Effect And Function Of Degq And Sfp On Fengycin Productivity Of Bacillus Subtilis

Abstract:Fengycin,a lipopeptide synthesized non-ribosomally by Bacillus spp.,plays an important role in controlling plant diseases.Research on the genes involved synthesis and regulation of the fengycin is conducive to building new genetic engineered strains,but also conducive to making new biological pesticides.The purpose of this study is to construct a genetic engineering strain which can produce fengycin and lay the theoretical foundation by studying the effect and function of degQ and sfp activation on fengycin productivity of Bacillus subtilis 168.Although genes specifying fengycin synthetase are conserved within the B.subtilis 168 genome,the corresponding antibiotics are not produced.Lipopeptide production depends on the 4'-phosphopantetheine transferase(PPtase)Sfp.However,the sfp allel of the 168 strain specifies an inactive protein due to a frameshift mutation.But the strain with a sfp gene remains a non-producer due to substitutional mutations in the-10 promoter region of degQ.And degQ,encoding a polypeptide of 46 amino acids,is a pleiotropic regulatory gene which controls the production of secreted enzymes enzymes and several antibiotics.However,how DegQ controlled?fengycin synthesis was unclear.In this study,the sfp gene from B.amyloliquefaciens FZB42 and the degQ gene from B.subtilis 168 were cloned to construct expression vectors pP43-degQ,pMK3-sfp and pP43-degQ-sfp for subsequently expressed in strain 168 which can't produce fengycin,constituted three genetically engineered bacterial strains.One of them can produce fengycin.By the method of MALDI-TOF-MS analysis,we detected that only 168-degQ-sfp can produce fengycin.Antimicrobial activities experiment represented that both the colony and the lipopeptide extracted from cell-culture medium of 168-degQ-sfp have the ability to inhibit the growth of Sclerotinia sclerotiorum on plates,whereas 168,168-degQ and 168-sfp have no activity like that.168-degQ-sfp has the potential to be an engineered stain which can inhibit the plant pathogenic fungi.We had known that sfp gene encodes a 4'-phosphopantetheine transferase,which is necessary for nonribosomal peptide synthetases.However,how DegQ controlled?fengycin synthesis was still unclear.As the aim of studying the effect and function of degQ activation on fengycin productivity of B.subtilis 168,we detected the transcription level of the pps operon.The Real-time qRT-PCR result showed that the relative expression of fengycin synthase gene cluster ppsABCDE in 168-degQ-sfp were expressed at a level of 6—16 fold higher than wild-type while 168-degQ and 168-sfp were not.This study indicated that the two functional genes degQ and sfp can jointly raise the expression of fengycin synthase gene cluster,increasing fengycin yields.The previous studies inferred that the DegQ regulated the production of bacilysin and?-PGA by DegS-DegU two-component regulatory system.DegQ stimulates phosphate transfer from phosphorylated DegS(DegS-Pi)to DegU.The expression of synthase genes are controlled directly by DegU or DegU-Pi by binding the promoter region.To testify whether the DegQ was involved in the interaction with the Fengycin biosynthetic gene cluster promoter,we constructed strain 168Pspac using Pspac promoter replaced the pps operon promoter of B.subtilis 168.Then,the sfp gene was introduced into the strain 168Pspac to construct strain 168Pspac.Unexpected,168Pspac can't produce fengycin.And when the sfp gene and degQ were both introduced into the strain 168Pspac,named 168P-degQ-sfp,MALDI-TOF-MS showed that the 168P-degQ-sfp can produce fengycin.The results showed that the DegQ was not involved in the interaction with the Fengycin biosynthetic gene cluster promoter,means that the mechanism of DegQ controlled fengycin synthesis was different from the bacilysin or the ?-PGA synthesis.The real reason of DegQ controlled?fengycin synthesis needs further research.