Triacylglycerol biosynthesis occurs via the glycerol-3-phosphate pathway in the insect Rhodnius prolixus.

Although triacylglycerol (TAG) stores play a critical role in organisms, mechanisms underlying TAG synthesis are poorly understood in invertebrates. In mammals, the synthesis of glycerolipids, including TAG, diacylglycerol (DAG) and phospholipids (PL), occurs predominantly by the glycerol-3-phosphate (G3P) pathway in most cell types, except for in enterocytes. In these cells, the monoacylglycerol (MAG) pathway accounts for the majority of glycerolipid production. The insect Rhodnius prolixus, a vector of Chagas' disease, exhibits a high capacity to produce glycerolipids in the midgut after a blood meal, providing substrates that are transferred to other organs, such as the fat body, which is specialized in TAG production and storage. In this report, the genes required for TAG synthesis were identified in the R. prolixus genome. The genomic data indicated that TAG is synthesized by the G3P pathway, which is the sole pathway for TAG synthesis in this organism. Furthermore, transcription of both the RpGpat and RpDgat genes were upregulated in a diverse number of organs at moments of highest lipid production. In the midgut and fat body, in vitro synthesis of glycerolipids required G3P, but not MAG, as the initial substrate. These results indicate that the G3P pathway is the only route for TAG synthesis in R. prolixus, and its regulation at the transcriptional level can be a determinant of glycerolipid synthesis and TAG formation in insect organs.

A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds.

Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45-50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to >50% of total fatty acids. In addition, >2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions.

The effect of excess cobalt on milk fatty acid profiles and transcriptional regulation of SCD, FASN, DGAT1 and DGAT2 in the mammary gland of lactating dairy cows.

The main objective of this study was to investigate the effect of excess cobalt (Co) on gene expression of stearoyl-CoA desaturase (SCD), fatty acid synthase (FASN), diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2) of lactating dairy cows in relation to milk fatty acid profile. Seven multiparous cows of the Norwegian Red cattle breed (NRF) had their basal diet supplemented with 1.4 g Co as a 24 g/l solution of Co-acetate per os twice daily for 7 days followed by a 9-day depuration period. Udder biopsies were performed prior to the treatment period, after 1 week of treatment and immediately after the depuration period. Excess Co reduced the proportion of all cis-9 monounsaturated fatty acids and increased the proportion of 18:0 in milk. However, gene expression levels of SCD, DGAT1, DGAT2 and FASN were not significantly altered. Our results indicate that the effect of Co on milk fatty acid profile is mediated at the post-transcriptional level by reduced activity of SCD in the mammary gland. Potential mechanisms explaining how Co might reduce stearoyl-CoA desaturation are discussed.

Type II diacylglycerol acyltransferase from Claviceps purpurea with ricinoleic acid, a hydroxyl fatty acid of industrial importance, as preferred substrate.

Claviceps purpurea, the fungal pathogen that causes the cereal disease ergot, produces glycerides that contain high levels of ricinoleic acid [(R)-12-hydroxyoctadec-cis-9-enoic acid] in its sclerotia. Recently, a fatty acid hydroxylase (C. purpurea FAH [CpFAH]) involved in the biosynthesis of ricinoleic acid was identified from this fungus (D. Meesapyodsuk and X. Qiu, Plant Physiol. 147:1325-1333, 2008). Here, we describe the cloning and biochemical characterization of a C. purpurea type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of ricinoleic acid into triglycerides. The CpDGAT2 gene was cloned by degenerate RT-PCR (reverse transcription-PCR). The expression of this gene restored the in vivo synthesis of triacylglycerol (TAG) in the quadruple mutant strain Saccharomyces cerevisiae H1246, in which all four TAG biosynthesis genes (DGA1, LRO1, ARE1, and ARE2) are disrupted. In vitro enzymatic assays using microsomal preparations from the transformed yeast strain indicated that CpDGAT2 prefers ricinoleic acid as an acyl donor over linoleic acid, oleic acid, or linolenic acid, and it prefers 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as an acyl acceptor. The coexpression of CpFAH with CpDGAT2 in yeast resulted in an increased accumulation of ricinoleic acid compared to the coexpression of CpFAH with the native yeast DGAT2 (S. cerevisiae DGA1 [ScDGA1]) or the expression of CpFAH alone. Northern blot analysis indicated that CpFAH is expressed solely in sclerotium cells, with no transcripts of this gene being detected in mycelium or conidial cells. CpDGAT2 was more widely expressed among the cell types examined, although expression was low in conidiospores. The high expression of CpDGAT2 and CpFAH in sclerotium cells, where high levels of ricinoleate glycerides accumulate, provided further evidence supporting the roles of CpDGAT2 and CpFAH as key enzymes for the synthesis and assembly of ricinoleic acid in C. purpurea.

Acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology.

Triacylglycerol (TG) is a storage lipid which serves as an energy reservoir and a source of signalling molecules and substrates for membrane biogenesis. TG is essential for many physiological processes and its metabolism is widely conserved in nature. Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the final step in the sn-glycerol-3-phosphate pathway leading to TG. DGAT activity resides mainly in two distinct membrane bound polypeptides, known as DGAT1 and DGAT2 which have been identified in numerous organisms. In addition, a few other enzymes also hold DGAT activity, including the DGAT-related acyl-CoA:monoacylglycerol acyltransferases (MGAT). Progress on understanding structure/function in DGATs has been limited by the lack of detailed three-dimensional structural information due to the hydrophobic properties of theses enzymes and difficulties associated with purification. This review examines several aspects of DGAT and MGAT genes and enzymes, including current knowledge on their gene structure, expression pattern, biochemical properties, membrane topology, functional motifs and subcellular localization. Recent progress in probing structural and functional aspects of DGAT1 and DGAT2, using a combination of molecular and biochemical techniques, is emphasized. Biotechnological applications involving DGAT enzymes ranging from obesity therapeutics to oilseed engineering are also discussed.

Cloning and molecular characterization of a novel acyl-CoA:diacylglycerol acyltransferase 1-like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum.

We have identified and isolated a cDNA encoding a novel acyl-CoA:diacylglycerol acyltransferase (DGAT)1-like protein, from the diatom microalga Phaeodactylum tricornutum (PtDGAT1). The full-length cDNA sequences of PtDGAT1 transcripts revealed that two types of mRNA, PtDGAT1short and PtDGAT1long, were transcribed from the single PtDGAT1 gene. PtDGAT1short encodes a 565 amino acid sequence that is homologous to several functionally characterized higher plant DGAT1 proteins, and 55% identical to the putative DGAT1 of the diatom Thalassiosira pseudonana, but shows little homology with other available putative and cloned algal DGAT sequences. PtDGAT1long lacks several catalytic domains, owing to a 63-bp nucleotide insertion in the mRNA containing a stop codon. Alternative splicing consisting of intron retention appears to regulate the amount of active DGAT1 produced, providing a possible molecular mechanism for increased triacylglycerol (TAG) biosynthesis in P. tricornutum under nitrogen starvation. DGAT mediates the last committed step in TAG biosynthesis, so we investigated the changes in expression levels of the two types of mRNA following nitrogen starvation inducing TAG accumulation. The abundance of both transcripts was markedly increased under nitrogen starvation, but much less so for PtDGAT1short. PtDGAT1 activity of PtDGAT1short was confirmed in a heterologous yeast transformation system by restoring DGAT activity in a Saccharomyces cerevisiae neutral lipid-deficient quadruple mutant strain (H1246), resulting in lipid body formation. Lipid body formation was only restored upon the expression of PtDGAT1short, and not of PtDGAT1long. The recombinant yeast appeared to display a preference for incorporating saturated C(16) and C(18) fatty acids into TAG.