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, 69 (18), 4395-4402

Towards the Synthetic Design of Camelina Oil Enriched in Tailored Acetyl-Triacylglycerols With Medium-Chain Fatty Acids

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Towards the Synthetic Design of Camelina Oil Enriched in Tailored Acetyl-Triacylglycerols With Medium-Chain Fatty Acids

Sunil Bansal et al. J Exp Bot.

Abstract

The ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs). Different transgenic camelina lines that had been genetically modified to produce MCFAs through the expression of MCFA-specific thioesterases and acyltransferases were retransformed with the Euonymus alatus gene for diacylglycerol acetyltransferase (EaDAcT) that synthesises acetyl-TAGs. Concomitant RNAi suppression of acyl-CoA:diacylglycerol acyltransferase increased the levels of acetyl-TAG, with up to 77 mole percent in the best lines. However, the total oil content was reduced. Analysis of the composition of the acetyl-TAG molecular species using electrospray ionisation mass spectrometry demonstrated the successful synthesis of acetyl-TAG containing MCFAs. Field growth of high-yielding plants generated enough oil for quantification of viscosity. As part of an ongoing design-test-learn cycle, these results, which include not only the synthesis of 'designer' lipids but also their functional analysis, will lead to the future production of such molecules tailored for specific applications.

Figures

Fig. 1.
Fig. 1.
Strategy for the production of MCFA acetyl-TAGs in transgenic oilseeds. (A) Structures of representative TAG, acetyl-TAG, and acetyl-TAG containing MCFA. (B) Metabolic pathways leading to the synthesis of acetyl-TAGs containing MCFAs. Compound abbreviations: acetyl-TAG, 3-acetyl-1,2-diacyl-sn-glycerol; CoA, co-enzyme A; DAG, diacylglycerol; G3P, glycerol-3-phosphate; LPC, lysophosphatidylcholine; MCFA, medium chain fatty acid; PC, phosphatidylcholine; TAG, triacylglycerol. Enzyme abbreviations: DAcT, diacylglycerol acetyltransferase; DGAT1, diacylglycerol acyltransferase; Fat, fatty acid thioesterase; LPAAT, lysophosphatic acid acyltransferase; PDAT1, phosphatidylcholine: diacylglycerol acyltransferase. Species abbreviations: Ch, Cuphea hookeriana; Cn, Cocos nucifera; Cp, Cuphea palustris; Ea, Euonymus alatus; Uc, Umbellularia californica.
Fig. 2.
Fig. 2.
Expression of EaDAcT combined with down-regulation of DGAT1 enhances acetyl-TAG accumulation. Scatter plots of the distribution of acetyl-TAG composition of homozygous T3 seeds from independent camelina lines expressing CpFatB2 and ChFatB2 or UcFatB1 and CnLPAAT, and transformed with EaDAcT alone or in combination with RNAi constructs targeting camelina DGAT1 and PDAT1 homeologues. Horizontal lines represent the mean values for each sample group.
Fig. 3.
Fig. 3.
Synthesis of medium chain acetyl-TAGs in camelina. (A) Positive electrospray ionisation tandem mass spectrometry (ESI-MS2) spectra scanning for the neutral loss of ammonium acetate to detect acetyl-TAG molecular species in neutral lipid extracts from T3 homozygous seeds of wild-type (WT) and MCFA-producing camelina plants expressing EaDAcT. Signal peaks possess the m/z value of the [M+NH4]+ adduct. For clarity, only the number of acyl carbons and not the number of double bonds (x) in each series of acetyl-TAG molecular species is indicated. (B) ESI-MS2 daughter scans of medium-chain acetyl-TAGs from camelina seed expressing EaDAcT. The data shown are the fragment peaks for acetyl-TAGs with [M+NH4]+ adducts with masses of 516.5 and 572.7.
Fig. 4.
Fig. 4.
Acetyl-TAGs contain lower levels of MCFAs than lcTAGs. Mean fatty acid composition of acetyl-TAGs and lcTAG fractions from T3 seed of independent homozygous lines in the CpFatB2+ChFatB2 (n=4) or UcFatB1+CnLPAAT (n=6) backgrounds expressing EaDAcT and RNAi cassettes targeting DGAT1 and PDAT1. Data are means (±SD). Significant differences as determined by Student’s t-test with Holm–Šidák correction method for multiple comparisons are indicated: *P<0.05.
Fig. 5.
Fig. 5.
Acetyl-TAGs possess low incorporation of MCFAs at sn-2. Mean fatty acid composition at sn-2 positions of lcTAGs and acetyl-TAGs from T4 seed of four independent homozygous camelina UcFatB1+CnLPAAT lines expressing EaDAcT in combination with DGAT1-RNAi, and of one independent homozygous camelina CpFatB2+ChFatB line expressing EaDAcT in combination with suppression of DGAT1 and PDAT1. Data are means (±SD). Significant differences as determined by Student’s t-test with Holm–Šidák correction method for multiple comparisons are indicated: *P<0.05.
Fig. 6.
Fig. 6.
MCFA acetyl-TAG lines possess a lower oil content. Mean oil content of T4 seed of independent homozygous lines in the CpFatB2+ChFatB2 and UcFatB1+CnLPAAT backgrounds expressing EaDAcT and DGAT1-RNAi. Data are means (±SD) of two independent gravimetric experiments.

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