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CN-121975709-A - Bacillus licheniformis recombinant strain for high-yield L-cysteine

CN121975709ACN 121975709 ACN121975709 ACN 121975709ACN-121975709-A

Abstract

The invention discloses a bacillus licheniformis recombinant strain for high-yield L-cysteine, belonging to the technical field of synthetic biology and microbial metabolic engineering. The invention strengthens the cysteine synthesis flux through multiple knockout and combined overexpression. By knocking out serine hydroxymethyl transferase coding genes glyA and L-serine ammonia lyase coding genes sdaAA of serine catabolism key paths and knocking out cysteine catabolism gene metC, simultaneously introducing feedback inhibition release mutant cysE f and transporter eamA through combined gene overexpression, a high-efficiency engineering strain is constructed, and finally, the L-cysteine yield reaches 1.075 g/L (the conversion rate is 18.69%) in shake flask fermentation, so that the application potential of bacillus licheniformis in sulfur metabolism engineering is highlighted.

Inventors

  • Li youran
  • YAN JING
  • Xiao Fengxu
  • SHI GUIYANG

Assignees

  • 江南大学

Dates

Publication Date
20260505
Application Date
20260127

Claims (10)

  1. 1. The bacillus licheniformis recombinant strain for producing the L-cysteine is characterized in that the recombinant strain knocks out serine hydroxymethyl transferase coding genes glyA and sdaAA of bacillus licheniformis and a cysteine catabolic gene metC, and simultaneously overexpresses and releases feedback inhibition of L-serine-O-acetyltransferase cysE f and L-cysteine transporter eamA, the nucleotide sequence of the gene cysE f is shown as SEQ ID NO.4, and the nucleotide sequence of the gene eamA is shown as SEQ ID NO. 5.
  2. 2. The bacillus licheniformis recombinant strain of claim 1, wherein the nucleotide genes of the genes glyA, sdaAA and metC are shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, respectively.
  3. 3. The recombinant bacillus licheniformis according to claim 1, wherein the promoter P2 is used for over-expression of the genes cysE f and the L-cysteine transporter coding gene eamA for the feedback inhibition released L-serine-O-acetyltransferase and the terminator ter is used for termination of the gene expression.
  4. 4. The recombinant Bacillus licheniformis according to claim 3, wherein the nucleotide sequence of the promoter P2 is shown in SEQ ID NO.6 and the nucleotide sequence of the terminator ter is shown in SEQ ID NO. 7.
  5. 5. The recombinant bacillus licheniformis of any of claims 1-4, wherein the bacillus licheniformis is bacillus licheniformis Bacillus licheniformis CICIM B1391,1391.
  6. 6. A method for constructing the bacillus licheniformis recombinant strain according to any one of claims 1-5, which is characterized by comprising the following steps of (1) taking bacillus licheniformis Bacillus licheniformis CICIM B-1391 as an original strain, knocking out a gene glyA, a gene sdaAA and a gene metC in a genome of the original strain to obtain knocked-out bacteria B. licheniformis CICIM B1391-1391 delta glyA delta sdaAA delta metC; (2) Cloning genes cysE f and eamA onto a plasmid epWBn to obtain a recombinant plasmid, and introducing the recombinant plasmid into a knock-out bacterium B. licheniformis CICIM B delta glyAdelta sdaAA delta metC in the step (1), thereby obtaining the bacillus licheniformis recombinant strain with high L-cysteine yield.
  7. 7. The method according to claim 6, wherein the nucleotide genes of gene glyA, gene sdaAA, gene metC, gene cysE f and gene eamA are shown as SEQ ID NO. 1-SEQ ID NO.5, respectively.
  8. 8. A method for producing L-cysteine by fermentation, which is characterized in that the recombinant bacterium according to any one of claims 1-5 is utilized for producing L-cysteine by fermentation.
  9. 9. The method according to claim 8, wherein the recombinant bacteria are inoculated according to an inoculum size of 3-5% after the activation culture, the fermentation temperature is 37 ℃, the rotation speed is 220rpm, and the culture time is 48-96 hours.
  10. 10. Use of a recombinant bacterium according to any one of claims 1 to 5, or a method according to any one of claims 6 to 7, or a method according to any one of claims 8 to 9, for the preparation of L-cysteine or an L-cysteine-containing product.

Description

Bacillus licheniformis recombinant strain for high-yield L-cysteine Technical Field The invention relates to a bacillus licheniformis recombinant strain for high-yield L-cysteine, belonging to the technical field of synthetic biology and microbial metabolic engineering. Background L-cysteine, as a sulfur-containing amino acid, plays an irreplaceable role in the living body. It is a junction molecule and protein constituent unit of sulfur-containing amino acid metabolism, not only directly involved in the folding and stability maintenance of protein (by forming disulfide bonds), but also a necessary precursor for biosynthesis of various key bioactive substances such as GSH, coenzyme A, S-adenosylmethionine (SAM) and the like. The L-cysteine has huge application value in industry and the market demand is continuously growing. In the medical field, it is used as an antidote, an expectorant and a nutritional supplement. In the cosmetic industry, it is commonly used in skin and hair care products by virtue of its antioxidant and skin metabolism promoting capabilities. In addition, in the food industry, it is both a highly effective dough conditioner and flavoring agent, as well as a natural antioxidant. However, in sharp contrast to its wide use, the industrial production of L-cysteine has long been severely dependent on chemical hydrolysis methods extracted from protein materials such as human hair, feathers, etc., which not only involve strong acid/alkali treatment, but also consume high energy, and generate large amounts of sulfur-and nitrogen-containing organic wastewater, causing serious environmental pollution, so the development of a green, sustainable, precisely controllable microbial fermentation process to replace the process has become a research hotspot and a necessary trend in this field. Bacillus licheniformis (Bacillus licheniformis) has important application value in the fields of industrial fermentation, biological medicine, environmental restoration and the like due to strong environmental adaptability (such as high temperature resistance, drying resistance and chemical stress resistance) and high-efficiency metabolic network. Although the sulfur metabolic pathways of E.coli and C.glutamicum have been widely studied, B.licheniformis, as a representative of gram-positive bacteria, has fundamental differences from gram-negative bacteria in cell structure, stress response and global regulatory networks. Thus, there is a need to provide a Bacillus licheniformis which can produce L-cysteine at high yield. Disclosure of Invention The invention aims to overcome the defects in the prior art, provides a bacillus licheniformis recombinant strain with high yield of L-cysteine and a construction method thereof, and applies the bacillus licheniformis recombinant strain to fermentation to produce L-cysteine so as to fill the blank of metabolizing the bacillus to produce L-cysteine. The first technical scheme provided by the invention is that the bacillus licheniformis recombinant strain with high yield of L-cysteine is obtained by knocking out serine hydroxymethyl transferase coding genes glyA and L-serine ammonia lyase coding genes sdaAA of bacillus licheniformis and knocking out cysteine catabolic gene metC, and simultaneously overexpressing and releasing feedback inhibition of L-serine-O-acetyl transferase cysE f and L-cysteine transporter eamA. In certain embodiments, the bacillus licheniformis is bacillus licheniformis Bacillus licheniformisCICIM B1391,1391. In certain embodiments, the nucleotide groups of gene glyA, gene sdaAA, and gene metC are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3. In certain embodiments, the feedback inhibition-released L-serine-O-acetyltransferase encoding gene cysE f and the L-cysteine transporter encoding gene eamA are overexpressed using promoter P2 and the expression of the genes is terminated using terminator. In certain embodiments, cysE and eamA are derived from E.coli ESCHERICHIA COLI and the gene cysE is mutated to cysE f, the nucleotide sequence of cysE f is shown in SEQ ID NO.4 and the nucleotide sequence of cysE eamA is shown in SEQ ID NO. 5. In certain embodiments, the nucleotide sequence of promoter P2 is shown in SEQ ID NO.6 and the nucleotide sequence of terminator is shown in SEQ ID NO. 7. The second technical scheme provided by the invention is a method for constructing a bacillus licheniformis recombinant strain for producing L-cysteine, which comprises the following steps of (1) taking bacillus licheniformis Bacillus licheniformisCICIM B1391,1391 as an initial strain, knocking out a gene glyA, a gene sdaAA and a gene metC in a genome of the bacillus licheniformis recombinant strain to obtain knocked-out bacteria B. licheniformisCICIM B1391 delta glyA delta sdaAA delta metC; (2) Cloning genes cysE f and eamA onto a plasmid epWBn to obtain a recombinant plasmid, and introducing the recombinant plasmid into a knock-out bacterium B. licheniformisCICIM B delta glyAde