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. Short and medium chain volatile esters have value as natural food additives (e.g. isoamyl acetate) and industrial solvents (e.g. ethyl acetate) and have been produced in native yeast strains and engineered E. coli. The biosynthetic pathways have a common reaction step, the condensation of an alcohol with an acetyl- or acyl-CoA by alcohol-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 fruit ripening. Long chain waxy esters are naturally produced in Acinetobacter and Marinobacter species. Here, we present the results of a series of experimental studies of AATases from S. cerevisiae, including 1) development of a rapid screening assay for AATase activity; 2) analysis of intracellular localization in S. cerevisiae and E. coli hosts; and, 3) structure-function analysis to identify protein domains responsible for membrane localization. Overexpression and structure-function studies revealed that Eht1, Eeb1, Atf1, and Atf2 initially localize to the endoplasmic reticulum. As cell growth progresses to stationary phase, Eht1, Atf1, and Atf2 traffic to the surface of lipid droplets, while Eeb1 localizes to the mitochondria. Activity assays revealed that membrane localization is important for function. With overexpression in E. coli, membrane localization of Atf1, Atf2, and Eeb1 is lost and activity is significantly reduced. In E. coli, Eht1 localizes to the plasma membrane and maintains function. Disruption of membrane localization by mutation to the N- and C-terminal domains also reduces function. Activity screening with a newly developed colorimetric assay for AATase activity revealed that Eht1 and Eeb1 exhibit high activity towards ethyl ester biosynthesis with acyl-CoAs with C4 to C12 carbon chain length. In comparison, Atf1 and Atf2 have significantly reduced activities, and the substrate specificity of Atf1 tends towards shorter chain length acyl-CoAs. Combined, these studies develop our overall understanding of ester biosynthesis in yeast and provide critical information to developing biochemical processes for ester biosynthesis.

Understanding short and medium chain volatile ester biosynthesis by AATase activity in yeast

251st National Meeting (2016)

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