Functional analysis of a malonyl-CoA:ACP transacylase-like domain from polyunsaturated fatty acid synthase in Thraustochytrium

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and eicosapentaenoic acid (EPA, 20:5-5,8,11,14,17) have been widely recognized for their health benefits. These omega-3 fatty acids are important because they are essential parts of cell membranes and are involved in mediating various physiological processes in humans and animals. The de novo biosynthesis of VLCPUFAs occurs only in certain microorganisms through either an aerobic or anaerobic pathway. In the aerobic pathway, various desaturases and elongases are involved for the introduction of double bonds and the elongation of chain length, while the anaerobic pathway is mediated by a polyunsaturated fatty acid synthase (PUFA synthase). In Thraustochytrium, the biosynthesis of VLCPUFAs is catalyzed by a PUFA synthase comprising three large subunits, each with multiple predicted catalytic domains. However, most of these domains are not biochemically characterized and their functions remain unknown. The objective of this project was to functionally characterize a malonyl-CoA:ACP transacylase (MAT)-like domain of a PUFA synthase from Thraustochytrium by the heterologous expression of the domain as a standalone protein in Escherichia coli. Bioinformatic analysis was first performed to dissect the MAT-like domain in the subunit-B of the PUFA synthase. After that, this domain was expressed as a standalone protein in E. coli mutant and wild type strains for complementation and activity assays. The results showed that the MAT-like domain could not complement a temperature-sensitive mutant (ΔfabDts) defective in malonyl-CoA:ACP transacylase activity, disallowing growth at a non-permissive temperature (42 ºC), while an authentic MAT domain from subunit-A of the synthase could complement the defective phenotype, allowing the growth at both permissive (37 ºC) and non-permissive temperature (42 ºC). This result indicated that the MAT-like domain does not function as a malonyl-CoA:ACP transacylase. Enzymatic assays showed that the MAT-like domain possessed thioesterase activity towards acyl-CoAs and that DHA-CoA was the preferred substrate among the acyl-CoAs tested. In addition, expression of this domain in an E. coli mutant ( ΔfadD) defective in acyl-CoA synthetase activity increased the accumulation of free fatty acids (FFAs). Furthermore, site-directed mutagenesis showed that the substitution of two residues, serine at 96 (S96) and histidine at 220 (H220), in the MAT-like domain with alanine significantly reduced its activity towards DHA-CoA and supressed the accumulation of FFAs in the ΔfadD mutant