Engineering of an oleate hydratase for efficient C10-Functionalization of oleic acid

Oleate hydratase catalyzes the hydration of unsaturated fatty acids, giving access to C₁₀-functionalization of oleic acid. The resultant 10-hydroxystearic acid is a key material for the synthesis of many biomass-derived value-added products. Herein, we report the engineering of an oleate hydratase from Paracoccus aminophilus (PaOH) with significantly improved catalytic efficiency (from 33 s^-1 mM^-1 to 119 s^-1 mM^-1), as well as 3.4 times increased half-life at 30 °C. The structural mechanism regarding the impact of mutations on the improved catalytic activity and thermostability was elucidated with the aid of molecular dynamics simulation. The practical feasibility of the engineered PaOH variant F233L/F122L/T15 N was demonstrated through the pilot synthesis of 10-hydroxystearic acid and 10-oxostearic acid via an optimized multi-enzymatic cascade reaction, with space-time yields of 540 g L^-1 day^-1 and 160 g L^-1 day^-1, respectively.