Method for constructing high-yield arginine strain based on argB mutant and application thereof
By modifying Corynebacterium glutamicum through metabolic engineering, a genetically stable high-yield L-arginine engineered strain was constructed, solving the problems of low efficiency and poor stability in existing technologies and realizing high yield and industrialization potential.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NINGXIA HENGLI BIOLOGICAL NEW MATERIAL CO LTD
- Filing Date
- 2026-04-23
- Publication Date
- 2026-06-26
Smart Images
Figure CN122278740A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of bioengineering and microbial fermentation technology, specifically relating to a method for constructing a high-yield L-arginine-producing Corynebacterium glutamicum engineered strain obtained through metabolic engineering and protein engineering, and its application in L-arginine production. Background Technology
[0002] L-arginine is a basic amino acid that is commonly used in skincare products due to its neutralizing, moisturizing, and antioxidant properties. It also has wide applications in the feed, pharmaceutical, and food industries. With the continued growth in market demand, especially the expansion of feed-grade L-arginine production capacity, developing efficient, low-cost, and environmentally friendly production methods has become a focus of industry attention.
[0003] Currently, microbial fermentation is the main method for producing L-arginine, with Corynebacterium glutamicum as the primary method. (Corynebacterium glutamicum ) and Escherichia coli ( Escherichia coli Engineered strains such as [specific strain name] are used as catalysts to produce [product name] using renewable starch sugar as raw material. This technology has become mainstream due to its mild reaction conditions and environmental friendliness. Early studies mostly used random mutagenesis screening to obtain high-yielding strains, but due to the uncertainty and non-directedness of mutations, the breeding efficiency was low, and the genetic stability of the strains was poor, which was not conducive to industrial application. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a genetically stable, high-yield L-arginine engineered strain constructed through a combinatorial metabolic engineering strategy, as well as its construction method and application.
[0005] To achieve the above objectives, this invention first constructs a chassis strain with an abundant supply of L-arginine precursors by knocking out non-essential competitive pathways and negative regulatory factors. Secondly, it rationally modifies the key rate-limiting enzyme in the synthetic pathway, N-acetylglutamate kinase (AGT), through protein engineering. argB This relieved the feedback inhibition of L-arginine. Finally, through promoter engineering and increase in the copy number of the target gene, the modified N-acetylglutamate kinase (Akinase) was enhanced. argB *) and arginine transporter lysE The expression of L-arginine was thus achieved, leading to its efficient accumulation.
[0006] The technical solution adopted in this invention is as follows: This invention provides a method for constructing a high-yield L-arginine-producing Corynebacterium glutamicum engineered bacterium, the method comprising the following steps: Step 1: Construction of chassis strain ARG5 Using Corynebacterium glutamicum ATCC 13032 as the starting strain, its genome was progressively edited using homologous recombination technology (such as the pK18mobsacB plasmid system), sequentially knocking out the following genes to obtain the chassis strain ARG5. ΔargR ΔfarR ΔproB ΔamtR ΔyggB ): (1) Knockout of the gene encoding the arginine biosynthesis repressor protein argR This relieves its transcriptional inhibition of the arginine synthesis pathway; (2) Knockout of regulatory genes farR This further relieves transcriptional repression; (3) Knockout of the gene encoding glutamate kinase proB This blocks the competitive pathway for the synthesis of the byproduct L-proline, directing more carbon flow towards L-arginine. (4) Knockout of global nitrogen metabolism regulators amtR It relieves nitrogen metabolism restriction, enhances nitrogen source supply, and promotes arginine synthesis; (5) Knockout of the gene encoding the mechanosensitive channel protein yggB By altering cell membrane permeability and reducing the leakage of byproducts (such as glutamate), it indirectly promotes arginine accumulation.
[0007] Step 2: Anti-feedback inhibition mutant argB * Construction (1) Construction argB Gene knockout strain: Using genome editing technology, pK18mobsacB- argB The plasmid was introduced into the ARG5 strain described in step one, and its own plasmid was knocked out. argB Genes were used to obtain the ArgB auxotrophic strain ARG5-ΔargB.
[0008] (2) Construction argB Mutants: argB Using the gene as a template, mutations were introduced at 94, 158, and 273 using mutagenesis technology. The mutant products were then ligated into the expression vector pXMJ19 to construct pXMJ19- argB *
[0009] Step 3: High-yield strain ARG6-Ptac- argB * / pXMJ19- lysE Construction (1) Construction of genome integration expression plasmid: Design and synthesize strong promoter Ptac and its downstream components obtained above. argB *Gene, construct recombinant plasmid pK18mobsacB- ldh ::Ptac- argB * This plasmid uses the lactate dehydrogenase gene ldhThe site is an integration target.
[0010] (2) Genome integration: The above plasmid was introduced into the ARG5 strain constructed in step one, and Ptac- was integrated through homologous recombination. argB *Expression cassettes are integrated into the genome ldh site, replacing the original ldh Genes, obtained strain ARG6-Ptac- argB * This operation not only enhances argB The expression of * also simultaneously knocked out the lactate dehydrogenase gene. ldh This blocks the pathway of lactic acid synthesis byproducts and further promotes the flow of carbon source to the target product.
[0011] (3) Introducing arginine export protein: The pXMJ19- used in step two was introduced into the arginine export protein. lysE plasmids (carrying) lysE Gene) electroconverted to ARG6-Ptac- argB *From the strain, positive transformants were selected through chloramphenicol resistance screening to obtain the final high-yield L-arginine-producing genetically engineered strain, named ARG6-Ptac- argB * / pXMJ19- lysE .
[0012] Step 4: Fermentation Verification The constructed engineered strain was fermented in shake flasks and fermenters to evaluate its L-arginine production capacity. Optimized media or other composite media suitable for the growth and acid production of Corynebacterium glutamicum were used for fermentation. Culture conditions were 28–32°C and 100–220 rpm. During fed-batch fermentation, the carbon source concentration was maintained by adding glucose. Beneficial effects
[0013] Comprehensive Strategy System: This invention comprehensively utilizes the knockout competition approach ( proB ), and release transcriptional repression ( argR , farR ), and deregulate global nitrogen regulation ( amtR ) and the efflux of weakened products ( yggB Through various metabolic engineering strategies, such as glutamate-producing Corynebacterium, the metabolic network of Corynebacterium glutamicum was systematically reconstructed, enabling carbon and nitrogen sources to flow more efficiently to L-arginine synthesis.
[0014] The key enzyme exhibits excellent performance: This invention, through mutation, yields a novel anti-feedback inhibition enzyme with independent intellectual property rights. argB *Mutants (T94S, I158V, R273K) maintain higher activity within cells, effectively overcoming bottlenecks in the synthetic pathway.
[0015] High yield and promising industrialization prospects: The engineered bacterium ARG6-Ptac- argB * / pXMJ19- lysE Under shake-flask fermentation conditions, the yield of L-arginine can reach 10.3 g / L, and under fed-batch fermentation conditions, the yield is as high as 105.2 g / L, far exceeding that of traditional strains, and has extremely high industrial production value and potential.
[0016] Stable heritable traits: key gene modification (e.g.) argR , farR Knockout, Ptac- argB *Integrations all occur on the genome, exhibiting good genetic stability, which is beneficial for long-term passaging and batch stability in large-scale industrial production. Attached Figure Description
[0017] Figure 1 ARG6-Ptac- argB * / pXMJ19- lysE Fermentation process diagram Detailed Implementation
[0018] The method of the present invention is described below through specific implementation schemes. Unless otherwise specified, the technical means used in the specific embodiments of the present invention are all methods known to those skilled in the art.
[0019] In specific embodiments of the present invention, the obtained mutants are defined as follows: The naming principle for mutants is as follows: The mutant obtained by replacing a specific amino acid residue (or substitution) is represented by "original amino acid residue + residue position + substituted amino acid residue" from the amino acid sequence. For example, "T94S" represents a mutant obtained by replacing the parental threonine (Thr) with serine (Ser) at position 94 of the amino acid sequence; and so on.
[0020] Table 1 shows the primer sequences involved in the examples.
[0021] The method for arginine determination is as follows: HPLC detection conditions: Chromatograph: Agilent 1260; Detector: Differential detector; Differential detector temperature: 30℃; Column: Waters Xbridge BEHAmide (2.5 μm, 4.6 × 250 mm); Column temperature: 45℃; Mobile phase: Acetonitrile-phosphate buffer (75:45); Flow rate: 1 mL / min.
[0022] Example 1: Chassis strain ARG5 ( ΔargR ΔfarR ΔproB ΔamtR ΔyggB Construction of ) (1) Construction of knockout plasmid Using the genomic DNA of Corynebacterium glutamicum ATCC 13032 as a template, primers were designed, such as... argR -Up-F / R and argR-Down-F / R amplification of the gene to be knocked out ( argR, farR, proB, amtR, yggB The upstream and downstream homologous arms (approximately 500 bp each) of the suicide plasmid pK18mobsacB were obtained. The upstream and downstream homologous arms were ligated using overlap extension PCR or restriction enzyme ligation methods, and then cloned into the corresponding restriction sites of the suicide plasmid pK18mobsacB to construct the recombinant plasmid pK18mobsacB-Δ. argR pK18mobsacB-Δ farR wait.
[0023] (2) Step-by-step elimination and screening: pK18mobsacB-Δ argR The cells were introduced into ATCC 13032 competent cells via electroporation.
[0024] Spread the plating on LBHIS plates containing kanamycin (25 μg / mL) and incubate at 30°C for 2-3 days. Select single-exchange colonies (plasmids integrated into the genome).
[0025] Single-exchange colonies were picked and inoculated into antibiotic-free LBG liquid medium and incubated overnight at 30°C for a second homologous recombination.
[0026] After appropriately diluting the culture medium, it was spread onto LBHIS plates containing 10% sucrose for reverse screening. Because... sacB The gene has a lethal effect on Gram-negative bacteria in the presence of sucrose. Colonies that can grow on sucrose plates are colonies that have lost their plasmid backbone, of which about 50% are mutants that have knocked out the target gene.
[0027] The correctly knocked-out mutant strain was identified by colony PCR and named ARG1(Δ). argR ).
[0028] (3) Perform multi-gene knockout sequentially: Using ARG1 as the starting strain, repeat the above steps and sequentially introduce pK18mobsacB-Δ farR pK18mobsacB-Δ proB pK18mobsacB-Δ amtR pK18mobsacB-Δ yggB Through a stepwise knockout process, a five-gene knockout strain was finally obtained and named ARG5. ΔargRΔfarRΔproB ΔamtRΔyggB ).
[0029] PCR amplification: Homologous arms were amplified using the 2 × Phanta Ultra Master Mix kit (purchased from Novizan Biotechnology Co., Ltd.). The total volume of the reaction system was 50 μL. The components of the reaction system are shown in Table 2.
[0030] Table 2 PCR amplification reaction system
[0031] The PCR reaction program was as follows: 98 °C, 3 min (pre-denaturation); 98 °C, 10 s (denaturation); 55 °C, 5 s (annealing); 72 °C, 20 s (extension); 30 cycles; 4 °C, ∞ (storage). The obtained PCR products were analyzed by 1% agarose gel electrophoresis, and then purified by gel extraction. The nucleic acid concentration was determined by NanoDrop assay to obtain the amplified gene fragment.
[0032] pK18mobsacB was double-digested with xbaI and HindIII, and the product was purified by gel extraction after detection by 1% agarose gel electrophoresis. The ligation was performed using the ClonExpress Ultra One Step Cloning Kit, with a total system volume of 10 μL. The components are shown in Table 3.
[0033] Table 3 Connection Reaction System
[0034] After gently mixing the above cyclization system, it was placed at 50°C for 30 min to react.
[0035] After the reaction is complete, the above reaction products are converted to... E. coli JM109 competent cells were then selected for plasmid extraction and DNA sequencing. Individuals with correctly sequenced DNA were considered successfully constructed vectors. This invention obtained pK18mobsacB-Δ through the aforementioned steps. argR pK18mobsacB-Δ farR pK18mobsacB-Δ proB pK18mobsacB-Δ amtR pK18mobsacB-Δ yggB , .
[0036] Example 2: Construction of the feedback-resistant argB* mutant Knockout strain ARG5-Δ argB Construction: Following the method in Example 1, pK18mobsacB- was constructed. argB Knock out the plasmid, introduce the ARG5 strain, and knock out its own... argB Genes. Due toargB It is an essential gene, and the knockout strain requires arginine-supplemented medium to grow, resulting in strain ARG5-Δ. argB .
[0037] argB Construction of mutants: Following the method in Example 1, using the ATCC 13032 genome as a template, high-fidelity enzymes were used for amplification. argB Gene. Using the purified argB fragment as a template, PCR amplification was performed using the mutation primer T94S-F / R. The mutant product was ligated with the pXMJ19 plasmid digested with BamHI / XbaI, and the ligation product was transformed into... E. coli DH5α was plated on chloramphenicol plates, bacteria were picked and cultured for sequencing, and iterative mutations were performed sequentially using I158V-F / R and R273K-F / R. The final plasmid was named... .
[0038] Example 3: High-yield strain Construction and fermentation verification Ptac- argB *Construction of the integrated expression plasmid: Synthesis of a plasmid containing the Ptac promoter, ribosome binding site (RBS), and plasmid obtained from Example 2. argB *Expression cassettes of genes and terminators. Simultaneously amplified. ldh Upstream and downstream homologous arms of a gene. ldh Upstream arm, Ptac- argB *Expression Box ldh The downstream arms were sequentially cloned into the pK18mobsacB vector to construct an integration expression plasmid. .
[0039] Genome integration and strains Obtaining: Electroporation was performed on strain ARG5. Following the screening method in Example 1, single exchangers were screened using kanamycin resistance, followed by double exchangers using sucrose plates. Colony PCR was used to verify that the ldh site was blocked by Ptac- argB * Expression box successfully replaced ( ldh (Gene knocked out), strain ARG6-Ptac- was obtained. argB *
[0040] Importing the lysE expression plasmid: The pXMJ19- used in Example 2 was introduced into the lysE expression plasmid. lysE plasmid electroconversion to ARG6-Ptac- argB *Competent cells were screened on LBHIS plates containing chloramphenicol (15 μg / mL) to obtain the final engineered strain, named ARG6-Ptac- argB * / pXMJ19-lysE *
[0041] Shake-flask fermentation verification: Pick ARG6-Ptac- from the tablet argB * / pXMJ19- lysE Single colonies were inoculated into 5 mL of BHI liquid medium (containing 15 μg / mL chloramphenicol) and cultured overnight at 30°C with shaking at 220 rpm to obtain the seed culture.
[0042] The seed culture was transferred at a 10% inoculum to a 500 mL baffled shaker flask containing 30 mL of fermentation medium. The fermentation medium formula (g / L) was: glucose 80, ammonium sulfate 20, urea 5, potassium dihydrogen phosphate 1, dipotassium hydrogen phosphate 1, magnesium sulfate heptahydrate 0.25, calcium chloride 0.01, ferric sulfate heptahydrate 0.01, manganese sulfate monohydrate 0.01, biotin 0.0002, vitamin B1 0.0002, MOPs 42, with the remainder being trace elements; pH 7.0.
[0043] The culture was carried out at 30℃ and 220 rpm with shaking for 72 h. During this period, the pH was adjusted with ammonia to maintain it at around 7.0. After fermentation, samples were taken to determine the L-arginine yield. HPLC analysis showed that the L-arginine yield reached 10.3 g / L at the shake-flask level.
[0044] Validation of fed-batch fermentation in a 5 L fermenter: Seed culture preparation is the same as shake-flask fermentation, and the culture is expanded to the required volume.
[0045] The seed culture was inoculated into a 5 L fermenter containing 3 L of fermentation medium at a 10% inoculation rate. The fermentation medium formulation was the same as that for shake flask fermentation, but the initial glucose concentration was increased to 40 g / L.
[0046] Fermentation conditions: temperature 30℃, pH controlled at 7.0 by automatic addition of ammonia water, dissolved oxygen (DO) controlled at above 30% by adjusting stirring speed (300-800 rpm) and aeration rate (1-2 vvm).
[0047] When the initial glucose is depleted, start adding an 800 g / L glucose solution to control the glucose concentration in the fermentation broth at 5-10 g / L.
[0048] The fermentation cycle was 72 hours. Samples were taken at regular intervals to measure OD600, residual sugar, and L-arginine yield.
[0049] After fermentation, the L-arginine yield was calculated. For example... Figure 1 The results showed that under fed-batch fermentation conditions, the final strain ARG6-Ptac- argB * / pXMJ19- lysEThe L-arginine yield is as high as 105.2 g / L, and the genetic traits are stable.
[0050] Example 4: Control Experiment To verify the effectiveness of each modification step, the following control strains were set up and fermented according to the shake-flask fermentation method of Example 3. The L-arginine yield was compared, and the results are shown in Table 4.
[0051] Starting strain: ATCC 13032 (original strain, which produces almost no arginine).
[0052] Chassis strain: ARG5 (only the negative regulatory and competitive pathways were knocked out).
[0053] Single integrated strain: ARG6-Ptac- argB * (Integrated Ptac- based on ARG5) argB *, but none lysE plasmids).
[0054] Final strain: ARG6-Ptac- argB * / pXMJ19- lysE .
[0055] Table 4 Results of shake-flask fermentation control
[0056] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A high-yield L-arginine-producing Corynebacterium glutamicum ( Corynebacterium glutamicum Engineered bacteria, characterized in that, Starting with Corynebacterium glutamicum ATCC 13032, its genome contains at least the following modifications: (1) Gene encoding arginine biosynthesis repressor protein argR Knocked out; (2) Regulatory genes farR Knocked out; (3) Glutamate kinase encoding gene proB It was knocked out.
2. The engineered bacteria according to claim 1, characterized in that, The genome of the engineered bacteria also contains one or more of the following modifications: (1) Global nitrogen metabolism regulators amtR Knocked out; (2) Mechanosensitive channel protein gene encoding gene yggB It was knocked out.
3. The engineered bacteria according to claim 2, characterized in that, The engineered bacteria had their genome simultaneously knocked out. argR , farR , proB , amtR and yggB The gene is from strain ARG5.
4. A method for constructing a mutant N-acetylglutamate kinase argB* resistant to feedback inhibition, characterized in that, The method includes: (1) Host bacteria argB The gene was knocked out; (2) Constructing pXMJ19- argB The plasmid, whose nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing; (3) For pXMJ19- argB Perform site-directed mutation; (4) Shake-flask fermentation.
5. The method according to claim 4, constructing a argB The mutant protein sequence is shown in SEQ ID NO.2 of the sequence listing, and the argB* mutation site combination is: T94S, I158V, R273K.
6. A high-yield L-arginine-producing Corynebacterium glutamicum engineered bacterium, characterized in that, The engineered bacteria express the mutant N-acetylglutamate kinase obtained by the method described in claim 5. argB * 7. The engineered bacteria according to claim 6, characterized in that, The engineered bacteria, using the ARG5 strain of claim 3 as a host, overexpress the mutant N-acetylglutamate kinase of claim 6 on the genome via a strong promoter. argB * 8. The engineered bacteria according to claim 7, characterized in that, The strong promoter is the Ptac promoter, and Ptac- is introduced through homologous recombination. argB *Lactate dehydrogenase gene expressed as an expression cassette integrated into the genome of ARG5 strain ldh The site was identified, and strain ARG6-Ptac-argB* was obtained.
9. The engineered bacteria according to claim 8, characterized in that, The engineered bacteria also contain an exogenously carried L-arginine exporter gene. lysE .
10. The engineered bacteria according to claim 9, characterized in that, The lysE The gene is located on the expression vector pXMJ19 and introduced into ARG6-Ptac- argB *Among the strains, the final engineered strain ARG6-Ptac- was obtained. argB * / pXMJ19- lysE .
11. A method for producing L-arginine, characterized in that, Includes the following steps: (1) Activation culture of the engineered strain of Corynebacterium glutamicum according to any one of claims 1-10; (2) The activated strain is inoculated into the fermentation medium for fermentation culture; (3) After fermentation, L-arginine was collected from the fermentation broth.
12. The method according to claim 11, characterized in that, The fermentation medium contains chloramphenicol, the fermentation temperature is 25-30℃, and the stirring speed is 100-220 rpm.
13. The method according to claim 11 or 12, characterized in that, The fermentation method is supplemental batch fermentation; The application of the engineered strain of Corynebacterium glutamicum according to any one of claims 1-10 or the method according to any one of claims 11-13 in the microbial fermentation preparation of L-arginine.