Cloning and molecular characterization of a novel acyl-CoA:diacylglycerol acyltransferase 1-like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum

Freddy Guihéneuf, Stefan Leu, Aliza Zarka, Inna Khozin-Goldberg, Ilkhom Khalilov, Sammy Boussiba

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82 Scopus citations

Abstract

We have identified and isolated a cDNA encoding a novel acyl-CoA:diacylglycerol acyltransferase (DGAT)1-like protein, from the diatom microalga Phaeodactylum tricornutum (PtDGAT1). The full-length cDNA sequences of PtDGAT1 transcripts revealed that two types of mRNA, PtDGAT1short and PtDGAT1long, were transcribed from the single PtDGAT1 gene. PtDGAT1short encodes a 565 amino acid sequence that is homologous to several functionally characterized higher plant DGAT1 proteins, and 55% identical to the putative DGAT1 of the diatom Thalassiosira pseudonana, but shows little homology with other available putative and cloned algal DGAT sequences. PtDGAT1long lacks several catalytic domains, owing to a 63-bp nucleotide insertion in the mRNA containing a stop codon. Alternative splicing consisting of intron retention appears to regulate the amount of active DGAT1 produced, providing a possible molecular mechanism for increased triacylglycerol (TAG) biosynthesis in P. tricornutum under nitrogen starvation. DGAT mediates the last committed step in TAG biosynthesis, so we investigated the changes in expression levels of the two types of mRNA following nitrogen starvation inducing TAG accumulation. The abundance of both transcripts was markedly increased under nitrogen starvation, but much less so for PtDGAT1short. PtDGAT1 activity of PtDGAT1short was confirmed in a heterologous yeast transformation system by restoring DGAT activity in a Saccharomyces cerevisiae neutral lipid-deficient quadruple mutant strain (H1246), resulting in lipid body formation. Lipid body formation was only restored upon the expression of PtDGAT1short, and not of PtDGAT1long. The recombinant yeast appeared to display a preference for incorporating saturated C16 and C18 fatty acids into TAG. Database Nucleotide sequence data are available in the GenBank/EMBL/DDBJ databases under accession number HQ589265, sequence to be released November 15 2011. The full-length cDNA sequences, encoding a novel acyl-CoA: diacylglycerol acyltransferase 1-like gene from Phaeodactylum tricornutum, revealed that two types of mRNA, PtDGAT1short and PtDGAT1long, were transcribed from PtDGAT1 gene. In Saccharomyces cerevisiae neutral lipid-deficient mutant, lipid body formation was only restored upon the expression of PtDGAT1short. Alternative-splicing mechanism appears to regulate the amount of active DGAT1 produced under nitrogen starvation.

Original languageEnglish
Pages (from-to)3651-3666
Number of pages16
JournalFEBS Journal
Volume278
Issue number19
DOIs
StatePublished - 1 Oct 2011

Keywords

  • Bacillariophyceae
  • Phaeodactylum tricornutum
  • diacylglycerol acyltransferase 1-like protein
  • intron-retention splicing
  • triacylglycerol

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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