Abstract
Thlaspi arvense L. (pennycress) is a cold-tolerant Brassicaceae that produces large amounts of seeds rich in triacylglycerols and protein, making it an attractive target for domestication into an offseason oilseed cash cover crop. Pennycress is easily genetically transformed, enabling synthetic biology approaches to tailor oil properties for specific biofuel and industrial applications. To test the feasibility in pennycress of producing TAGs and acetyl-TAGs rich in medium-chain fatty acids (MCFAs; C6–C14) for industrial, biojet fuel and improved biodiesel applications, we generated transgenic lines with seed-specific expression of unique acyltransferase (LPAT and diacylglycerol acyltransferase) genes and thioesterase (FatB) genes isolated from Cuphea viscosissima, Cuphea avigera var. pulcherrima, Cuphea hookeriana, Coco nucifera, and Umbellularia californica. Wild-type pennycress seed TAGs accumulate no fatty acids shorter than 16C and less than 5 mol percent C16 as palmitic acid (16:0). Co-expressing UcFatB and CnLPAT produced up to 17 mol% accumulation of lauric acid (12:0) in seed TAGs, whereas CvFatB1 CvLPAT2 CpDGAT1 combinatorial expression produced up to 27 mol% medium chain FAs Medium Chain Fatty Acids mostly in the form of capric acid (10:0). CpFatB2 ChFatB2 combinatorial expression predominantly produced, in equal parts, up to 28 mol% myristic acid (14:0) and palmitic acid. Genetically crossing the combinatorial constructs into a fatty acid elongation1 (fae1) mutant that produced no 22:1 erucic acid, and with an Euonymus alatus diacylglycerol acetyltransferase (EaDAcT)-expressing line that produced 60 mol% acetyl-TAGs, had no or relatively minor effects on MCFAs accumulation, suggesting fluxes to MCFAs were largely unaltered. Seed germination assays revealed no or minor delays in seed germination for most lines, the exception being CpFatB2 ChFatB2-expressing lines, which had substantially slower seed germination rates. Taken together, these data show that pennycress can be engineered to produce seeds accumulating modest amounts of MCFAs of varying carbon-chain length in TAGs and acetyl-TAGs, with rates of seed germination being delayed in only some cases. We hypothesize that increasing MCFAs further may require functional reductions to endogenous transferases and/or other FA elongases.
Highlights
Brassicaceae seeds accumulate high levels of triacylglycerols (TAGs, oil) in embryos where they function as an energy source and carbon skeletons for the germinating seedlings (Murphy and Cummins, 1989; Chapman and Ohlrogge, 2012)
We examined how Medium Chain Fatty Acids (MCFAs) accumulation is affected by knockout of erucic acid production and/or in combination with synthesis of acetyl-TAGs
Our results showed that MCFAs of various chain lengths (C8–C16) can be produced and incorporated into pennycress seed oil at modest levels, but may result in reductions in total seed oil content and reduced rates of seed germination when MCFAs are incorporated in TAGs at high levels
Summary
Brassicaceae seeds accumulate high levels of triacylglycerols (TAGs, oil) in embryos where they function as an energy source and carbon skeletons for the germinating seedlings (Murphy and Cummins, 1989; Chapman and Ohlrogge, 2012). To expand genetic diversity for producing novel fatty acids that add value to pennycress oil, we took a biotechnological approach to introduce genes from non-related and non-agronomic plant species that produce extreme amounts of MCFAs and acetylTAGs that are not naturally accumulated in pennycress seeds. In this regard, some plant species, including the genus Cuphea which grow in temperate climates, contain seed TAGs with as much as 94 mol percent MCFAs (Graham and Kleiman, 1992). Our results showed that MCFAs of various chain lengths (C8–C16) can be produced and incorporated into pennycress seed oil at modest levels, but may result in reductions in total seed oil content (from 9 to 43 percent reductions under our conditions) and reduced rates of seed germination when MCFAs are incorporated in TAGs at high levels
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