Stability evaluation and genome mining of antimicrobial metabolites of Bacillus velezensis PJP10

Abstract:

[Background] Food-borne pathogens are easy to grow and reproduce in food and have high pathogenicity, posing critical threats to food safety and public health.[Objective] To analyze the antimicrobial stability of Bacillus velezensis PJP10 from multiple perspectives, discover the genes encoding antimicrobial enzymes and involved in the biosynthesis of antimicrobial substances by genome sequencing, and measure the in vitro activities of enzymes and active metabolites, so as to provide data for the application of this strain in industry and agriculture. [Methods] The microbial inhibition tests were carried out to examine the effects of pH, temperature, protease, metal ions, ultraviolet (UV), and salt concentration on the antimicrobial stability of the strain. The potential genes encoding antimicrobial enzymes of PJP10 were investigated by whole genome sequencing and the enzyme-producing abilities assessed by enzyme activity assays. The secondary metabolites of PJP10 were predicted by antiSMASH. HPLC and microbial inhibition tests were employed to analyze the components of the crude extract obtained by the acid precipitation and ethyl acetate extraction. [Results] The cell-free fermentation supernatant exhibited excellent stability at high temperatures, strong acid, strong alkali, high salt and UV, and was sensitive to trypsin and metal ions, but not proteinase K or pepsin. The genome of PJP10 carried abundant genes encoding amylase, aminopeptidase, protease, chitinase, cellulase, esterase, pectinase, glucanase, and amidase, and the strain could produce protease, amylase, cellulase, and esterase. The antiSMASH predicted that strain PJP10 had a variety of gene clusters for the synthesis of secondary metabolites, including bacillaene, difficidin, macrolactin H, surfactin, bacillomycin D, fengycin, bacilysin, bacillibactin, amylocyclicin, and plantazolicin. In addition, the extract obtained by acidification exhibited strong inhibitory activities against Salmonella enteritidis and Escherichia coli but no activity against Ralstonia solanacearum. The ethyl acetate extract showed strong inhibitory effects on Staphylococcus aureus and R. solanacearum and weak inhibitory effects on S. enteritidis and E. coli. According to HPLC results, we hypothesized that the antimicrobial substances in the cell-free supernatant of PJP10 were iturin, fengycin, and surfactin. [Conclusion] The strain PJP10 shows high antimicrobial stability and carries rich genes encoding active enzyme and abundant gene clusters for the synthesis of antimicrobial substances, demonstrating a promising prospect of application in industry and agriculture.