Targeted engineering of camelina and pennycress seeds for ultrahigh accumulation of acetyl-TAG

Abstract

Acetyl-TAG (3-acetyl-1,2-diacylglycerol), unique triacylglycerols (TAG) possessing an acetate group at the sn-3 position, exhibit valuable properties, such as reduced viscosity and freezing points. Previous attempts to engineer acetyl-TAG production in oilseed crops did not achieve the high levels found in naturally producing Euonymus seeds. Here, we demonstrate the successful generation of camelina and pennycress transgenic lines accumulating nearly pure acetyl-TAG at 93 mol% and 98 mol%, respectively. These ultrahigh acetyl-TAG synthesizing lines were created using gene-edited FATTY ACID ELONGASE1 (FAE1) mutant lines as an improved genetic background to increase levels of acetyl-CoA available for acetyl-TAG synthesis mediated by the expression of EfDAcT, a high-activity diacylglycerol acetyltransferase isolated from Euonymus fortunei. Combining EfDAcT expression with suppression of the competing TAG-synthesizing enzyme DGAT1 further enhanced acetyl-TAG accumulation. These ultrahigh levels of acetyl-TAG exceed those in earlier engineered oilseeds and are equivalent or greater than those in Euonymus seeds. Imaging of lipid localization in transgenic seeds revealed that the low amounts of residual TAG were mostly confined to the embryonic axis. Similar spatial distributions of specific TAG and acetyl-TAG molecular species, as well as their probable diacylglycerol (DAG) precursors, provide additional evidence that acetyl-TAG and TAG are both synthesized from the same tissue-specific DAG pools. Remarkably, this ultrahigh production of acetyl-TAG in transgenic seeds exhibited minimal negative effects on seed properties, highlighting the potential for production of designer oils required for economical biofuel industries.

Keywords: biofuels; oil seeds; synthetic biology; triacylglycerols.

Fig. 1.

Pennycress fae1 mutant seeds possess increased levels of acetyl-CoA. Seed fatty acid composition (A), seed weight (B), seed fatty acid content (C), and seed acetyl-CoA levels (D and E) of camelina and pennycress wild-type and fae1 mutant seed. Data represent mean ± SE, n ≥ 3 biological replicates. Asterisks indicate statistical difference from wild-type seeds (Student’s t test; *P ≤ 0.05, ***P ≤ 0.001).

Fig. 2.

Mutation of fae1 has a modest impact on acetyl-TAG accumulation in transgenic camelina lines. (A) Scatter plot of acetyl-TAG content in segregating T2 seeds of transgenic fae1 lines expressing either EfDAcT or EfDAcT+DGAT1 RNAi. Gray boxes represent previously quantified acetyl-TAG values in wild-type seeds, with the top and bottom edges indicating the maximum and minimum values, respectively (10). Horizontal lines represent mean values, n ≥ 3 independent transgenic lines. (B) Scatter plot of acetyl-TAG content in homozygous wild-type and fae1 transgenic lines expressing EfDAcT or EfDAcT+DGAT1 RNAi that were grown together. Each independent line is represented by a distinct color. Horizontal lines represent mean values, n ≥ 3 biological replicates. Asterisks indicate statistical difference between genotypes (one-way ANOVA followed by Sidak's multiple comparisons test; *P ≤ 0.05 and ****P ≤ 0.0001). (C and D) Linear regression analysis of acetyl-TAG accumulation and 20:1 VLCFA levels in segregating F2 seed from crosses between fae1 and transgenic wild-type seeds expressing EfDAcT (C) or EfDAcT+DGAT1 RNAi (D). The red data point indicates 20:1 and acetyl-TAG accumulation in the original transgenic parent segregating for EfDAcT (C) or EfDAcT+DGAT1 RNAi (D). All other data points represent 20:1 and acetyl-TAG content in segregating F2 seed.

Fig. 3.

The fae1 mutation enhances acetyl-TAG accumulation in transgenic pennycress seeds. (A) Scatter plot of acetyl-TAG content in homozygous transgenic wild-type and fae1 lines expressing EfDAcT or EfDAcT+DGAT1 RNAi. Each independent line is represented by a distinct color. Horizontal lines represent mean values, n = three independent transgenic lines with ≥3 biological reps. Asterisks indicate statistical difference from wild-type or fae1 seeds (one-way ANOVA followed by Sidak's multiple comparisons test; ***P ≤ 0.001 and ****P ≤ 0.0001). (B) Positive ESI-MS spectra of neutral lipids from fae1 mutant control and homozygous pennycress seeds expressing EfDAcT+DGAT1 RNAi in fae1 background. Signal peaks represent m/z values of the [M + NH₄]⁺ adduct. The number of acyl carbons in each series of acetyl-TAG and TAG molecular species is indicated; the number of double bonds (x) is not delineated. (C) Scatter plot of acetyl-TAG levels obtained from homozygous F3 seed crosses between fae1 or wild-type seeds and transgenic seeds expressing EfDAcT+DGAT1 RNAi. Red data points represent acetyl-TAG levels in the transgenic parent used in each cross.

Fig. 4.

Assessment of gene expression levels of TAG-related genes in transgenic pennycress lines. Quantitative real-time PCR analysis of DGAT1 (A), PDAT1 (B), FAE1 (C), and EfDAcT (D) in transgenic wild-type and fae1 developing seeds expressing EfDAcT or EfDAcT+DGAT1 RNAi 20 d after flowering. Gene expression levels were normalized to the geometric mean of the reference genes TIP4 and UBC. Data represent mean ± SE, n = 3 biological reps. Different letters indicate statistical difference between genotypes (one-way ANOVA followed by Tukey's multiple comparisons test; P ≤ 0.05).

Fig. 5.

MALDI-MS imaging of TAG and acetyl-TAG in pennycress mature seeds. MALDI-MS images of TAG potassium adducts [M + K]⁺ in mature pennycress seeds from wild-type, fae1 mutant, and homozygous pennycress transgenic lines expressing EfDAcT or EfDAcT+DGAT1 RNAi. Images shown are summed ion images of acetyl-TAG and TAG molecular species with the same number of acyl carbons regardless of the number of double bonds (x). Normalized ion intensities are shown using a jet color scale, with red representing 100% of the total TAG signal. Minimal signals observed as acetyl-TAG for WT and fae1 are from unknown chemical interferents. The scale bar represents 500 μm. Optical images are included for reference.

Fig. 6.

Seed germination and plant traits of pennycress high acetyl-TAG-producing lines. (A) Germination rates of seed from homozygous transgenic lines expressing EfDAcT or EfDAcT+DGAT1 RNAi in both wild-type and fae1 backgrounds. Solid and dashed lines indicate different independent lines. Data represent mean ± SE, n = 3 biological replicates. (B) Time in days until first flower opening. (C) Plant height measurements at 21, 37, and 51 d after planting. Data represent mean ± SE of two independent transgenic lines with three biological replicates. Different letters indicate statistical difference between genotypes (one-way ANOVA followed by Tukey's multiple comparisons test; P ≤ 0.05).

Fig. 7.

Strategies for engineering ultrahigh levels of acetyl-TAG in camelina and pennycress. In wild-type seeds, TAGs are mostly produced through the activity of DGAT1, which transfers an acyl group to the sn-3 position of DAG to produce TAG. To engineer ultrahigh levels of acetyl-TAG in transgenic plants, multiple strategies were used including 1) the expression of a high-activity acetyltransferase (EfDAcT) which transfers an acetate group from acetyl-CoA to the sn-3 position of DAG to produce acetyl-TAG; 2) RNAi to inhibit DGAT1 expression and reduce TAG synthesis; and 3) mutating FAE1 to increase acetyl-CoA levels available for EfDAcT. In pennycress, the absence of VLCFA in the acyl-CoA pool caused by fae1 mutation could also reduce the ability of DGAT1 to use the remaining acyl-CoA, as illustrated by the dashed arrow. Abbreviations: Acetyl-TAG, acetyl-triacylglycerol; DAG, diacylglycerol; DGAT1, diacylglycerol acyltransferase1; EfDAcT, Euonymus fortunei diacylglycerol acetyltransferase; FAE1, fatty acid elongase1; RNAi, RNA interference; TAG, triacylglycerol.