Thomas Vanhercke

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

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.

Synergistic effect of WRI1 and DGAT1 coexpression on triacylglycerol biosynthesis in plants

Metabolic engineering approaches to increase plant oil levels can generally be divided into categories which increase fatty acid biosynthesis (‘Push’), are involved in TAG assembly (‘Pull’) or increase TAG storage/decrease breakdown (‘Accumulation’). In this study, we describe the surprising synergy when Push (WRI1) and Pull (DGAT1) approaches are combined. Co‐expression of these genes in the Nicotiana benthamiana transient leaf expression system resulted in TAG levels exceeding those expected from an additive effect and biochemical tracer studies confirmed increased flux of carbon through fatty acid and TAG synthesis pathways. Leaf fatty acid profile also synergistically shifts from polyunsaturated to monounsaturated fatty acids.

Recruiting a new substrate for triacylglycerol synthesis in plants: the monoacylglycerol acyltransferase pathway.

Background Monoacylglycerol acyltransferases (MGATs) are predominantly associated with lipid absorption and resynthesis in the animal intestine where they catalyse the first step in the monoacylglycerol (MAG) pathway by acylating MAG to form diacylglycerol (DAG). Typical plant triacylglycerol (TAG) biosynthesis routes such as the Kennedy pathway do not include an MGAT step. Rather, DAG and TAG are synthesised de novo from glycerol-3-phosphate (G-3-P) by a series of three subsequent acylation reactions although a complex interplay with membrane lipids exists. Methodology/Principal Findings We demonstrate that heterologous expression of a mouse MGAT acyltransferase in Nicotiana benthamiana significantly increases TAG accumulation in vegetative tissues despite the low levels of endogenous MAG substrate available. In addition, DAG produced by this acyltransferase can serve as a substrate for both native and coexpressed diacylglycerol acyltransferases (DGAT). Finally, we show that the Arabidopsis thaliana GPAT4 acyltransferase can produce MAG in Saccharomyces cerevisiae using oleoyl-CoA as the acyl-donor. Conclusions/Significance This study demonstrates the concept of a new method of increasing oil content in vegetative tissues by using MAG as a substrate for TAG biosynthesis. Based on in vitro yeast assays and expression results in N. benthamiana, we propose that co-expression of a MAG synthesising enzyme such as A. thaliana GPAT4 and a MGAT or bifunctional M/DGAT can result in DAG and TAG synthesis from G-3-P via a route that is independent and complementary to the endogenous Kennedy pathway and other TAG synthesis routes.

Metabolic engineering of plant oils and waxes for use as industrial feedstocks

Society has come to rely heavily on mineral oil for both energy and petrochemical needs. Plant lipids are uniquely suited to serve as a renewable source of high-value fatty acids for use as chemical feedstocks and as a substitute for current petrochemicals. Despite the broad variety of acyl structures encountered in nature and the cloning of many genes involved in their biosynthesis, attempts at engineering economic levels of specialty industrial fatty acids in major oilseed crops have so far met with only limited success. Much of the progress has been hampered by an incomplete knowledge of the fatty acid biosynthesis and accumulation pathways. This review covers new insights based on metabolic flux and reverse engineering studies that have changed our view of plant oil synthesis from a mostly linear process to instead an intricate network with acyl fluxes differing between plant species. These insights are leading to new strategies for high-level production of industrial fatty acids and waxes. Furthermore, progress in increasing the levels of oil and wax structures in storage and vegetative tissues has the potential to yield novel lipid production platforms. The challenge and opportunity for the next decade will be to marry these technologies when engineering current and new crops for the sustainable production of oil and wax feedstocks.

Metabolic engineering of medium-chain fatty acid biosynthesis in Nicotiana benthamiana plant leaf lipids

Various research groups are investigating the production of oil in non-seed biomass such as leaves. Recently, high levels of oil accumulation have been achieved in plant biomass using a combination of biotechnological approaches which also resulted in significant changes to the fatty acid composition of the leaf oil. In this study, we were interested to determine whether medium-chain fatty acids (MCFA) could be accumulated in leaf oil. MCFA are an ideal feedstock for biodiesel and a range of oleochemical products including lubricants, coatings, and detergents. In this study, we explore the synthesis, accumulation, and glycerolipid head-group distribution of MCFA in leaves of Nicotiana benthamiana after transient transgenic expression of C12:0-, C14:0-, and C16:0-ACP thioesterase genes. We demonstrate that the production of these MCFA in leaf is increased by the co-expression of the WRINKLED1 (WRI1) transcription factor, with the lysophosphatidic acid acyltransferase (LPAAT) from Cocos nucifera being required for the assembly of tri-MCFA TAG species. We also demonstrate that the newly-produced MCFA are incorporated into the triacylglycerol of leaves in which WRI1 + diacylglycerol acyltransferase1 (DGAT1) genes are co-expressed for increased oil accumulation.

Mechanistic and structural insights into the regioselectivity of an acyl-CoA fatty acid desaturase via directed molecular evolution

Membrane-bound fatty acid desaturases and related enzymes play a pivotal role in the biosynthesis of unsaturated and various unusual fatty acids. Structural insights into the remarkable catalytic diversity and wide range of substrate specificities of this class of enzymes remain limited due to the lack of a crystal structure. To investigate the structural basis of the double bond positioning (regioselectivity) of the desaturation reaction in more detail, we relied on a combination of directed evolution in vitro and a powerful yeast complementation assay to screen for Δx regioselectivity. After two selection rounds, variants of the bifunctional Δ12/Δ9-desaturase from the house cricket (Acheta domesticus) exhibited increased Δ9-desaturation activity on shorter chain fatty acids. This change in specificity was the result of as few as three mutations, some of them near the putative active site. Subsequent analysis of individual substitutions revealed an important role of residue Phe-52 in facilitating Δ9-desaturation of shorter chain acyl substrates and allowed for the redesign of the cricket Δ12/Δ9-desaturase into a 16:0-specific Δ9-desaturase. Our results demonstrate that a minimal number of mutations can have a profound impact on the regioselectivity of acyl-CoA fatty acid desaturases and include the first biochemical data supporting the acyl-CoA acyl carrier specificity of a desaturase able to carry out Δ12-desaturation.

Step changes in leaf oil accumulation via iterative metabolic engineering

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.

Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy

Summary Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber‐specific (patatin) and constitutive (CaMV‐35S) promoters. This coordinated metabolic engineering approach resulted in over a 100‐fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild‐type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof‐of‐concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.

Deep Sequencing of the Fruit Transcriptome and Lipid Accumulation in a Non-Seed Tissue of Chinese Tallow, a Potential Biofuel Crop.

Chinese tallow (Triadica sebifera) is a valuable oilseed-producing tree that can grow in a variety of conditions without competing for food production, and is a promising biofuel feedstock candidate. The fruits are unique in that they contain both saturated and unsaturated fat present in the tallow and seed layer, respectively. The tallow layer is poorly studied and is considered only as an external fatty deposition secreted from the seed. In this study we show that tallow is in fact a non-seed cellular tissue capable of triglyceride synthesis. Knowledge of lipid synthesis and storage mechanisms in tissues other than seed is limited but essential to generate oil-rich biomass crops. Here, we describe the annotated transcriptome assembly generated from the fruit coat, tallow and seed tissues of Chinese tallow. The final assembly was functionally annotated, allowing for the identification of candidate genes and reconstruction of lipid pathways. A tallow tissue-specific paralog for the transcription factor gene WRINKLED1 (WRI1) and lipid droplet-associated protein genes, distinct from those expressed in seed tissue, were found to be active in tallow, underpinning the mode of oil synthesis and packaging in this tissue. Our data have established an excellent knowledge base that can provide genetic and biochemical insights for engineering non-seed tissues to accumulate large amounts of oil. In addition to the large data set of annotated transcripts, the study also provides gene-based simple sequence repeat and single nucleotide polymorphism markers.

Thioesterase overexpression in Nicotiana benthamiana leaf increases the fatty acid flux into triacylgycerol

Increasing the oil content of leafy biomass is emerging as a sustainable source of vegetable oil to meet global demand. Transient gene expression in leaf provides a reproducible platform to study the effect of transgenes on lipid biosynthesis. We first generated a transgenic Nicotiana benthamiana line containing high levels of triacylglycerol in the leaf tissue (31.4% by dry weight) by stably expressing WRI1, DGAT1 and OLEOSIN. We then used this line as a platform to test the effect of three Arabidopsis thaliana thioesterases (FATA1, FATA2 and FATB). Further increases in leaf oil content were observed with biochemical and lipid assays revealing an increase in the export of fatty acids from the chloroplast and a modification in the oil profile.