Abstract
Baeyer-Villiger monooxygenases (BVMOs) can be used for the biosynthesis of lactones and esters from ketones. However, the BVMO-based biocatalysts are not so stable under process conditions. Thereby, this study focused on enhancing stability of the BVMO-based biocatalysts. The biotransformation of ricinoleic acid into (Z)-11-(heptanoyloxy)undec-9-enoic acid by the recombinant Escherichia coli expressing the BVMO from Pseudomonas putida and an alcohol dehydrogenase from Micrococcus luteus was used as a model system. After thorough investigation of the key factors to influence stability of the BVMO, Cys302 was identified as an engineering target. The substitution of Cys302 to Leu enabled the engineered enzyme (i.e., E6BVMOC302L) to become more stable toward oxidative and thermal stresses. The catalytic activity of E6BVMOC302L-based E. coli biocatalysts was also greater than the E6BVMO-based biocatalysts. Another factor to influence biocatalytic performance of the BVMO-based whole-cell biocatalysts was availability of carbon and energy source during biotransformations. Glucose feeding into the reaction medium led to a marked increase of final product concentrations. Overall, the bioprocess engineering to improve metabolic stability of host cells in addition to the BVMO engineering allowed us to produce (Z)-11-(heptanoyloxy)undec-9-enoic acid to a concentration of 132 mM (41 g/L) from 150 mM ricinoleic acid within 8 h.