A distinct DGAT with sn -3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds

Abstract

Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-snglycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O-acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications.3-acetyl-1,2-diacyl-sn-glycerols | biodiesel | diacylglycerol acyl-CoA acyltransferase | membrane-bound O-acyltransferase | triacylglycerols

Seasonal accumulation of acetylated triacylglycerols by a freeze-tolerant insect

“…This distinct structure results in the reduced viscosity and the significantly lower energy density of acTAGs compared with lcTAGs (Durrett et al, 2010). Trace amounts of acTAGs have been described in a broad range of organisms, including the antlers of Japanese deer (Cervus nippon) (Yang et al, 2004), the udders of domestic cows (Bos primigenius) (Limb et al, 1999) and the seeds of the burning bush plant (Euonymus alatus) (Durrett et al, 2010).…”

“…This distinct structure results in the reduced viscosity and the significantly lower energy density of acTAGs compared with lcTAGs (Durrett et al, 2010). Trace amounts of acTAGs have been described in a broad range of organisms, including the antlers of Japanese deer (Cervus nippon) (Yang et al, 2004), the udders of domestic cows (Bos primigenius) (Limb et al, 1999) and the seeds of the burning bush plant (Euonymus alatus) (Durrett et al, 2010). acTAGs constitute a very small proportion of the total lipid pool in the two mammal species investigated [<0.1% by mass Limb et al, 1999;Yang et al, 2004)], but predominate (92% by moles) in E. alatus seed oils (Durrett et al, 2010).…”

“…Trace amounts of acTAGs have been described in a broad range of organisms, including the antlers of Japanese deer (Cervus nippon) (Yang et al, 2004), the udders of domestic cows (Bos primigenius) (Limb et al, 1999) and the seeds of the burning bush plant (Euonymus alatus) (Durrett et al, 2010). acTAGs constitute a very small proportion of the total lipid pool in the two mammal species investigated [<0.1% by mass Limb et al, 1999;Yang et al, 2004)], but predominate (92% by moles) in E. alatus seed oils (Durrett et al, 2010). Euonymus alatus synthesises acTAGs via an unusual diacylglycerol acyltransferase (DGAT) by esterifying an acetyl group, rather than a third long-chain fatty acid, to diacylglycerol (DAG) (Durrett et al, 2010).…”

Bioengineering Recombinant Diacylglycerol Acyltransferases

“…DGAT genes have been isolated from many organisms. At least two forms of DGATs are present in mammals (Cases et al, 1998;Cases et al, 2001) and plants Shockey et al, 2006) with additional forms reported in burning bush (Euonymus alatus) (Durrett et al, 2010), peanut (Saha et al, 2006), and Arabidopsis (Rani et al, 2010). Plants and animals deficient in DGATs accumulate less TAG (Smith et al, 2000;Stone et al, 2004;Zou et al, 1999).…”

“…Animals with reduced DGAT activity are resistant to diet-induced obesity Smith et al, 2000) and lack milk production (Smith et al, 2000). Over-expression of DGAT enzymes increases TAG content in plants (Andrianov et al, 2010;Bouvier-Nave et al, 2000;Burgal et al, 2008;Durrett et al, 2010;Jako et al, 2001;Lardizabal et al, 2008;Xu et al, 2008), animals (Kamisaka et al, 2010;Liu et al, 2009;Liu et al, 2007;Roorda et al, 2005), and yeast (Kamisaka et al, 2007). DGATs have nonredundant functions in TAG biosynthesis in species such as mice (Stone et al, 2004) and tung tree (Vernicia fordii) (Shockey et al, 2006).…”