Engineering of Elizabethkingia meningoseptica Oleate Hydratase with Enhanced Activity: High-Yielding Synthesis of 10-Hydroxy Fatty Acids

Oleate hydratase from Elizabethkingia meningoseptica ( Em -OhyA) catalyzes the 10-hydration of the carbon–carbon double bond of oleic acid, offering a green, clean, and efficient route to produce valuable 10-hydroxy fatty acids. To enhance Em -OhyA activity, directed evolution was performed using a multi-regional combination mutagenesis strategy. Based on molecular docking, 31 mutation sites across five functional regions—A (E122), B (T436/N438), C (Y241), D (FAD-binding), and E (substrate tail)—were targeted. Four rounds of evolution were carried out: iterative saturation mutagenesis of regions A–C, combination of top variants, and successive saturation mutagenesis of regions D and E. The best variant, L98M/F134L/F435G, exhibited a 9.6-fold increase in activity and a 7.1-fold increase in catalytic efficiency ( k cat / K M ) relative to the wild-type, while maintaining high regioselectivity (>99%) and high enantioselectivity (97%). Mutational analysis indicated that the B, D, and E regions play a more critical role in hydration than the A and C regions. Docking of oleic acid into catalytic pockets of the enzyme and mutant suggested shortened distance from substrate to the carboxylate binding region (N438) and to the water activation/addition region (E122) in the mutant, and molecular dynamics simulations showed reduced energy of the substrate bound in the catalytic pocket of the mutant, thereby boosting the 10-hydration activity. The triple mutant was well expressed in Escherichia coli, and whole-cell biotransformation achieved 98% isolation yield at 600 mM substrate loading and achieved the highest reported product concentration (176.4 g/L) for aerobic hydratases. Moreover, the Em -OhyA mutants displayed significantly improved activity toward other fatty acids, including palmitoleic acid (3.7-fold), myristoleic acid (6.0-fold), and linoleic acid (8.5-fold), affording the highest yields (75−85%) for the corresponding 10-hydroxy fatty acids among reported whole-cell biotransformation. The success of engineering Em -OhyA provides mechanistic insight into oleate hydratase structure–function relationships, guidance for engineering this enzyme family, and simple and high-yielding biocatalytic routes to hydroxy fatty acids of industrial relevance.