Study On The Gene Expression Regulation Of Fengycin Synthetases In Bacillus Amyloliquefaciens

Bacillus amyloliquefaciens is a kind of Bacillus genus.Fengycin,which is metabolized and synthesized by non-ribosomal synthesis,is a ring lipopeptide composed of 10 amino acids and14?18 C of cap-hydroxy fatty acids.Fengycin is produced by multimodular proteins termed non-ribosomal peptide synthetases in the cell.Fengycin,which is not easy to develop drug resistance and high safety,has the characteristics of good stability and the potential to develop into a new food preservative and antibacterial drug.The application of fengycin of the low content produced by wild bacteria is restricted,because of lacking of systematic knowledge about fengycin synthetic regulatory mechanism prevent the researchers from effectively modifying it at the molecular level.In the previous study,the research group has successfully cloned a section of non-coding RNA?FenSr3?related to biosynthesis of fengycin,which is involved in the biosynthesis of fengycin.In this research,B.amyloliquefaciens LPB-18 was selected for the research strain and the regulatory effect of nc RNA on the synthesis of B.almyloliquefaciens LPB-18 fengycin by knocking out and overexpressing the nc RNA sequence related to fengycin synthetases were detected.The research results provide a theoretical basis for improving the anabolism of fengycin of B.alloliquefaciens LPB-18 at the molecular level.In addition,the coating preservation experiment of Agaricus bisporus with fengycin was carried out to further study the preservation effect of fengycin,which laid a theoretical foundation for further exploring the application value of fengycin in the preservation of fruits and vegetables.The main findings were as follows:1.The regulatory effect of nc RNA on the synthesis of Bacillu almyloliquefaciens LPB-18fengycin by knocking out the nc RNA sequenceTo study the effect of fermentation on the synthesis of lipopeptide fengycin,the FenSr3gene was koncked out,by the DNA homologous recombination technique.The Bacillus.almyloliquefaciens LPB-18N strain was successfully constructed,and the FenSr3 gene was inactivated.Compared with the B.almyloliquefaciens LPB-18,the yield of fengycin was increased from 190.908 mg/L to 327.598 mg/L,and the yield of fengycin was increased by 72%.Transcriptome sequencing results showed that the fengycin synthase gene fen A?fen B and fen E in the mutant strain was up-regulated by 10.91 times,11.83 times and 9.43 times.Meanwhile the catabolism pathway of fatty acids was significantly up-regulated,and the long-chain fatty acids were oxidized to short-chain fatty acids,generating CO₂and H₂O and releasing a large amount of ATP,which provided energy for the synthesis of fengycin.The tricarboxylic acid cycling pathway is cycled by carbon source,which provided a large amount of energy and substrate for the synthesis of fengycin.The up-regulated expression of transcription cofactor sigma H promoted the up-regulated expression of spo0A gene,resulting in the inhibition of Abr B gene.As a negative regulatory protein,the signaling protein Abr B binds with the fengycin promoter to inhibit the expression of the fengycin synthase gene.When the expression of Abr B was down-regulated,the concentration of intracellular repressing protein Abr B decreased,which can effectively reduce the inhibition of transcriptional regulation of fengycin,produce a large number of fengycin synthase.Significantly increased the yield of antibacterial lipopeptide fengycin.The results showed that FenSr3 gene was involved in regulating the anabolism of fengycin and had a negative regulatory effect.The deletion of this gene significantly improved thegene expression of fengycin synthase in B.amyloliquefaciens LPB-18N.2.The regulatory effect of nc RNA on the synthesis of Bacillus almyloliquefaciens LPB-18fengycin by overexpressing the nc RNA sequenceIn order to clarify the regulatory mode of FenSr3 gene regulating fengycin anabolism,PCR cloning was successfully used to construct an engineered strain of B.amyloliquefaciens LPB-18P,and the FenSr3 gene was overexpressed.Compared with B.amyloliquefaciens LPB-18,the yield of fengycin decreased from 190.464 mg/L to 38.6mg/L,and the yield of fengycin was significantly reduced?p<0.05?.The obtained overexpressed plasmids were electrotransformed into the knocked out competent cells of B.amyloliquefaciens LPB-18N,and the recovery strain was constructed.Under the same fermentation conditions,the yield of fengycin reached 182.6mg/L,basically returning to the fermentation level of the original strain.Therefore,it can be concluded that FenSr3 gene plays a negative regulatory role in the anabolism of fengycin.3.Application of fengycin in the preservation experiment of Agaricus bisporusIn order to study the application value of fengycin in the preservation of fruits and vegetables,this study carried out the coating preservation experiment of Agaricus bisporus with fengycin.The results showed that,In the Agaricus bisporus during storage,coating containing fengycin treatment group,the quality index and physiological and biochemical indexes were higher than control group.The concentration of fengycin added by the C treatment group was300 mg/L,its whiteness value was 90.7 and its sensory score was 96,both of which were higher than those of the other treatment groups.And there was no obvious bacterial staining in appearance.It could be seen that fengycin had a certain antibacterial effect in the coating test.Therefore,within a certain concentration range,the more antibacterial lipopeptide fengycin was added,the more significant its antibacterial and fresh-keeping effect would be.