Engineering triacylglycerol accumulation in duckweed (Lemna japonica)

Abstract

Duckweeds are amongst the fastest growing of higher plants, making them attractive highbiomass targets for biofuel feedstock production. Their fronds have high rates of fatty acid synthesis to meet the demand for new membranes, but triacylglycerols (TAG) only accumulate to very low levels. Here we report the engineering of Lemna japonica for the synthesis and accumulation of TAG in its fronds. This was achieved by expression of an estradiol-inducible cyan fluorescent protein-Arabidopsis WRINKLED1 fusion protein (CFP-AtWRI1), strong constitutive expression of a mouse diacylglycerol:acyl-CoA acyltransferase2 (MmDGAT), and a sesame oleosin variant (SiOLE(*)). Individual expression of each gene increased TAG accumulation by 1-to 7-fold relative to controls, while expression of pairs of these genes increased TAG by 7-to 45-fold. In uninduced transgenics containing all three genes, TAG accumulation increased by 45fold to 3.6% of dry weight (DW) without severely impacting growth, and by 108-fold to 8.7% of DW after incubation on medium containing 100 μM estradiol for four days. TAG accumulation was accompanied by an increase in total fatty acids of up to 3-fold to approximately 15% of DW. Lipid droplets from fronds of all transgenic lines were visible by confocal microscopy of BODIPY-stained fronds. At a conservative 12 tonnes (dry matter) per acre and 10% (DW) TAG, duckweed could produce 350 gallons of oil/acre/year, approximately 7-fold the yield of soybean, and similar to that

WRINKLED1 Positively Regulates Oil Biosynthesis in Carya cathayensis

“…In addition to the use of single gene overexpression, the multigene strategies are now clearly leading the way, eventually attaining oilseed-like TAG levels in nonseed tissues. This has been successfully achieved by simultaneously expressing WRI1, an active regulator of FA synthesis (“Push”), diacylglycerol acyltransferase (DGAT), and phospholipid:diacylglycerol acyltransferase (PDAT), which catalyzes the final TAG assembly step (“Pull”), and oleosin lipid droplet protein (“Protect”) in some species. − Further work will be done to improve oil content in plants by the overexpression of hickory multigenes.…”

Dynamic Regulation of Lipid Droplet Biogenesis in Plant Cells and Proteins Involved in the Process

“…At the transcriptional level, lipid synthesis and partition are regulated by several key transcription factors, including WRINKLED1 (WRI1) and LEAFY COTYLEDON2 (LEC2) [ 39 ]. TAG content in transgenic Lemna japonica fronds overexpressing AtWRI1 was significantly increased by about 7-fold compared to WT, indicating that WRI1 is a positive regulator of lipid synthesis [ 40 ]. The ectopic expression of a seed-specific LEC2 promotes accumulation of TAGs in leaf tissues.…”

A toolkit for plant lipid engineering: Surveying the efficacies of lipogenic factors for accumulating specialty lipids

“…Future efforts to develop improved strategies for mitigated growth impairment and further enhancement of vegetative TAG production will focus on the identification of alternative lipogenic factors that can more efficiently incorporate FAs to TAGs and have reduced negative impacts on plant growth. Additional promising approaches include restricting the expression of lipogenic factors to certain tissues or growth stages using inducible or tissue-specific promoters ( Andrianov et al., 2010 ; Kim et al., 2015c ; Liang et al, 2022 ) , or the expression of factors such as purple acid phosphatase2 ( Cai et al, 2022 ). Despite the challenges, vegetative biomass represents a sustainable and economical platform for lipid accumulation and the success in engineering TAG accumulation therein will facilitate increased yields per unit land area of high-value lipids containing specialty FAs in vegetative tissues by introducing additional specialized lipogenic factors.…”