Rapid ester biosynthesis screening reveals a high activity alcohol‐O‐acyltransferase (AATase) from tomato fruit

Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl‐CoA with an alcohol by alcohol‐O‐acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short‐ and medium‐chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf‐S.l). Atf1‐S.l exhibited broad specificity towards acyl‐CoAs with chain length from C₄ to C₁₀ and was specific towards 1‐pentanol. The AATase screen also revealed new acyl‐CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf‐C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester‐based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels.