Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri

Wagner, Martin; Hoppe, Katharina; Czabany, Tibor; Heilmann, Mareike; Daum, Günther; Feußner, Ivo; Fulda, Martin

doi:10.1016/j.plaphy.2010.03.008

Cited by 101 publications

(81 citation statements)

References 45 publications

(36 reference statements)

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“…Nile red staining and microscopy Intracellular lipid bodies as an indicator for TAG formation were visualised by fluorescent microscopy using Nile red (Invitrogen) staining as described previously (Wagner et al 2010). Aliquots of yeast cells (500 mL) grown to stationary phase were harvested, washed twice with 1 Â PBS, dissolved in 500 mL of 1 Â PBS, stained with 10 mL Nile red dye (2 mg mL -1 in DMSO), and then incubated in a dark room for 15 min at room temperature.…”

Section: Yeast Lipid Analysismentioning

confidence: 99%

Characterisation of DGAT1 and DGAT2 from Jatropha curcas and their functions in storage lipid biosynthesis

Yang

Wang

et al. 2014

Functional Plant Biol.

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Abstract. Diacylglycerol acyltransferases (DGATs) catalyse the final step of triacylglycerol (TAG) biosynthesis of the Kennedy pathway, and play a critical role during TAG accumulation in developing oleaginous seeds. In this study, the molecular cloning and characterisation of two DGAT genes, JcDGAT1 and JcDGAT2, from jatropha (Jatropha curcas L., a potential biodiesel plant) is presented. Using heterogonous overexpression techniques, both JcDGAT1 and JcDGAT2 were able to restore TAG biosynthesis in a yeast mutant H1246 strain, and enhance the quantity of TAG biosynthesis by 16.6 and 14.3%, respectively, in strain INVSc1. In transgenic tobacco, overexpression of JcDGAT1 and JcDGAT2 resulted in an increase in seed oil content of, respectively, 32.8 and 31.8%. Further, the functional divergence of JcDGAT1 and JcDGAT2 in TAG biosynthesis was demonstrated by comparing the fatty acid compositions in both the transgenic yeast and tobacco systems. In particular, JcDGAT2 incorporated a 2.5-fold higher linoleic acid content into TAG than JcDGAT1 in transgenic yeast and exhibited a significant linoleic acid substrate preference in both yeast and tobacco. This study provides new insights in understanding the molecular mechanisms of DGAT genes underlying the biosynthesis of linoleic acids and TAG in plants.

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Section: Yeast Lipid Analysismentioning

confidence: 99%

Characterisation of DGAT1 and DGAT2 from Jatropha curcas and their functions in storage lipid biosynthesis

Yang

Wang

et al. 2014

Functional Plant Biol.

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“…So far, microalgal DGATs have been characterized by comparative genomics from limited species (Wagner et al 2010). DGAT1 has been characterized from the diatom Phaeodactylum tricornutum (Guihéneuf et al 2011); however DGAT3 has not been experimentally validated.…”

Section: Introductionmentioning

confidence: 99%

Cloning and characterization of a novel diacylglycerol acyltransferase from the diatom Phaeodactylum tricornutum

et al. 2013

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In this study, a cDNA encoding a novel acylCoA:diacylglycerol acyltransferase (DGAT)-like protein is identified and isolated from the diatom microalga Phaeodactylum tricornutum (PtDGAT3). Analysis of the sequence reveals that ptDGAT3 cDNA encodes a protein of 504 amino acids with a molecular mass of 64.5 KDa. The putative ptDGAT3 protein has two catalytic domains: a wax ester synthase-like acyl-CoA acyltransferase domain and a bacteria-specific acyltransferase domain, which shows higher similarity to the DGAT3 of Acinetobacter calcoaceticus than reported DGAT1 or DGAT2 from high plants or algae. Its activity was confirmed by heterologous expression of PtDGAT3 in a neutral lipid-deficient quadruple mutant yeast Saccharomyces cerevisiae H1246. The recombinant yeast restored the formation of a lipid body and displayed a preference to the incorporation of unsaturated C 18 fatty acids into triacyglycerol (TAG). This is the first characterized algal DGAT3 gene, giving further evidence to the occurrence of a DGAT3-mediated TAG biosynthesis pathway.

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“…Similarly, the reverse construct Dgat1-GFP from plasmid 752, flanked by a T4-blunted PstI site and an overhang originating from XhoI digestion, was ligated into the Ecl136II and XhoI sites of pYES2, yielding plasmid 1067. Transformation, expression, and analysis of TAG formation were performed as described previously (28).…”

Section: Methodsmentioning

confidence: 99%

Dictyostelium discoideum Dgat2 Can Substitute for the Essential Function of Dgat1 in Triglyceride Production but Not in Ether Lipid Synthesis

Herrfurth

Gottlieb

et al. 2014

Eukaryot Cell

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b Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium discoideum are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, its natural food substrate. Fat accumulation is not only a serious health problem in Western societies but also a desired trait in dairy cattle and crop plants. The biochemical pathway of fat synthesis is largely conserved in all organisms and governed by an enzyme activity that performs the last and committed step in fat production, namely, the conversion of diacylglycerol (DAG) to triacylglycerol (TAG) or triglycerides. These so-called acyl coenzyme A (CoA):diacylglycerol acyltransferases (DGATs) are therefore targets for therapeutic drug development and represent objects of intense genetic and biochemical studies (recently reviewed by reference 1).Despite their relevance, the two enzymes mainly responsible for TAG production in mammals, DGAT1 and DGAT2, have been identified only about a decade ago. After the DGAT1 gene was cloned (2) and knocked out (3), the remaining enzyme activity suggested the existence of another enzyme. When the second DGAT was cloned (4), it became obvious that DGAT1 and DGAT2 are divergent in size (roughly 55 versus 43 kDa), differ in the number of transmembrane domains (6 or more versus only 2), and are largely unrelated in sequence (aligned in reference 1).Although both proteins are expressed in virtually all tissues in mammals, their distinguishing features strongly influence their fate in the cell. Whereas DGAT1 is a resident protein of the endoplasmic reticulum (ER) (5), DGAT2 localizes to the ER (6) and to lipid droplets (7). The protein targeting of mammalian DGAT2 to both subcellular locations is even separable by mutations in the primary sequence (8) and thus appears to occur independently.In newborn humans, a homozygous mutation of DGAT1 causes a severe diarrheal disorder that may even be fatal (9). In contrast, tra...

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Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri

Cited by 101 publications

References 45 publications

Characterisation of DGAT1 and DGAT2 from Jatropha curcas and their functions in storage lipid biosynthesis

Characterisation of DGAT1 and DGAT2 from Jatropha curcas and their functions in storage lipid biosynthesis

Cloning and characterization of a novel diacylglycerol acyltransferase from the diatom Phaeodactylum tricornutum

Dictyostelium discoideum Dgat2 Can Substitute for the Essential Function of Dgat1 in Triglyceride Production but Not in Ether Lipid Synthesis

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