UC Riverside

Production of renewable, non-toxic and environmental-friendly biofuels and chemicals has been the focus of metabolic engineering. To achieve high yield production by microbial cell factories, it is necessary to identify highly active biocatalysts and engineer efficient biosynthetic pathways. Alcohol-O-actyl/acyltransferase (AATase) is responsible for synthesis of fatty acid ethyl esters (FAEEs) by condensing acetyl/acyl-CoAs and ethanol in yeast and plants. This work demonstrated that S. cerevisiae is a suitable host for FAEE production, because AATase has higher specific enzymatic activity compared to that in E. coli. To enable the rapid profiling of AATase activities, a spectrophotometric-based coupled enzyme assay was developed for high throughput screening of AATase enzymatic activity. With this assay, a library of AATases was characterized for the substrate specificity towards acyl-CoAs and Atf-Sl from tomato was discovered with high activity towards various alcohols. Enzyme co-localization and substrate channeling are strategies to improve enzyme cascade reaction rate and yield. A protein-based scaffold based on oleosin-cohesin-dockerin was developed for co-localizing multienzyme pathways on the surface of intracellular lipid droplets (LDs). The upstream enzyme in yeast ester biosynthesis was recruited to the native localization LDs of the terminal reaction step, AATase.

Fluorescent microscopy studies show that most of the endogenous AATases in S. cerevisiae are localized to LDs. To understand the localization mechanism and trafficking pathway of AATase, structure-function analysis and protein-protein interaction studies were performed for Eht1 and its paralogue Eeb1, which arose from genome duplication. N- and C-terminal regions of Eht1 are necessary for initially targeting to the ER membrane and subsequent sorting to LDs. Eht1 is a peripheral membrane protein on ER with both termini exposed to cytosol. The translocation from ER to LDs of Eht1 is related to translocons on the ER. Immunoprecipitation and MS analysis have identified possible physical protein interactions of Eht1 to assist in the trafficking from the ER to LDs. Combined, this work not only engineers AATase in S. cerevisiae by investigating highly active AATase and spatially organizing multienzyme pathways, but also understand the mechanism of AATase ER-dependent LDs targeting.