Identification and biochemical characterization of five long-chain acyl-coenzyme A synthetases from the diatom Phaeodactylum tricornutum

Abstract

Long-chain acyl-CoA synthetase (ACSL; EC 6.2.1.3) catalyzes the conversion of free fatty acid to acyl-CoA ester, which is necessary for many pathways of fatty acid and lipid metabolism. The diatom Phaeodactylum tricornutum genome encodes five putative ACSLs (PtACSL1-5) that contain several highly conserved motifs and share limited sequence similarities with each other and with other known ACSLs. To verify their long-chain acyl-CoA synthetase activities, five cDNAs encoding these PtACSLs were cloned, expressed, and tested for their ability to complement the Saccharomyces cerevisiae double mutant FAA1ΔFAA4Δ. Only two of five PtACSLs were able to restore growth, facilitate exogenous fatty acid uptake, and enhance storage lipid accumulation. We also found that P. tricornutum cells are capable of importing long-chain fatty acids from extracellular environment. The identification of P. tricornutum ACSLs will provide molecular basis for the study of ACSL-mediated lipid synthesis and metabolism in diatoms.

Keywords: 4,4-Difluro-5-methyl-4-bora-3,4-diaza-s-indacene-3-dodecanoic acid; ACP; ACSL; BSA; C(1)-BODIPY-C(12); Fatty acids; Phaeodactylum tricornutum; QRT-PCR; RACE; TAG; VLCS/FATP; Yeast; acyl–acyl carrier protein; bovine serum albumin; long-chain acyl-CoA synthetase; quantitative reverse transcription PCR; rapid amplification of cDNA end; triacylglycerol; very long-chain acyl-CoA synthetase/fatty acid transport protein.