Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene

Summary N ootkatone is one of the major terpenes in the heartwood of the N ootka cypress C allitropsis nootkatensis . It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large‐scale EST sequencing from N ootka cypress wood revealed a valencene synthase gene ( C n VS ) . C n VS expression in different tissues from the tree correlates well with nootkatone content, suggesting that C n VS represents the first dedicated gene in the nootkatone biosynthetic pathway in C . nootkatensis The gene belongs to the gymnosperm‐specific TPS ‐d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro , C n VS displays high robustness under different p H and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from C itrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of C n VS in S accharomyces cerevisiae indicated potential for higher yields. In an optimized R hodobacter sphaeroides strain, expression of C n VS increased valencene yields 14‐fold to 352 mg/L, bringing production to levels with industrial potential.