(781d) Understanding Medium Chain Ester Biosynthesis in Yeast through Alcohol-o-Acyltransferase Structure-Function Studies

Fatty acid ethyl esters are secondary metabolites that are produced during microbial fermentation and fruit ripening. Short and medium chain volatile esters have value as natural food additives (e.g. isoamyl acetate) and industrial solvents (e.g. ethyl acetate), and long chain waxy esters have potential for use as diesel fuel substitutes. The biosynthetic pathways in yeast that produce these compounds have a common reaction step, the condensation of an alcohol with an acetyl- or acyl-CoA by alcohol-O-acetyltransferse (AATase, E.C. 2.3.1.84 and 2.3.1.74). Previous activity screening revealed that Eht1 and Eeb1 exhibit high activity towards ethyl ester biosynthesis with acyl-CoAs with C4 to C12 carbon chain length. However, our understanding of medium chain fatty acid ester synthesis in yeast is limited. Here, we present the results of a series of structure-function studies of medium chain fatty acid ester synthase, Eht1 and Eeb1 from S. cerevisiae, including 1) analysis of subcellular localization in S. cerevisiae and E. coli hosts; 2) truncation and alanine scanning studies to identify the protein domains responsible for membrane localization; 3) membrane topology study of Eht1 and Eeb1. Pairwise alignment suggests Eht1 and Eeb1 are paralogs and closely related, with 58% sequence identity and 73% sequence similarity at the amino acid level. Overexpression studies revealed that Eht1 and Eeb1 initially localize to the endoplasmic reticulum. As cell growth progresses to stationary phase, Eht1 traffic to the surface of lipid droplets, while Eeb1 localizes to mitochondria. With overexpression in E. coli, membrane localization of Eeb1 is lost, in comparison to that Eht1 localizes to plasma membrane. The truncation and alanine scanning studies suggested that both N and C termini are important for their localization and trafficking. The membrane topology study of them on endoplasmic reticulum membrane indicated that both termini are exposed to the cytosolic side of ER. Combined, these studies develop our overall understanding of medium chain fatty acid ester biosynthesis in yeast and provide critical information to developing biochemical processes for ester biosynthesis.