Discovery of fungal onoceroid triterpenoids through domainless enzyme-targeted global genome mining

Genomics-guided methodologies have been widely adopted and proven beneficial for the discovery of natural products. However, a major challenge in the field of genome mining is determining how to selectively extract biosynthetic gene clusters (BGCs) for untapped natural products from a substantial number of available genome sequences. In this study, we developed a fungal genome mining pipeline that extracts BGCs encoding enzymes that lack a detectable protein domain and are not recognized as biosynthetic proteins by existing bioinformatic tools. We searched for BGCs encoding a homologue of Pyr4-family terpene cyclases, which are representative examples of domainless enzymes, in approximately 2,000 fungal genomes and discovered several BGCs with unique features. The subsequent characterization of these BGCs led to the discovery of the first fungal onoceroid triterpenoids and unprecedented onoceroid synthases. Our genome mining pipeline has broad applicability in fungal genome mining and can serve as a beneficial tool for accessing diverse, unexploited natural products.