Step changes in leaf oil accumulation via iterative metabolic engineering

Abstract

Synthesis and accumulation of plant oils in the entire vegetative biomass offers the potential to deliver yields surpassing those of oilseed crops. However, current levels still fall well short of those typically found in oilseeds. Here we show how transcriptome and biochemical analyses pointed to a futile cycle in a previously established Nicotiana tabacum line, accumulating up to 15% (dry weight) of the storage lipid triacylglycerol in leaf tissue. To overcome this metabolic bottleneck, we either silenced the SDP1 lipase or overexpressed the Arabidopsis thaliana LEC2 transcription factor in this transgenic background. Both strategies independently resulted in the accumulation of 30-33% triacylglycerol in leaf tissues. Our results demonstrate that the combined optimization of de novo fatty acid biosynthesis, storage lipid assembly and lipid turnover in leaf tissue results in a major overhaul of the plant central carbon allocation and lipid metabolism. The resulting further step changes in oil accumulation in the entire plant biomass offers the possibility of delivering yields that outperform current oilseed crops.

Up‐regulation of lipid biosynthesis increases the oil content in leaves of Sorghum bicolor

“…Starch is a major carbon and energy storage form in leaves and some source tissues such as tubers and therefore expected to compete with lipids for photosynthetic carbon. Previous attempts at engineering elevated levels of storage lipids in a variety of species and tissues did negatively impact transitory or storage starch metabolism (Zale et al ., ; Vanhercke et al ., ; Hofvander et al ., ; Liu et al ., ; Mitchell et al ., ; Vanhercke et al ., ). Quantification of starch levels in transgenic sorghum leaves showed a distinct difference between the two transgenic populations.…”

“…All transgenic plants exhibited a shift in fatty acid profile towards a lower degree of desaturation. This was mainly due to a reduction in α‐linoleic acid, similar to previous results obtained in tobacco (Vanhercke et al ., ; Vanhercke et al ., ). However, plants transformed with either the Z. mays Ubiquitin‐1 (pOIL102+pOIL197) or PEPC promoter driving WRI1 expression (pOIL103+pOIL197) displayed distinct changes in the proportions of palmitic acid and linoleic acid in the TAG and TFA fractions.…”

“…Each 25 μL reaction volume contained 12.5 μL of ddPCR Supermix master mix, 400 n m of each primer, 200 n m of the probe and 1 μL cDNA. Oil droplet generation, PCR amplification and data analysis were done as described (Vanhercke et al ., ). The S. bicolor actin gene served as reference for gene expression analysis (Shen et al ., b).…”

“…A growing number of transgenic metabolic engineering studies have demonstrated the feasibility of increasing the oil content in various nonseed tissues (Weselake, ; Xu and Shanklin, ). Amongst these reports, multigene strategies are now clearly leading the way, culminating in the engineering of oilseed‐like TAG levels in leaves of tobacco (Vanhercke et al ., ). This was achieved by the simultaneous overexpression of WRINKLED1 ( WRI1 ), a positive regulator of fatty acid synthesis (‘Push’), acyl CoA:diacylglycerol acyltransferase 1 ( DGAT1) which catalyses the final TAG assembly step (‘Pull’), and Oleosin‐L oil droplet protein (‘Package’) while reducing lipid turnover by overexpression of the LEAFY COTYLEDON2 ( LEC2 ) embryogenic transcription factor or silencing of the SUGAR DEPENDENT1 ( SDP1 ) lipase (‘Protect’).…”

Oil Accumulation in Transgenic Potato Tubers Alters Starch Quality and Nutritional Profile

“…It may even be possible to generate crops that provide both a conventional grain harvest as well as a biomass harvest for oil. Significant progress has already been made in the production of biomass oil crops with TAG accumulating to 2 and 35% (DW, dry weight basis) in the vegetative tissue of sugar cane and tobacco, respectively (Zale et al, 2016; Vanhercke et al, 2017). Alternative metabolic engineering approaches focusing on increasing the lipid content of storage organs such as potato tubers have also proved successful, with TAG content ranging from 0.3 to 1% DW (Klaus et al, 2004; Hofvander et al, 2016).…”