Cloning and Molecular Characterization of the Acyl‐CoA:Diacylglycerol Acyltransferase 1 (DGAT1) Gene from Echium

Abstract

Boraginaceae species, such as those from the genus Echium, contain high levels of the Delta(6)-desaturated gamma-linolenic (18:3n-6) and octadecatetraenoic (18:4n-3) acids. These are unusual fatty acids among the plant kingdom that are gaining interest due to their benefits to human health. The potential utility of acyltransferases aimed at an increase in oil yield and fatty acid profiling has been reported. In this work, a gene encoding an acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) was cloned from Echium pitardii. Genomic and cDNA sequences obtained revealed a gene structure composed of 16 exons, yielding a protein (EpDGAT) of 473 amino acids with high similarity to DGAT1 enzymes of plants. Protein features such as a predicted structure with a highly hydrophilic N-terminus followed by 10 transmembrane domains, as well as the presence of diverse specific signatures, also indicate that EpDGAT belongs to the DGAT1 family. indeed. DGAT activity of the protein encoded by EpDGAT was confirmed by heterologous expression of the full-length cDNA in a yeast mutant (H1246) defective in the synthesis of triacylglycerols. Fatty acid composition of the triacylglycerols synthesized by EpDGAT in H1246 yeast cultures supplemented with polyunsaturated fatty acids suggest a substrate preference for the trienoic fatty acids alpha-linolenic acid (18:3n-3) and gamma-linolenic acid over the dienoic linoleic acid (18:2n-6). Site-directed mutagenesis has revealed the presence of a critical residue (P(178) in EpDGAT) within a reported thiolase signature for binding of acyl-enzyme intermediates that might be involved in the active site of the enzyme. Transcript analysis for EpDGAT shows an ubiquitous expression of the gene which is increased in leaves during senescence.

Cloning and molecular characterization of a glycerol-3-phosphate O-acyltransferase (GPAT) gene from Echium (Boraginaceae) involved in the biosynthesis of cutin polyesters

“…This hypothesis is supported by the fact that some unusual fatty acids are found in both polyesters and seed oils, and by the observation that several enzymes of unusual fatty acid synthesis are divergent forms of enzymes of normal fatty acid synthesis. A number of molecular studies on lipid biosynthesis have been performed in Echium plants (García-Maroto et al 2002;Mañas-Fernández et al 2009) which are characterized by the accumulation of unusual D 6 -desaturated fatty acids (c-linolenic and stearidonic acids) in the seed oil. Here, we report on the cloning and molecular characterization of EpGPAT1, a homolog of Arabidopsis polyestertype GPATs which was isolated from the seeds of Echium pitardii (Boraginaceae).…”

An Improved Variant of Soybean Type 1 Diacylglycerol Acyltransferase Increases the Oil Content and Decreases the Soluble Carbohydrate Content of Soybeans

“…Aside from these three DGAT types, bifunctional wax ester synthases also can catalyze the DGAT reaction . Depending on the species, DGAT enzymes can have strong preferences for different acyl-CoA or DAG substrates Huang, 1986, 1987;Griffiths and Harwood, 1991;Mañas-Fernández et al, 2009;Furmanek et al, 2014).…”

Identification and characterization of a type-2 diacylglycerol acyltransferase (DGAT2) from Rhodosporidium diobovatum

“…(ii) Hydrophobicity analysis showed that RdD-GAT had four transmembrane domains, which was very different from DGAT1 and DGAT3. Members of DGAT3 family are cytosolic enzymes which lack any transmembrane domains in the protein structure, while members of DGAT1 family were predicted to contain at least eight highly hydrophobic transmembrane regions (Guihéneuf et al 2011;Mañas-Fernández et al 2009 Fig. 2 Phylogenetic analysis of homolog sequences of RdD-GAT across the organisms.…”