Characterization of type-2 diacylglycerol acyltransferases in Haematococcus lacustris reveals their functions and engineering potential in triacylglycerol biosynthesis and possible roles in astaxanthin esterification

Abstract Background: Haematococcus lacustris is an ideal source of astaxanthin (AST) which is stored at oil bodies containing esterified AST (EAST) and triacylglycerol (TAG). Diacylglycerol acyltransferases (DGATs) catalyze the last step of the acyl-CoA-dependent TAG biosynthesis and are also considered as the crucial enzymes involving in EAST biosynthesis in H. lacustris. Previous studies have identified four putative DGAT2 encoding genes in H. lacustris and only the HpDGAT2D allowed the recovery of TAG biosynthesis, but the engineering potential of HpDGAT2s in TAG biosynthesis, especially possible roles in AST esterification, remains ambiguous.Results: Five putative DGAT2 genes (HpDGAT2A, 2B, 2C, 2D, and 2E) were identified in H. lacustris. Transcription analysis showed that the expression levels of HpDGAT2A, 2B, and 2E genes markedly increased under high light and nitrogen deficient conditions with distinct patterns, which led to significant TAG and EAST accumulation. Functional complementation demonstrated HpDGAT2A, 2B, 2D, and 2E had the capability to restore TAG synthesis in a TAG-deficient yeast strain (H1246) with the large difference in enzymatic activity. Fatty acids (FAs) profiles assays revealed that HpDGAT2A, 2D, and 2E except for 2B preferred monounsaturated fatty acyl-CoA (MUFA) for TAG synthesis in yeast cells, and also showed polyunsaturated fatty acyl-CoA (PUFA) preference by feeding strategy. The over-expression of HpDGAT2D in Arabidopsis thaliana and Chlamydomonas reinhardtii significantly increased the TAG content and obviously promoted the MUFA and PUFA contents. Interestingly, molecular docking analysis implied that HpDGAT2s structures contained AST binding sites, which provides strong evidence for AST esterification function in H. lacustris.Conclusions: Our study represents a pioneering work on the characterization of HpDGAT2s by systematically integrating expression pattern, AST/TAG accumulation, functional complementation, molecular docking, and over-expression in yeast, plants, and algae. These results (1) update the gene models of HpDGAT2s, (2) prove TAG biosynthesis capacity of HpDGAT2s, (3) show the strong preference for MUFA and PUFA, (4) offer target genes to modulate TAG biosynthesis by genetic engineering method, and (5) provide new evidence for HpDGAT2s roles in AST esterification.