Engineering an ancestral Baeyer-Villiger monooxygenase for efficient oxidative C-C bond cleavage of long-chain keto-fatty acids.

Baeyer-Villiger monooxygenases (BVMOs) catalyze regiospecific oxygenation of ketones into esters and lactones by using molecular oxygen and NADPH. However, the enzymes are often deactivated by oxidative and thermal stresses. Therefore, this study has focused on enhancement of structural stability of the BVMOs to improve whole-cell biotransformation performance. BVMOs, which catalyze oxidative C-C bond cleavage of keto-fatty acids (e.g., 10-ketostearic acid produced from oleic acid) into the corresponding esters, were used as model enzymes. Large scale sequence mining, clustering, and ancestral sequence reconstruction (ASR) led to the generation of an ancestral enzyme, which showed greater whole-cell biotransformation performance with high regioselectivity toward normal ester product than the extant enzymes (e.g., BVMO from Pseudomonas putida KT2440). Furthermore, molecular dynamics simulation-based loop engineering of the ancestral enzyme enabled the generation of highly active variants. One of the loop-engineered enzymes allowed the recombinant Escherichia coli cells coexpressing a fatty acid double bond hydratase and secondary alcohol dehydrogenase to produce 9-(nonanoyloxy)nonanoic acid to 26 mM with a bioconversion of 87% from oleic acid. The ester concentration was 13-fold greater than the extant enzyme-based whole-cell biocatalysts (e.g., PpBVMO). Therefore, this study will contribute to engineering of the BVMOs to improve the structural stability and whole-cell biotransformation performance.