Method for Promoting Bacillus subtilis to Synthesize Surfactin based on Multi-gene Synergy

• US 20200131229A1
• Filed: 12/14/2018
• Published: 04/30/2020
• Est. Priority Date: 12/14/2018
• Status: Active Grant

• Current Assignee: Jiangnan University
• Original Assignee: Jiangnan University
• Inventors: WU, Qun, XU, Yan
• Granted Patent: US 11,254,709 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 14/32 from Bacillus (G)
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/64 Cyclic peptides containing only normal peptide links
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/75 for Bacillus
• CPC Class Codes: C12R 2001/125 Bacillus subtilis ; Hay bacillus; Grass bacillus

Abstract

The present invention discloses a method for promoting B. subtilis to synthesize surfactin based on multi-gene synergy, and belongs to the field of genetic engineering. Firstly, B. subtilis is enabled to obtain the ability to synthesize surfactin by integrant expression of sfp protein derived from a high-yield strain, on this basis, by knocking out genes associated with a competitive pathway, overexpressing genes associated with the surfactin tolerance of B. subtilis, strengthening genes associated with a branched chain fatty acid synthesis pathway or improving the intracellular srfA gene transcription level, the synergy among genes is realized, and systemic metabolic engineering transformation is performed on the B. subtilis, thereby greatly improving the ability of B. subtilis genetically engineered bacteria to synthesize surfactin. Compared with a starting strain B. subtilis 168, the amount of extracellular accumulation of surfactin of the B. subtilis genetically engineered bacteria increases from 0 g/L to 12.8 g/L. The present invention obtains the recombinant B. subtilis with significantly improved ability to synthesize surfactin by multi-gene cooperative transformation of B. subtilis, and has a good application prospect.

Claims

1. A method for promoting B. subtilis to synthesize surfactin, characterized by replacing original sfp genes of B. subtilis with other sfp genes capable of complete expression;

and performing transformation as follows;

knocking out at least one of gene clusters associated with biofilm formation, NRPS/PKS gene clusters, or genes associated with negative regulatory factors, and/or, overexpressing at least one of surfactin efflux and resistance genes, genes associated with a branched chain fatty acid synthesis pathway, genes associated with a glycolytic pathway, and a gene associated with an exogenous cluster effect system.

2. The method of claim 1, characterized in that starting from B. subtilis 168, the original sfp genes of B. subtilis 168 are replaced with the sfp genes derived from B. amyloliquefaciens MT45 by homologous recombination.

3. The method of claim 1, characterized in that the gene clusters associated with biofilm formation comprise epsA-O and tasA-sipW-yqxM;

the NRPS/PKS gene clusters comprise dhb operon expressing a siderophore bacillibactin synthase, pks operon expressing an unknown polyketide synthase, and pps operon expressing a fengycin synthase;

the genes associated with the negative regulatory factors comprise codY, rapCFH, sinl, spx, perR and phoP.

4. The method of claim 3, characterized by knocking out at least two genes of the gene clusters associated with biofilm formation, the NRPS/PKS gene clusters or the genes associated with the negative regulatory factors.

5. The method of claim 1, characterized in that the surfactin efflux and resistance genes comprise swrC, liaRSFGHI and acrB;

the genes associated with the branched chain fatty acid synthesis pathway comprise fabHB, IpdV, bkdAA, bkdAB, bkdB, lipALM, aisS, ilvD, ilvC, leuABCD, fabD, accDABC, fabF, fabG, fabZ, fabl and tesA;

the genes associated with the glycolytic pathway comprise pfkA, gapA, pgk and pdhABCD;

the gene associated with the exogenous cluster effect system comprises comQXPA.

6. The method of claim 5, characterized by overexpressing at least two genes of the surfactin efflux and resistance genes, the genes associated with the branched chain fatty acid synthesis pathway, the genes associated with the glycolytic pathway and the gene associated with the exogenous cluster effect system.

7. The method of claim 1, characterized by knocking out at least one gene of the gene clusters associated with biofilm formation, the NRPS/PKS gene clusters or the genes associated with the negative regulatory factors, and overexpressing at least one gene of the surfactin efflux and resistance genes, the genes associated with the branched chain fatty acid synthesis pathway, the genes associated with the glycolytic pathway and the gene associated with an exogenous cluster effect system;

the gene clusters associated with biofilm formation comprise epsA-O and tasA-sipW-yqxM;

the NRPS/PKS gene clusters comprise dhb operon expressing a siderophore bacillibactin synthase, pks operon expressing an unknown polyketide synthase, and pps operon expressing a fengycin synthase;

the genes associated with the negative regulatory factors comprise codY, rapCFH, sinl, spx, perR and phoP;

the surfactin efflux and resistance genes comprise swrC, liaRSFGHI and acrB;

the genes associated with the branched chain fatty acid synthesis pathway comprise fabHB, IpdV, bkdAA, bkdAB, bkdB, lipALM, aisS, ilvD, ilvC, leuABCD, fabD, accDABC, fabF, fabG, fabZ, fabl and tesA;

the genes associated with the glycolytic pathway comprise pfkA, gapA, pgk and pdhABCD;

the gene associated with the exogenous cluster effect system comprises comQXPA.

8. The method of claim 7, characterized in that the comQXPA and srfA promoters are combined for expression.

9. The method of claim 1, characterized in that the knockout is performed by a homologous recombination knockout method, and the length of a homologous arm used for knocking out long fragment genes is 1000-2000 bp.

10. The method of claim 1, characterized in that fusion of multiple gene fragments is performed by using a two-step approach.

11. The method of claim 1, characterized by replacing the sfp genes in B. subtilis 168 with sfp genes derived from B. amyloliquefaciens MT45, knocking out epsA-O, tasA-sipW-yqxM, dhb operon, pks operon, pps operon, codY, rapCFH, sinl, spx, perR and phoP genes, and overexpressing fabHB, IpdV, bkdAA, bkdAB, bkdB, lipALM, alsS, ilvD, ilvC, leuABCD, fabD, accDABC, fabF, fabG, fabZ, fabl, tesA, pfkA, gapA, pgk, pdhABCD and comQXPA genes.

12. The method of claim 11, characterized in that the Gene ID of the sfp genes is WP_088612131.1.

13. The method of claim 12, characterized in that the wherein said B. subtilis comprises B. subtilis 168, B. subtilis WB400, B. subtilis WB600, B. subtilis WB800, B. subtilis WB800N.

14. Recombinant B. subtilis prepared by the method of claim 1.

15. A method for synthesizing surfactin, characterized in that the B. subtilis of claim 13 is applied as a production strain for fermentation production of surfactin.

16. The method of claim 15, characterized in that a fermentation medium used for fermentation contains 60-65 g/L of sucrose, 5-10 g/L of ammonium nitrate, 3-5 g/L of peptone, 2-5 g/L of potassium dihydrogen phosphate, 8-12 g/L of disodium hydrogen phosphate, 0.05-0.15 g/L of magnesium sulfate, 5-8 μ

M of calcium chloride, 2-5 μ

M of ferrous sulfate and 2-5 μ

M of EDTA.

17. Application of the method of claim 13 in the preparation of products containing surfactin.

18. The application of claim 17, characterized in that the products containing surfactin include surfactants, oil extraction aids, cosmetics, drugs, bioremediation agents and food products.