Heterologous Expression and Characterization of Plant Wax Ester Producing Enzymes

Wax esters are widely distributed among microbes, plants, and mammals, and they serve protective and energy storage functions. Three classes of enzymes catalyze the reaction between a fatty acyl alcohol and a fatty acyl-CoA, generating wax esters. Multiple isozymes of two of these enzyme classes, the membrane-bound O-acyltransferase class of wax synthase (WS) and the bifunctional wax synthase/diacylglycerol acyl transferase (WSD), co-exist in plants. Although WSD enzymes are known to produce the wax esters of the plant cuticle, the functionality of plant WS enzymes is less well characterized. In this study, we investigated the phylogenetic relationships among the 12 WS and 11 WSD isozymes that occur in Arabidopsis, and established two in vivo heterologous expression systems, in the yeast Saccharomyces cerevisiae and in Arabidopsis seeds to investigate the catalytic abilities of the WS enzymes. These two refactored wax assembly chassis were used to demonstrate that WS isozymes show distinct differences in the types of esters that can be assembled. We also determined the cellular and subcellular localization of two Arabidopsis WS isozymes. Additionally, using publicly available Arabidopsis transcriptomics data, we identified the co-expression modules of the 12 Arabidopsis WS coding genes. Collectively, these analyses suggest that WS genes may function in cuticle assembly and in supporting novel photosynthetic function(s).