Identification and Characterization of a Novel Multifunctional Terpene Synthase from the Pathogenic Fungus <i>Cochliobolus heterostrophus</i>

Sesquiterpenes, a structurally diverse class of natural products, play pivotal roles in the pharmaceutical, fragrance, and flavor industries. In this study, we first identified and functionally characterized a multifunctional sesquiterpene synthase, ChTPS6, from the plant-pathogenic fungus <i>Cochliobolus heterostrophus</i>. When incubated with FPP, ChTPS6 primarily catalyzed the formation of cubebol as the main product, which is widely used as a sustained cooling and refreshing agent in the food industry. Remarkably, ChTPS6 exhibited substrate promiscuity, efficiently utilizing GPP and GGPP as alternative substrates. Moreover, through protein modeling and site-directed mutagenesis, we pinpointed key amino acid residues essential for the catalytic activity of ChTPS6, and successfully re-engineered the enzyme to alter its product specificity. These findings provide novel insights into the catalytic mechanism and functional versatility of ChTPS6 while broadening its potential applications in the biosynthesis of valuable sesquiterpenes.