Abstract
N-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, 22:6n-3), have been reported to play roles in preventing cardiovascular diseases. The major source of DHA is fish oils but a recent increase in the global demand of DHA and decrease in fish stocks require a substitute. Thraustochytrids, unicellular marine protists belonging to the Chromista kingdom, can synthesize large amounts of DHA, and, thus, are expected to be an alternative to fish oils. DHA is found in the acyl chain(s) of phospholipids as well as triacylglycerols in thraustochytrids; however, how thraustochytrids incorporate DHA into phospholipids remains unknown. We report here a novel lysophospholipid acyltransferase (PLAT1), which is responsible for the generation of DHA-containing phosphatidylcholine and phosphatidylethanolamine in thraustochytrids. The PLAT1 gene, which was isolated from the genomic DNA of Aurantiochytrium limacinum F26-b, was expressed in Saccharomyces cerevisiae, and the FLAG-tagged recombinant enzyme was characterized after purification with anti-FLAG affinity gel. PLAT1 shows wide specificity for donor substrates as well as acceptor substrates in vitro, i.e, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates. In contrast to the in vitro experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in plat1-knockout mutants, resulting in a decrease of 16:0-DHA-phosphatidylcholine (PC) [PC(38∶6)] and 16:0-DHA-phosphatidylethanolamine (PE) [PE(38∶6)], which are two major DHA-containing phospholipids in A. limacinum F26-b. However, the amounts of other phospholipid species including DHA-DHA-PC [PC(44∶12)] and DHA-DHA-PE [PE(44∶12)] were almost the same in plat-knockout mutants and the wild-type. These results indicate that PLAT1 is the enzyme responsible for the generation of 16:0-DHA-PC and 16:0-DHA-PE in the thraustochytrid.
Highlights
N-3 polyunsaturated fatty acids (n-3PUFA), such as docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), have been reported to play roles in preventing cardiovascular diseases [1,2]
The neighbor-joining tree of 18S rRNA gene clearly shows that the strain F26-b forms a monophyletic group with the originally descripted ex-type strain (ATCC MYA 1381) of A. limacinum [22,23] and four related strains that is strongly supported by the highest bootstrap value (100%) (Figure 1)
We cloned the PL AcylTransferase 1 (PLAT1) gene from the genomic DNA of A. limacinum F26-b, which is genealogically related to A. limacinum ATCC MYA-1381 (Figure 1)
Summary
N-3 polyunsaturated fatty acids (n-3PUFA), such as docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), have been reported to play roles in preventing cardiovascular diseases [1,2]. DHA is endogenously converted to resolvin D1, and protectin D1 by the actions of 12/15-lipoxygenase. These autacoids were shown to exert strong pro-resolving and antiinflammatory actions [6,7,8]. It was reported that dietary n-3 PUFAs administered in phospholipid (PL) form were superior to those in triacylglycerol form for maintaining a healthy metabolic profile [10]. Fish oils include n-3 PUFA-containing triacylglycerols but not PLs, and, a source of n-3 PUFA-containing PLs is required
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