Broad‐Spectrum Aqueous Esterification Using the Adenylation Domain of a Carboxylic Acid Reductase Coupled With ATP Regeneration

Biocatalytic esterification in water is a green alternative to chemical synthesis but often faces challenges such as low enzyme efficiency, poor substrate solubility, and expensive cofactors. Here, we present a streamlined aqueous esterification system utilizing the adenylation domain of carboxylic acid reductase (A-domain_CAR), a minimal catalyst that efficiently activates carboxylic acids. A-domain_CAR exhibited superior catalytic performance over full-length CARs, achieving up to 96% yield of methyl cinnamate under optimized aqueous conditions. To improve cost-efficiency and scalability, the system was coupled with a Class III polyphosphate kinase 2 (Class III PPK2) from Deinococcus proteolyticus for in situ ATP regeneration using AMP and polyphosphate. This two-enzyme platform enabled high-yield esterification across a broad range of cinnamic and benzoic acid derivatives and various alcohols. Incorporating micellar media further enhanced the conversion of poorly soluble aromatic alcohols such as benzyl and phenethyl alcohol. Preparative-scale esterification of methyl caffeate, a bioactive antioxidant ester, was successfully demonstrated with a 66% yield in a 500 mL aqueous reaction. This study highlights A-domain_CAR as a modular, efficient, and scalable biocatalyst, advancing sustainable ester synthesis for applications in pharmaceuticals, fine chemicals, and bio-based materials.