Non-Canonical Type II Terpene Cyclases Deliver Rare Terpenoid Architectures

Terpene cyclases (TCs) catalyze the generation of complex polycyclic terpenoids through elaborate mechanistic cascades. Very little is known about the unusual type II TCs responsible for generation of a large diverse family of meroterpenoids known as indole diterpenoids (IDTs). The radarins are insecticidal and cytotoxic IDTs that possess an unusual core architecture for which the catalytic machinery is unknown. Here, we interrogate the genome of <i>Aspergillus fresenii</i> to reveal the biosynthetic machinery responsible for delivering the specific radarin scaffold. We demonstrate that a bespoke regioselective IDT epoxidase (RadM) acts at the terminal olefin of precursor 3'-geranylgeranylindole, providing access to this distinct class of IDT architectures. Coexpression of this epoxidase with a noncanonical type II TC RadB led to the biosynthesis of radarin C, in a complex skeletal rearrangement that proceeds via rare alcoholic deprotonation and ketone formation. Our modeling of RadB reveals that catalysis by RadB is enabled by a unique His residue that acts as both the proton donor and acceptor. Intriguingly, coexpression of RadM with an alternative <i>A. fresenii</i> IDT cyclase gene, which does not share this catalytic His, revealed an alternate partial cyclization of 14,15-epoxy-3'-GGI, producing a new decalin IDT. This work solves the final piece of the biosynthetic puzzle to access the full suite of known IDT architectures, informs type II cyclase engineering efforts, and expands the repertoire of impressive chemistry catalyzed by noncanonical TCs.