Evaluation of two thioesterases from Marinobacter aquaeolei VT8: Relationship to wax ester production.

The biosynthesis of lipid-based biofuels is an important aspect of developing sustainable alternatives to conventional oils derived from fossil fuel reserves. Many biosynthetic approaches to biodiesel fuels and oils involve fatty acid derivatives as a precursor, and thioesterases have been employed in various strategies to increase fatty acid pools. Thioesterases liberate fatty acids from fatty acyl-coenzyme A or fatty acyl-acyl carrier protein substrates. The role played by thioesterases has not been extensively studied in model bacteria that accumulate elevated levels of biological oils based on fatty acid precursors. In this report, two primary thioesterases from the wax ester accumulating bacterium Marinobacter aquaeolei VT8 were heterologously expressed, isolated and characterized. These genes were further analyzed at the transcriptional level in the native bacterium during wax ester accumulation, and their genes were disrupted to determine the effect these changes had on wax ester levels. Combined, these results indicate that these two thioesterases do not play an integral role in wax ester accumulation in this natural lipid-accumulating model bacterium.

Altering small and medium alcohol selectivity in the wax ester synthase.

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT or wax ester synthase) catalyzes the terminal reaction in the bacterial wax ester biosynthetic pathway, utilizing a range of alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. The wild-type wax ester synthase Maqu_0168 from Marinobacter aquaeolei VT8 exhibits a preference for longer fatty alcohols, while applications with smaller alcohols would yield products with desired biotechnological properties. Small and medium chain length alcohol substrates are much poorer substrates for the native enzyme, which may hinder broad application of the wax ester synthase in many proposed biosynthetic schemes. Developing approaches to improve enzyme activity toward specific smaller alcohol substrates first requires a clear understanding of which amino acids of the primary sequences of these enzymes contribute to substrate specificity in the native enzyme. In this report, we surveyed a range of potential residues and identified the leucine at position 356 and methionine at position 405 in Maqu_0168 as residues that affected selectivity toward small, branched, and aromatic alcohols when substituted with different amino acids. This analysis provides evidence of residues that line the binding site for wax ester synthase, which will aid rational approaches to improve this enzyme with specific substrates.