(271e) High Throughput Screening Assay for Alcohol Acetyl/Acyltransferase

The biological synthesis of esters has recently been the focus of a number of metabolic engineering efforts. Long chain waxy esters have potential as diesel fuel substitutes and volatile esters are valuable as natural food additives (e.g. isoamyl acetate and phenyl ethyl acetate) and industrial solvents (e.g. ethyl acetate). The biosynthetic pathways that produce these compounds share a common terminal reaction step, the condensation of an alcohol with an acetyl- or acyl-CoA by alchohol-O-acetyltransferase (AATase, E.C. 2.3.1.84 and 2.3.1.74). With respect to enzyme engineering, rational design and directed evolution approaches to improve AATase activity have been limited due to the low throughput nature of gas chromatography (GC) and high performance liquid chromatography (HPLC). To address this bottleneck, we developed a high throughput screening assay to evaluate AATase activity. The spectrophotometric assay can be performed in 96-well plate format and is applicable to high throughput screening (Z ≥ 0.5). AATases orthologs from Saccharomyces and non-Saccharomyces yeast and also fruit species were screened with various short- and medium-chain acyl-coA and ethanol as substrates. A key finding from these screening experiments was the identifications of a high activity AATase from tomato fruit. The developed assay is robust and easy to use, thus providing new tool for rational design and directed evolution studies of AATases.