Identification and characterization of a novel type-2 DGAT homologue gene from the green microalga Haematococcus pluvialis

Abstract

Background: The unicellular green microalga Haematococcus pluvialis is an ideal source of astaxanthin (AST) which is stored at oil bodies contain both esterified astaxanthin (EAST) and triacylglycerol (TAG). Diacylglycerol acyltransferase (DGAT) catalyzes the last step of the acyl-CoA-dependent TAG biosynthesis and is considered as the crucial enzyme involving in EAST biosynthesis in H. pluvialis. However, the function of DGAT in H. pluvialis has not been reported. Results: A full-length cDNA sequences encoding a putative DGAT2 (HaeDGAT2E) was obtained from H. pluvialis . It contained an open reading frame (ORF) of 1,017-bp encoding a protein of 338 amino acid residues. The isolated HaeDGAT2E protein shared high identity of 57.6% and 54.1% with DGAT2E from Chlamydomonas reinhardtii and Chromochloris zofingiensis respectively. There were 7 conserved motifs and 3 trans-membrane regions in HaeDGAT2E. The phylogenetic analysis suggested that HaeDGAT2E belonged to DGAT2E subfamily. HaeDGAT2E activity was confirmed in the TAG deficient yeast strain (H1246) by restoring its ability to produce TAG. Upon expression of HaeDGAT2E , C16:0 and C18:1 fatty acid contents were 190.2% and 132.4% higher respectively than that of the H1246 strain. In addition, over-expression of HaeDGAT2E in transgenic Nicotiana Benthamiana resulted in increased contents of C16:0 (113.5%) and C18:1 (234.5%). Conclusions: A novel gene encoding HaeDGAT2E was identified in H. pluvialis . This is the first functional analysis of DGAT2 in Haematococcus . This information is important for understanding TAG accumulation and for further elucidating EAST biosynthesis in H. pluvialis .