Genomic determinants of antifungal activity of Streptomyces melanosporofaciens STM-2 revealed by genome mining, comparative genomics and evolutionary analysis

Introduction Streptomyces species are prolific producers of bioactive compounds, and they play a role in biological control in agricultural systems. Materials and methods In the current study, we employed an integrated approach that involved physiological and biochemical characterization, genome mining, comparative genomics, and evolutionary analysis to elucidate the specific genomic determinants underlying the antifungal activity exhibited by STM-2, a novel Streptomyces melanosporofaciens strain, isolated from Chiayi County, Taiwan. Results and discussion Initial physiological and biochemical characterization, complemented by enzymatic screening, revealed the secretion of antifungal hydrolytic enzymes, including protease, CMCase, pectinase and xylanase, which actively play a role in fungal growth inhibition and fungal cell wall degradation. To elucidate the molecular basis of this activity, a wholegenome sequence was obtained and annotated, revealing a typical high-GC (71%) linear chromosome. Genome mining using antiSMASH predicted an extensive repertoire of 52 biosynthetic gene clusters (BGCs), encompassing type I and II PKS, NRPS, and hybrid PKS–NRPS systems, RiPPs, siderophores, terpenes, and diverse tailoring enzymes (P450s, glycosyltransferases, halogenase candidates). Again, we identified genes tentatively responsible for antifungal activity, including chitinases, β-1,3-glucanases, xylanses, pectinases, and proteases. Comparative pangenomic analysis, supported by high-resolution Average Nucleotide Identity (ANI) and digital DNA–DNA hybridization (dDDH) scores, revealed the evolutionary uniqueness of the strain, identifying an accessory genome containing unique genes specifically associated with specialized antifungal activity. Further in silico structural analysis of these unique gene products using AlphaFold2 yielded threedimensional protein models with very high pLDDT scores, providing high-confidence structural evidence for their specialized functional roles. Collectively, these results provide a comprehensive understanding of the genomic determinants required for potent biocontrol activity, positioning STM-2 as a promising candidate for biocontrol and further research in biotechnological applications.