Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves

Abstract

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.

Keywords: DGAT1; Nicotiana tabacum; WRI1; leaf; oleosin; triacylglycerol.

Figure 1

Increasing triacylglycerol (TAG) levels in leaf tissue by an integrated metabolic engineering approach. (a) Fatty acids are produced and exported from the chloroplast (Push) into the endoplasmic reticulum where they are assembled into TAG (Pull). TAG accumulates either as oil droplets or oleosin-coated oil bodies (Protect). Previously tested genes for each approach are listed and referenced. (b) Map of the binary T-DNA region used to co-express the Arabidopsis thaliana (Arath) WRI1, A. thaliana DGAT1 and Sesamum indicum (Sesin) OLEOSIN genes. Red lines show the terminator regions; promoter regions are represented by green and brown arrows; orange arrows indicate the coding regions.

Figure 2

Leaf triacylglycerol (TAG) content, fatty acid profile and phenotype of transgenic Nicotiana tabacum T₁ plants. (a) TAG content on a dry weight (DW) in leaves of control (wild-type and null segregant) plants and the transgenic tobacco line exhibiting maximum TAG levels. ‘Vegetative’ stage was harvested 69 days after sowing (DAS), ‘Flowering’ at 104 DAS, ‘Seed Setting’ at 139 DAS and fully ‘Senesced’ leaves at 207 DAS. Error bars represent standard deviations of minimum three samples. (b) Seedling establishment was delayed, but no discernible negative phenotype was observed in mature transgenic T₁ events compared to null segregants or wild-type plants. (c) Fatty acid profile of TAG from both transgenic and wild-type tobacco leaves at the onset of senescence. Error bars represent standard deviations (n = 3). (d) Total ion scan of TAG and DAG from triplicate samples, normalized on a dry weight basis, of transgenic and wild-type leaf total lipids by LC-MS. Detailed LC-MS analysis of different PC, DAG and TAG molecular species is shown in Figure S4.

Figure 3

Triacylglycerol (TAG) and starch quantification in wild-type and T₁ transgenic tissues. (a) TAG quantification on a dry weight (DW) basis in young green vs. mature leaf at the onset of senescence. (b) TAG quantification on a DW basis in young stem, mature stem and mature root tissues. (c) Leaf starch on a fresh weight (FW) basis in mature leaf tissue. Error bars represent standard deviations (n = 3).

Figure 4

Accumulation of lipid droplets and spatial distribution of different triacylglycerol (TAG) molecular species within transgenic leaf tissue. (a) 3D reconstructed z-stacks of confocal images from wild-type (left) and transgenic (right) leaf tissue. Neutral lipids including TAG, stained with BODIPY, appear as green droplets within the leaf mesophyll cells that contain visible chloroplasts (red autofluorescence). Note that non-TAG features also fluorescing green include the vascular bundle and leaf epidermis. (b) Bright-field microscopy images of wild-type (upper) and transgenic (lower) leaf cross-sections subsequently used for MALDI-Orbitrap imaging. Scale bars correspond to 1000 microns. (c) Ratio of TAG total ion counts (TIC; all TAG molecular species summed at each analysed position) to PC-TIC as detected by MALDI-Orbitrap imaging in wild-type (upper) and transgenic (lower) leaf cross-sections. (d) Spatial distribution of selected dominant TAG and PC species across a transgenic leaf cross-section as observed by MALDI-Orbitrap.

Figure 5

Triacylglycerol (TAG) extraction efficiencies from fresh transgenic leaf tissue obtained with solvents of different polarity. Subsequent Bligh and Dyer extractions were performed on the fresh leaf samples to recover any residual lipid and to determine overall extraction efficiency. Extraction efficiencies are calculated using the formula 100 × % lipid extracted/combined yield following extraction with acetone and subsequent BD. Error bars represent standard deviations (n = 3).