Purification,Identification And Biosynthesis Pathway Analysis Of Antimicrobial Compounds Produced By Bacillus Siamensis JFL15

Agricultural production and aquaculture are major industry in China.However,the problems of pathogen invasion and the abuse of antibiotics in the production process have seriously affected the healthy development of these industries,which has caused major economic losses and security risks,so searching for a safe and effective substitute of antibiotics becomes the urgent matter.In order to provided theoretical basis and technical support for the further development and application of Bacillus siamensis JFL15,isolated from from the gastrointestinal tract of hairtail(Trichiurus haumela)and suppressing various phytopathogens and multidrug-resistant aquatic bacterial pathogens,the purification,identification and biosynthesis pathway analysis of antimicrobial compounds were investigated in detail.Main results were as following:(1)The strain JFL15 was identified as Bacillus siamensis,and designated as B.siamensis JFL15 according to the morphological,physiological and biochemical characteristic and 16 S rDNA sequence.(2)The culture medium and culture conditions was optimized.The optimal culture medium was MSM medium and optimal culture conditions were that: loaded liquid 100 mL,inoculum amount 1%,initial pH 7.2,fermentation temperature 30 °C,fermentation time 72 h,rotation speed 220 r/min.Meanwhile,the physicochemical properties and extraction methods for antimicrobial substances were preliminarily researched.Results showed the fermentation liquor was stable to enzyme,high temperature(up to 100°C),pH range from 3-9 and ultraviolet radiation,and the antimicrobial activity maintained after storage for 28 days under 4°C.The crude extract from ammonium sulfate precipitation,hydrochloric acid precipitation methanol extraction and ethyl acetate extraction methods both presented strong antimicrobial activity against 5 phytopathogens and 15 multidrug-resistant aquatic bacterial pathogens.(3)The antimicrobial compounds of B.siamensis JFL15 were purified and identified,which were successfully identified as volatile antifungal compounds(keton es,hydrocarbons,alcohols,esters,etc.),lipopeptides(surfactin,fengycin,iturin A,ba cillomycin F),bacillibactin,polyketides and cyclic dipeptides,and there are multiple homologues in each class of compounds.(4)The minimum inhibitory concentration(MIC)and antifungal mechanism of iturin A and bacillomycin F were researched.Results showed the MICs of iturin A and bacillomycin F against M.grisea,R.solani,C.gloeosporioides,P.litchi,and C.gloeosporioides were 62.50,31.25,62.50,31.25,62.50 and 125.00,62.50,125.00,62.50,125.00 ?g/mL,respectively.The SEM results indicated that the hyphae on the cell surface of M.grisea underwent severe ultrastructural changes in the presence of iturin A or bacillomycin F,thereby causing a sunk,lumpy,and wrinkled appearance.(5)The genome of B.siamensis JFL15 was sequenced.Results showed the complete genome sequence of strain JFL15 is characterized by a circular chromosome of 3,841,225 bp with a 46.36% G+C content,and 3600 protein-coding DNA sequences,82 tRNA genes,25 rRNA operons,9 sRNA genes,and 123 tandem repeat sequences were predicted.Meanwhile,the functional gene annotation and metabolic analysis of B.siamensis JFL15 were studied,including COG,GO and KEGG,etc.(6)According to the antiSMASH4.0 analysis of B.siamensis JFL15 genome,at least 9 antimicrobial compounds biosynthesis gene clusters were predicted,including 2 lipopeptides biosynthesis gene clusters(surfactin and fengycin),1 bacillibactin biosynthesis gene clusters,2 polyketides biosynthesis gene clusters(bacillaene and difficidin),2 antibiotics biosynthesis gene clusters(plantathiazolicin and butirosin),1 lantipeptide biosynthesis gene clusters,and a new type of polyketide biosynthesis gene cluster.Meanwhile,the metabolic pathways and regulatory factors of these antimicrobial substances were further analyzed.(7)Eight antimicrobial protein genes(c,g,s,t.a2,n,ap and b1)were successful mining from the genome of B.siamensis JFL15 by bioinformatics.These genes were then expressed in the prokaryotic expression,and the antimicrobial activities were further detected.Results showed all eight proteins were successfully expressed,and the recombinant protein C,G,T and AP exhibited relatively weak antifungal activity against C.gloeosporioides,while the recombinant protein S,A2,N and B1 showed no antimicrobial activity.(8)The genetic transformation system of B.siamensis was constructed.The optimal electro-transformation conditions were as follows: 1% DL-threonine,2% glycine,0.1% tryptophan and 0.03% Tween 80 were added to the preparation of competent cell;the electroporation buffer only contain 0.5 M sorbitol,0.5 M mannitol,0.5 M trehalose,and without any salt ions;the concentration of the plasmid is better than 600 ng/?L,and the optimal electric shock parameters are voltage 2.1 kv,electric shock time 5 ms,capacitor 25 uF and resistance 200 ?.(9)The homologous knockout plasmid pHT08-cody-up-down and gene knockout plasmid pHT08-Cas9-gRNA based on CRISPR were successfully constructed to target the cody gene.Both of these plasmids were confirmed to be transferred into B.siamensis JFL15,and the Cas9 protein was successfully expressed in B.siamensis JFL15,but the cody gene was not been knocked out through the above two methods.