Isolation of a Gene Encoding a 1,2-Diacylglycerol-sn-acetyl-CoA Acetyltransferase from Developing Seeds of Euonymus alatus

Abstract

1,2-Diacyl-3-acetyl-sn-glycerols (ac-TAG) are unusual triacylglycerols that constitute the major storage lipid in the seeds of Euonymus alatus (Burning Bush). These acTAGs have long-chain acyl groups esterified at both the sn-1 and sn-2 positions of glycerol. Cell-free extracts of developing seeds of E. alatus contain both long-chain acyl-CoA and acetyl-CoA sn-1,2-diacylglycerol acyltransferase (DGAT) activity. We have isolated a gene from developing seeds of Euonymus alatus that shows a very high sequence similarity to the members of the DGAT1 gene family (i.e. related to acyl-CoA:cholesterol acyltransferases). This Euonymus DGAT1 gene, when expressed in wild type yeast, results in a 5-fold enhancement of longchain triacylglycerol (lc-TAG) accumulation, as well as the appearance of low levels of ac-TAG. Hydrogenated ac-TAG molecular species were identified by gas chromatography-mass spectrometry. Microsomes isolated from this transformed yeast show diacylglycerol:acetyl-CoA acetyltransferase activity, which is about 40-fold higher than that measured in microsomes prepared from yeast transformed with the empty vector or with the Arabidopsis thaliana DGAT1 gene. The specific activity of this microsomal acetyltransferase activity is of the same order of magnitude as the microsomal long-chain DGAT activities measured for yeast lines transformed with the empty vector or either the Arabidopsis or Euonymus DGAT1 genes. Despite this, ac-TAG accumulation in yeast transformed with the Euonymus DGAT1 gene was very low (0.26% of lc-TAG), whereas lc-TAG accumulation was enhanced. Possible reasons for this anomaly are discussed. Expression of the Euonymus DGAT1-like gene in yeast lines where endogenous TAG synthesis has been deleted confirmed that the gene product has both long-chain acyland acetyltransferase activity.

Tung Tree DGAT1 and DGAT2 Have Nonredundant Functions in Triacylglycerol Biosynthesis and Are Localized to Different Subdomains of the Endoplasmic Reticulum

“…DGAT enzyme activity was measured by in vitro assay (Zou et al, 1999) containing 100 mM Tris-HCl, pH 8.0, 400 mM diacylglycerol (delivered from a 40 mM stock in methanol), 20 mM [1-14 C]acyl-CoA (specific activity, 20,000 cpm/mmol; prepared enzymatically as described by Rajasekharan et al [1993]), and 25 mg of yeast microsomal membrane protein in a total volume of 100 mL. The mixture was incubated at 308C for 1 h and then extracted twice with 400 mL of hexane:isopropanol (3:2, v/v) (Milcamps et al, 2005). The upper hexane phases were pooled, dried under N 2 , redissolved in a small volume of chloroform containing 200 mg of carrier trilinolein, and chromatographed on silica gel TLC plates using n-hexane:diethyl ether:acetic acid (80:20:1, v/v) as the mobile phase.…”

“…The type 1 class of DGAT enzymes (DGAT1) was discovered first in mouse based on homology with mammalian acyl-CoA:cholesterol acyltransferase genes (Cases et al, 1998); subsequently, other DGAT1 genes were identified and characterized in several plant species (Hobbs et al, 1999;Routaboul et al, 1999;Zou et al, 1999;Bouvier-Navé et al, 2000;Nykiforuk et al, 2002;He et al, 2004b;Milcamps et al, 2005). For instance, the Arabidopsis thaliana DGAT1 gene has been shown to contribute significantly to seed TAG biosynthesis, both by overexpression (Jako et al, 2001) and through mutational downregulation studies (Katavic et al, 1995;Routaboul et al, 1999).…”

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

“…Microsomal membranes were prepared by a published method (Milcamps et al, 2005) with minor modifications. Yeast cultures expressing DGAT variants were grown in 100-mL volumes to early stationary phase, harvested, and resuspended in 4 mL of 20 mM Tris-HCl, pH 8, 10 mM MgCl 2 , 1 mM EDTA, 5% glycerol, 1 mM dithiothreitol, and 300 mM (NH 4 ) 2 SO 4 .…”

Efficiency for increasing seed oil content using WRINKLED1 and DGAT1 under the control of two seed-specific promoters, FAE1 and Napin

“…DGAT1 has been characterized from several plant species (Routaboul et al 1999;Zou et al 1999;Milcamps et al 2005). Seed-specific overexpression of DGAT1 results in an increase in the seed size and oil content (Jako et al 2001;Taylor et al 2009), but disruption of DGAT1 led to a reduced oil content in seeds (Routaboul et al 1999;Zou et al 1999).…”