Expression of a high‐activity diacylglycerol acetyltransferase results in enhanced synthesis of acetyl‐TAG in camelina seed oil

Abstract

Acetyl-triacylglycerols (acetyl-TAG) contain an acetate group in the sn-3 position instead of the long-chain fatty acid present in regular triacylglycerol (TAG). The acetate group confers unique physical properties such as reduced viscosity and a lower freezing point to acetyl-TAG, providing advantages for use as emulsifiers, lubricants, and 'drop-in' biofuels. Previously, the synthesis of acetyl-TAG in the seeds of the oilseed crop camelina (Camelina sativa) was achieved through the heterologous expression of the diacylglycerol acetyltransferase gene EaDAcT, isolated from Euonymus alatus seeds that naturally accumulate high levels of acetyl-TAG. Subsequent work identified a similar acetyltransferase, EfDAcT, in the seeds of Euonymus fortunei, that possesses higher in vitro activity compared to EaDAcT. In this study, the seed-specific expression of EfDAcT in camelina led to a 20 mol% increase in acetyl-TAG levels over that of EaDAcT. Coupling EfDAcT expression with suppression of the endogenous competing enzyme DGAT1 further enhanced acetyl-TAG accumulation, up to 90 mol% in the best transgenic lines. Accumulation of high levels of acetyl-TAG was stable over multiple generations, with minimal effect on seed size, weight, and fatty acid content. Slight delays in germination were noted in transgenic seeds compared to the wild type. EfDAcT transcript and protein levels were correlated during seed development with a limited window of EfDAcT protein accumulation. In high acetyl-TAG producing lines, EfDAcT protein expression in developing seeds did not reflect the eventual acetyl-TAG levels in mature seeds, suggesting that other factors limit acetyl-TAG accumulation.

A toolkit for plant lipid engineering: Surveying the efficacies of lipogenic factors for accumulating specialty lipids

“…Heterologous expression of EaDAcT resulted in accumulation of 40% and 52% of acTAG in seeds of Arabidopsis and Camelina, respectively ( Durrett et al., 2010 ). EfDAcT, functioning more efficiently than EaDAcT, produced an average of 72% acTAG in transgenic Camelina seeds ( Alkotami et al., 2021 ). Interestingly, co-expression of CnLPAT from MCFA-containing coconut and EaDAcT from acTAG-enriched E. alatus in camelina plants expressing UcFATB1 produced acTAGs with MCFAs, which have yet to be found in nature, suggesting a potential synthetic-biology strategy for creating novel lipid structures in plants ( Bansal et al., 2018 ).…”

Functional analysis of alternative castor bean DGAT enzymes

“…Beyond this activity, EaDAcT can also acetylate fatty alcohols in vitro ( Bansal and Durrett, 2016 ). Heterologous expression of this enzyme in Arabidopsis yielded 40% of acTAGs in its oil, showing an attractive application of DAcT in the formation process of low viscosity oils for the production of biofuels ( Durrett et al , 2010 ; Liu et al, 2015 ; Tran et al, 2017a ); however, no homologous DAcT was identified and characterized for oilseed crops, or plants with relevant oil in industry applications ( Alkotami et al, 2021 ; Mihálik et al, 2020 ; Tran et al , 2017b ).…”

Defining the physical properties of blends of acetyl‐triacylglycerols derived from transgenic oil seeds

“…Transformation of EaDAcT into the emerging oil seed crop Camelina sativa resulted in acetyl‐TAG levels of about 65 mol% in the best transgenic lines. Combining EaDAcT expression with the RNAi‐mediated suppression of the endogenous camelina DGAT1 enzymes which synthesize lcTAG and thus compete with EaDAcT, resulted in acetyl‐TAG levels as high as 81 mol% (Alkotami et al, 2021; Liu et al, 2015). Higher levels of acetyl‐TAG were subsequently achieved by expressing EfDAcT, a higher activity acetyltransferase enzyme isolated from E. fortunei (Tran, Shelton, et al, 2017).…”