Engineering Novel Wax Ester Synthases for Biodiesel Production

Microbial conversion of sugars to biodiesels has recently been demonstrated by other research groups. One particular method is to catalyze the transfer of biomass-derived acyl-CoAs to alcohols in vivo to create fatty acid ethyl esters (FAEEs) for use as biodiesel. One enzyme that can perform this reaction within microorganisms is wax ester synthase (WS). However, a major problem is that WS cannot be expressed in vivo at high enough intracellular concentrations in its natural form to create biodiesel with the desired product titers. Modifying wax synthase to maintain specific activity but improve its soluble expression could improve microbial production of FAEEs and lead to higher biodiesel production. For this project we used a combination of computational modeling and experimental verification to improve solubility. A structural model was computationally created from enzyme homologs, and various point mutations replaced surface hydrophobic amino acid residues with hydrophilic residues to increase solubility. The first experimental verification used western blots of cell lysates to compare the relative amounts of mutagenized WS to that of the wild-type enzyme. We extended this method to measure the solubility of a WS-meGFP construct by quantifying relative green fluorescence of soluble protein within cell lysates. Both methods yielded results with a high variance, which is unfavorable for drawing meaningful conclusions. Currently we are bench-marking an assay that uses the twin-arginine translocation (TAT) quality control (QC) pathway coupled with growth selection to screen for enzyme solubility enhancements. Through our current work we hope to significantly increase the soluble expression of WS and learn how different mutations affect its solubility.