Identification and characterization of an efficient acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1) gene from the microalga Chlorella ellipsoidea

Abstract

BackgroundOil in the form of triacylglycerols (TAGs) is quantitatively the most important storage form of energy for eukaryotic cells. Diacylglycerol acyltransferase (DGAT) is considered the rate-limiting enzyme for TAG accumulation. Chlorella, a unicellular eukaryotic green alga, has attracted much attention as a potential feedstock for renewable energy production. However, the function of DGAT1 in Chlorella has not been reported.ResultsA full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Chlorella ellipsoidea. The 2,142 bp open reading frame of this cDNA, designated CeDGAT1, encodes a protein of 713 amino acids showing no more than 40% identity with DGAT1s of higher plants. Transcript analysis showed that the expression level of CeDGAT1 markedly increased under nitrogen starvation, which led to significant triacylglycerol (TAG) accumulation. CeDGAT1 activity was confirmed in the yeast quadruple mutant strain H1246 by restoring its ability to produce TAG. Upon expression of CeDGAT1, the total fatty acid content in wild-type yeast (INVSc1) increased by 142%, significantly higher than that transformed with DGAT1s from higher plants, including even the oil crop soybean. The over-expression of CeDGAT1 under the NOS promoter in wild-type Arabidopsis thaliana and Brassica napus var. Westar significantly increased the oil content by 8–37% and 12–18% and the average 1,000-seed weight by 9–15% and 6–29%, respectively, but did not alter the fatty acid composition of the seed oil. The net increase in the 1,000-seed total lipid content was up to 25–50% in both transgenic Arabidopsis and B. napus.ConclusionsWe identified a gene encoding DGAT1 in C. ellipsoidea and confirmed that it plays an important role in TAG accumulation. This is the first functional analysis of DGAT1 in Chlorella. This information is important for understanding lipid synthesis and accumulation in Chlorella and for genetic engineering to enhance oil production in microalgae and oil plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-0995-5) contains supplementary material, which is available to authorized users.

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

“…As shown in Additional le 7: Figure S3, there were two completely conserved amino acid residues (P and F) among all DGAT2s, which is consistent with previous reports that these two highly conserved residues maybe located at the active sites of the enzymes and make signi cant contribution to the enzymatic activities [49]. The phylogenetic analysis of the HpDGAT2s and other DGATs orthologs from eukaryotic algae and plants was illustrated in Additional le 8: Figure S4, which is consistent with most of previous results [20][21][22][23][24][25][26]. Brie y, all HpDGAT2s clustered with the algal DGAT2s orthologs, which are distinct from other DGAT subfamilies including DGAT1, DGAT3, and DGAT/WSD.…”

“…DGATs catalyze the terminal step in acyl-CoA-dependent TAG production pathway and represent a key target in manipulating TAG production [11]. At present, DGATs from different algal species have been widely studied, which indicates that the diversity microalgae are prominent candidates for DGATs and the function of distinct DGATs is unique or species-speci c [19][20][21][22][23][24][25][26][27][28]. Obviously, the HpDGAT2s genes were differentially regulated by HL, ND, and double HL-ND stresses conditions with distinct patterns, suggesting that these enzymes are together involved in the AST and TAG biosynthesis ( MiDGAT2A was regulated by ND stress, which lead to TAG accumulation [28].…”

“…4c). Guo et al (2017) indicated that the CeDGAT1 gene can stimulate FAs biosynthesis and enhance seed weight and oil content when expressed in A. thaliana and B. napus [21].…”

“…Usually, only one or two copies of DGAT1s are identi ed in a number of microalgae, whereas multiple copies of DGAT2s are typically present, suggesting that DGAT2s may play an important function in TAG biosynthesis [12][13][14][19][20][21][22][23][24][25][26][27]. Recently, most of the current knowledge about the algal DGATs is derived from the limited algal species including Chlamydomonas reinhardtii, Chlorella ellipsoidea, Nannochloropsis oceanica, Lobosphaera incise, Chlorella/Chromochloris zo ngiensis, Myrmecia incise, and Phaeodactylum tricornutum, in which DGATs have been focused on the molecular cloning, biochemical identi cation, functional characterization, and engineering potential in modulating TAG biosynthesis [19][20][21][22][23][24][25][26][27][28]. It is not di cult to nd some interesting conclusions that the diversity microalgae are prominent candidates for DGATs and the function of distinct DGATs is unique or species-speci c. Therefore, it derived the research interest to other industrially special astaxanthin (AST) producing alga such as Haematococcus lacustris [29].…”

Properties and Biotechnological Applications of Acyl‐CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae

“…Following these aforementioned studies, many groups have further used DGAT1 from different species to boost seed content in various crops such as G. max (Hatanaka et al, ; Roesler et al, ), B. juncea (Savadi et al, ), Z. mays (Alameldin et al, ), C. sativa (Kim et al, ), and Jatropa curcas (Maravi et al, ). Moreover, overexpression of DGAT1 from microalgae, such as Chlorella ellipsoidea and Nannochloropsis oceanica , also led to increased oil content in Arabidopsis and B. napus (Guo et al, ; Zienkiewicz et al, ). Furthermore, DGAT1 has been used to increase the proportion of unusual fatty acids in seed oil, particularly epoxy fatty acid in G. max (coexpressed with an EPOXYGENASE gene; Li et al, ) and capric acid in C. sativa (in combination with fatty acyl‐ACP thioesterase B1 and LPAAT from Cuphea viscosissima ; Iskandarov et al, ).…”