Stereoselective decarboxylation of β-functionalized carboxylic acids enabled by engineered fatty acid photodecarboxylase

Abstract

Here, light-driven fatty acid photodecarboxylase (CvFAP) was engineered to broaden its substrate scope for the stereoselective decarboxylation of β-functionalized carboxylic acids, producing chiral carboxylic acids and chiral amines/alcohols. Initially, saturation mutagenesis was performed at V463, a residue within 5 Å of the substrate, which was identified by AFISHED scanning (AFISHED: seven representative amino acids ). Two beneficial variants, V463T and V463A, were obtained, showing 210% and 150% increases, respectively, in yield over wild-type (WT) CvFAP in the stereoselective decarboxylation of a medium-chain substrate, 10 mM β-hydroxydecanoic acid. Molecular dynamics simulations and interaction analysis reveal that the distance between the substrate and FAD cofactor in the above variants is 1.41–1.56 Å shorter than in the case of WT, due to microperturbation-induced “hydrogen bond network remodeling”. Moreover, clustering analysis reveals that for the short-chain substrate β-hydroxyhexanoic acid, the (R)-isomer preferentially forms an intramolecular hydrogen bond, facilitating the proper anchoring of the carboxyl group within the active site. This mechanism explains the strict enantioselectivity of CvFAP, as evidenced by achieving (S)-2-pentanol in 99% ee with V463A. Furthermore, the photostability of CvFAP was proven to be a vital factor influencing its activity. This work expands the catalytic scope of CvFAP toward the decarboxylation of β-hydroxy carboxylic acids with varying chain lengths and provides a promising strategy for the further resolution of β-functionalized carboxylic acids and synthesis of chiral chemicals by utilizing renewable solar energy.