrogenase. good A and glutamate-pyruvate aminotransferase good A, promotes glutamate production and overexpresses the gene encoding the ATP-binding subunit of the L-arginine ABC transporter. art P enhances the extracellular transport of citrulline, while CRISPR-Cas9 gene editing technology is used to target the enzyme encoding arginine succinate synthase. arg G and arg The H gene was deleted to inhibit the further synthesis of L-arginine from L-citrulline. A modified strain W3110-G capable of accumulating L-citrulline was constructed, and its production performance was studied, providing an industrial basis for the direct fermentation production of L-citrulline. Attached Figure Description

[0024] Figure 1 This is a pCas9 plasmid map;

[0025] Figure 2 This is a pGBR plasmid map;

[0026] Figure 3 yes argG Construction and validation electrophoresis images of gene knockout fragments;

[0027] Figure 4 This is an electrophoresis diagram showing the construction and verification of the argH gene knockout fragment;

[0028] Figure 5 This is a diagram of the fermentation process in a 30L fermenter. Detailed Implementation

[0029] This invention uses Escherichia coli (E. coli) Escherichia coli W3110 was genetically engineered to serve as the host bacterium. This was applied to *Escherichia coli* (E. coli). Escherichia coli W3110 encodes glutamate dehydrogenase. good Gene A, glutamate-pyruvate aminotransferase goodA and the L-arginine ABC transporter ATP-binding subunit. art The P gene was overexpressed; simultaneously, the enzyme encoding argininosuccinate synthase was expressed. arg The G gene and its encoding argininosuccinate lyase arg The H gene was knocked out, and the resulting recombinant strain was used to study the fermentation production of L-citrulline.

[0030] First, the host bacteria were deprived of argininosuccinate synthase and argininosuccinate lyase activities, thus blocking the further degradation of L-citrulline into argininosuccinate and arginine. Then, the gene encoding the ATP-binding subunit of the L-arginine ABC transporter was overexpressed in plasmid form. art P, gene encoding glutamate dehydrogenase good A and glutamate-pyruvate aminotransferase good A. Enhances the transport of citrulline and the synthesis and expression of its precursor glutamate.

[0031] In this invention, the further degradation of citrulline produced by the host bacteria into arginine is achieved by knocking out the host's arginine succinate synthase. arg G and argininosuccinate lyase arg H implementation.

[0032] In this invention, enhancing the synthesis of glutamate, the precursor of citrulline, in the host bacteria is mainly achieved by overexpressing glutamate dehydrogenase in plasmid form. good A and glutamate-pyruvate aminotransferase good Gene A is achieved.

[0033] In this invention, enhancing the host bacterium's transport of citrulline is primarily achieved by overexpressing the L-arginine ABC transporter ATP-binding subunit gene in plasmid form. art P gene realization.

[0034] In this invention, the genetically engineered bacteria are modified at specific sites in the host genome using CRISPR-Cas9 gene editing technology.

[0035] The present invention also provides a method for preparing L-citrulline, wherein fermentation is carried out under conditions suitable for the growth of engineered bacteria, and L-citrulline is collected from the fermentation broth.

[0036] The preferred culture medium composition in the method is as follows: glucose: 10-30 g / L, yeast extract 2-8 g / L, peptone 2-8 g / L, magnesium sulfate 1-5 g / L, ammonium sulfate 1-5 g / L, dipotassium hydrogen phosphate 3-7 g / L, ferrous sulfate and manganese sulfate 15-30 mg / L, arginine 0.1-0.3 g / L. The remainder is water, pH 6.8-7.2.

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0038] Example 1: Construction of genetically engineered bacteria W3110-G.

[0039] 1. arg G, arg H gene knockout.

[0040] Using CRISPR-Cas9 gene editing technology, E. coli W3110 group was subjected to... arg G and arg The H gene is knocked out. This system uses two tool plasmids; a plasmid map is attached. Figure 1 and 2 The pCas9 contains the elimination system of the sgRNA expression plasmid pGBR, the Red recombination system, and the Cas9 protein expression system, and is resistant to kanamycin (working concentration: 50 mg / L), and is cultured at 32°C; pGBR contains the J23119(SpeI) promoter and pBR322 replicon, the sgRNA-Cas9 protein binding region sequence, and is resistant to streptomycin (working concentration: 50 mg / L).

[0041] The specific steps of this method are as follows:

[0042] 1.1 Construction of pGBR plasmid.

[0043] 1.1.1 Construction of plasmids.

[0044] The pGBR plasmid was constructed to transcribe the corresponding sgRNA (sequence shown in Table 1) in bacterial cells, which then binds to the Cas9 protein to form a complex. This complex recognizes the target gene through base pairing and cleaves it, causing a double-strand break in the target gene's DNA. The target sequence was designed using Cas-Designer, and the pGBR plasmid was constructed using PCR.

[0045] Using primer Sg- arg GF, Sg- arg GR and Sg- arg HF, Sg- arg HR reverse PCR amplifies the signal containing the target sequence. arg G-pGRB and argH-pGRB plasmid. Digested with DPNⅠ at 37℃ for 40 min to remove template plasmid.

[0046] 1.1.2 Plasmid transformation.

[0047] Take 10 μL of PCR reaction solution and add it to 100 μL of Jm109 competent cells. Mix well and incubate on ice for 30 min. Heat shock at 42°C for 60-100 s, then immediately incubate on ice for 3 min. Add 800 μL of LB medium and incubate at 37°C for 60-90 min. Centrifuge at 5000 rpm for 5 min, discard the supernatant, and resuspend the bacterial cells in 100 μL. Spread the resuspended bacterial cells onto a plate containing 50 mg / L streptomycin. Invert the plate and incubate overnight at 37°C. After bacterial growth, select positive recombinants.

[0048] The selected positive recombinants were inoculated into a medium containing streptomycin resistance and cultured overnight to extract plasmids for later use.

[0049] 1.2 Preparation of recombinant DNA fragments.

[0050] The recombinant DNA fragment consists of upstream and downstream homologous arms of the knockout gene. Using the primer design software Snapgene 5.2, primers for the upstream and downstream homologous arms (approximately 500 bp in length) were designed using the upstream and downstream sequences of the gene to be knocked out as templates; primers for the integration gene sequence were designed using the gene to be integrated as a template. The upstream and downstream homologous arms were amplified separately by PCR, and the recombinant fragment was prepared by overlapping PCR.

[0051] 1.2.1 Knockout arg Preparation of G recombinant DNA fragments

[0052] First, use primers. arg G-F1 / arg G-R1 and primers arg G-F2 / arg G-R2 was amplified using the E. coli W3110 genome as a template to create a knockout gene. arg The upstream and downstream homologous arm fragments required for the G gene were obtained, and then these two fragments were purified and recovered separately. Primers were used... arg G-F1 / arg G-R2 uses fusion PCR to ligate two fragments to create a recombinant DNA fragment, which is then purified and recovered for later use.

[0053] 1.2.2 Knockout arg Preparation of H recombinant DNA fragment

[0054] First, use primers. arg H-F1 / arg H-R1 and primers arg H-F2 / argH-R2 was amplified using the E. coli W3110 genome as a template to create a knockout gene. arg The upstream and downstream homologous arm fragments required for the H gene were obtained, and then these two fragments were purified and recovered separately. Primers were used... arg H-F1 / arg H-R2 combines two fragments via fusion PCR to create a recombinant DNA fragment, which is then purified and recovered for later use.

[0055] The primer sequences are shown in Table 1 below.

[0056] 1.3 Transformation of plasmids and recombinant fragments.

[0057] 1.3.1 Transformation of pCas plasmid.

[0058] The plasmid was transformed into the prepared W3110 competent cells using chemical transformation. After the cells were revived and cultured at 32°C, they were plated onto LB agar plates containing kanamycin and incubated overnight. Single colonies growing on the resistant plates were identified to screen for the correct recombinants.

[0059] 1.3.2 Preparation of electrocompetent cells of strain W3110 containing pCas9 plasmid.

[0060] The W3110 strain containing the pCas9 plasmid was cultured at 32℃. When the OD≈0.15, arabinose was added to induce the expression of the pCas9 plasmid recombinant enzyme system. The culture was continued until the OD≈0.3, at which point competent cells were prepared.

[0061] 1.3.3 Electroporation of pGRB and recombinant DNA fragments.

[0062] pGRB and the recombinant DNA fragment were simultaneously electroporated into electroporated competent cells of strain W3110 containing the pCas9 plasmid. After recovery for 60-90 min, the cells were plated onto plates containing kanamycin and streptomycin and incubated overnight at 32°C. Colony PCR was performed using the upstream primer of the upper homologous arm and the downstream primer of the lower homologous arm to screen for positive recombinants.

[0063] 1.4 Elimination of tool plasmids.

[0064] 1.4.1 Elimination of pGBR plasmids.

[0065] Positive recombinants were placed in LB medium containing IPTG and kanamycin resistance and cultured at 32°C. After three consecutive subcultures, the cultures were diluted appropriately and plated onto plates containing kanamycin, and incubated at 32°C. Single colonies that grew on kanamycin plates but not on streptomycin plates were selected for preservation.

[0066] 1.4.2 Elimination of pCas9 plasmid.

[0067] Positive recombinants that eliminated the pGBR plasmid were inoculated into antibiotic-free LB medium and cultured at 42°C. After three consecutive passages, the cultures were diluted appropriately and plated onto LB agar plates and incubated at 37°C. Single colonies that grew on antibiotic-free LB agar plates but did not grow on kanamycin agar plates were selected for preservation.

[0068] 2. good A, good A and art Construction of P gene overexpression plasmid

[0069] Escherichia coli (E. coli) Escherichia coli Using the W3110 genome and plasmid PBR332 as templates, good AF, good AR art PF, art PR, good AF, good AR, PBR-F, and PBR-R are primers for amplification. good A, good A, art The P gene fragment and the PBR332 plasmid fragment were then used to construct a gene containing P gene fragments and PBR332 plasmid fragments. good A, good A and art A new plasmid for the P gene was created. The recombinant plasmid was then transformed into W3110-Δ. arg G-△ arg H competent cells.

[0070] good The nucleotide sequence of gene A is shown in SEQ ID NO: 01; alaA The nucleotide sequence of the gene is shown in SEQ ID NO: 02; art The nucleotide sequence of the P gene is shown in SEQ ID NO: 03.

[0071] Table 1: Primer sequence listing.

[0072]

[0073] Example 2: Shake flask fermentation of genetically engineered strain W3110-G.

[0074] Take the bacterial strain stored at -80℃, streak it on an agar slant, and incubate at 37℃ for 12 hours. Subculture twice to restore the strain viability. Use a sterile inoculation loop to inoculate the strain into seed culture medium and incubate at 37℃ for 7-10 hours. Inoculate the seed culture medium into the fermentation medium at a rate of 10%, making the fermentation medium volume 10% of the shake flask. Incubate at 37℃ with shaking at 200 rpm. During fermentation, add dilute ammonia and glucose solution to maintain normal fermentation and pH. The fermentation cycle is 30 hours.

[0075] The slant culture medium consisted of 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the remainder was water, with a pH of 6.9-7.1.

[0076] The seed culture medium consisted of 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and the remainder was water, with a pH of 6.9-7.1.

[0077] The fermentation medium consisted of: glucose 20 g / L, yeast extract 5 g / L, peptone 5 g / L, magnesium sulfate 2 g / L, ammonium sulfate 2 g / L, dipotassium hydrogen phosphate 3.5 g / L, ferrous sulfate and manganese sulfate 20 mg / L, and arginine 0.2 g / L. The remainder was water, with a pH of 6.8-7.2.

[0078] After 30 hours of shake-flask culture, the L-citrulline accumulation in the fermentation broth of the genetically engineered strain W3110-G reached 3.1 g / L, indicating that enhancing the synthesis and expression of L-citrulline precursors and blocking the degradation pathway of L-citrulline can increase the accumulation of L-citrulline.

[0079] Example 3: Fermentation of genetically engineered strain W3110-G in a 30L fermenter

[0080] Take the bacterial strain stored at -80℃, streak it on an agar slant, and incubate at 37℃ for 12 hours. Subculture twice to restore the strain's viability. Use a sterile inoculation loop to inoculate the strain into seed culture medium and incubate at 37℃ for 7-10 hours.

[0081] Take 50 mL of the cultured seeds and inoculate them into a sterilized seed tank, bringing the total volume of the seed tank to 7 L. The seed tank was calibrated to 100% dissolved oxygen under the following conditions: 37℃, pH 7.0, agitator speed of 500 rpm, and aeration rate of 0.5 m³ / h. 3 / h, tank pressure 0.05MPa; 0% dissolved oxygen calibration conditions: 37℃, pH 7.0, rotation speed 200rpm, air flow rate 0.2m³ / h. 3 / h, tank pressure 0.05MPa. During fermentation, dissolved oxygen is controlled to be >30% by controlling tank pressure, aeration rate and stirring speed. When OD600>25, the inoculum is transferred at a rate of 2L.

[0082] The fermenter was calibrated to a volume of 15L with 100% dissolved oxygen under the following conditions: 37℃, pH 7.0, rotation speed 500rpm, and aeration rate 0.9m³ / h. 3 / h, tank pressure 0.05MPa; 0% dissolved oxygen calibration conditions: 37℃, pH 7.0, rotation speed 200rpm, air flow rate 0.4m³ / h. 3 / h, tank pressure 0.05MPa. During fermentation, dissolved oxygen is controlled to >30% by controlling tank pressure, aeration rate and stirring speed, and the fermentation cycle is 60h.

[0083] Slant culture medium: yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L, agar powder 20g / L, the remainder is water, pH 6.9-7.1.

[0084] Seed culture medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, the remainder being water, pH 6.9-7.1.

[0085] Seed culture medium: glucose 20 g / L, yeast extract 5 g / L, peptone 4 g / L, magnesium sulfate 1.5 g / L, dipotassium hydrogen phosphate 2 g / L. The remainder is water, pH 6.8-7.2.

[0086] Fermentation medium: glucose 20 g / L, yeast extract 5 g / L, peptone 5 g / L, magnesium sulfate 2 g / L, ammonium sulfate 2 g / L, dipotassium hydrogen phosphate 3.5 g / L, ferrous sulfate and manganese sulfate 20 mg / L, arginine 0.2 g / L. The remainder is water, pH 6.8-7.2.

[0087] After 65 hours of fermentation, the accumulation of L-citrulline in the fermentation broth of engineered strain W3110-G reached 81.86 g / L. The OD and citrulline yields during fermentation were as follows: Figure 5 As shown, after extraction, the fermentation broth contains no other impurities, and the product purity is high, reaching 99.9%. Based on current technology, the preliminary estimated cost is 78,000 yuan / ton, while the current market price is 110,000 yuan / ton. With a production scale of 3,000 tons, the gross profit could reach 96 million yuan. This indicates that the strain constructed in this invention can directly ferment and produce L-citrulline, which can be applied to industrial production.

[0088] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.

Claims

1. A genetically engineered *Escherichia coli* strain for fermenting and producing L-citrulline, characterized by: The genetically engineered bacteria were created using Escherichia coli W3110 as the host bacterium through genetic engineering. CRISPR-Cas9 gene editing technology was employed to modify the host bacterium's genome, specifically targeting the enzyme encoding arginine succinate synthase. arg G gene and encoding argininosuccinate lyase arg The H gene was knocked out; simultaneously, the enzyme encoding glutamate dehydrogenase was inhibited. gdh Gene A, glutamate-pyruvate aminotransferase ala A and the ATP-binding subunit of the L-arginine ABC transporter. art P gene overexpression; The method is as follows: using primer Sg- arg GF, Sg- arg GR and Sg- arg HF, Sg- arg HR, constructing PCR-containing target sequences respectively arg G-pGRB and arg H-pGRB plasmid; wherein, primer Sg- arg GF, Sg- arg GR and Sg- arg HF, Sg- arg The nucleotide sequences of HR are shown in SEQ ID NO: 04~07; The knockout was amplified separately using PCR. arg G gene and knockout arg The upstream and downstream homologous arms of the H gene were extracted, and then the upstream and downstream homologous fragments were ligated to prepare knockout samples. arg G recombinant DNA fragment and knockout arg H recombinant DNA fragment; Will arg G-pGRB plasmid, arg H-pGRB plasmid, knockout arg G recombinant DNA fragment and knockout arg The recombinant DNA fragment H was transformed into competent cells of strain W3110 containing the pCas9 plasmid to obtain W3110-Δ. arg G-△ arg H mutant strain; then containing gdh A, ala A and art The recombinant plasmid containing the P gene was transformed into W3110-△ arg G-△ arg The genetically engineered bacteria were obtained from the H mutant strain.

2. The method for constructing the *Escherichia coli* genetically engineered strain for fermenting and producing L-citrulline according to claim 1, characterized in that: The construction method employs CRISPR-Cas9 gene editing technology to modify the host bacterial genome. arg G and arg The H gene is knocked out and then integrated. gdh A, ala A and art Plasmid containing the P gene fragment.

3. The construction method according to claim 2, characterized in that: Includes the following steps: Step 1: Construction of pGBR plasmid: Using primer Sg- arg GF, Sg- arg GR and Sg- arg HF, Sg- arg HR, constructing PCR-containing target sequences respectively arg G-pGRB and arg H-pGRB plasmid; wherein, primer Sg- arg GF, Sg- arg GR and Sg- arg HF, Sg- arg The nucleotide sequences of HR are shown in SEQ ID NO: 04~07; Step 2: Preparation of recombinant DNA fragments: The recombinant DNA fragments consist of the upstream and downstream homologous arms of the knockout gene; the knockout gene is amplified separately using PCR. arg G gene and knockout arg The upstream and downstream homologous arms of the H gene were extracted, and then the upstream and downstream homologous fragments were ligated to prepare knockout samples. arg G recombinant DNA fragment and knockout arg H recombinant DNA fragment; Step 3: Transform the pCas9 plasmid into Escherichia coli W3110 to prepare competent cells of W3110 strain containing the pCas9 plasmid; Step 4: Simultaneously transform the pGBR plasmid constructed in Step 1 and the recombinant DNA fragment constructed in Step 2 into competent cells of strain W3110 containing the pCas9 plasmid obtained in Step 3. Verify and screen for positive recombinants, eliminate the tool plasmid, and obtain W3110-Δ. arg G-△ arg H mutant strain; Step 5: Amplify separately gdh A, ala A, art Using the P gene fragment and the PBR332 plasmid fragment, a gene recombination method was employed to construct a structure containing... gdh A, ala A and art The recombinant plasmid of the P gene was then transferred into the W3110-△ gene obtained in step four. arg G-△ arg The genetically engineered Escherichia coli strain that produces L-citrulline through fermentation was obtained from competent cells of the H mutant strain through positive verification.

4. The application of the genetically engineered Escherichia coli as described in claim 1 in the fermentation production of L-citrulline.

5. A method for producing L-citrulline by fermentation, characterized in that: The Escherichia coli genetically engineered strain described in claim 1 is used for fermentation production.

6. The method for producing L-citrulline by fermentation according to claim 5, characterized in that: The fermentation medium used for fermentation is formulated as follows: glucose: 10-30 g / L, yeast powder 2-8 g / L, peptone 2-8 g / L, magnesium sulfate 1-5 g / L, ammonium sulfate 1-5 g / L, dipotassium hydrogen phosphate 3-7 g / L, ferrous sulfate and manganese sulfate 15-30 mg / L, arginine 0.1-0.3 g / L; the remainder is water, pH 6.8-7.

2.

7. The method for producing L-citrulline by fermentation according to claim 5, characterized in that: The fermentation process conditions are as follows: inoculum size 10%-15%, pH 6.5-7.5, culture temperature 30-40℃, and aeration rate 0.2-1.0 m³ / h. 3 The stirring speed is 100-600 r / min, and the fermentation time is 30-65 h.

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