(615h) Developing Biocatalysts for Volatile Ester Synthesis

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. With respect to enzyme engineering, rational design is challenging as there are no known crystal structures of AATases and there are no suitable templates to generate high confidence homology models. Directed evolution approaches are limited to low throughput by GC and HPLC analysis of enzyme activity. These or other enzyme engineering approaches are needed as reported activities are low (~1 nM/min/mg) and, in general, stability is poor (~50% deactivation in 60 mins at 30 °C).

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 optimal enzyme and host selection for bioester synthesis. These studies include: 1) activity screening of AATase orthologs from Saccharomyces and non-Saccharomyces yeasts and various fruit species; 2) structure-function and cellular localization studies of the most active AATase, Atf1 from S. cerevisiae, and 3) identification of orthologs with membrane-dependent activity. Eight different alcohol substrates (methanol, ethanol, propanol, isopropanol, butanol, isobutanol, pentanol, and isopentanol) were screened against AATases from S. cerevisiae, S. pastorianus, K. lactis, P. anomala, S. lycopersicum, and C. melo for the production of the corresponding esters. Enzyme screening revealed substrate profiles for each enzyme, from which we can identify the most suitable candidates to develop biosynthetic pathways for the production of linear and branched esters from C3 to C7 chain lengths. Studies of these same orthologs revealed a new lipid droplet localization mechanism common to S. cerevisiae and S. pastorianus orthologs. Screening also identified a high activity AATase from tomato fruit that is soluble and is cytosolically localized when overexpressed in a yeast host. Finally, we have evaluated and compared the expression and activity of membrane-dependent and non-membrane-dependent orthologs in yeast and E. coli hosts. From these studies of the AATase family we aim to develop a better understanding of these enzymes and provide insight into the proper selection of enzymes appropriate for various hosts and desired ester syntheses. We also aim to use these studies to help develop effective activity screens for future directed evolution studies to improve activity and stability of selected orthologs.