Systematic mining of fungal chimeric terpene synthases using an efficient precursor-providing yeast chassis

Abstract

Chimeric terpene synthases, which consist of C-terminal prenyltransferase (PT) and N-terminal class I terpene synthase (TS) domains (termed PTTSs here), is unique to fungi and produces structurally diverse di- and sesterterpenes. Prior to this study, 20 PTTSs had been functionally characterized. Our understanding of the origin and functional evolution of <i>PTTS</i> genes is limited. Our systematic search of sequenced fungal genomes among diverse taxa revealed that <i>PTTS</i> genes were restricted to Dikarya. Phylogenetic findings indicated different potential models of the origin and evolution of <i>PTTS</i> genes. One was that <i>PTTS</i> genes originated in the common Dikarya ancestor and then underwent frequent gene loss among various subsequent lineages. To understand their functional evolution, we selected 74 <i>PTTS</i> genes for biochemical characterization in an efficient precursor-providing yeast system employing chassis-based, robot-assisted, high-throughput automatic assembly. We found 34 <i>PTTS</i> genes that encoded active enzymes and collectively produced 24 di- and sesterterpenes. About half of these di- and sesterterpenes were also the products of the 20 known PTTSs, indicating functional conservation, whereas the PTTS products included the previously unknown sesterterpenes, sesterevisene (1), and sesterorbiculene (2), suggesting that a diversity of PTTS products awaits discovery. Separating functional PTTSs into two monophyletic groups implied that an early gene duplication event occurred during the evolution of the PTTS family followed by functional divergence with the characteristics of distinct cyclization mechanisms.