Breeding High-yield Macrolactin Strains With Mutation And Antibacterial Activity Of Macrolactin Against Botrytis Cinerea

Gray mold caused by Botrytis cinerea is a destructive disease of strawberry.The infection mechanism of B.cinerea is complex,the range of parasitism is wide and the genetic variation is easy to occur.Therefore,it is important to find an antibacterial substance with high efficiency,environmental and no drug resistance.Macrolactin is the secondary metabolite of Bacillus siamensis in this paper.Basic studies have shown that it has obvious antibacterial effect on B.cinerea and other plant pathogens.On this basis,Macrolactin was isolated,purified and identified.Its control effect on B.cinerea was further studied.However,the yield of Macrolactin produced by wild strain is low,and the related regulation mechanism of biosynthesis is still unclear,which limits the research and application of this compound.Therefore,artificial mutation breeding technology was adopted to improve the production of Macrolactin and the regulatory pathway related to Macrolactin synthesis was analyzed by proteomics.The main results are as follows:（1）Macrolactin in B.siamensis YB304 fermentation products was isolated and purified by silica gel column chromatography,TLC and semi-HPLC.NJ08-3was identified by LC-MS,¹³C-NMR and ¹H-NMR spectra as Macrolactin R,with structure formula of C₃₀H₄₄O₁₀.The antibacterial activity of the compound was stable for a long time at p H5-7,lower than 80℃,and was not sensitive to most conventional organic reagents.At the same time,Macrolactin R had good inhibitory effect on some pathogenic bacteria and fungi.（2）In order to improve the yield of Macrolactin R from wild strains,the mutant Mut-K53 was obtained by UV-ARTP mutagenesis combined with screening of antibacterial zone activity and HPLC.Compared with the wild strain,the fermentation performance of Mut-K53 was significantly improved,and the Macrolactin R yield was increased by 3.95 times,which is 156.5μg/m L.（3）TMT proteomics combined with PRM was used to analyze the metabolic pathway changes caused by differential proteins and the regulatory mechanism of Macrolactin R.The results showed that the utilization of carbon sources such as sucrose was enhanced in Mut-K53;the glycolysis pathway was positively up-regulated;the amino acid metabolism pathway such as aromatic amino acids was significantly up-regulated.The production of various precursor substances such as acyl-Co A and amino acids was one of the main reasons for the increase of Macrolactin production.At the same time,the key proteins of Macrolactin biosynthesis are mostly up-regulated,which is the direct reason for the increase of Macrolactin R production.（4）The antibacterial activity of Macrolactin R on B.cinerea was studied.The results showed that the EC₅₀value of Macrolactin R to B.cinerea mycelia was 2.88μg/ml and was 1.93μg/ml to B.cinerea spores,which was equal to that of Natamycin and Pyrimethanil.Meanwile,Macrolactin R had significant antibacterial effect on strawberry gray mold in vitro;Macrolactin R could cause cell apoptosis and content leakage by destroying cell membrane structure.In conclusion,Macrolactin R has well antibacterial activity against Botrytis cinerea.