Analysis of triglyceride synthesis unveils a green algal soluble diacylglycerol acyltransferase and provides clues to potential enzymatic components of the chloroplast pathway

Abstract

Background: Microalgal triglyceride (TAG) synthesis has attracted considerable attention. Particular emphasis has been put towards characterizing the algal homologs of the canonical rate-limiting enzymes, diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). Less work has been done to analyze homologs from a phylogenetic perspective. In this work, we used HMMER iterative profiling and phylogenetic and functional analyses to determine the number and sequence characteristics of algal DGAT and PDAT, as well as related sequences that constitute their corresponding superfamilies. We included most algae with available genomes, as well as representative eukaryotic and prokaryotic species. Results: Amongst our main findings, we identified a novel clade of DGAT1-like proteins exclusive to red algae and glaucophyta and a previously uncharacterized subclade of DGAT2 proteins with an unusual number of transmembrane segments. Our analysis also revealed the existence of a novel DGAT exclusive to green algae with moderate similarity to plant soluble DGAT3. The DGAT3 clade shares a most recent ancestor with a group of uncharacterized proteins from cyanobacteria. Subcellular targeting prediction suggests that most green algal DGAT3 proteins are imported to the chloroplast, evidencing that the green algal chloroplast might have a soluble pathway for the <i>de novo</i> synthesis of TAGs. Heterologous expression of <i>C. reinhardtii</i> DGAT3 produces an increase in the accumulation of TAG, as evidenced by thin layer chromatography. Conclusions: Our analysis contributes to advance in the knowledge of complex superfamilies involved in lipid metabolism and provides clues to possible enzymatic players of chloroplast TAG synthesis.