37 results on '"Vanhercke T"'
Search Results
2. A transcriptional journey from sucrose to endosperm oil bodies in triple transgene oily wheat grain
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Larkin, P.J., primary, Zhou, X.R., additional, Liu, Q., additional, Reynolds, K., additional, Vanhercke, T., additional, Ral, J.P., additional, Li, Z., additional, Wu, X.B., additional, Yu, R., additional, Luo, J.X., additional, Newberry, M., additional, and Howitt, C.H., additional more...
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- 2021
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3. Deep sequencing of the fruit transcriptome and lipid accumulation in a non-seed tissue of Chinese tallow, a potential biofuel crop
- Author
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Divi, U.K., Zhou, X-R, Wang, P., Butlin, J., Zhang, D-M, Liu, Q., Vanhercke, T., Petrie, J.R., Talbot, M., White, R.G., Taylor, J.M., Larkin, P., Singh, S.P., Divi, U.K., Zhou, X-R, Wang, P., Butlin, J., Zhang, D-M, Liu, Q., Vanhercke, T., Petrie, J.R., Talbot, M., White, R.G., Taylor, J.M., Larkin, P., and Singh, S.P. more...
- Abstract
Chinese tallow (Triadica sebifera) is a valuable oilseedproducing 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. more...
- Published
- 2015
4. Metabolic engineering of biomass for high energy density: Oilseed-like triacylglycerol yields from plant leaves
- Author
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Vanhercke, T., El Tahchy, A., Liu, Q., Zhou, XueRong, Shrestha, Pushkar, Divi, U. K., Ral, Jean-Philippe, Mansour, M. P., Nichols, Peter D., James, C. N., Horn, P. J., Chapman, K. D., Beaudoin, Frédéric, Ruiz-López, Noemí, Larkin, P. J., Feyter, R. C. de, Singh, Surinder P., Petrie, James R., Vanhercke, T., El Tahchy, A., Liu, Q., Zhou, XueRong, Shrestha, Pushkar, Divi, U. K., Ral, Jean-Philippe, Mansour, M. P., Nichols, Peter D., James, C. N., Horn, P. J., Chapman, K. D., Beaudoin, Frédéric, Ruiz-López, Noemí, Larkin, P. J., Feyter, R. C. de, Singh, Surinder P., and Petrie, James R. more...
- 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. © 2013 CSIRO. more...
- Published
- 2014
5. Transgenic wheat with increased endosperm lipid – Impacts on grain composition and baking quality
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<p>Commonwealth Scientific and Industrial Research Organisation</p>, Larkin, P. J., Liu, Q., Vanhercke, T., Zhou, X. R., Bose, U., Broadbent, J. A., Colgrave, Michelle L., Ral, J. P., Reynolds, K. B., Sun, M., El Tahchy, A., Shrestha, P., Li, Z. Y., Jobling, S. A., Lonergan, P., Wu, X. B., Yu, R., Luo, J. X., Howitt, C. A., Newberry, M., <p>Commonwealth Scientific and Industrial Research Organisation</p>, Larkin, P. J., Liu, Q., Vanhercke, T., Zhou, X. R., Bose, U., Broadbent, J. A., Colgrave, Michelle L., Ral, J. P., Reynolds, K. B., Sun, M., El Tahchy, A., Shrestha, P., Li, Z. Y., Jobling, S. A., Lonergan, P., Wu, X. B., Yu, R., Luo, J. X., Howitt, C. A., and Newberry, M. more...
- Abstract
Larkin, P. J., Liu, Q., Vanhercke, T., Zhou, X. R., Bose, U., Broadbent, J. A., ... Newberry, M. (2021). Transgenic wheat with increased endosperm lipid – Impacts on grain composition and baking quality. Journal of Cereal Science, 101, article 103289. https://doi.org/10.1016/j.jcs.2021.103289 more...
6. Pooled effector library screening in protoplasts rapidly identifies novel Avr genes.
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Arndell T, Chen J, Sperschneider J, Upadhyaya NM, Blundell C, Niesner N, Outram MA, Wang A, Swain S, Luo M, Ayliffe MA, Figueroa M, Vanhercke T, and Dodds PN
- Abstract
Crop breeding for durable disease resistance is challenging due to the rapid evolution of pathogen virulence. While progress in resistance (R) gene cloning and stacking has accelerated in recent years
1-3 , the identification of corresponding avirulence (Avr) genes in many pathogens is hampered by the lack of high-throughput screening options. To address this technology gap, we developed a platform for pooled library screening in plant protoplasts to allow rapid identification of interacting R-Avr pairs. We validated this platform by isolating known and novel Avr genes from wheat stem rust (Puccinia graminis f. sp. tritici) after screening a designed library of putative effectors against individual R genes. Rapid Avr gene identification provides molecular tools to understand and track pathogen virulence evolution via genotype surveillance, which in turn will lead to optimized R gene stacking and deployment strategies. This platform should be broadly applicable to many crop pathogens and could potentially be adapted for screening genes involved in other protoplast-selectable traits., (© 2024. The Author(s).) more...- Published
- 2024
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7. Perspectives on Future Protein Production.
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Colgrave ML, Dominik S, Tobin AB, Stockmann R, Simon C, Howitt CA, Belobrajdic DP, Paull C, and Vanhercke T
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- Amino Acids, Animals, Aquaculture, Diet, Diet, Healthy, Meat, Microalgae
- Abstract
An increasing world population, rising affluence, urbanization, and changing eating habits are all contributing to the diversification of protein production. Protein is a building block of life and is an essential part of a healthy diet, providing amino acids for growth and repair. The challenges and opportunities for production of protein-rich foods from animals (meat, dairy, and aquaculture), plant-based sources (pulses), and emerging protein sources (insects, yeast, and microalgae) are discussed against the backdrop of palatability, nutrition, and sustainability. more...
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- 2021
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8. Sesamum indicum Oleosin L improves oil packaging in Nicotiana benthamiana leaves.
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Yee S, Rolland V, Reynolds KB, Shrestha P, Ma L, Singh SP, Vanhercke T, Petrie JR, and El Tahchy A
- Abstract
Plant oil production has been increasing continuously in the past decade. There has been significant investment in the production of high biomass plants with elevated oil content. We recently showed that the expression of Arabidopsis thaliana WRI1 and DGAT1 genes increase oil content by up to 15% in leaf dry weight tissue. However, triacylglycerols in leaf tissue are subject to degradation during senescence. In order to better package the oil, we expressed a series of lipid droplet proteins isolated from bacterial and plant sources in Nicotiana benthamiana leaf tissue. We observed further increases in leaf oil content of up to 2.3-fold when we co-expressed Sesamum indicum Oleosin L with At WRI1 and At DGAT1. Biochemical assays and lipid droplet visualization with confocal microscopy confirmed the increase in oil content and revealed a significant change in the size and abundance of lipid droplets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2021 The Commonwealth Scientific and Industrial Research Organisation. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.) more...
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- 2021
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9. Corrigendum: Editorial: Proceedings of ASPL2019 - 8th Asian-Oceanian Symposium on Plant Lipids.
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Zhou XR, Nishida I, Suh MC, and Vanhercke T
- Abstract
[This corrects the article DOI: 10.3389/fpls.2020.617094.]., (Copyright © 2021 Zhou, Nishida, Suh and Vanhercke.)
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- 2021
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10. Editorial: Proceedings of ASPL2019 - 8th Asian-Oceanian Symposium on Plant Lipids.
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Zhou XR, Nishida I, Suh MC, and Vanhercke T
- Abstract
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
- Published
- 2020
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11. Increasing growth and yield by altering carbon metabolism in a transgenic leaf oil crop.
- Author
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Mitchell MC, Pritchard J, Okada S, Zhang J, Venables I, Vanhercke T, and Ral JP
- Abstract
Engineering high biomass plants that produce oil (triacylglycerol or TAG) in vegetative rather than seed-related tissues could help meet our growing demand for plant oil. Several studies have already demonstrated the potential of this approach by creating transgenic crop and model plants that accumulate TAG in their leaves and stems. However, TAG synthesis may compete with other important carbon and energy reserves, including carbohydrate production, and thereby limit plant growth. The aims of this study were thus: first, to investigate the effect of TAG accumulation on growth and development of previously generated high leaf oil tobacco plants; and second, to increase plant growth and/or oil yields by further altering carbon fixation and partitioning. This study showed that TAG accumulation varied with leaf and plant developmental stage, affected leaf carbon and nitrogen partitioning and reduced the relative growth rate and final biomass of high leaf oil plants. To overcome these growth limitations, four genes related to carbon fixation (encoding CBB cycle enzymes SBPase and chloroplast-targeted FBPase) or carbon partitioning (encoding sucrose biosynthetic enzyme cytosolic FBPase and lipid-related transcription factor DOF4) were overexpressed in high leaf oil plants. In glasshouse conditions, all four constructs increased early growth without affecting TAG accumulation while chloroplast-targeted FBPase and DOF4 also increased final biomass and oil yields. These results highlight the reliance of plant growth on carbon partitioning, in addition to carbon supply, and will guide future attempts to improve biomass and TAG accumulation in transgenic leaf oil crops., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.) more...
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- 2020
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12. Consensus Mutagenesis and Ancestral Reconstruction Provide Insight into the Substrate Specificity and Evolution of the Front-End Δ6-Desaturase Family.
- Author
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Li D, Damry AM, Petrie JR, Vanhercke T, Singh SP, and Jackson CJ
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- Chlorophyta chemistry, Chlorophyta classification, Chlorophyta genetics, Fatty Acids, Omega-3 chemistry, Fatty Acids, Omega-3 metabolism, Fatty Acids, Omega-6 chemistry, Fatty Acids, Omega-6 metabolism, Linoleoyl-CoA Desaturase metabolism, Multigene Family, Mutagenesis, Phylogeny, Protein Conformation, Substrate Specificity, Chlorophyta enzymology, Linoleoyl-CoA Desaturase chemistry, Linoleoyl-CoA Desaturase genetics
- Abstract
Marine algae are a major source of ω-3 long-chain polyunsaturated fatty acids (ω3-LCPUFAs), which are conditionally essential nutrients in humans and a target for industrial production. The biosynthesis of these molecules in marine algae requires the desaturation of fatty acids by Δ6-desaturases, and enzymes from different species display a range of specificities toward ω3- and ω6-LCPUFA precursors. In the absence of a molecular structure, the structural basis for the variable substrate specificity of Δ6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the Δ6-desaturase family, focusing on the ω3-specific Δ6-desaturase from Micromonas pusilla (MpΔ6des) and the bispecific (ω3/ω6) Δ6-desaturase from Ostreococcus tauri (OtΔ6des). Our characterization of consensus amino acid substitutions in MpΔ6des revealed that residues in diverse regions of the protein, such as the N-terminal cytochrome b
5 domain, can make important contributions to determining substrate specificity. Ancestral protein reconstruction also suggests that some extant Δ6-desaturases, such as OtΔ6des, could have adapted to different environmental conditions by losing specificity for ω3-LCPUFAs. This data set provides a map of regions within Δ6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology. more...- Published
- 2020
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13. A Versatile High Throughput Screening Platform for Plant Metabolic Engineering Highlights the Major Role of ABI3 in Lipid Metabolism Regulation.
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Pouvreau B, Blundell C, Vohra H, Zwart AB, Arndell T, Singh S, and Vanhercke T
- Abstract
Traditional functional genetic studies in crops are time consuming, complicated and cannot be readily scaled up. The reason is that mutant or transformed crops need to be generated to study the effect of gene modifications on specific traits of interest. However, many crop species have a complex genome and a long generation time. As a result, it usually takes several months to over a year to obtain desired mutants or transgenic plants, which represents a significant bottleneck in the development of new crop varieties. To overcome this major issue, we are currently establishing a versatile plant genetic screening platform, amenable to high throughput screening in almost any crop species, with a unique workflow. This platform combines protoplast transformation and fluorescence activated cell sorting. Here we show that tobacco protoplasts can accumulate high levels of lipid if transiently transformed with genes involved in lipid biosynthesis and can be sorted based on lipid content. Hence, protoplasts can be used as a predictive tool for plant lipid engineering. Using this newly established strategy, we demonstrate the major role of ABI3 in plant lipid accumulation. We anticipate that this workflow can be applied to numerous highly valuable metabolic traits other than storage lipid accumulation. This new strategy represents a significant step toward screening complex genetic libraries, in a single experiment and in a matter of days, as opposed to years by conventional means., (Copyright © 2020 Pouvreau, Blundell, Vohra, Zwart, Arndell, Singh and Vanhercke.) more...
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- 2020
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14. A Synergistic Genetic Engineering Strategy Induced Triacylglycerol Accumulation in Potato ( Solanum tuberosum ) Leaf.
- Author
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Xu XY, Akbar S, Shrestha P, Venugoban L, Devilla R, Hussain D, Lee J, Rug M, Tian L, Vanhercke T, Singh SP, Li Z, Sharp PJ, and Liu Q
- Abstract
Potato is the 4th largest staple food in the world currently. As a high biomass crop, potato harbors excellent potential to produce energy-rich compounds such as triacylglycerol as a valuable co-product. We have previously reported that transgenic potato tubers overexpressing WRINKLED1 , DIACYLGLYCEROL ACYLTRANSFERASE 1 , and OLEOSIN genes produced considerable levels of triacylglycerol. In this study, the same genetic engineering strategy was employed on potato leaves. The overexpression of Arabidopsis thaliana WRINKED1 under the transcriptional control of a senescence-inducible promoter together with Arabidopsis thaliana DIACYLGLYCEROL ACYLTRANSFERASE 1 and Sesamum indicum OLEOSIN driven by the Cauliflower Mosaic Virus 35S promoter and small subunit of Rubisco promoter respectively, resulted in an approximately 30- fold enhancement of triacylglycerols in the senescent transgenic potato leaves compared to the wild type. The increase of triacylglycerol in the transgenic potato leaves was accompanied by perturbations of carbohydrate accumulation, apparent in a reduction in starch content and increased total soluble sugars, as well as changes of polar membrane lipids at different developmental stages. Microscopic and biochemical analysis further indicated that triacylglycerols and lipid droplets could not be produced in chloroplasts, despite the increase and enlargement of plastoglobuli at the senescent stage. Possibly enhanced accumulation of fatty acid phytyl esters in the plastoglobuli were reflected in transgenic potato leaves relative to wild type. It is likely that the plastoglobuli may have hijacked some of the carbon as the result of WRINKED1 expression, which could be a potential factor restricting the effective accumulation of triacylglycerols in potato leaves. Increased lipid production was also observed in potato tubers, which may have affected the tuberization to a certain extent. The expression of transgenes in potato leaf not only altered the carbon partitioning in the photosynthetic source tissue, but also the underground sink organs which highly relies on the leaves in development and energy deposition., (Copyright © 2020 Xu, Akbar, Shrestha, Venugoban, Devilla, Hussain, Lee, Rug, Tian, Vanhercke, Singh, Li, Sharp and Liu.) more...
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- 2020
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15. Reorganization of Acyl Flux through the Lipid Metabolic Network in Oil-Accumulating Tobacco Leaves.
- Author
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Zhou XR, Bhandari S, Johnson BS, Kotapati HK, Allen DK, Vanhercke T, and Bates PD
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- Acyltransferases genetics, Acyltransferases metabolism, Fatty Acids metabolism, Gene Expression Regulation, Plant, Lipid Metabolism, Metabolic Networks and Pathways, Microsomes metabolism, Nicotiana genetics, Triglycerides biosynthesis, Membrane Lipids metabolism, Metabolic Engineering methods, Plant Leaves metabolism, Plant Oils metabolism, Plants, Genetically Modified metabolism, Nicotiana metabolism, Triglycerides metabolism
- Abstract
The triacylglycerols (TAGs; i.e. oils) that accumulate in plants represent the most energy-dense form of biological carbon storage, and are used for food, fuels, and chemicals. The increasing human population and decreasing amount of arable land have amplified the need to produce plant oil more efficiently. Engineering plants to accumulate oils in vegetative tissues is a novel strategy, because most plants only accumulate large amounts of lipids in the seeds. Recently, tobacco ( Nicotiana tabacum ) leaves were engineered to accumulate oil at 15% of dry weight due to a push (increased fatty acid synthesis)-and-pull (increased final step of TAG biosynthesis) engineering strategy. However, to accumulate both TAG and essential membrane lipids, fatty acid flux through nonengineered reactions of the endogenous metabolic network must also adapt, which is not evident from total oil analysis. To increase our understanding of endogenous leaf lipid metabolism and its ability to adapt to metabolic engineering, we utilized a series of in vitro and in vivo experiments to characterize the path of acyl flux in wild-type and transgenic oil-accumulating tobacco leaves. Acyl flux around the phosphatidylcholine acyl editing cycle was the largest acyl flux reaction in wild-type and engineered tobacco leaves. In oil-accumulating leaves, acyl flux into the eukaryotic pathway of glycerolipid assembly was enhanced at the expense of the prokaryotic pathway. However, a direct Kennedy pathway of TAG biosynthesis was not detected, as acyl flux through phosphatidylcholine preceded the incorporation into TAG. These results provide insight into the plasticity and control of acyl lipid metabolism in leaves., (© 2020 American Society of Plant Biologists. All Rights Reserved.) more...
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- 2020
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16. Upregulated Lipid Biosynthesis at the Expense of Starch Production in Potato ( Solanum tuberosum ) Vegetative Tissues via Simultaneous Downregulation of ADP-Glucose Pyrophosphorylase and Sugar Dependent1 Expressions.
- Author
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Xu X, Vanhercke T, Shrestha P, Luo J, Akbar S, Konik-Rose C, Venugoban L, Hussain D, Tian L, Singh S, Li Z, Sharp PJ, and Liu Q
- Abstract
Triacylglycerol is a major component of vegetable oil in seeds and fruits of many plants, but its production in vegetative tissues is rather limited. It would be intriguing and important to explore any possibility to expand current oil production platforms, for example from the plant vegetative tissues. By expressing a suite of transgenes involved in the triacylglycerol biosynthesis, we have previously observed substantial accumulation of triacylglycerol in tobacco ( Nicotiana tabacum ) leaf and potato ( Solanum tuberosum ) tuber. In this study, simultaneous RNA interference (RNAi) downregulation of ADP-glucose pyrophosphorylase (AGPase) and Sugar-dependent1 (SDP1), was able to increase the accumulation of triacylglycerol and other lipids in both wild type potato and the previously generated high oil potato line 69. Particularly, a 16-fold enhancement of triacylglycerol production was observed in the mature transgenic tubers derived from the wild type potato, and a two-fold increase in triacylglycerol was observed in the high oil potato line 69, accounting for about 7% of tuber dry weight, which is the highest triacylglycerol accumulation ever reported in potato. In addition to the alterations of lipid content and fatty acid composition, sugar accumulation, starch content of the RNAi potato lines in both tuber and leaf tissues were also substantially changed, as well as the tuber starch properties. Microscopic analysis further revealed variation of lipid droplet distribution and starch granule morphology in the mature transgenic tubers compared to their parent lines. This study reflects that the carbon partitioning between lipid and starch in both leaves and non-photosynthetic tuber tissues, respectively, are highly orchestrated in potato, and it is promising to convert low-energy starch to storage lipids via genetic manipulation of the carbon metabolism pathways., (Copyright © 2019 Xu, Vanhercke, Shrestha, Luo, Akbar, Konik-Rose, Venugoban, Hussain, Tian, Singh, Li, Sharp and Liu.) more...
- Published
- 2019
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17. Metabolic engineering for enhanced oil in biomass.
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Vanhercke T, Dyer JM, Mullen RT, Kilaru A, Rahman MM, Petrie JR, Green AG, Yurchenko O, and Singh SP
- Subjects
- Triglycerides metabolism, Biomass, Metabolic Engineering, Plant Oils metabolism
- Abstract
The world is hungry for energy. Plant oils in the form of triacylglycerol (TAG) are one of the most reduced storage forms of carbon found in nature and hence represent an excellent source of energy. The myriad of applications for plant oils range across foods, feeds, biofuels, and chemical feedstocks as a unique substitute for petroleum derivatives. Traditionally, plant oils are sourced either from oilseeds or tissues surrounding the seed (mesocarp). Most vegetative tissues, such as leaves and stems, however, accumulate relatively low levels of TAG. Since non-seed tissues constitute the majority of the plant biomass, metabolic engineering to improve their low-intrinsic TAG-biosynthetic capacity has recently attracted significant attention as a novel, sustainable and potentially high-yielding oil production platform. While initial attempts predominantly targeted single genes, recent combinatorial metabolic engineering strategies have focused on the simultaneous optimization of oil synthesis, packaging and degradation pathways (i.e., 'push, pull, package and protect'). This holistic approach has resulted in dramatic, seed-like TAG levels in vegetative tissues. With the first proof of concept hurdle addressed, new challenges and opportunities emerge, including engineering fatty acid profile, translation into agronomic crops, extraction, and downstream processing to deliver accessible and sustainable bioenergy., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.) more...
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- 2019
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18. Up-regulation of lipid biosynthesis increases the oil content in leaves of Sorghum bicolor.
- Author
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Vanhercke T, Belide S, Taylor MC, El Tahchy A, Okada S, Rolland V, Liu Q, Mitchell M, Shrestha P, Venables I, Ma L, Blundell C, Mathew A, Ziolkowski L, Niesner N, Hussain D, Dong B, Liu G, Godwin ID, Lee J, Rug M, Zhou XR, Singh SP, and Petrie JR more...
- Subjects
- Amino Acids analysis, Amino Acids metabolism, Lipid Metabolism, Lipids analysis, Plant Leaves chemistry, Plant Oils metabolism, Plants, Genetically Modified metabolism, Sorghum chemistry, Starch analysis, Starch metabolism, Triglycerides metabolism, Up-Regulation, Lipids biosynthesis, Plant Leaves metabolism, Plant Oils analysis, Sorghum metabolism
- Abstract
Synthesis and accumulation of the storage lipid triacylglycerol in vegetative plant tissues has emerged as a promising strategy to meet the world's future need for vegetable oil. Sorghum (Sorghum bicolor) is a particularly attractive target crop given its high biomass, drought resistance and C
4 photosynthesis. While oilseed-like triacylglycerol levels have been engineered in the C3 model plant tobacco, progress in C4 monocot crops has been lagging behind. In this study, we report the accumulation of triacylglycerol in sorghum leaf tissues to levels between 3 and 8.4% on a dry weight basis depending on leaf and plant developmental stage. This was achieved by the combined overexpression of genes encoding the Zea mays WRI1 transcription factor, Umbelopsis ramanniana UrDGAT2a acyltransferase and Sesamum indicum Oleosin-L oil body protein. Increased oil content was visible as lipid droplets, primarily in the leaf mesophyll cells. A comparison between a constitutive and mesophyll-specific promoter driving WRI1 expression revealed distinct changes in the overall leaf lipidome as well as transitory starch and soluble sugar levels. Metabolome profiling uncovered changes in the abundance of various amino acids and dicarboxylic acids. The results presented here are a first step forward towards the development of sorghum as a dedicated biomass oil crop and provide a basis for further combinatorial metabolic engineering., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.) more...- Published
- 2019
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19. From plant metabolic engineering to plant synthetic biology: The evolution of the design/build/test/learn cycle.
- Author
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Pouvreau B, Vanhercke T, and Singh S
- Subjects
- Agriculture, Crops, Agricultural metabolism, Plants metabolism, Crops, Agricultural genetics, Genetic Engineering, Metabolic Engineering, Metabolic Networks and Pathways genetics, Plants genetics, Synthetic Biology
- Abstract
Genetic improvement of crops started since the dawn of agriculture and has continuously evolved in parallel with emerging technological innovations. The use of genome engineering in crop improvement has already revolutionised modern agriculture in less than thirty years. Plant metabolic engineering is still at a development stage and faces several challenges, in particular with the time necessary to develop plant based solutions to bio-industrial demands. However the recent success of several metabolic engineering approaches applied to major crops are encouraging and the emerging field of plant synthetic biology offers new opportunities. Some pioneering studies have demonstrated that synthetic genetic circuits or orthogonal metabolic pathways can be introduced into plants to achieve a desired function. The combination of metabolic engineering and synthetic biology is expected to significantly accelerate crop improvement. A defining aspect of both fields is the design/build/test/learn cycle, or the use of iterative rounds of testing modifications to refine hypotheses and develop best solutions. Several technological and technical improvements are now available to make a better use of each design, build, test, and learn components of the cycle. All these advances should facilitate the rapid development of a wide variety of bio-products for a world in need of sustainable solutions., (Copyright © 2018 Elsevier B.V. All rights reserved.) more...
- Published
- 2018
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20. Robust genetic transformation of sorghum ( Sorghum bicolor L.) using differentiating embryogenic callus induced from immature embryos.
- Author
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Belide S, Vanhercke T, Petrie JR, and Singh SP
- Abstract
Background: Sorghum ( Sorghum bicolor L.) is one of the world's most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Despite several decades of study, sorghum has been widely considered as a recalcitrant major crop for transformation due to accumulation of phenolic compounds, lack of model genotypes, low regeneration frequency and loss of regeneration potential through sub-cultures. Among different explants used for genetic transformation of sorghum, immature embryos are ideal over other explants. However, the continuous supply of quality immature embryos for transformation is labour intensive and expensive. In addition, transformation efficiencies are also influenced by environmental conditions (light and temperature). Despite these challenges, immature embryos remain the predominant choice because of their success rate and also due to non-availability of other dependable explants without compromising the transformation efficiency., Results: We report here a robust genetic transformation method for sorghum (Tx430) using differentiating embryogenic calli (DEC) with nodular structures induced from immature embryos and maintained for more than a year without losing regeneration potential on modified MS media. The addition of lipoic acid (LA) to callus induction media along with optimized growth regulators increased callus induction frequency from 61.3 ± 3.2 to 79 ± 6.5% from immature embryos (1.5-2.0 mm in length) isolated 12-15 days after pollination. Similarly, the regeneration efficiency and the number of shoots from DEC tissue was enhanced by LA. The optimized regeneration system in combination with particle bombardment resulted in an average transformation efficiency (TE) of 27.2 or 46.6% based on the selection strategy, 25% to twofold higher TE than published reports in Tx430. Up to 100% putative transgenic shoots were positive for npt - II by PCR and 48% of events had < 3 copies of transgenes as determined by digital droplet PCR. Reproducibility of this method was demonstrated by generating ~ 800 transgenic plants using 10 different gene constructs., Conclusions: This protocol demonstrates significant improvements in both efficiency and ease of use over existing sorghum transformation methods using PDS, also enables quick hypothesis testing in the production of various high value products in sorghum. more...
- Published
- 2017
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21. High-performance variants of plant diacylglycerol acyltransferase 1 generated by directed evolution provide insights into structure function.
- Author
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Chen G, Xu Y, Siloto RMP, Caldo KMP, Vanhercke T, Tahchy AE, Niesner N, Chen Y, Mietkiewska E, and Weselake RJ
- Subjects
- Amino Acid Sequence, Amino Acid Substitution genetics, Brassica napus enzymology, Catalytic Domain genetics, Catalytic Domain physiology, Diacylglycerol O-Acyltransferase metabolism, Diacylglycerol O-Acyltransferase physiology, Directed Molecular Evolution methods, Plant Leaves metabolism, Protein Conformation, Nicotiana enzymology, Triglycerides biosynthesis, Diacylglycerol O-Acyltransferase genetics
- Abstract
Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the acyl-CoA-dependent biosynthesis of triacylglycerol, the predominant component of seed oil. In some oil crops, including Brassica napus, the level of DGAT1 activity can have a substantial effect on triacylglycerol production. Structure-function insights into DGAT1, however, remain limited because of the lack of a three-dimensional detailed structure for this membrane-bound enzyme. In this study, the amino acid residues governing B. napus DGAT1 (BnaDGAT1) activity were investigated via directed evolution, targeted mutagenesis, in vitro enzymatic assay, topological analysis, and transient expression of cDNA encoding selected enzyme variants in Nicotiana benthamiana. Directed evolution revealed that numerous amino acid residues were associated with increased BnaDGAT1 activity, and 67% of these residues were conserved among plant DGAT1s. The identified amino acid residue substitution sites occur throughout the BnaDGAT1 polypeptide, with 89% of the substitutions located outside the putative substrate binding or active sites. In addition, cDNAs encoding variants I447F or L441P were transiently overexpressed in N. benthamiana leaves, resulting in 33.2 or 70.5% higher triacylglycerol content, respectively, compared with native BnaDGAT1. Overall, the results provide novel insights into amino acid residues underlying plant DGAT1 function and performance-enhanced BnaDGAT1 variants for increasing vegetable oil production., (© 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.) more...
- Published
- 2017
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22. Comparative Lipidomics and Proteomics of Lipid Droplets in the Mesocarp and Seed Tissues of Chinese Tallow ( Triadica sebifera ).
- Author
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Zhi Y, Taylor MC, Campbell PM, Warden AC, Shrestha P, El Tahchy A, Rolland V, Vanhercke T, Petrie JR, White RG, Chen W, Singh SP, and Liu Q
- Abstract
Lipid droplets (LDs) are composed of a monolayer of phospholipids (PLs), surrounding a core of non-polar lipids that consist mostly of triacylglycerols (TAGs) and to a lesser extent diacylglycerols. In this study, lipidome analysis illustrated striking differences in non-polar lipids and PL species between LDs derived from Triadica sebifera seed kernels and mesocarp. In mesocarp LDs, the most abundant species of TAG contained one C18:1 and two C16:0 and fatty acids, while TAGs containing three C18 fatty acids with higher level of unsaturation were dominant in the seed kernel LDs. This reflects the distinct differences in fatty acid composition of mesocarp (palmitate-rich) and seed-derived oil (α-linoleneate-rich) in T. sebifera . Major PLs in seed LDs were found to be rich in polyunsaturated fatty acids, in contrast to those with relatively shorter carbon chain and lower level of unsaturation in mesocarp LDs. The LD proteome analysis in T. sebifera identified 207 proteins from mesocarp, and 54 proteins from seed kernel, which belong to various functional classes including lipid metabolism, transcription and translation, trafficking and transport, cytoskeleton, chaperones, and signal transduction. Oleosin and lipid droplets associated proteins (LDAP) were found to be the predominant proteins associated with LDs in seed and mesocarp tissues, respectively. We also show that LDs appear to be in close proximity to a number of organelles including the endoplasmic reticulum, mitochondria, peroxisomes, and Golgi apparatus. This comparative study between seed and mesocarp LDs may shed some light on the structure of plant LDs and improve our understanding of their functionality and cellular metabolic networks in oleaginous plant tissues. more...
- Published
- 2017
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23. Step changes in leaf oil accumulation via iterative metabolic engineering.
- Author
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Vanhercke T, Divi UK, El Tahchy A, Liu Q, Mitchell M, Taylor MC, Eastmond PJ, Bryant F, Mechanicos A, Blundell C, Zhi Y, Belide S, Shrestha P, Zhou XR, Ral JP, White RG, Green A, Singh SP, and Petrie JR more...
- Subjects
- Arabidopsis physiology, Arabidopsis Proteins genetics, Plant Oils isolation & purification, Transcription Factors genetics, Genetic Enhancement methods, Metabolic Engineering methods, Metabolic Networks and Pathways physiology, Plant Leaves physiology, Plant Oils metabolism, Nicotiana physiology
- 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., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.) more...
- Published
- 2017
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24. Thioesterase overexpression in Nicotiana benthamiana leaf increases the fatty acid flux into triacylgycerol.
- Author
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El Tahchy A, Reynolds KB, Petrie JR, Singh SP, and Vanhercke T
- Subjects
- Acetates metabolism, Arabidopsis Proteins metabolism, Gene Expression, Plants, Genetically Modified, Nicotiana genetics, Fatty Acids metabolism, Plant Leaves metabolism, Plant Proteins metabolism, Nicotiana metabolism, Triglycerides metabolism
- Abstract
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., (© 2016 Federation of European Biochemical Societies.) more...
- Published
- 2017
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25. Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy.
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Liu Q, Guo Q, Akbar S, Zhi Y, El Tahchy A, Mitchell M, Li Z, Shrestha P, Vanhercke T, Ral JP, Liang G, Wang MB, White R, Larkin P, Singh S, and Petrie J
- Subjects
- Carbohydrates analysis, Fatty Acids analysis, Fatty Acids chemistry, Fatty Acids metabolism, Galactolipids metabolism, Genes, Plant, Microscopy, Electron, Scanning, Microscopy, Electron, Transmission, Microscopy, Fluorescence, Phospholipids metabolism, Plant Oils analysis, Plant Oils chemistry, Plant Proteins genetics, Plant Proteins metabolism, Plant Tubers cytology, Plants, Genetically Modified, Solanum tuberosum cytology, Starch analysis, Starch metabolism, Transformation, Genetic, Triglycerides metabolism, Gene Expression Regulation, Plant genetics, Genetic Enhancement methods, Metabolic Engineering methods, Plant Oils metabolism, Plant Tubers genetics, Plant Tubers metabolism, Solanum tuberosum genetics
- Abstract
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., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.) more...
- Published
- 2017
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26. Classification and substrate head-group specificity of membrane fatty acid desaturases.
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Li D, Moorman R, Vanhercke T, Petrie J, Singh S, and Jackson CJ
- Abstract
Membrane fatty acid desaturases are a diverse superfamily of enzymes that catalyze the introduction of double bonds into fatty acids. They are essential in a range of metabolic processes, such as the production of omega-3 fatty acids. However, our structure-function understanding of this superfamily is still developing and their range of activities and substrate specificities are broad, and often overlapping, which has made their systematic characterization challenging. A central issue with characterizing these proteins has been the lack of a structural model, which has been overcome with the recent publication of the crystal structures of two mammalian fatty acid desaturases. In this work, we have used sequence similarity networks to investigate the similarity among over 5000 related membrane fatty acid desaturase sequences, leading to a detailed classification of the superfamily, families and subfamilies with regard to their function and substrate head-group specificity. This work will facilitate rapid prediction of the function and specificity of new and existing sequences, as well as forming a basis for future efforts to manipulate the substrate specificity of these proteins for biotechnology applications. more...
- Published
- 2016
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27. Deep Sequencing of the Fruit Transcriptome and Lipid Accumulation in a Non-Seed Tissue of Chinese Tallow, a Potential Biofuel Crop.
- Author
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Divi UK, Zhou XR, Wang P, Butlin J, Zhang DM, Liu Q, Vanhercke T, Petrie JR, Talbot M, White RG, Taylor JM, Larkin P, and Singh SP
- Subjects
- Biofuels, Euphorbiaceae metabolism, Euphorbiaceae ultrastructure, Fatty Acids analysis, Fruit genetics, Fruit metabolism, Fruit ultrastructure, Gene Expression Regulation, Plant, High-Throughput Nucleotide Sequencing, Lipid Metabolism, Lipids analysis, Molecular Sequence Annotation, Organ Specificity, Plant Oils analysis, Plant Proteins genetics, Seeds genetics, Seeds metabolism, Seeds ultrastructure, Sequence Analysis, DNA, Euphorbiaceae genetics, Fatty Acids metabolism, Plant Oils metabolism, Transcriptome
- Abstract
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., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.) more...
- Published
- 2016
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28. Expression of Mouse MGAT in Arabidopsis Results in Increased Lipid Accumulation in Seeds.
- Author
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El Tahchy A, Petrie JR, Shrestha P, Vanhercke T, and Singh SP
- Abstract
Worldwide demand for vegetable oil is projected to double within the next 30 years due to increasing food, fuel, and industrial requirements. There is therefore great interest in metabolic engineering strategies that boost oil accumulation in plant tissues, however, efforts to date have only achieved levels of storage lipid accumulation in plant tissues far below the benchmark to meet demand. Monoacylglycerol acyltransferase (MGAT) is predominantly associated with lipid absorption and resynthesis in the animal intestine where it catalyzes monoacylglycerol (MAG) to form diacylglycerol (DAG), and then triacylglycerol (TAG). In contrast plant lipid biosynthesis routes do not include MGAT. Rather, DAG and TAG are either synthesized from glycerol-3-phosphate by a series of three subsequent acylation reactions, or originated from phospholipids via an acyl editing pathway. Mouse MGATs 1 and 2 have been shown to increase oil content transiently in Nicotiana benthamiana leaf tissue by 2.6 fold. Here we explore the feasibility of this approach to increase TAG in Arabidopsis thaliana seed. The stable MGAT2 expression resulted in a significant increase in seed oil content by 1.32 fold. We also report evidence of the MGAT2 activity based on in vitro assays. Up to 3.9 fold increase of radiolabeled DAG were produced in seed lysate which suggest that the transgenic MGAT activity can result in DAG re-synthesis by salvaging the MAG product of lipid breakdown. The expression of MGAT2 therefore creates an independent and complementary TAG biosynthesis route to the endogenous Kennedy pathway and other glycerolipid synthesis routes. more...
- Published
- 2015
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29. Metabolic engineering of medium-chain fatty acid biosynthesis in Nicotiana benthamiana plant leaf lipids.
- Author
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Reynolds KB, Taylor MC, Zhou XR, Vanhercke T, Wood CC, Blanchard CL, Singh SP, and Petrie JR
- Abstract
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. more...
- Published
- 2015
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30. Transcriptional and biochemical responses of monoacylglycerol acyltransferase-mediated oil synthesis and associated senescence-like responses in Nicotiana benthamiana.
- Author
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Divi UK, El Tahchy A, Vanhercke T, Petrie JR, Robles-Martinez JA, and Singh SP
- Abstract
Triacylglycerol (TAG) accumulates in plant seeds as a major renewable source of carbon for food, fuel and industrial feedstock. Approaches to enhance TAG content by altering lipid pathways and genes in vegetative parts have gained significant attention for biofuel and other applications. However, consequences of these modifications are not always studied in detail. In an attempt to increase TAG levels in leaves we previously demonstrated that a novel substrate, monoacylglycerol (MAG), can be used for the biosynthesis of diacylglycerol (DAG) and TAG. Transient expression of the Mus musculus monoacylglycerol acyltransferases MGAT1 and 2 in the model plant Nicotiana benthamiana increased TAG levels at 5 days post-infiltration (dpi). Here we show that increased TAG and DAG levels can be achieved as early as 2 dpi. In addition, the MGAT1 infiltrated areas showed senescence-like symptoms from 3 dpi onwards. To unravel underlying molecular mechanisms, Illumina deep sequencing was carried out (a) for de-novo assembling and annotation of N. benthamiana leaf transcripts and (b) to characterize MGAT1-responsive transcriptome. We found that MGAT1-responsive genes are involved in several processes including TAG biosynthesis, photosynthesis, cell-wall, cutin, suberin, wax and mucilage biosynthesis, lipid and hormone metabolism. Comparative analysis with transcript profiles from other senescence studies identified characteristic gene expression changes involved in senescence induction. We confirmed that increased TAG and observed senescence-symptoms are due to the MAG depletion caused by MGAT1 activity and suggest a mechanism for MGAT1 induced TAG increase and senescence-like symptoms. The data generated will serve as a valuable resource for oil and senescence related studies and for future N. benthamiana transcriptome studies. more...
- Published
- 2014
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31. Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves.
- Author
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Vanhercke T, El Tahchy A, Liu Q, Zhou XR, Shrestha P, Divi UK, Ral JP, Mansour MP, Nichols PD, James CN, Horn PJ, Chapman KD, Beaudoin F, Ruiz-López N, Larkin PJ, de Feyter RC, Singh SP, and Petrie JR more...
- Subjects
- Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Biofuels, Biomass, Diacylglycerol O-Acyltransferase genetics, Diacylglycerol O-Acyltransferase metabolism, Fatty Acids analysis, Fatty Acids metabolism, Gene Expression, Phenotype, Plant Leaves metabolism, Plant Oils analysis, Plant Proteins genetics, Plant Proteins metabolism, Plants, Genetically Modified, Time Factors, Nicotiana genetics, Transcription Factors genetics, Transcription Factors metabolism, Transgenes, Triglycerides analysis, Gene Expression Regulation, Plant, Metabolic Engineering, Plant Oils metabolism, Nicotiana metabolism, Triglycerides metabolism
- 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., (© 2013 CSIRO. Plant Biotechnology Journal published by Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.) more...
- Published
- 2014
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32. Synergistic effect of WRI1 and DGAT1 coexpression on triacylglycerol biosynthesis in plants.
- Author
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Vanhercke T, El Tahchy A, Shrestha P, Zhou XR, Singh SP, and Petrie JR
- Subjects
- Arabidopsis Proteins genetics, Diacylglycerol O-Acyltransferase genetics, Fatty Acids metabolism, Fatty Acids, Monounsaturated metabolism, Fatty Acids, Unsaturated biosynthesis, Fatty Acids, Unsaturated metabolism, Gene Expression, Gene Transfer Techniques, Plant Leaves enzymology, Plants, Genetically Modified enzymology, Plants, Genetically Modified metabolism, Recombinant Proteins biosynthesis, Nicotiana enzymology, Transcription Factors genetics, Triglycerides metabolism, Up-Regulation, Arabidopsis Proteins biosynthesis, Diacylglycerol O-Acyltransferase biosynthesis, Fatty Acids biosynthesis, Plant Leaves metabolism, Nicotiana metabolism, Transcription Factors biosynthesis, Triglycerides biosynthesis
- Abstract
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., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.) more...
- Published
- 2013
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33. Metabolic engineering of plant oils and waxes for use as industrial feedstocks.
- Author
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Vanhercke T, Wood CC, Stymne S, Singh SP, and Green AG
- Subjects
- Fatty Acids biosynthesis, Plants metabolism, Animal Feed, Metabolic Engineering, Plant Oils metabolism, Waxes metabolism
- Abstract
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., (© 2012 CSIRO Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.) more...
- Published
- 2013
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34. Recruiting a new substrate for triacylglycerol synthesis in plants: the monoacylglycerol acyltransferase pathway.
- Author
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Petrie JR, Vanhercke T, Shrestha P, El Tahchy A, White A, Zhou XR, Liu Q, Mansour MP, Nichols PD, and Singh SP
- Subjects
- Animals, Arabidopsis metabolism, Diacylglycerol O-Acyltransferase metabolism, Glycerol-3-Phosphate O-Acyltransferase metabolism, Glycerophosphates metabolism, Metabolic Networks and Pathways, Mice, Monoglycerides metabolism, Saccharomyces cerevisiae enzymology, Nicotiana enzymology, Acyltransferases metabolism, Triglycerides biosynthesis
- Abstract
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. more...
- Published
- 2012
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35. Mechanistic and structural insights into the regioselectivity of an acyl-CoA fatty acid desaturase via directed molecular evolution.
- Author
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Vanhercke T, Shrestha P, Green AG, and Singh SP
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Animals, Catalytic Domain, Fatty Acid Desaturases genetics, Fatty Acid Desaturases metabolism, Fatty Acids chemistry, Fatty Acids genetics, Fatty Acids metabolism, Gryllidae genetics, Insect Proteins genetics, Insect Proteins metabolism, Molecular Sequence Data, Mutation, Missense, Saccharomyces cerevisiae, Structure-Activity Relationship, Substrate Specificity physiology, Directed Molecular Evolution, Fatty Acid Desaturases chemistry, Gryllidae enzymology, Insect Proteins chemistry
- Abstract
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. more...
- Published
- 2011
- Full Text
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36. Reducing mutational bias in random protein libraries.
- Author
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Vanhercke T, Ampe C, Tirry L, and Denolf P
- Subjects
- Bacillus thuringiensis Toxins, Bacterial Proteins metabolism, Bacterial Toxins metabolism, Cloning, Molecular, Endotoxins metabolism, Hemolysin Proteins, Mutagenesis, Plasmids, Polymerase Chain Reaction, Taq Polymerase metabolism, Bacillus thuringiensis chemistry, Bacterial Proteins genetics, Bacterial Toxins genetics, DNA genetics, Endotoxins genetics, Gene Library, Proteins genetics
- Abstract
The success of protein optimization through directed molecular evolution depends to a large extent on the size and quality of the displayed library. Current low-fidelity DNA polymerases that are commonly used during random mutagenesis and recombination in vitro display strong mutational preferences, favoring the substitution of certain nucleotides over others. The result is a biased and reduced functional diversity in the library under selection. In an effort to reduce mutational bias, we combined two different low-fidelity DNA polymerases, Taq and Mutazyme, which have opposite mutational spectra. As a first step, random mutants of the Bacillus thuringiensis cry9Ca1 gene were generated by separate error-prone polymerase chain reactions (PCRs) with each of the two polymerases. Subsequent shuffling by staggered extension process (StEP) of the PCR products resulted in intermediate numbers of AT and GC substitutions, compared to the Taq or Mutazyme error-prone PCR libraries. This strategy should allow generating unbiased libraries or libraries with a specific degree of mutational bias by applying optimal mutagenesis frequencies during error-prone PCR and controlling the concentration of template in the shuffling reaction while taking into account the GC content of the target gene. more...
- Published
- 2005
- Full Text
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37. Rescue and in situ selection and evaluation (RISE): a method for high-throughput panning of phage display libraries.
- Author
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Vanhercke T, Ampe C, Tirry L, and Denolf P
- Subjects
- Enzyme-Linked Immunosorbent Assay, Bacteriophages genetics, Bacteriophages metabolism, Peptide Library
- Abstract
Phage display has proven to be an invaluable instrument in the search for proteins and peptides with optimized or novel functions. The amplification and selection of phage libraries typically involve several operations and handling large bacterial cultures during each round. Purification of the assembled phage particles after rescue adds to the labor and time demand. The authors therefore devised a method, termed rescue and in situ selection and evaluation (RISE), which combines all steps from rescue to binding in a single microwell. To test this concept, wells were precoated with different antibodies, which allowed newly formed phage particles to be captured directly in situ during overnight rescue. Following 6 washing steps, the retained phages could be easily detected in an enzyme-linked immunosorbent assay (ELISA), thus eliminating the need for purification or concentration of the viral particles. As a consequence, RISE enables a rapid characterization of phage-displayed proteins. In addition, this method allowed for the selective enrichment of phages displaying a hemagglutinin (HA) epitope tag, spiked in a 10(4)-fold excess of wild-type background. Because the combination of phage rescue, selection, or evaluation in a single microwell is amenable to automation, RISE may boost the high-throughput screening of smaller sized phage display libraries. more...
- Published
- 2005
- Full Text
- View/download PDF
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