Genome mining and UHPLC–QTOF–MS/MS illuminate the potential antimicrobial active compounds and specificity of biosynthetic gene clusters in Bacillus subtilis NCD-2

Abstract

Background Bacillus subtilis strain NCD-2 is an excellent biocontrol agent against plant soil-borne diseases and shows broad-spectrum antifungal activities. This study aimed to explore some of the secondary metabolite biosynthetic gene clusters and related bioactive compounds in strain NCD-2. An integrative approach, which combined genome mining with structural identification technologies using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-MS/MS), was conducted to interpret the chemical origins of the significant biological activities in strain NCD-2. Results Genome mining revealed that strain NCD-2 contained nine gene clusters having predicted functions involving secondary metabolites with bioactive abilities. They encoded six known products including fengycin, surfactin, bacillaene, subtilosin, bacillibactin, bacilysin and three unknown products. Fengycin, surfactin, bacillaene and bacillibactin were successfully detected from the fermentation broth of strain NCD-2 by UHPLC-QTOF-MS/MS. Bacillaene, subtilosin, bacillibactin, and bacilysin related biosynthetic gene clusters showed 100% amino acid sequence similarity with B. velezensis strain FZB42，however, the biosynthetic gene clusters for surfactin and fengycin showed 83% and 92%, respectively. Further comparison of gene clusters encoding fengycin and surfactin revealed that strain NCD-2 had lost the fenC and fenD genes in the fengycin biosynthetic operon. Moreover, biosynthetic enzyme-related gene srfAB for surfactin had divided into two parts. Bioinformatics analysis predicted that FenE function in strain NCD-2 was same to that of FenE and FenC in strain FZB42, and FenA function in strain NCD-2 was same to that of FenA and FenD in strain FZB42. Five kinds of fengycin, with 26 homologs, and surfactin, with 4 homologs, were detected from strain NCD-2. To the best of our knowledge, this is the first report of a non-typical and unique gene cluster related to fengycin synthesis. Conclusions It was found that there were many gene clusters encoding antimicrobial compounds in the genome of strain NCD-2, and the fengycin synthetic gene cluster might be unique by using genome mining and UHPLC–QTOF–MS/MS. The production of fengycin, surfactin, bacillaene and bacillibactin might explain the biological activities of strain NCD-2.