Bacillus licheniformis recombinant strain with high yield of L-cysteine

Abstract

The invention discloses a bacillus licheniformis recombinant strain with high yield of L-cysteine, and belongs to the technical field of synthetic biology and microbial metabolism engineering. The cysteine synthesis flux is enhanced through multiple knockout and combined overexpression. An efficient engineering strain is constructed by knocking out a serine hydroxymethyltransferase coding gene glyA and an L-serine ammonolysis enzyme coding gene sdaAA of a serine catabolism key pathway, knocking out a cysteine catabolism gene metC and combining gene overexpression to introduce a feedback inhibition removal mutant cysEf and a transporter eamA, and the efficient engineering strain is used for preparing a recombinant strain. Finally, the yield of the L-cysteine reaches 1.075 g / L in shake flask fermentation (the conversion rate is 18.69%), and the application potential of the bacillus licheniformis in sulfur metabolism engineering is highlighted.

Description

Technical Field

[0001] This invention relates to a recombinant strain of Bacillus licheniformis that produces high levels of L-cysteine, belonging to the fields of synthetic biology and microbial metabolic engineering. Background Technology

[0002] L-cysteine, as a sulfur-containing amino acid, plays an irreplaceable role in living organisms. It is a pivotal molecule in the metabolism of sulfur-containing amino acids and a building block of proteins. It not only directly participates in protein folding and stability maintenance (through the formation of disulfide bonds), but is also an essential precursor for the biosynthesis of many key bioactive substances such as GSH, coenzyme A, and S-adenosylmethionine (SAM).

[0003] L-cysteine ​​has significant industrial applications, and market demand continues to grow. In the pharmaceutical field, it is used as an antidote, expectorant, and nutritional supplement. In the cosmetics industry, due to its antioxidant properties and ability to promote skin metabolism, it is frequently used in skincare and haircare products. Furthermore, in the food industry, it serves as a highly effective dough improver and flavoring agent, as well as a natural antioxidant. However, in stark contrast to its wide range of applications, the industrial production of L-cysteine ​​has long relied heavily on chemical hydrolysis from keratin raw materials such as human hair and feathers. This process not only involves strong acid / alkali treatment, resulting in high energy consumption, but also generates large amounts of sulfur- and nitrogen-containing organic wastewater, causing serious environmental pollution. Therefore, developing green, sustainable, and precisely controllable microbial fermentation as an alternative process has become a research hotspot and an inevitable trend in this field.

[0004] Bacillus licheniformis ( Bacillus licheniformis Due to its strong environmental adaptability (such as resistance to high temperatures, dryness, and chemical stress) and efficient metabolic network, *Bacillus licheniformis* has significant application value in fields such as industrial fermentation, biomedicine, and environmental remediation. Although the sulfur metabolism pathways of *Escherichia coli* and *Corynebacterium glutamicum* have been extensively studied, *Bacillus licheniformis*, as a representative of Gram-positive bacteria, differs fundamentally from Gram-negative bacteria in cell structure, stress response, and global regulatory network.

[0005] Therefore, there is an urgent need to provide a Bacillus licheniformis strain that can produce high levels of L-cysteine. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology, provide a recombinant Bacillus licheniformis strain that produces high levels of L-cysteine ​​and its construction method, and apply it to fermentation to produce L-cysteine, thereby filling the gap in the production of L-cysteine ​​by Bacillus metabolism.

[0007] The first technical solution provided by this invention is a recombinant Bacillus licheniformis strain that produces high levels of L-cysteine, wherein the recombinant strain has the serine hydroxymethyltransferase encoding gene of Bacillus licheniformis knocked out. glyA, L-serine aminolyase encoding gene sdaAA and knocking out cysteine ​​catabolism genes metC Simultaneously, overexpression of L-serine-O-acetyltransferase, which relieves feedback inhibition. cysE f With L-cysteine ​​transporter eamA .

[0008] In some embodiments, the Bacillus licheniformis is Bacillus licheniformis. Bacillus licheniformis CICIM B1391.

[0009] In some implementations, genes glyA, Gene sdaAA and genes metC The nucleotide genes are SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0010] In some embodiments, promoter P2 is used to target the L-serine-O-acetyltransferase encoding gene that has been de-inhibited from feedback inhibition. cysE f and L-cysteine ​​transporter encoding gene eamA Overexpression was performed, and gene expression was terminated using the terminator ter.

[0011] In some implementations, cysE and eamA Derived from Escherichia coli Escherichia coli and the gene cysE Mutate into cysE f The gene cysE f The nucleotide sequence of the gene is shown in SEQ ID NO.4. eamA The nucleotide sequence is shown in SEQ ID NO.5.

[0012] In some embodiments, the nucleotide sequence of promoter P2 is shown in SEQ ID NO.6, and the nucleotide sequence of terminator ter is shown in SEQ ID NO.7.

[0013] The second technical solution provided by the present invention is a method for constructing a recombinant Bacillus licheniformis producing L-cysteine, the method comprising the following steps: (1) using Bacillus licheniformis... Bacillus licheniformis CICIMB1391 was used as the starting strain, and genes in its genome were knocked out.glyA ,Gene sdaAA and genes metC Knockout bacteria were obtained B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC; (2) Genes cysE f and eamA The plasmid was cloned into the plasmid epWBn to obtain the recombinant plasmid, which was then introduced into the knockout bacteria in step (1). B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC Thus, a recombinant strain of Bacillus licheniformis with high L-cysteine ​​production was obtained.

[0014] In some implementations, genes glyA, Gene sdaAA and genes metC The nucleotide genes are SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0015] In some embodiments, promoter P2 is used to target the L-serine-O-acetyltransferase encoding gene that has been de-inhibited from feedback inhibition. cysE f and L-cysteine ​​transporter encoding gene eamA Overexpression was performed, and gene expression was terminated using the terminator ter.

[0016] In some implementations, cysE and eamA Derived from Escherichia coli Escherichia coli and the gene cysE Mutate into cysE f The gene cysE f The nucleotide sequence of the gene is shown in SEQ ID NO.4. eamA The nucleotide sequence is shown in SEQ ID NO.5.

[0017] In some embodiments, the nucleotide sequence of promoter P2 is shown in SEQ ID NO.6, and the nucleotide sequence of terminator ter is shown in SEQ ID NO.7.

[0018] The third technical solution provided by the present invention is a method for producing L-cysteine ​​by fermentation, wherein the method utilizes the recombinant bacteria described in the first technical solution to produce L-cysteine ​​by fermentation.

[0019] In some embodiments, the recombinant strain of Bacillus licheniformis is inoculated into a fermentation medium and fermented at 37°C and 220 rpm for 48 h.

[0020] In some embodiments, the fermentation culture is first cultured in 15 mL LB medium at 37°C and 220 rpm for 20 h before being inoculated into the fermentation medium.

[0021] Specifically, the recombinant strain was first inoculated into 15 ml of LB medium and cultured on a shaker at 37°C and 220 rpm for 20 h. Then, it was inoculated into 30 mL of fermentation medium at a volume concentration of 3-5% to start fermentation. Fermentation was carried out for 48-96 h at 37°C and 220 rpm. After fermentation, the fermentation broth was collected to detect L-cysteine.

[0022] In some embodiments, the fermentation medium comprises: 30 g / L glucose, 10 g / L dipotassium hydrogen phosphate, 1.36 g / L potassium dihydrogen phosphate, 5 g / L ammonium sulfate, 10 g / L urea, 2 g / L magnesium sulfate heptahydrate, 10 g / L monosodium glutamate, 2 g / L MKF, 20 g / L peptone, 10 g / L yeast extract, 10 g / L L-serine, 7 g / L sodium sulfate, and 4 g / L sodium thiosulfate.

[0023] In some embodiments, the MKF composition is: 54.4 g / L ferric ammonium citrate, 9.8 g / L manganese chloride tetrahydrate, 1.6 g / L cobalt chloride hexahydrate, 1 g / L copper chloride dihydrate, 1.9 g / L boric acid, 9 g / L zinc sulfate heptahydrate, 1.1 g / L sodium molybdate dihydrate, 1.5 g / L sodium selenite, 1.5 g / L nickel sulfate hexahydrate, and deionized water as the solvent.

[0024] The fourth technical solution provided by this invention is the application of the recombinant bacteria described in the first technical solution, or the method described in the second technical solution, or the method described in the third technical solution in the preparation of L-cysteine ​​or products containing L-cysteine.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes the exogenous addition of serine, a precursor in L-cysteine ​​biosynthesis, to simultaneously weaken precursor shunting and product degradation, and introduces feedback inhibition through gene overexpression to eliminate mutants. cysE f With transport proteins eamA This study enhanced L-cysteine ​​biosynthesis in Bacillus licheniformis by knocking out key genes in the serine catabolism pathway of Bacillus licheniformis. glyA(Encodes serine hydroxymethyltransferase, which catalyzes the production of glycine from serine) and sdaAA (Encoding L-serine ammonia-lyase, catalyzing the conversion of L-serine to pyruvate), thereby reducing the conversion of carbon to the pyruvate and glycine pathways and increasing the metabolic flux of the precursor L-serine biosynthesis. Then, the cysteine ​​catabolism gene is knocked out. metC (Encoding an L-cysteine ​​desulfatase, catalyzing the conversion of L-cysteine ​​to pyruvate, ammonia, and hydrogen sulfide; and encoding a cystathionine-β-lyase, catalyzing the conversion of cystathionine to L-homocysteine), this enzyme effectively protects the target product L-cysteine. Feedback inhibition is relieved through overexpression. cysE f and eamA The gene enables the enzyme to maintain high catalytic activity even when the target product accumulates, while promoting the secretion of intracellularly synthesized L-cysteine ​​to the extracellular environment, reducing the intracellular concentration of L-cysteine, and further mitigating the feedback inhibition effect.

[0026] Therefore, this invention enhances cysteine ​​synthesis throughput through multiple knockouts and combined overexpression. This is achieved by knocking out the serine hydroxymethyltransferase encoding gene, a key pathway in serine catabolism. glyA and L-serine ammonia-lyase encoding gene sdaAA Furthermore, knocking out the cysteine ​​catabolism gene metC Simultaneously, the introduction of feedback inhibition through gene overexpression relieves the mutant. cysE f With transport proteins eamA A highly efficient engineered strain was constructed, ultimately achieving an L-cysteine ​​yield of 1.075 g / L (conversion rate of 18.69%) in shake-flask fermentation, highlighting the application potential of Bacillus licheniformis in sulfur metabolism engineering. Attached Figure Description

[0027] Figure 1 for glyA Gene knockout plasmid pJOE8999- glyA Atlas. Figure 2 for sdaAA Gene knockout plasmid pJOE8999- sdaAA Atlas.

[0028] Figure 3 for metC Gene knockout plasmid pJOE8999- metC Atlas.

[0029] Figure 4 To knock out plasmid pJOE8999- glyA pJOE8999- sdaAA pJOE8999- metCEnzyme digestion verification diagram.

[0030] Figure 5 For epWBn-P2- cysE f -eamA Plasmid map.

[0031] Figure 6 strain B. licheniformis CICIM B1391Δ glyA , B. licheniformis CICIMB1391Δ glyA Δ sdaAA , B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC ,epWBn-P2- cysE f -eamA / B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC :: cysE f :: eamA OD of four strains fermented in shake flasks for 48 hours 600 Condition.

[0032] Figure 7 strain B. licheniformis CICIM B1391Δ glyA , B. licheniformis CICIMB1391Δ glyA Δ sdaAA , B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC ,epWBn-P2- cysE f CICIM B1391Δ -eamA / B. licheniformis Δ glyA Δ sdaAA :: metC f :: cysE L-cysteine ​​production during shake-flask fermentation of four strains for 48 hours.

[0033] eamA strain epWBn-P2- Figure 8 f cysE CICIM B1391Δ -eamA / B. licheniformis Δ glyA Δ sdaAA :: metCf :: cysE L-serine uptake and L-cysteine ​​production during shake-flask fermentation for 84 hours.

[0034] eamA At 72h, strain epWBn-P2- Figure 9 f cysE CICIM B1391Δ -eamA / B. licheniformis Δ glyA Δ sdaAA :: [[ID=]121]metC f :: cysE OD 600 And the highest production of L-cysteine. Detailed Implementation

[0035] refer to eamA The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0036] Test method: Determination of L-cysteine ​​content in fermentation broth during fermentation process (1) During the fermentation process, 0.5 mL of fermentation broth was placed in a 1.5 mL EP tube, and 0.04 M TCEP-HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) reducing agent was added in equal proportion. The reaction was carried out at 37℃ for 30 min.

[0037] (2) Sample derivatization: After the reaction, the sample was diluted 4-fold with water. 50 μL of the diluted sample was added to 300 μL of 0.5 M Na₂CO₃-NaHCO₃ (pH=9.0) buffer, followed by 50 μL of 0.5% DNFB derivatizing agent (2,4-dinitrofluorobenzene, using acetonitrile as solvent). The reaction was carried out at 60℃ for 1 h, and then 600 μL of 0.05 M sodium acetate solution (pH=6.0) was added to terminate the reaction. The sample was centrifuged, filtered through a membrane, and placed in a liquid chromatography vial for analysis.

[0038] (3) The instrument used was an Agilent HPLC high-performance liquid chromatograph. The column was an Alphasil XD-C18AQ column (4.6×250mm, 5μm); the UV detector wavelength was 360 nm; the injection volume was 5 μL; the column temperature was 35℃; the flow rate was 1 mL / min; the mobile phase consisted of two phases, A and B. Mobile phase A: 50% acetonitrile; mobile phase B: 4.1 g sodium acetate, 900 mL purified water, 100 mL acetonitrile, 1 mL triethylamine, 1 mL 1 g / L EDTA, and the pH was adjusted to 6.0 with glacial acetic acid. The gradient elution program is shown in Table 1.

[0039] Table 1 Gradient elution program

[0040] Raw materials used in the examples: 1. LB medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride. Yeast extract and peptone were sourced from OXOID (UK), and sodium chloride was sourced from Shanghai Aladdin Biochemical Technology Co., Ltd. (China).

[0041] 2. The fermentation medium consists of: 30 g / L glucose, 10 g / L dipotassium hydrogen phosphate, 1.36 g / L potassium dihydrogen phosphate, 5 g / L ammonium sulfate, 10 g / L urea, 2 g / L magnesium sulfate heptahydrate, 10 g / L monosodium glutamate, 2 g / L MKF, 20 g / L peptone, 10 g / L yeast extract, 10 g / L L-serine, 7 g / L sodium sulfate, and 4 g / L sodium thiosulfate. All reagents in the fermentation medium are sourced from Shanghai Aladdin Biochemical Technology Co., Ltd. (China).

[0042] 3. The MKF composition is as follows: 54.4 g / L ferric ammonium citrate, 9.8 g / L manganese chloride tetrahydrate, 1.6 g / L cobalt chloride hexahydrate, 1 g / L copper chloride dihydrate, 1.9 g / L boric acid, 9 g / L zinc sulfate heptahydrate, 1.1 g / L sodium molybdate dihydrate, 1.5 g / L sodium selenite, and 1.5 g / L nickel sulfate hexahydrate, with deionized water as the solvent. All reagents in the fermentation medium are sourced from Shanghai Aladdin Biochemical Technology Co., Ltd. (China).

[0043] 4. Bacillus licheniformis Figures 1 - 9 CICIM B1391 was published in the paper “Identification of a strong constitutive promoter of Bacillus licheniformis and verification of its expression effect”. The applicant promised to distribute the strain to the public through legal channels within 20 years from the date of application.

[0044] 5. Plasmid pJOE8999 is a commercially available plasmid.

[0045] Example 1: Construction of the knockout plasmid (1) Construction of plasmid pJOE8999- Bacillus licheniformis Using the Bacillus licheniformis genome as a template, primers were used... glyA -STY-F / R and glyA -XTY-F / R were subjected to polymerase chain reaction (PCR) to amplify the product. glyA Two 500bp fragments, one above and one below the gene, were used as templates, and primers were applied. glyA -STY-F / glyA-XTY-R, a 1000bp polymerase chain reaction was obtained. glyA -TY homologous arm fragment. Using a laboratory-obtained vector containing the PN2 promoter, target gene site, and repetitive sequence as a template, primer pairs PN2- glyA -F / PN2- glyA -R and glyA -F / glyA Polymerase chain reaction (PCR) was performed using primer PN2-R to amplify two fragments, PN2 and BD1. Then, using the PN2 and BD1 fragments as templates, primer PN2-R was used to amplify the PN2 fragments. glyA -F / glyA -R, through another round of overlapping polymerase chain reaction, the PN2-BD1 fragment is obtained. Finally, with glyA -TY and PN2-BD1 are used as templates, and primers PN2- are used. glyA -F and glyA -XTY-R undergoes another overlapping polymerase chain reaction to obtain glyA Knockout box fragment. glyA Knockout box and glyA RI 44]Eco Homologous recombination of the shuttle vector pJOE8999 by HI double digestion was performed and transformed into E. coli. / Bam In JM109, the cells were plated on kanamycin-resistant plates, and single colonies were picked for colony PCR verification (primer PN2-). E.coli -F and glyA Sequencing confirmed that the plasmid pJOE8999- contained the complete knockout cassette fragment (-XTY-R). glyA .

[0046] (2) Construction of plasmid pJOE8999- glyA Using the Bacillus licheniformis genome as a template, primers were used... sdaAA -STY-F / R and sdaAA -XTY-F / R were subjected to polymerase chain reaction (PCR) to amplify the product. sdaAA Two 500bp fragments, one above and one below the gene, were used as templates, and primers were applied. sdaAA -STY-F / sdaAA -XTY-R, a 1000bp polymerase chain reaction was obtained. sdaAA -TY homologous arm fragment. Using a laboratory-obtained vector containing the PN2 promoter, target gene site, and repetitive sequence as a template, primer pairs PN2- sdaAA -F / PN2- sdaAA -R and sdaAA -F / sdaAAPolymerase chain reaction (PCR) was performed using primer PN2-R to amplify two fragments, PN2 and BD2. Then, using the PN2 and BD2 fragments as templates, primer PN2-R was used to amplify the PN2 fragments. sdaAA -F / sdaAA -R, through another round of overlapping polymerase chain reaction, the PN2-BD2 fragment is obtained. Finally, with sdaAA -TY and PN2-BD2 are used as templates, and primers PN2- are used. sdaAA -F and sdaAA -XTY-R undergoes another overlapping polymerase chain reaction to obtain sdaAA Knockout box fragment. sdaAA Knockout box and sdaAA RI Eco Homologous recombination of the shuttle vector pJOE8999 by HI double digestion was performed and transformed into E. coli. / Bam In JM109, the cells were plated on kanamycin-resistant plates, and single colonies were picked for colony PCR verification (primer PN2-). E.coli -F and sdaAA Sequencing confirmed that the plasmid pJOE8999- contained the complete knockout cassette fragment (-XTY-R). sdaAA .

[0047] (3) Construction of plasmid pJOE8999- sdaAA Using the Bacillus licheniformis genome as a template, primers were used... metC -STY-F / R and metC -XTY-F / R were subjected to polymerase chain reaction (PCR) to amplify the product. metC metC Two 500bp fragments, one above and one below the gene, were used as templates, and primers were applied. metC -STY-F / metC -XTY-R, a 1000bp polymerase chain reaction was obtained. metC -TY homologous arm fragment. Using a laboratory-obtained vector containing the PN2 promoter, target gene site, and repetitive sequence as a template, primer pairs PN2- metC -F / PN2- metC -R and metC -F / metC Polymerase chain reaction (PCR) was performed using primer PN2-R to amplify two fragments, PN2 and BD3. Then, using the PN2 and BD3 fragments as templates, primer PN2-R was used to amplify the PN2 fragments. metC -F / metC -R, through another round of overlapping polymerase chain reaction, the PN2-BD3 fragment is obtained. Finally, with metC -TY and PN2-BD3 are used as templates, and primers PN2- metC -F and metC-XTY-R undergoes another overlapping polymerase chain reaction to obtain metC Knockout box fragment. metC Knockout box and Eco RI / Bam Homologous recombination of the shuttle vector pJOE8999 by HI double digestion was performed and transformed into E. coli. E.coli In JM109, the cells were plated on kanamycin-resistant plates, and single colonies were picked for colony PCR verification (primer PN2-). metC -F and metC Sequencing confirmed that the plasmid pJOE8999- contained the complete knockout cassette fragment (-XTY-R). metC .

[0048] Table 2 Primers for Example 1

[0049] Example 2: Construction of strain B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC (1) Construction of recombinant bacteria B. licheniformis CICIM B1391Δ glyA Plasmid pJOE8999- glyA Electroporation to Bacillus licheniformis B . licheniformis Recombinant bacteria with gene knockout plasmids were constructed from CICIM B1391 competent cells. The constructed recombinant strain was first activated on a plate, and then a single colony was inoculated into a shake flask containing 15 mL of LB medium and cultured at 37°C and 220 rpm for 16 h. Subsequently, the cultured bacterial solution was inoculated into a new shake flask containing 15 mL of LB medium and cultured at 42°C and 220 rpm for 24 h. The bacterial solution was then diluted 10% with sterile water. -6 , 10- 7 Spread kanamycin resistance plates and incubate at 37°C for 16 h. Then use primer pairs... glyA -YZ-F / glyA -YZ-R knockout verification was performed. Transformants exhibiting double crossover were selected and inoculated into 15 mL LB medium, incubated at 42°C for 16 h, and single colonies were streaked onto both kanamycin-resistant and non-resistant plates. Strains that grew normally on non-resistant plates but not on kanamycin-resistant plates were considered plasmid-eliminated strains. Recombinant Bacillus licheniformis was obtained. B. licheniformis CICIM B1391Δ glyA .

[0050] (2) Construction of recombinant bacteria B. licheniformis CICIM B1391Δ glyA Δ sdaAA Plasmid pJOE8999- sdaAA Electroporation to Bacillus licheniformis B. licheniformis CICIM B1391Δ glyA Recombinant bacteria with gene knockout plasmids were constructed from competent cells. The constructed recombinant strain was first activated on a plate, and then a single activated colony was inoculated into a shake flask containing 15 mL of LB medium and cultured at 37°C and 220 rpm for 16 h. Subsequently, the cultured bacterial solution was inoculated into a new shake flask containing 15 mL of LB medium and cultured at 42°C and 220 rpm for 24 h. The bacterial solution was then diluted 10% with sterile water. -6 , 10- 7 Spread kanamycin resistance plates and incubate at 37°C for 16 h. Then use primer pairs... sdaAA -YZ-F / sdaAA -YZ-R knockout verification was performed. Transformants exhibiting double crossover were selected and inoculated into 15 mL LB medium, incubated at 42°C for 16 h, and single colonies were streaked onto both kanamycin-resistant and non-resistant plates. Strains that grew normally on non-resistant plates but not on kanamycin-resistant plates were considered plasmid-eliminated strains. Recombinant Bacillus licheniformis was obtained. B. licheniformis CICIM B1391Δ glyA Δ sdaAA .

[0051] (3) Construction of recombinant bacteria B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC Plasmid pJOE8999- metC Electroporation to Bacillus licheniformis B. licheniformis CICIM B1391Δ glyA Δ sdaAA Recombinant bacteria with gene knockout plasmids were constructed from competent cells. The constructed recombinant strain was first activated on a plate, and then a single activated colony was inoculated into a shake flask containing 15 mL of LB medium and cultured at 37°C and 220 rpm for 16 h. Subsequently, the cultured bacterial solution was inoculated into a new shake flask containing 15 mL of LB medium and cultured at 42°C and 220 rpm for 24 h. The bacterial solution was then diluted 10% with sterile water. -6 , 10- 7Spread kanamycin resistance plates and incubate at 37°C for 16 h. Then use primer pairs... metC -YZ-F / metC -YZ-R knockout verification was performed. Transformants exhibiting double crossover were selected and inoculated into 15 mL LB medium, incubated at 42°C for 16 h, and single colonies were streaked onto both kanamycin-resistant and non-resistant plates. Strains that grew normally on non-resistant plates but not on kanamycin-resistant plates were considered plasmid-eliminated strains. Recombinant Bacillus licheniformis was obtained. B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC .

[0052] Table 3 Primers for Example 2

[0053] Example 3: Strain epWBn-P2- cysE f -eamA / B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC :: cysE f :: eamA The first step is to use the synthetic material from Shanghai Sangon Biotech Co., Ltd. cysE f Genes and eamA Using genes as templates, cysE f -F / cysE f -R and eamA -F / eamA -R primer pair, PCR amplification of fragment cysE f Genes and eamA Gene.

[0054] The second step is to use the two amplified target fragments as templates and apply primer pairs. cysE f -F / eamA -R, obtained via overlapping polymerase chain reaction cysE f - eamA Fragment. Using primer pairs cysE f -F / ter-ter-R, by adding the terminator ter to the end of the combinatorial gene, obtains... cysE f - eamA -ter fragment.

[0055] The third step involved using the pwB980 plasmid as a backbone and employing molecular cloning technology to add a high-copy E. coli replicon from the pUC19 plasmid to the corresponding site, thus constructing the basic vector epWBn. Subsequently, the P2 promoter fragment was directionally inserted into a specific multiple cloning site of the epWBn plasmid via enzyme digestion and ligation, ultimately yielding the recombinant plasmid epWBn-P2.

[0056] Step 4 Kpn I / Bam The recombinant plasmid epWBn-P2 was digested with two restriction endonucleases (HI) to obtain the linearized vector epWBn-P2. The obtained vector fragment was then purified by gel extraction. The purified vector epWBn-P2 was compared with... cysE f - eamA -ter fragment ligation. Subsequently, the successfully constructed recombinant DNA molecule was transformed into E. coli. E.coli JM109 competent cells were evenly spread on kanamycin-resistant plates. A suitable number of transformants were selected and colony PCR was performed using primers epWBn-F / epWBn-R. Transformed strains with positive PCR results were inoculated into 15 mL LB broth for preservation, and plasmids were further extracted and sent for Sanger sequencing. After confirming correct plasmid construction, the plasmid was electroporated into recombinant Bacillus licheniformis. B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ [[ID=8*]]metC The transformed bacteria were obtained from competent cells. The transformed bacteria were plated on kanamycin-resistant plates, and colony PCR was performed using epWBn-F / epWBn-R primers. The positive transformants were then inoculated into 15 mL LB broth for preservation, ultimately yielding the recombinant Bacillus licheniformis strain epWBn-P2-. cysE f -eamA / B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC :: cysE f :: eamA .

[0057] Table 4 Primers for Example 3

[0058] Example 4: Detection of L-cysteine ​​content during fermentation Recombinant bacteria B. licheniformis CICIM B1391Δ glyA ,B. licheniformis CICIM B1391Δ glyA Δ sdaAA , B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC Scribing on a non-resistance-resistant plate, epWBn-P2- cysE f -eamA / B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC :: cysE f :: eamA After streaking kanamycin resistance plates and incubating at 37°C for 20 hours, single colonies were picked and inoculated into 15 mL LB shake flasks. After overnight incubation, 3% of the culture was inoculated into 250 mL shake flasks containing 30 mL of fermentation medium. Fermentation was carried out for 48 hours, with samples taken every 12 hours to measure the OD value of the fermentation broth. 600 And L-cysteine ​​content, such as Figure 6 and 7 As shown.

[0059] according to Figure 6 The growth of bacteria can be observed. sdaAA Gene knockout had the most significant inhibitory effect on the growth of the strain. Compared to B. licheniformis CICIM B1391Δ glyA (It enters a stable period after 36 hours). B. licheniformis CICIM B1391Δ glyA Δ sdaAA , B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC ,epWBn-P2- cysE f -eamA / B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC :: cysE f :: eamA The growth rate of the three strains decreased significantly, and they had not entered the stationary phase by 48 hours. 600 The highest values ​​reached were 28.06, 29.5, and 20.24, respectively. The overexpression plasmid epWBn-P2- was introduced. cysE f -eamA The recombinant strain epWBn-P2- cysE f-eamA / B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC :: cysE f :: eamA Growth was further inhibited.

[0060] like Figure * As shown, within 48 hours B. licheniformis CICIM B1391Δ glyA , B. licheniformis CICIM B1391Δ glyA Δ sdaAA , B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ [[ID=*48]]metC ,epWBn-P2- cysE f -eamA / B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC :: cysE f :: eamA The highest L-cysteine ​​yields of the four strains were 123.681 mg / L, 160.890 mg / L, 150.083 mg / L, and 952.246 mg / L, respectively. The overexpression plasmid epWBn-P2- cysE f -eamA Mediated recombinant strain epWBn-P2- cysE It seems there is an error in the original text where "8*" and "*48" are present. Please check and correct the original text for a more accurate translation. f -eamA / B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC :: cysE f :: eamA The ability of the fermenter to synthesize cysteine ​​was significantly enhanced, with L-cysteine ​​production increasing from an initial 150.08 mg / L to 952.25 mg / L after 48 hours of fermentation. This result indicates that relieving the effect of L-cysteine ​​on… cysE Feedback inhibition of the gene-encoded L-serine-O-acetyltransferase, which simultaneously alleviates the toxic effects of L-cysteine ​​on bacterial cells, is a key regulatory strategy for enhancing the L-cysteine ​​synthesis capacity of bacterial strains. Figures 8 - 9 As shown, the recombinant Bacillus licheniformis strain epWBn-P2- cysE f -eamA / B. licheniformisCICIM B1391Δ glyA Δ sdaAA Δ metC :: cysE f :: eamA After 84 hours of shake-flask fermentation, the L-cysteine ​​yield reached its peak of 1.075 g / L at 72 hours, at which point the 5.752 g / L serine was completely consumed, and the substrate conversion rate reached 18.69%.

[0061] 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 recombinant Bacillus licheniformis strain producing L-cysteine, characterized in that, The recombinant bacteria had the serine hydroxymethyltransferase encoding gene of Bacillus licheniformis knocked out. glyA、 L-serine aminolyase encoding gene sdaAA and knocking out cysteine ​​catabolism genes metC Simultaneously, overexpression of L-serine-O-acetyltransferase, which relieves feedback inhibition. cysE f With L-cysteine ​​transporter eamA The gene cysE f The nucleotide sequence of the gene is shown in SEQ ID NO.

4. eamA The nucleotide sequence is shown in SEQ ID NO.

5.

2. The recombinant Bacillus licheniformis according to claim 1, characterized in that, Gene glyA、 Gene sdaAA and genes metC The nucleotide genes are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

3. The recombinant Bacillus licheniformis according to claim 1, characterized in that, Using promoter P2 to target the L-serine-O-acetyltransferase encoding gene that relieves feedback inhibition cysE f and L-cysteine ​​transporter encoding gene eamA Overexpression was performed, and gene expression was terminated using the terminator ter.

4. The recombinant Bacillus licheniformis according to claim 3, characterized in that, The nucleotide sequence of promoter P2 is shown in SEQ ID NO.6, and the nucleotide sequence of terminator ter is shown in SEQ ID NO.

7.

5. The recombinant Bacillus licheniformis according to any one of claims 1 to 4, characterized in that, The Bacillus licheniformis is Bacillus licheniformis. Bacillus licheniformis CICIM B1391.

6. A method for constructing recombinant Bacillus licheniformis according to any one of claims 1 to 5, characterized in that, The method includes the following steps: (1) using Bacillus licheniformis Bacillus licheniformis CICIM B1391 was used as the starting strain, and genes in its genome were knocked out. glyA ,Gene sdaAA and genes metC Knockout bacteria were obtained B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC; (2) Genes cysE f and eamA The plasmid was cloned into the plasmid epWBn to obtain the recombinant plasmid, which was then introduced into the knockout bacteria in step (1). B. licheniformis CICIM B1391Δ glyA Δ sdaAA Δ metC Thus, a recombinant strain of Bacillus licheniformis with high L-cysteine ​​production was obtained.

7. The method according to claim 6, characterized in that, Gene glyA、 Gene sdaAA、 Gene metC Gene cysE f and genes eamA The nucleotide genes are shown in SEQ ID NO.1 to SEQ ID NO.5, respectively.

8. A method for producing L-cysteine ​​by fermentation, characterized in that, The method involves producing L-cysteine ​​by fermentation using the recombinant bacteria described in any one of claims 1 to 5.

9. The method according to claim 8, characterized in that, After activating and culturing the recombinant bacteria, inoculate at a rate of 3-5%, ferment at 37℃, rotate at 220 rpm, and culture for 48-96 hours.

10. The use of the recombinant bacteria according to any one of claims 1 to 5, or the method according to any one of claims 6 to 7, or the method according to any one of claims 8 to 9 in the preparation of L-cysteine ​​or L-cysteine-containing products.

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