Exploring endophytic actinomycetes from Shiraia fruiting bodies to enhance fungal hypocrellin A biosynthesis

The medicinal fruiting bodies of Shiraia harbor diverse microbial communities that are increasingly recognized as important regulators of fungal secondary metabolism. While endophytic bacteria and fungi associated with Shiraia have recently attracted considerable attention, the diversity and functional roles of endophytic actinomycetes remain largely unexplored. Here, we combined culture-dependent isolation with high-throughput 16 S rRNA amplicon sequencing to investigate the endophytic actinomycete community associated with Shiraia fruiting bodies. A total of 28 actinomycete isolates representing four genera were recovered, with Streptomyces being the predominant culturable genus. Functional screening identified the native endophyte S. pseudogriseolus A6 as the most effective elicitor of fungal hypocrellin A (HA) biosynthesis. Genome analysis revealed abundant carbohydrate-active enzymes and 27 secondary metabolite biosynthetic gene clusters, indicating a strong genetic potential for fungal colonization and metabolite-mediated communication. Co-cultivation with live strain A6 markedly enhanced both intracellular and extracellular HA accumulation without significantly affecting fungal biomass. Under the optimized co-cultivation conditions, total HA production reached 296.0 mg/L, representing a threefold increase over the monoculture control. Furthermore, A6 coordinately upregulated the expression of HA biosynthetic genes and metabolite transport genes. This study provides the first characterization of endophytic actinomycetes associated with Shiraia fruiting bodies and identifies actinomycete elicitors capable of stimulating fungal HA biosynthesis. These findings expand current knowledge of the Shiraia microbiome and highlight endophytic actinomycetes as promising microbial resources for enhancing fungal perylenequinone production.