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

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.

Overexpression of diacylglycerol acetyltransferase from Euonymus europaeus in Yarrowia lipolytica leads to the production of single-cell oil enriched with 3-acetyl-1,2-diacylglycerols.

The 3-acetyl-1,2-diacylglycerols (acTAGs) are the molecules that are structurally similar to triacylglycerols (TAGs). They are naturally produced by plants of the family Celastraceae and animals such as Cervus nippon and Eurosta solidaginis. The presence of acetate in the sn-3 position of the glycerol backbone confers advantages to these compounds, for example, lower viscosity and calorific value compared to classical TAGs. In this work, the gene EeDAcT, which encodes diacylglycerol acetyltransferase in a species of bush (Euonymus europaeus), was overexpressed in strains Po1d (capable of accumulating storage lipids) and JMY1877 (incapable of accumulating storage lipids) of Yarrowia lipolytica, to test the activity of the gene EeDAcT and the production of acTAGs in oleaginous and nonoleaginous genetic backgrounds. It was observed that both the strains containing the gene EeDAcT (YL33 and YL35 for Po1d and JMY1877 strains, respectively) produced acTAGs. The strain YL33 accumulated up to 20% intracellular lipids, 20% of which was acTAGs, and 40% was TAGs. On the other hand, the strain YL35, which showed interrupted TAGs accumulation, produced up to 10% acTAGs as the only storage lipid. Unfortunately, the quantity of acTAGs produced in YL35 was insignificant, as the overall lipid accumulated in the strain was not more than 4% of the biomass. The fatty acid profile of acTAGs produced by the YL33 strain was remarkably similar to TAGs, and both of these structures were rich in oleic (45%) and palmitic (25%) acids.

Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop.

Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run-off on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and creating an edible seed oil comparable to that of canola. We also stably transformed pennycress with the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene, producing low-viscosity acetyl-triacylglycerol-containing seed oil suitable as a diesel-engine drop-in fuel. Adoption of pennycress as a model system will accelerate oilseed-crop translational research and facilitate pennycress' rapid domestication to meet the growing sustainable food and fuel demands.

Membrane topology and identification of key residues of EaDAcT, a plant MBOAT with unusual substrate specificity.

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl-sn-glycerol (acetyl-TAG). EaDAcT belongs to a small, plant-specific subfamily of the membrane bound O-acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed that EaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)-specific marker. By mapping the membrane topology of EaDAcT, we obtained an experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains (TMDs), with both the N- and C-termini orientated toward the lumen of the ER. In addition, there is a large cytoplasmic loop between the first and second TMDs, with the MBOAT signature region of the protein embedded in the third TMD close to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl-TAG-producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential for EaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity of EaDAcT, suggesting that multiple amino acids are important for substrate recognition.

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

Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-sn-glycerols (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.

Isolation of a gene encoding a 1,2-diacylglycerol-sn-acetyl-CoA acetyltransferase from developing seeds of Euonymus alatus.

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 ac-TAGs 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 long-chain 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 acyl- and acetyltransferase activity.