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

Abstract

Background Haematococcus lacustris is an ideal source of astaxanthin (AST), which is stored in oil bodies containing esterified AST (EAST) and triacylglycerol (TAG). Diacylglycerol acyltransferases (DGATs) catalyze the last step of acyl-CoA-dependent TAG biosynthesis and are also considered as crucial enzymes involved in EAST biosynthesis in H. lacustris. Previous studies have identified four putative DGAT2-encoding genes in H. lacustris, and only HpDGAT2D allowed the recovery of TAG biosynthesis, but the engineering potential of HpDGAT2s in TAG biosynthesis remains ambiguous. Results Five putative DGAT2 genes (HpDGAT2A, HpDGAT2B, HpDGAT2C, HpDGAT2D, and HpDGAT2E) were identified in H. lacustris. Transcription analysis showed that the expression levels of the HpDGAT2A, HpDGAT2D, and HpDGAT2E genes markedly increased under high light and nitrogen deficient conditions with distinct patterns, which led to significant TAG and EAST accumulation. Functional complementation demonstrated that HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E had the capacity to restore TAG synthesis in a TAG-deficient yeast strain (H1246) showing a large difference in enzymatic activity. Fatty acid (FA) profile assays revealed that HpDGAT2A, HpDGAT2D, and HpDGAT2E, but not HpDGAT2B, preferred monounsaturated fatty acyl-CoAs (MUFAs) for TAG synthesis in yeast cells, and showed a preference for polyunsaturated fatty acyl-CoAs (PUFAs) based on their feeding strategy. The heterologous expression of HpDGAT2D in Arabidopsis thaliana and Chlamydomonas reinhardtii significantly increased the TAG content and obviously promoted the MUFAs and PUFAs contents. Conclusions Our study represents systematic work on the characterization of HpDGAT2s by integrating expression patterns, AST/TAG accumulation, functional complementation, and heterologous expression in yeast, plants, and algae. These results (1) update the gene models of HpDGAT2s, (2) prove the TAG biosynthesis capacity of HpDGAT2s, (3) show the strong preference for MUFAs and PUFAs, and (4) offer target genes to modulate TAG biosynthesis by using genetic engineering methods.

The oleaginous astaxanthin-producing alga Chromochloris zofingiensis: potential from production to an emerging model for studying lipid metabolism and carotenogenesis

“…In general, microalgae harbor a much larger number of DGAT isoforms than vascular plants (e.g., one versus eleven for the type II), pointing to more complex regulations of microalgal TAG synthesis. Although why microalgae need such high dose of DGATs remains less understood, functional characterization of DGATs from multiple aspects has been conducted for many species including C. reinhardtii [ 183 , 187 , 188 ], C. zofingiensis [ 189 – 191 ], H. pluvialis [ 192 , 193 ], N. oceanica [ 194 – 196 ], and P. tricornutum [ 197 – 199 ]. C. zofingiensis harbors ten putative DGAT isoforms, two type I (DGAT1A and DGAT1B) and eight type II (DGTT1 through DGTT8); all are predicted to be extrachloroplast-targeted [ 189 ].…”

“…It is worth noting that in C. reinhardtii and some vascular plants, albeit multiple DGATs are present in their genomes, the reconstruction of astaxanthin biosynthesis pathways in them leads to the accumulation of free astaxanthin rather than ester [ 263 , 264 , 270 , 271 ], questioning the role of DGATs in astaxanthin esterification. Since both C. zofingiensis and H. pluvialis harbor multiple DGAT gene copies yet lack well-established genetic tools [ 189 , 192 , 193 ], it is challenging to validate the esterification function of these DGAT genes in vivo via genetic manipulations. Functional analysis in free astaxanthin-producing yeast may represent an option.…”

Grand Challenges in Microalgae Domestication

“…Transformation of S. obliquus via A. tumefasciens is feasible (Suttangkakul et al, 2019 ). Both nuclear and chloroplastic transformations have been described for H. pluvialis (Yuan et al, 2019 ; Wang et al, 2020 ; Cui et al, 2021 ). Finally, Charophyta are Green Algae close to Embryophyta, propagating by haploid asexual division; sexual reproduction is known.…”

Characterization of a Haematococcus pluvialis Diacylglycerol Acyltransferase 1 and Its Potential in Unsaturated Fatty Acid-Rich Triacylglycerol Production

“…To date, one DGAT1 and five DGAT2 ( HpDGAT2A , HpDGAT2B , HpDGAT2C , HpDGAT2D , and HpDGAT2E ) genes have been identified in H. pluvialis ( Ma et al, 2018 ; Cui et al, 2021 ). Except for HpDGAT2C (HpDGTT2), the other four HpDGAT2s displayed the enzymatic activity to restore TAG biosynthesis in a TAG-deficient yeast strain when heterologously overexpressed in the host ( Nguyen et al, 2020 ; Cui et al, 2021 ; Ma et al, 2021 ).…”

“…To date, one DGAT1 and five DGAT2 ( HpDGAT2A , HpDGAT2B , HpDGAT2C , HpDGAT2D , and HpDGAT2E ) genes have been identified in H. pluvialis ( Ma et al, 2018 ; Cui et al, 2021 ). Except for HpDGAT2C (HpDGTT2), the other four HpDGAT2s displayed the enzymatic activity to restore TAG biosynthesis in a TAG-deficient yeast strain when heterologously overexpressed in the host ( Nguyen et al, 2020 ; Cui et al, 2021 ; Ma et al, 2021 ). Furthermore, two DGAT2 genes ( HpDGTT2 and HpDGAT2D ) were examined as the potential targets in genetic engineering for valued oil production in a number of microalgae and higher plants ( Cui et al, 2021 ; Ma et al, 2021 ).…”