| Fengycin is a new lipopeptide antimicrobial substance.At present,little is known about the synthesis and regulation mechanism of fengycin.How to analyze the synthesis regulation mechanism and increase the yield of wild-type strains with extremely low yield of fengycin through various biological methods is of great value to promote its research and application.In this paper,we realized the synthesis of fengycin from scratch and put forward the feasible method to increase the yield of fengycin.The first aspect is the construction and enhancement of fengycin biosynthetic strain.(1)B.subtilis 168,which has the ability to synthesize complete gene cluster and highly efficient protein synthesis and secretion,was used as the base cell.Because of the mutation of 4’-phosphopanonyl mercaptoethylamine transferase gene(sfp),it cannot complete the activation of non-ribosomal peptide synthetase,so it has no catalytic activity.So non-ribosomal peptide synthase activation module which was composed of strong promoter P43 and sfp and regulation of protein synthesis and secretion of gene regulation factors secreted degQ was integrated into the genome through the CRISPR synthetase activation technology.Then B.subtilis 168 had the ability to synthesize fengycin,and the synthesis process of fengycin was completed from scratch.At this time,the yield of fengycin was 10.43 mg/L,and the constructed strain was named B.subtilis ds.(2)β-hydroxyl fatty acid chain with 16-18 carbon atoms is the main structural component of peptides.By regulating the synthesis of fatty acid chain,the synthesis of some lipopeptides,such as surfactin.However,the effect on the synthesis of fengycin is still unclear.Therefore,genes related to the rate-limiting pathway of fatty acid synthesis(acs,birA and accACD)were overexpressed in B.subtilis ds,which increased the yield of fengycin to 24.73 mg/L,2.37 times that of B.subtilis ds.The newly constructed strain was named B.subtilis dspabacd.In the second aspect,the optimization of medium and the synthesis of fengycin mainly affect the analysis of metabolites.(3)In order to study the optimum conditions for the synthesis of Bacillus subtilis,the types of fermentation medium and the important fermentation conditions of B.subtilis ds and B.subtilis dspabacd were investigated.The medium type,temperature,rotation speed and carbon source were optimized.The yield of fengycin in B.subtilis ds was increased to 37.92 mg/L,which was 3.64 times higher than the original strain.The yield of fengycin in B.subtilis dspabacd was increased to 130.11 mg/L,which was 5.26 times of the original strain.The maximum yield medium was XMN medium,and the fermentation conditions were 30℃,180 r/min and 5%inoculation amount.(4)The metabolites of B.subtilis ds and B.subtilis dspabacd under optimal conditions were analyzed by metabonomic analysis technique.It was confirmed that accACD,birA and acs promoted the synthesis of fengycin by increasing the amount of intracellular fatty acids.In addition,it was found that intracellular amino acids,such as alanine,glutamate and aspartate,had an important effect on the synthesis of fengycin,which provided theoretical guidance for further improving the yield of fengycin. |
