Molecular Regulatory Of Biosynthesis Mechanism And Application In Wheat Storage Of Bacillomycin D

Bacillus amyloliquefaciens,widely distributed in natural environment,is an aerobiotic and gram-positive bacteria that could produce spores.As a biocontrol microorganism,it mainly produces secondary metabolites to resist pathogenic microorganism.Bacillomycin D is a cyclic antifungal lipopeptide produced by secondary metabolism of bacillus.It is a cyclic lipopeptide structure composed of a ?-amino fatty acid chain and a peptide chain with seven amino acids which was catalyzed by a non-ribosomal synthetase.In general,the yield of bacillomycin D in bacillus was low.As a kind of secondary metabolite,the regulation mechanism of bacillomycin D is complex.There are many studies on its synthase genes globally,but the regulation mechanism in gene level has not been clarified,which has affected the improvement of the high-yield bacillomycin D strains at the gene level.Fusarium graminearum,a kind of filamentous fungus,could caused head blight which was one of the main diseases of wheat.It could also produced deoxynivalenol contaminated wheat and threaten to human health.Bacillomycin D has high antifungal bioactivity.Therefore,it has a promising potential application in food storage.In the current study,based on the results of whole genome sequencing of B.amyloliquefaciens fmbJ,we explored the influences of knockout and overexpression signal transduction genes(rapC,comA,degU,spo0A,and codY)on synthesis of bacillomycin D.Then,the regulating mechanism of the signal transduction genes in bacillomycin D synthesis was illustrated by transcriptome sequencing.Furthermore,the antifungal activity of F.graminearum by bacillomycin D was investigated.Finally,the performance of bacillomycin D in controlling F.graminearum contamination and toxin production in wheat were evaluated under the storage conditions of high temperature and humidity.The aim of this study was to select high-yield bacillomycin D strains at the gene level,and build a foundation for bacillomycin D industrial production and application in food storage and preservation.The main research results were as follows:1.Whole genome sequencing of B.amyloliquefaciens fmbJThe whole genome of B.amyloliquefaciens fmbJ was combination sequenced by two high-throughput sequencing technologies,Illumina Hiseq 4000 and Pacbio RSII.The genome size of fmbJ was 4193344 bp,and the GC content was 45.98%which contained 4249 genes.Gene functional annotation of COG categories was applied for these genes.The results indicated that 64 genes were related to controlling the bacterial motility,199 genes were connected with signal transduction mechanism,and 121 genes were associated with transport,catabolism,and biosynthesis of secondary metabolites.In regard to gene structure and content,the genome of fmbJ was similar to that of other bacillus,but it had its own characteristics.The difference between fmbJ and B.subtilis 168 was the greatest,while it was closest to B.amyloliquefaciens NAU-B3.The fmbJ genome contained synthase genes clusters of bacillomycin D,fengycin and surfactin,as well as many genes(including comA,degU,spoOA,codY,rapC,abrB,sigA,sigH,and so on)involved in the regulation of lipopeptide synthesis,which lay a foundation for the following studies on the synthesis regulation mechanism of bacillomycin D.2.Signal factor RapC regulated bacillomycin D biosynthesisTo clarify the influence of rapC regulating the synthesis of lipopeptide on the yield of bacillomycin D,rapC gene in fmbJ was successfully deleted by the marker-free method.Compared to wild strain finbJ,it was found that the deletion of rapC gene in fmbJ significantly improved bacillomycin D production from 240.7 ± 18.9 to 360.8± 30.7 mg/L.Expression profile of the relative synthetase genes and the signal proteins for bacilomycin D production was conducted by RT-PCR and the regulatory mechanisms were elucidated.The results showed that rapC gene knockout in fmbJ significantly improved bacillomycin D production attributed to the increased the expression of bacillomycin D synthesis-related genes through enhancing the transcriptional level of comA,comP,and phrC.These results showed that the production of bacillomycin D in B.amyloliquefaciens fmbJ might be regulated by the RapC-PhrC system.3.Signal factors ComA,DegU and SpoOA regulated bacillomycin D biosynthesisThree non-traced knockout comA,degU and spoOA mutant strains(fmbJ?comA,fmbJ ?degU,and fmbj?spoOA)were constructed by the synergistic recombination technique.The knockout of these genes resulted in obvious differences in colony morphology and the slower growth of the mutant strains.Under 33?,180 rpm and 72 h fermentation,the yield of bacillomycin D in mutant strains and the inhibition of Aspergillus ochraceus were reduced compared to the wild strain fmbJ.Transcriptome analysis of gene expression differences between mutant strains fmbJ?comA,fmbJ?degU,fmbJ?spo0A and wild strain fmbJ were dectected.The results showed that the number of genes had significant differences between mutant strains and fmbJ with 315(fmbJ?comA),654(fmbJ?degU)and 1034(fmbJ?spo0A),respectively.Compared with fmbJ,in the mutant strains fmbJ?comA,fmbJAdegU,and fmbJ?spoOA,104,377 and 466 genes were significantly up-regulated,while the number of genes significantly down-regulated was 211,277 and 568,respectively.GO function enrichment analysis and metabolic pathway analysis showed that ComA,DegU and Spo0A mainly involved in quorum sensing system,secondary metabolite synthesis,two-component system and substance transport system in B.amyloliquefaciens fmbJ.Moreover,bmyA,bmyB,bmyC and bmyD genes involved in the synthesis of bacillomycin D were all down-regulated.Then,constitutive overexpressed strains of fmbJcomA(amyE::PamyJ-comA),fmbJdegU(amyE::PamyJ-degU)and fmbJspo0A(amyE::PamyJ-spo0A)were constructed.The production of bacillomycin D in compositive overexpressed strains was significantly higher than that of the wild strain fmbJ,which was 1.77,1.91 and 1.82 times higher,respectively.Third,fmbJcomA,fmbJdegU and fmbJspo0A were constructed as dissociative overexpressed strains and were fermented for 72 h in the medium with different concentrations of the inducer(IPTG).The results showed that degU and spoOA played a direct regulatory role in bacillomycin D biosynthesis,and may directly act on the regulatory region of the synthesis of bacillomycin D gene promoter.While comA may have an indirect regulatory effect on the synthesis of bacillomycin D,and the active protein may be phosphorylated ComA.4.Global regulator CodY regulated bacillomycin D biosynthesisNon-traced knockout codY gene mutant strain of fmbJ?codY was constructed by the synergistic recombination technique.Compared to the wild strain fmbJ,the yeild of bacillomycin D by fmbJ?codY decreased from 236.7 ± 12.4 mg/L to 154.6 ± 8.4 mg/L in the condition of 33? and 180 rpm with fermentation culture 72 h.Under the same fermentation conditions,the yield of bacillomycin D by complement expression strain fmbJ?codY-codY was 224.9 ± 10.6 mg/L,which returned same to the level of wild strain.The results showed that the deletion of codY gene was the main reason for the decreased bacillomycin D production.Transcriptome analysis showed that 315 genes were significantly differentially expressed between the mutant strain fmbJ?codY and wild strain fmbJ,in which 159 up-regulated genes for codY transcriptional inhibition and 156 down-regulated genes for codY transcriptional activation.GO functional enrichment analysis and metabolic pathway analysis showed that CodY mainly involved in amino acid metabolism,secondary metabolite synthesis,two-component control system and ABC transport system in B.amyloliquefaciens fmbJ.In the mutant strain fmbJ?codY,the genes involving the synthesis of amino acids were up-regulated,while the bacillomycin D synthase genes(bmyA,bmyB,bmyC and bmyD)were down-regulated.Then,the overexpressed strain fmbJcodY was constructed,and different concentrations of inducer IPTG were added during 72 h fermentation.The results showed that proper concentration of CodY could promote the synthesis of bacillomycin D.When CodY accumulated excessively,the yield of bacillomycin D did not increase further.It could be inferred that the global regulator CodY had a concentration dependence on the synthesis of bacillomycin D.5.Application of bacillomycin D in wheat storageThe inhibition effect of bacillomycin D on the growth of F.graminearum was investigated.The hyphal growth and sporulation of F.graminearum were restrained dramatically in the presence of 75 ?g/mL bacillomycin D,and the inhibition rate reached to 94.6%and 97.5%,respectively.Ultrastructural observation of the hyphae showed bacillomycin D caused distorting and deforming the mycelia of F.graminearum,destroying cell walls and cell membranes,thus made organelles and cytoplasm irregular and empty.Furthermore,the actual application performance of bacillomycin D in controlling F.graminearum contamination was studied by simulating the storage conditions of grain.Bacillomycin D could lower the free fatty acid value and total antioxidant capacity and delay the decline of wheat quality,then exhibit an effective protection for wheat infection by F.graminearum.Bacillomycin D could effectively inhibit mold growth and DON production during wheat seeds storage,thus enhancing the quality and shelf life of the kernel.The addition of bacillomycin D(75?g/g wheat)remarkably inhibited the amount of F.graminearum growth,and reduced the DON production to 47.5-71.5%.These results indicated that bacillomycin D might be a promising natural and effective fungicide,and would have potential for reducing mycotoxins in food and feed.