Unearthing a sesterterpene biosynthetic repertoire in the Brassicaceae through genome mining reveals convergent evolution

Abstract

Sesterterpenoids are a rare terpene class harboring untapped chemodiversity and bioactivities. Their structural diversity originates primarily from the scaffold-generating sesterterpene synthases (STSs). In fungi, all six known STSs are bifunctional, containing C-terminal <i>trans</i>-prenyltransferase (PT) and N-terminal terpene synthase (TPS) domains. In plants, two colocalized PT and TPS gene pairs from <i>Arabidopsis thaliana</i> were recently reported to synthesize sesterterpenes. However, the landscape of PT and TPS genes in plant genomes is unclear. Here, using a customized algorithm for systematically searching plant genomes, we reveal a suite of physically colocalized pairs of PT and TPS genes for the biosynthesis of a large sesterterpene repertoire in the wider Brassicaceae. Transient expression of seven TPSs from <i>A. thaliana</i>, <i>Capsella rubella</i>, and <i>Brassica oleracea</i> in <i>Nicotiana benthamiana</i> yielded fungal-type sesterterpenes with tri-, tetra-, and pentacyclic scaffolds, and notably (-)-<i>ent</i>-quiannulatene, an enantiomer of the fungal metabolite (+)-quiannulatene. Protein and structural modeling analysis identified an amino acid site implicated in structural diversification. Mutation of this site in one STS (<i>AtTPS19</i>) resulted in premature termination of carbocation intermediates and accumulation of bi-, tri-, and tetracyclic sesterterpenes, revealing the cyclization path for the pentacyclic sesterterpene (-)-retigeranin B. These structural and mechanistic insights, together with phylogenetic analysis, suggest convergent evolution of plant and fungal STSs, and also indicate that the colocalized PT-TPS gene pairs in the Brassicaceae may have originated from a common ancestral gene pair present before speciation. Our findings further provide opportunities for rapid discovery and production of sesterterpenes through metabolic and protein engineering.