Structure‐guided engineering of oleate hydrates for the synthesis of small chiral alcohols without decoy molecule

Small and chiral secondary alcohols are sought‐after compounds that are frequently used in the synthesis of biologically active compounds. However, their stereochemically correct synthesis remains a challenge for the chemical industry. Synthetic routes are restrained by multiple steps and thus low atom efficiency. Here we employ engineered variants from the oleate hydratase from E. meningoseptica to produce chiral C5‐C10 secondary alcohols within a single step. First, we established a two‐phase system (2‐PS) to trap the volatile substrates/products which facilitated semi‐rational mutagensis in the active site and tunnel of the enzyme. These efforts led to variants with an up to 20‐fold increase in catalytic activity and >99% ee, notably, without the use of a decoy molecule. Computational analysis indicated structural changes in the tunnel radius, ligand transport and energy profiles, which gave us hints to explain the enhanced performance of the variants. Interestingly, scaling up the reaction volume demonstrated significant increases in product concentrations, leading up to a 100‐fold increase in comparison to the current benchmark. This new 2‐PS and the engineered enzyme variants offer a promising approach for a scalable, asymmetric hydration of small unactivated alkenes, which would drastically ease the access to chiral alcohol‐based pheromones or drugs.