Deciphering temporal antifungal dynamics of a rare actinomycete via integrated omics

ABSTRACT Fungal phytopathogens pose a persistent threat to global crop production, and widespread use of fungicides has driven resistance development and environmental concerns, necessitating sustainable alternatives. Actinomycetes produce diverse bioactive metabolites, yet natural product discovery has disproportionately focused on Streptomyces , leaving rare actinomycete taxa underexplored. Saccharomonospora xinjiangensis XJ-54 is a rare actinomycete exhibiting antifungal activity against Fusarium phytopathogens, including Fusarium oxysporum f. sp . cucumerinum (FORC), and harbors numerous biosynthetic gene clusters (BGCs) whose products remain uncharacterized. However, as in many rare actinomycetes, BGCs may be transcriptionally silent under standard laboratory conditions, and their expression dynamics remain poorly understood. To elucidate the molecular basis of antifungal activity in S. xinjiangensis XJ-54, we integrated genomic, transcriptomic, and metabolomic analyses. Cell-free supernatants inhibited FORC after 5 days of fermentation, with activity increasing by day 7. Time-resolved RNA sequencing demonstrated that all genomically identified BGCs were transcriptionally active but exhibited distinct growth-phase-dependent expression patterns, with approximately half upregulated during exponential growth and the remainder following transition to stationary phase. We observed temporal variations in transcriptional coupling between cluster-specific regulators and biosynthetic genes. Liquid chromatography-mass spectrometry-based metabolomics showed growth-phase-dependent metabolite shifts, including stationary phase accumulation of secoiridoid-like monoterpenoids, N-acyl amines, and alkaloids (imidazoles, pyridines, and indoles), correlating with the observed antifungal phenotype. Bioactivity-guided fractionation subsequently yielded an active fraction containing a predicted halogenated alkaloid that induced hyphal damage in FORC. These findings indicate that antifungal activity in S. xinjiangensis XJ-54 arises from temporally coordinated biosynthetic programs, providing a framework for optimizing growth conditions and prioritizing BGCs for functional characterization. IMPORTANCE The discovery of new antifungal compounds is critical for sustainable agriculture, yet most natural product research has focused on a narrow range of well-studied microorganisms. Rare actinomycetes represent an untapped reservoir of chemical diversity, but their biosynthetic potential often remains hidden. By integrating time-resolved transcriptomics with metabolomics, we show that the rare actinomycete Saccharomonospora xinjiangensis XJ-54 produces antifungal metabolites through temporally coordinated biosynthetic programs. Contrary to the prevailing assumption that the majority of biosynthetic gene clusters (BGCs) are silent, all BGCs in this strain showed detectable transcriptional activity under standard laboratory conditions, with expression patterns that were strongly growth-phase dependent. These findings provide a framework for linking gene cluster expression to metabolite production and bioactivity, enabling more rational prioritization of BGCs and optimization of cultivation strategies. More broadly, this work provides a roadmap for unlocking the biosynthetic potential of rare actinomycetes and accelerating the discovery of antifungal natural products relevant to sustainable crop protection.