Targeted identification of new phaterpenes and elucidation of the relevant biosynthetic pathway in Streptomyces...

Sesquiterpenes have highly diverse chemical structures and biological activities, resulting in considerable interest in terms of their application and exploration of new analogs. In this study, we identified a new cryptic bacterial terpenoid biosynthetic gene cluster (i.e., pha) in the genome of the soil microorganism Streptomyces phaeochromogenes OSK-123 (strain OSK-123). According to online BiG-FAM and sequence similarity network analyses, pha was revealed to span an approximately 18 kb DNA region with 14 open reading frames, which likely include sequences encoding enzymes catalyzing the production of new sesquiterpenes. To facilitate the identification of target compounds, strong constitutive promoters were incorporated into pha. Additionally, we conducted a comparative analysis of different fermentation extracts for wild-type and promoter-substituted strains as well as RT-qPCR and LC-ESI-MS analyses to efficiently detect and identify target compounds. An examination of spectroscopic data identified four new 6/5-fused bicyclic sesquiterpenoid compounds, designated as phaterpene A-D (compounds 1-4). The terpene synthase PhaA catalyzed the formation of a six-membered ring sesquiterpene skeleton via heterologous expression in Escherichia coli. The complete pha sequence was incorporated into plasmid pLQ1512 and heterologously expressed in Streptomyces albus J1074 to elucidate the phaterpene biosynthetic pathway. Candidate modification genes were disrupted and functionally validated in S. albus J1074. The protoporphyrinogen/coproporphyrinogen oxidase PhaB was identified to catalyze the formation of a 6,5-fused bicyclic sesquiterpenoid scaffold. On the basis of these findings, the new phaterpene biosynthetic pathway was established. This research not only presented a practical approach for discovery of the targeted compounds through integration of multiple pipelines, but also enriched the understanding of chemical diversity and biosynthetic machinery of the new sesquiterpenes.