Biosynthetic production of esters: Fundamental studies of the key pathway enzyme, alcohol acetyltransferase

The microbial production of esters including flavor compounds, fragrances, and biofuels, has been the focus of a number of metabolic engineering efforts. Long chain waxy esters have potential for use as diesel fuel substitutes and have been produced in engineered E. coli. Volatile aliphatic and alicyclic esters have value as natural food additives (e.g. isoamyl acetate and phenyl ethyl acetate) and industrial solvents (e.g. ethyl acetate) and have been produced in native yeast strains and engineered E. coli. 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). AATases that produce short chain esters have been identified in various yeast and fruit species and are active during fermentation and during fruit ripening. Long chain waxy esters are naturally produced in Acinetobacter species. While there have been successes in metabolic engineering of ester biosynthesis via this reaction step, a poor understanding of the structure, function, and kinetic characteristics of the AATase family have limited these efforts to low yields and titers. In an effort to develop a better understanding of the structure-function relationships and behavior of this class of enzyme, we have undertaken a series of fundamental biochemical studies on the AATase enzyme family. From these studies we aim to provide insight into proper enzyme and host selection for bioester synthesis.