Discovery of leucine analogues of tyrocidines and tryptocidines from Brevibacillus brevis B011 through genome mining and in vitro biosynthesis

Brevibacillus brevis B011 is an endophytic isolate from tobacco roots with significant antifungal activity, yet the metabolites responsible for this phenotype and their biosynthetic mechanisms have not been clearly resolved. Genome mining predicted 13 biosynthetic gene clusters, including nonribosomal peptide clusters for edeine, tyrocidine, and gramicidin. Gene knockout, confrontation assays and High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) indicated that edeines is the primary determinant of antifungal activity, with tyrocidine contributing secondarily, whereas gramicidin plays a minimal role under the tested conditions. Absolute quantitative shows that the total yield of edeine is 19.2 mg/L. Based on LC–MS total ion chromatogram peak areas, the relative abundances of tyrocidines F, G, H, and I were normalized to the sum of these four derivatives as 100%, giving proportions of 25.6%, 18.4%, 34.4%, and 21.6%, respectively, with tyrocidine H being the most abundant. MS/MS putative the primary structure of edeines and four tyrocidine derivatives (each containing leucine as the third residue). The derivatives and their biosynthetic pathways were further verified through in vitro one-pot system. Comparative analysis revealed that tyrocidine F is a previously unreported derivative, whereas the remaining derivatives are consistent with reported streptocidins. Multiple sequence alignment of the adenylation (A) domains showed that TycB_M3 had only 34.7% identity with the canonical Phe-specific A domain (1AMU), showing higher similarity (39.1–56.9%) to Leu-specific A domains and exclusively recognizing leucine. Molecular docking predicted Leu-specificity-conferring residues (S401, Q404, G424, K427, S429, S591, and D593), with G424 (G301 in 1AMU) likely playing a key role in the substrate shifts from Phe to Leu. This study discovery new antimicrobial peptides through genome mining and established an antifungal phenotype-metabolite-BGC framework for B . brevis B011, providing a stronger experimental basis for its future application in plant disease biocontrol.