This study reveals a highly regioselective and cofactor-independent enzymatic approach to terpene and terpenoid hydration using a carotenoid 1,2-hydratase from Rubrivivax gelatinosus. Beyond detailing reaction optimization strategies, an extensive substrate scope, and scale-up potential, it explores mutagenesis and structural modeling to identify key catalytic residues and improve enzymatic activity.

Abstract

Terminally hydrated terpenes are highly sought-after compounds in the flavor and fragrance industries. However, their selective synthesis remains a considerable challenge in catalysis. Regioselective hydration of non-activated C─C double bonds is typically hindered by poor selectivity and low atom efficiency in conventional methods. In this study, we harness the underexplored potential of the acyclic carotenoid 1,2-hydratase from Rubrivivax gelatinosus IL144, employing it as a whole-cell biocatalyst for cofactor-independent terminal hydration of a diverse range of terpenes. This enzyme demonstrates exceptional activity across more than 20 C₁₂─C₂₀ terpenes and shows notable tolerance to various functional groups, establishing it as a valuable tool for sustainable organic synthesis. We emphasize the critical influence of expression system choice in maximizing enzymatic performance, enabling high-yield transformations on the gram scale. Through a combination of homology modeling, consensus analysis, and targeted mutagenesis, essential residues involved in catalytic activity were identified. Notably, enhanced catalytic efficiency was only achievable through the epistatic effect of three specific mutations. These findings highlight the biocatalytic potential of acyclic carotenoid hydratase, offering a green and efficient route to the production of valuable tertiary alcohols.