Generating Pennycress (Thlaspi arvense) Seed Triacylglycerols and Acetyl-Triacylglycerols Containing Medium-Chain Fatty Acids

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.

Chromosome‐level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates

“…It is amenable to genetic transformation using the floral dip method (McGinn et al ., 2019 ), and its diploid nature with many one‐to‐one gene correspondence with A. thaliana (Chopra et al ., 2018 ) could provide an avenue for gene discovery followed by field‐based phenotypic validation. Indeed, several agronomic and biochemical traits have already been identified in pennycress using this translational approach, including traits crucial for de novo domestication of T. arvense such as transparent testa phenotypes (Chopra et al ., 2018 ), early flowering (Chopra et al ., 2020 b), reduced shatter (Chopra et al ., 2020 b) and seed oil composition traits (Chopra et al ., 2020 b; Esfahanian et al ., 2021 ; Jarvis et al ., 2021 ; McGinn et al ., 2019 ). Field pennycress could thus serve as a de novo ‐domesticated oilseed crop for the cooler climates of the world and at the same time as a new dicotyledonous model for functional genetics studies.…”

CRISPR/Cas9-Induced fad2 and rod1 Mutations Stacked With fae1 Confer High Oleic Acid Seed Oil in Pennycress (Thlaspi arvense L.)

“…Two well-established oilseed crops are soybean (Glycine max L.) and canola (Brassica napus), producing over 400 million metric tons of seed, annually, rich in both oil and protein (USDA, United States Department of Agriculture Foreign Agriculture Services, 2013). The oils from these crops can be used in a plethora of products including foodstuffs, feed, cosmetics, and industrial lubricants along with biodiesel and biojet fuels (Dyer et al, 2008;Volkova et al, 2016). However, it is important to balance needs for human nutrition with those of bio-based products.…”

“…Erucic acid has multiple industrial uses and is suitable for hydrocracking to produce dodecane (C11) and other medium and long chain fatty acids (MCFAs, LCFAs) for aviation fuel and biodiesel (Bezergianni and Kalogianni, 2009;Sotelo-Boyás et al, 2012;Isbell et al, 2015). Pennycress can also be engineered to produce seed TAGs and acetyl-TAGs containing MCFAs suitable for industrial, biojet fuel and improved biodiesel applications (Esfahanian et al, 2021). It has also been shown in different plant species that specific mutations and mutation combinations can substantially decrease VLCFAs and polyunsaturated fatty acids (PUFAs, e.g., 18:2 linoleic acid and 18:3 linolenic acid) thereby increasing 18:1 oleic acid content in seed oil, which is a desirable biodiesel component (Sivaraman et al, 2004;Kang et al, 2011;McGinn et al, 2019;Chopra et al, 2020;Neumann et al, 2021).…”

Chromosome-level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates

“…It is amenable to genetic transformation via the floral dip method (McGinn et al, 2019), and its diploid nature with many one-to-one gene correspondences with A. thaliana (Chopra et al, 2018) could provide an avenue for gene discovery followed by field-based phenotypic validation. Indeed, several agronomic and biochemical traits have already been identified in pennycress using this translational approach, including traits crucial for de novo domestication of T. arvense such as transparent testa phenotypes (Chopra et al, 2018), early flowering (Chopra et al, 2020b), reduced shatter (Chopra et al, 2020b), and seed oil composition traits (McGinn et al, 2019;Jarvis et al, 2021;Esfahanian et al, 2021;Chopra et al, 2020b). Field pennycress could thus serve as a de novo domesticated oilseed crop for the cooler climates of the world and at the same time as a new dicotyledonous model for functional genetics studies.…”