Computational Mining of a Novel Acetyl-CoA Synthetase for the Biosynthesis of Unnatural Phenylalanine Butyramide

Phenylalanine butyramide (FBA) is an unnatural amide with the potential to be a postbiotic derivative due to its improved organoleptic and physicochemical properties compared to butyric acid and its salts. However, a green biosynthesis method to prepare FBA has not yet been studied. In this study, we mined a novel acetyl-CoA synthetase from Methanocella arvoryzae (MethACS) for the synthesis of FBA using a sequence- and structure-guided database mining approach that combines solubility prediction with the binding free energy of the protein. MethACS exhibits excellent catalytic performance, pH stability, and good thermophilicity. Using MethACS, 0.35 g/L of FBA could be produced with a 100% conversion rate when the concentration ratio of butyric acid to phenylalaninamide was >1:4. Through density-functional theory calculations and molecular dynamics simulation, we gained insight into the mechanism of the synthesis of FBA catalyzed by MethACS. Trp264 was further identified as a crucial residue influencing substrate specificity by saturation mutagenesis. Substrate profiling revealed that MethACS can catalyze the formation of amide derivatives of short- and medium-chain fatty acids. This study offers an efficient approach for investigating the biosynthesis of unnatural substances.