Genomics-driven Discovery Of NRPS And Type ? PKS Metabolites From Bacillus Subtilis Fmb60 And Their Bioactivity

Food safety has been a publiuc issue for many years.Due to microbiological contamination and bacterial resistance in food,as well as the prohibition of some chemical preservatives,food safety is facing a great threat.Therefore,it is necessary to develop novel,natural and safe food preservatives.As a kind of generally recognized safe(GRAS)strain,Bacillus subtilis has a wide range of applications in industry,agriculture,animal husbandry,biological control and food industry.Studies have shown that B.subtilis has a strong ability of secretion and expression.Genome sequencing results found that it has the potential to produce a variety of antimicrobial substances.In recent years,new and safe antimicrobial substances have been isolated from B.subtilis continuously.As an important microbial resource pool,biological compost contains a large microbial community,which has a wide application in the promotion of crop growth and the prevention of plant diseases and insect pests.Therefore,it can be used for bacterial screening.In this thesis,a B.subtilis strain with broad-spectrum antimicrobial activity was screened from plant straw composting.The NRPS and type I PKS gene cluster information were analyzed by genome and bioinformatics analysis.Next,by means of separation and purification techniques and spectroscopy tools,the structure and biological activity of the active substances associated with NRPS and type I PKS gene cluster were identified.The main results are shown as follows:1.Species identification and genomic sequencing of fmb60 with broad spectrum antimicrobial activityFmb60 strain was screened from the composting of plant stalks.The antibacterial test results showed that the fermentation supernatant has a strong inhibitory effect against gram-positive bacteria(B.cereus ATCC 14579,M.luteus CMCC 28001,B.pumilus CMCC 63202,Aureus ATCC 25923,C.difficile CICC 22951 and B.megaterium CICC 10448)and Gram-negative bacteria(E.coli ATCC 25922,P.fluorescens ATCC 49642).Based on the physiological and biochemical identification and 16S rDNA homology analysis,fmb60 strain was identified as B.subtilis.After that,the phylogenetic tree of this strain was constructed.The genome of B.subtilis fmb60 was extracted and three scaffold sequences were obtained by genome sequencing.3045 genes were identified from gene sequences by glimmer software.This strain contained 17 secondary metabolites synthesis gene clusters,including three NRPS gene clusters,one type I PKS gene clusters,and one NRPS-PKS gene cluster.Moreover,three NRPS gene clusters were found to be highly homologous with surfactin(srf),fengycin(fen)and bacillibactin(dhb)synthase by 78%,100%and 92%,respectively.The PKS gene cluster Bacillus genus calyculin synthesis gene cluster had a certain homology(64%),while NRPS/PKS synthetic gene cluster xenocoumacin synthetic gene cluster had a certain homology(28%).2.Identification of metabolites relating to NRPS gene cluster and antioxidant activity of bacillibactinThree antimicrobial substances were obtained from B.subtilis fmb60 fermentation supernatant through acid precipitation,Sephhadex LH-20 chromatography and HPLC purification,successively.HPLC-MS/MS and NMR analysis showed that the substances were surfactin,fengycin and bacillibactin.The MIC of surfactin,fengycin and bacillibactin against different indicators were determined by broth dilution method.The results showed that surfactin and bacillibactin had noticeably inhibitory activity against Gram-positive and Gram-negative bacteria.On the other hand,fengycin had no inhibitory activity against the aforementioned six bacteria.The antioxidant activity of bacillibactin was studied by chemical methods(DPPH free radical,hydroxyl radical,superoxide anion radical,reducing power and total antioxidant capacity)and the protection of oxidative damage in PC 12 cells induced by H2O2.The results showed that bacillibactin had higher antioxidant activity than Vc,and its antioxidant activity was dependent on the concentration.At the same time,cell oxidative damage experiments showed that cell pretreatment by bacillibactin(<200 ?g/mL)had a significant inhibitory effect on H2O2-induced oxidative damage in PC 12 cells.3.Identification of Aurantinin B,C,D and their bacteriostatic mechanism against S.aureusIn this chapter,three kinds of antimicrobial substances aurantinin B,C and D were obtained from the B.subtilis fmb60 fermentation broth by acid precipitation,ethyl acetate extraction,Sephhadex LH-20 chromatography and HPLC.The structures were elucidated by LC-HRMS and NMR data analysis.In additon,aurantinins C and D were identified as novel antimicrobial compounds.Broth dilution method was used to test the MIC of aurantinin B,C,D to various indicator bacteria.The results showed that aurantinin B,C,D had better antibacterial effects of S.aureus ATCC 25923,M.luteus CMCC 28001,B.pumilus CMCC 63202,B.cereus ATCC 14579,B.subtilis ATCC 168,L.monocytogenes CICC 21662?E.faecalis ATCC 29212,P.fluorescens ATCC 49642 and S.aureus(MRSA),especially for anaerobic bacteria.However,the antibacterial effect of E.coli ATCC 25922 was not significant.Furthemore,the toxicity induced in hepatocyte L02 and Caco2 by aurantinin B,C and D were assayed.The results suggested that aurantinin B,C,D were low cytotoxicity against the hepatocyte L02 and Caco2(IC50>100 ?g/mL).In addition,the antibacterial mechanism of aurantinin B,C and D against S.aureus ATCC 25923 was studied.The results showed that when S.aureus ATCC 25923 were treated with aurantinin B,C,D for 1 h,the bacteria growth could be inhitied,and the cell's intracellular material began to leak.Furthermore,the mycellial morphological of S.aureus ATCC 25923 were changed under the action of aurantinin B,C and D.Therefore,it was presumed that aurantinin B,C and D could act on the cell membrane of bacteria to form cavity.As a result,intracellular substances were leaked and cell membrane was depolarized rapidly,eventually leading to the cell death.Through the analysis of the type I PKS clusters of the genome of B.subtilis fmb60,an antiSMASH and literature search,as well as MS and NMR data,it was suggested that this gene cluster was the most possbile candidate gene responsible for the synthesis of aurantinins.4.Separation,purification and structural identification of Amicoumacins compounds and biological activity researchSixteen compounds were isolated from B.subtilis fmb60 through the ethyl acetate extraction,Sephhadex LH-20 and HPLC.The molecular structures of products were comfirmed by HPLC-MS/MS and NMR.The compounds were lipoamide D,E,F,bacillmacin A,B,N-acetylmethy amicoumacin C,amicoumacin A,B and C,bacilosarcin A,B and C,N-acetylamicoumacin B,C,AI-77-H and lipoamicoumacins B.Among them,bacillmacin A,B were as new compounds,and N-acetylmethy amicoumacin C was a new natural compound.Then the biological activities of amicoumacins were analyzed.Broth dilution method was used to test the MIC of compounds 4-16 to different indicator bacteria.The results showed that amicoumacin A and bacilosarcin B had better antibacterial effects against S.aureus ATCC 25923,M.luteus CMCC 28001,B.pumilus CMCC 63202,B.cereus ATCC 14579,L.monocytogenes CICC 21662,P.fluorescens ATCC 49642and S.aureus(MRSA).The MIC value of bacilosarcin B against S.aureus(MRSA)was merely 3.13 ?g/mL.The compounds were assayed for IC50 towards different cell lines by MTT assay.The results showed that the amicoumacin A and bacilosarcin B exhibited significant antiproliferative activity with IC50 values of 3.5 to 19.3 ?M.The IC50 value of bacillmacin B was 28.2 to 39.1 ?M,and other compounds showed only limited activity to cancer cells.FRAP activities and the ATBS+ scavenging capacities of the amicoumacins were investigated.Bacillmacin B,N-acetylmethyamicoumacin C,N-acetylamicoumacin B,AI-77-H,and lipoamicoumacins B showed notable antioxidant activities.Then,the protective effect of amicoumacins compounds on H2O2 induced oxidative-stress damage in PC 12 cells was studied.The resulting data suggested that amicoumacins compounds could protect the oxidative injury of PC 12 cells induced by H2O2.In addition,the analysis of B.subtilis fmb60 genome showed that genes related to amicoumacins compounds syntheses in chromosome were NRPS/PKS synthetic gene cluster.Through sequence aligment and literature review,amicoumacins compounds were demonstrated to be secondary metabolites of NRPS/PKS synthetic gene cluster.