Genome Mining and Characterization of an Uncharacterized Monofunctional Class I Diterpene Synthase From a Marine‐Derived Fungus

The first marine-derived fungal monofunctional class I diterpene synthase RousA was characterized by a combination of bioinformatics, phylogenetic analysis, and heterologous coexpression. RousA resides on a remote branch of phylogenetic tree, indicating it is an uncharacterized terpene cyclase. Heterologous coexpression of RousA and RousB in engineered yeast led to the production of a 5-8-5 tricyclic diterpene scaffold 1. Its absolute configuration was first established by ECD calculation. Structural modeling and site-directed mutagenesis demonstrated that several amino acid residues in nonconserved motif have a strong influence on 5-8-5 accumulation and F65 residue serves as pivotal "bidirectional functional switch" between tricyclic and bicyclic systems. A cyclization process of tricyclic product 1 exhibits a deprotonation mechanism distinct from those of known enzymes. The antimicrobial and plant immune activities were also evaluated.