Abstract

Biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFA) such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and docosapentaenoic acid (DPA, 22:5-4,7,10,13,16) in protist Thraustochytrium is catalyzed by a polyunsaturated fatty acids (PUFA) synthase comprising three large subunits, each with multiple catalytic domains. This study used complementation test, in vitro assays, and functional expression to characterize an acyltransferase (AT)-like domain in Subunit-B of a PUFA synthase from Thraustochytrium. Complementation test in Escherichia coli showed that the AT-like domain could not restore the growth phenotype of a temperature-sensitive mutant (∆fabDts) defective in malonyl-CoA:ACP transacylase activity. In vitro assays showed that the AT-like domain possessed thioesterase activity towards a few acyl-CoAs tested where docosahexaenoyl-CoA (DHA-CoA) was the preferred substrate. Expression of this domain in an E. coli mutant (∆fadD) defective in acyl-CoA synthetase activity resulted in the increased accumulation of free fatty acids. Site-directed mutagenesis showed that the substitution of two putative active site residues, serine at 96 (S96) and histidine at 220 (H220), in the AT-like domain significantly reduced its activity towards DHA-CoA and accumulation of free fatty acids in the ∆fadD mutant. These results indicate that the AT-like domain of the PUFA synthase does not function as a malonyl-CoA:ACP transacylase, rather it functions as a thioesterase. It might catalyze the last step of the VLCPUFA biosynthesis by releasing freshly synthesized VLCPUFAs attached to ACP domains of the PUFA synthase in Thraustochytrium.

Highlights

  • Very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3) are essential components of cell membranes and precursors for eicosanoids and docosanoids involved in mediating various physiological processes in humans and animals [1]

  • DHA-CoA and accumulation of free fatty acids in the ∆fadD mutant. These results indicate that the AT-like domain of the PUFA synthase does not function as a malonyl-CoA:acyl carrier protein (ACP) transacylase, rather it functions as a thioesterase

  • A malonyl-CoA:ACP transacylase (MAT) domain in Subunit-A was highly conserved, while a AT-like domain found in Subunit-B of the PUFA synthase was less conserved in terms of sequence similarity and domain organization among PUFA synthases

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Summary

Introduction

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3) are essential components of cell membranes and precursors for eicosanoids and docosanoids involved in mediating various physiological processes in humans and animals [1]. Humans and animals are encouraged to supplement these fatty acids in diets for better health, as they cannot be de novo synthesized [2,3]. The current sources of VLCPUFAs for humans and animals are oils from marine fish and oleaginous VLCPUFA-producing microorganisms. Metabolic engineering of oilseed plants using the biosynthetic genes from VLCPUFA-producing microorganisms has been considered as a potential alternative way to supply these fatty acids [4,5]

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