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

ABSTRACT 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.