Table 2_Genomic determinants of antifungal activity of Streptomyces melanosporofaciens STM-2 revealed by genome mining, comparative genomics and evolutionary analysis.xlsx

IntroductionStreptomyces species are prolific producers of bioactive compounds, and they play a role in biological control in agricultural systems.
Materials and methodsIn 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 discussionInitial 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.

引言：链霉菌属（Streptomyces）物种可大量产生生物活性化合物，在农业生态系统中发挥生物防治作用。
材料与方法：本研究采用整合研究策略，结合生理生化特性表征、基因组挖掘、比较基因组学及进化分析，旨在阐明从中国台湾省嘉义县分离得到的新型深孢链霉菌（Streptomyces melanosporofaciens）菌株STM-2所展现的抗真菌活性背后的特定基因组决定因子。
结果与讨论：初始生理生化表征结合酶活筛选实验显示，该菌株可分泌抗真菌水解酶类，包括蛋白酶、羧甲基纤维素酶（CMCase）、果胶酶及木聚糖酶，这些酶可有效抑制真菌生长并降解真菌细胞壁。为阐明该抗真菌活性的分子基础，本研究完成了全基因组测序与注释，结果显示其基因组为GC含量高达71%的典型线性染色体。通过antiSMASH进行基因组挖掘，预测到该菌株拥有52个丰富的生物合成基因簇（biosynthetic gene clusters，BGCs），涵盖I型与II型聚酮合酶（PKS）、非核糖体肽合成酶（NRPS）、聚酮合酶-非核糖体肽合成酶杂合系统、核糖体合成并经翻译后修饰的肽（RiPPs）、铁载体、萜类化合物及多种修饰酶，包括细胞色素P450单加氧酶、糖基转移酶及候选卤化酶。此外，本研究还初步鉴定出若干与抗真菌活性相关的基因，包括几丁质酶、β-1,3-葡聚糖酶、木聚糖酶、果胶酶及蛋白酶。结合高分辨率平均核苷酸一致性（Average Nucleotide Identity，ANI）与数字DNA-DNA杂交（digital DNA–DNA hybridization，dDDH）分析结果开展比较泛基因组学分析，结果揭示了该菌株的进化独特性，并鉴定出其附属基因组中存在与特异性抗真菌活性紧密相关的独有基因。进一步利用AlphaFold2对这些独有基因产物开展计算机结构分析，获得了具有极高pLDDT得分的三维蛋白质结构模型，为其专属功能角色提供了高可信度的结构证据。综上，本研究结果全面阐明了该菌株高效生物防治活性所需的基因组决定因子，表明STM-2是极具应用潜力的生物防治候选菌株，可用于后续生物技术相关研究与应用。