Identification And Synthetic Pathway Of Novel Bioactive Secondary Metabolites In Bacillus Amyloliquefaciens SQR9

Bacillus amyloliquefaciens SQR9,isolated from the rhizosphere of cucumber,was proved to be an outstanding plant growth-promoting rhizobacteria(PGPR)strain,which could inhibit the growth of Fusarium oxysporum f.sp.cucumerinum J.H.Owen(FOC).Bacillus amyloliquefaciens SQR9 synthesized a large number of secondary metabolites which could inhibit the growth of plant pathogen.For SQR9,approximately 7.9%of its genome encode a variety of antibiotics,such as lipopeptides and polyketides.This study proceeded deep data mining in the whole-genome of B.amyloliquefaciens SQR9 for the discovery of new bioactive natural products,and found a unique genomic island GI3 associated with the biosynthesis of a novel natural product.The function of GI3 was find out through gene knock-out technology and used the activity tracking method to isolate and purify the extraction of B.amyloliquefaciens SQR9,and obtain the target substance for molecular structure determination.Finally,we investigated the biosynthesis and self-immunity through transcriptional analysis,gene knockout and gene fusion expression.The main results were summarized as follows.1.Genomic island GI3 synthesis of a novel bioactive secondary metabolites.The whole-genome of B.amyloliquefaciens SQR9 was obtained,and the biosynthetic gene clusters(BGCs)and associated secondary metabolites(SMs)were discovered by antibiotics&secondary metabolite analysis shell(antiSMASH),and 12 clusters were found involved in the biosynthesis of secondary metabolites.Some clusters were related to the biosynthesis pathways of the known secondary metabolites,but the biosynthesis pathway and the synthetic substance of Cluster 2 was unknown.Domain of cluster 2 includes such as acyltransferase(AT),acyl carrier protein(ACP),?-ketoacyl synthase(KS),?-keto reductase(KR),dehydratase(DH),thioesterase(TE),and these typical domains were belonged to type I fatty acid synthase or type I polyketide synthase,and the Cluster 2 was located at the third genomic island GI3 of SQR9.GI3 start from 656129 to 738610,about 82.3kb,composed of 26 ORFs.Linear alignment analysis of the genome showed that the genes around GI3 were present in other homologous strains,but ant the location of genomic islands GI3,these strains were varied.GC content of GI3 is significantly lower than the whole genome(40.1%vs.46.1%),suggesting that GI3 might be acquired by horizontal gene transfer(HGT).The 26 genes of GI3 were partitioned in to four sections,three polycistronic mRNAs and a monocistronic mRNA.The first operon was a polycistronic mRNA,contained 5 genes(bnaA,bnaB,bnaC,bnaD,bnaE),and the gene bnaA and bnaB belonged to ABC-transporter superfamily.The second operon was a polycistronic mRNA,contained 6 genes(bnaF,bnaG,bnaH,bnal,bnaJ,bnaK),and the gene bnaH was a typical type I fatty synthase.The third operon was a polycistronic mRNA,contained 14 genes(bnaL,bnaM,bnaN,bnaO,bnaP,bnaQ,bnaR,bnaS,bnaT,bnaU,bnaV,bnaW,bnaX,bnaY),and belonged to type I polyketide synthase.The fourth operon was a monocistronic mRNA,only contain one genes,bnaZ,and on the negative strand,may encode for TE,and end the extension of the carbon chain.2.The production of GI3 can inhibit the growth of the same specie strain of SQR9.B.amyloliquefaciens.SQR9 has a broad bacteriostatic spectrum,can inhibit the growth of various plant pathogens and same relatives Bacillus.To explore the function of GI3,a mutant SQR9MGI3 with a deletion of the whole GI3 was obtained.For tested phytopathogens and some bacteria,such as Bacillus,SQR9MGI3 showed slightly lower antagonistic activity compared with wild type SQR9,indicating GI3 product has weak activity against these strains.For phylogenetically closed strain B.amyloliquefaciens FZB42,that is the same specie strain of SQR9,the mutant SQR9MGI3 completely lost this antagonistic activity,indicating that the product of this genome island is functional for interspecies competition.To verify the conclusion,another of B.amyloliquefaciens-strain NJN6 was tested,the results were consistent with FZB42.These results confirmed the hypothesis that the production of GI3 plays a major role in the antagonistic activity against the homologous strains,that is B.amyloliquefaciens.3.The production of GI3 were long-chain fatty acids with antibacterial activity.A large number activity substance was produced by GI3 during stable phase,which had good thermostability,photo stability and pH-stability,so we used the activity tracking strategy to isolate and purify these products.The wild type B.amyloliquefaciens SQR9 was grown in Landy medium,then the supernatant was extracted by methanol,and separated into fractions by silica gel chromatography,and the active fractions through MPLC(medium pressure liquid chromatography),reversed-phase HPLC for further separation and purification.Two active pure compounds were obtained,11-hydroxy-12-methyltetradecanonic acid and its homologue 13-hydroxy-14-methylhexadecanonic acid,named the fatty acids Baillunoic acids.To further verify their activity,Bacillunoic acid A(C17H34O3)was chemically synthesized and has antagonistic activity against FZB42.4.The second operon and the third operon work together to synthesis Bacillunoic acids.The second operon was a type I fatty acid synthase(FAS),the third operon was belonged to a typical type I polyketide synthase(PKS)and contained large number domains such like AT?KS?ACP?KR?DH?TE.Two mutants,SQR9M2-PKS with a deletion of the second operon and keep the active of PKS,SQR9M3-FAS with a deletion of the third operon and keep the active of FAS were constructed.SQR9M2-PKS and SQR9M3-FAS lost the antagonistic ability against FZB42,but when SQR9M3-FAS were co-cultured with SQR9M2-PKS,the extraction of the supernatant could inhibit the growth of B.amyloliquefaciens FZB42.When the cell pellets of SQR9M2-PKS were added into the supernatant of SQR9M3-FAS,the supernatant could inhibit the growth of B.amyloliquefaciens FZB42,but added the cell pellets of SQR9M3-FAS into the supernatant of SQR9M2-PKS did not have the antibacterial activity.These experimental results verified the hypothesis that the hybrid type I fatty acid-type I polyketide synthase product the Bacillunoic acids.What's more,the second operon synthase work first to synthesise a primer,and the third operon work later to finish the synthesis.5.The self-immunity of GI3 was through ABC-transporter.The mutant SQR9MGI3 lost the antibacterial activity to FZB42,and we also find that the mutant SQR9MGI3 was also sensitive to wild type SQR9,suggested that GI3 is not only responsible for the interspecies bioactive substance production,but also responsible for its self-immunity of this product.Gene bnaA and bnaB were two genes of the first operon,and BlastP showed that these two genes belong to ABC transporter family.The bnaA and bnaB genes were knocked out individually and got two mutants SQR9MA and SQR9MB.On LB agar platesand in liquid LB medium,SQR9MA arise autolysis phenomenon,and in liquid LB medium SQR9MB also had the same phenomenon,but not obvious as SQR9MA.Collect the intracellular active substances and extracellular active substances of SQR9MA,the intracellular extraction could inhibit the growth of FZB42,but extracellular extraction did not have the antimicrobial activity.The result could prove that that first operon could transport Bacillunoic acids to extracellular to protect itself from the damage of the product.To confirm the result,the plasmid pMarAc with gene bnaA,bnaB and their promoter was constructed,and transformed into FZB42 to obtain a strain FZB42cAB,FZB42cAB could protect itself from the damage of Bacillunoic acids to some extent when incubated by Baillunoic acids.In this study,discovery of a novel bioactive natural product synthesized by genomic island GI3 in Bacillus amyloliqueficien SQR9,Bacillunoic acids,were identified as a long-chain saturated fatty acid,13-hydroxy-14-methylhexadecanonic acid and 11-hydroxy-12-methyltetradecanonic acid,and were synthesized by a hybrid type I fatty acid synthase(FAS)-polyketide synthase(PKS)system.Bacillunoic acids were also toxic to SQR9 itself,but in the presence of bacillunoic acids,the expression of the ABC transporter was induced and bacillunoic acids were secreted to protect SQR9 cells from damage.