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4. Step changes in leaf oil accumulation via iterative metabolic engineering

Author

Vanhercke, Thomas, Divi, Uday K., El Tahchy, Anna, Liu, Qing, Mitchell, Madeline, Taylor, Matthew C., Eastmond, Peter J., Bryant, Fiona, Mechanicos, Anna, Blundell, Cheryl, Zhi, Yao, Belide, Srinivas, Shrestha, Pushkar, Zhou, Xue-Rong, Ral, Jean-Philippe, White, Rosemary G., Green, Allan, Singh, Surinder P., and Petrie, James R.

Published
2017
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12. Development of a Brassica napus (Canola) Crop Containing Fish Oil-Like Levels of DHA in the Seed Oil

Author

Petrie, James R., primary, Zhou, Xue-Rong, additional, Leonforte, Antonio, additional, McAllister, Jason, additional, Shrestha, Pushkar, additional, Kennedy, Yoko, additional, Belide, Srinivas, additional, Buzza, Greg, additional, Gororo, Nelson, additional, Gao, Wenxiang, additional, Lester, Geraldine, additional, Mansour, Maged P., additional, Mulder, Roger J., additional, Liu, Qing, additional, Tian, Lijun, additional, Silva, Claudio, additional, Cogan, Noel O. I., additional, Nichols, Peter D., additional, Green, Allan G., additional, de Feyter, Robert, additional, Devine, Malcolm D., additional, and Singh, Surinder P., additional

Published
2020
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13. Engineering Trienoic Fatty Acids into Cottonseed Oil Improves Low-Temperature Seed Germination, Plant Photosynthesis and Cotton Fiber Quality

Author

Gao, Lihong, primary, Chen, Wei, additional, Xu, Xiaoyu, additional, Zhang, Jing, additional, Singh, Tanoj K, additional, Liu, Shiming, additional, Zhang, Dongmei, additional, Tian, Lijun, additional, White, Adam, additional, Shrestha, Pushkar, additional, Zhou, Xue-Rong, additional, Llewellyn, Danny, additional, Green, Allan, additional, Singh, Surinder P, additional, and Liu, Qing, additional

Published
2020
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14. A Synergistic Genetic Engineering Strategy Induced Triacylglycerol Accumulation in Potato (Solanum tuberosum) Leaf

Author

Xu, Xiao-yu, primary, Akbar, Sehrish, additional, Shrestha, Pushkar, additional, Venugoban, Lauren, additional, Devilla, Rosangela, additional, Hussain, Dawar, additional, Lee, Jiwon, additional, Rug, Melanie, additional, Tian, Lijun, additional, Vanhercke, Thomas, additional, Singh, Surinder P., additional, Li, Zhongyi, additional, Sharp, Peter J., additional, and Liu, Qing, additional

Published
2020
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16. Up-regulation of lipid biosynthesis increases the oil content in leaves of Sorghum bicolor

Author

Vanhercke, Thomas, Belide, Srinivas, Taylor, Matthew C, El Tahchy, Anna, Okada, Shoko, Rolland, Vivien, Liu, Qing, Mitchell, Madeline, Shrestha, Pushkar, Venables, Ingrid, Ma, Lina, Blundell, Cheryl, Mathew, Anu, Ziolkowski, Lisa, Niesner, Nathalie, Hussain, Dawar, Dong, Bei, Liu, Guoquan, Godwin, Ian D, Lee, Jiwon, Rug, Melanie, Zhou, Xue-Rong, Singh, Surinder P, Petrie, James R, Vanhercke, Thomas, Belide, Srinivas, Taylor, Matthew C, El Tahchy, Anna, Okada, Shoko, Rolland, Vivien, Liu, Qing, Mitchell, Madeline, Shrestha, Pushkar, Venables, Ingrid, Ma, Lina, Blundell, Cheryl, Mathew, Anu, Ziolkowski, Lisa, Niesner, Nathalie, Hussain, Dawar, Dong, Bei, Liu, Guoquan, Godwin, Ian D, Lee, Jiwon, Rug, Melanie, Zhou, Xue-Rong, Singh, Surinder P, and Petrie, James R

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 C4 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.

Published
2019

17. 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

Xu, Xiaoyu, primary, Vanhercke, Thomas, additional, Shrestha, Pushkar, additional, Luo, Jixun, additional, Akbar, Sehrish, additional, Konik-Rose, Christine, additional, Venugoban, Lauren, additional, Hussain, Dawar, additional, Tian, Lijun, additional, Singh, Surinder, additional, Li, Zhongyi, additional, Sharp, Peter J., additional, and Liu, Qing, additional

Published
2019
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19. Rapid expression of transgenes driven by seed-specific constructs in leaf tissue: DHA production

Author

Zhou Xue-Rong, Wood Craig C, Mansour Maged P, Liu Qing, Shrestha Pushkar, Petrie James R, Nichols Peter D, Green Allan G, and Singh Surinder P

Subjects
Plant culture, SB1-1110, Biology (General), QH301-705.5
Abstract

Abstract Background Metabolic engineering of seed biosynthetic pathways to diversify and improve crop product quality is a highly active research area. The validation of genes driven by seed-specific promoters is time-consuming since the transformed plants must be grown to maturity before the gene function can be analysed. Results In this study we demonstrate that genes driven by seed-specific promoters contained within complex constructs can be transiently-expressed in the Nicotiana benthamiana leaf-assay system by co-infiltrating the Arabidopsis thaliana LEAFY COTYLEDON2 (LEC2) gene. A real-world case study is described in which we first assembled an efficient transgenic DHA synthesis pathway using a traditional N. benthamiana Cauliflower Mosaic Virus (CaMV) 35S-driven leaf assay before using the LEC2-extended assay to rapidly validate a complex seed-specific construct containing the same genes before stable transformation in Arabidopsis. Conclusions The LEC2-extended N. benthamiana assay allows the transient activation of seed-specific promoters in leaf tissue. In this study we have used the assay as a rapid preliminary screen of a complex seed-specific transgenic construct prior to stable transformation, a feature that will become increasingly useful as genetic engineering moves from the manipulation of single genes to the engineering of complex pathways. We propose that the assay will prove useful for other applications wherein rapid expression of transgenes driven by seed-specific constructs in leaf tissue are sought.

Published
2010
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20. Engineering docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in Brassica juncea.

Author

Belide, Srinivas, Shrestha, Pushkar, Kennedy, Yoko, Leonforte, Antonio, Devine, Malcolm D., Petrie, James R., Singh, Surinder P., and Zhou, Xue‐Rong

Subjects
*DOCOSAHEXAENOIC acid, *FISH oils, *BRASSICA juncea, *CANOLA, *COMPOSITION of seeds, *UNSATURATED fatty acids
Abstract

(c) Fatty acid profile of BjDHA and BjDPA pooled seeds derived from Line 4, and their crossing progeny (n >= 6). gl I Brassica juncea i was transformed with the binary vector GA7 ModB (Figure 1a) used previously to develop DHA canola (Petrie I et al i ., 2020). Keywords: 3 LC-PUFA; DPA; DHA; Brassica juncea EN 3 LC-PUFA DPA DHA Brassica juncea 19 21 3 12/29/21 20220101 NES 220101 Omega-3 long-chain polyunsaturated fatty acids ( 3 LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are important for human health. [Extracted from the article]

Published
2022
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22. Up-regulation of lipid biosynthesis increases the oil content in leaves ofSorghum bicolor

Author

Vanhercke, Thomas, primary, Belide, Srinivas, additional, Taylor, Matthew C., additional, El Tahchy, Anna, additional, Okada, Shoko, additional, Rolland, Vivien, additional, Liu, Qing, additional, Mitchell, Madeline, additional, Shrestha, Pushkar, additional, Venables, Ingrid, additional, Ma, Lina, additional, Blundell, Cheryl, additional, Mathew, Anu, additional, Ziolkowski, Lisa, additional, Niesner, Nathalie, additional, Hussain, Dawar, additional, Dong, Bei, additional, Liu, Guoquan, additional, Godwin, Ian D., additional, Lee, Jiwon, additional, Rug, Melanie, additional, Zhou, Xue-Rong, additional, Singh, Surinder P., additional, and Petrie, James R., additional

Published
2018
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23. Comparative Lipidomics and Proteomics of Lipid Droplets in the Mesocarp and Seed Tissues of Chinese Tallow (Triadica sebifera)

Author

Zhi, Yao, primary, Taylor, Matthew C., additional, Campbell, Peter M., additional, Warden, Andrew C., additional, Shrestha, Pushkar, additional, El Tahchy, Anna, additional, Rolland, Vivien, additional, Vanhercke, Thomas, additional, Petrie, James R., additional, White, Rosemary G., additional, Chen, Wenli, additional, Singh, Surinder P., additional, and Liu, Qing, additional

Published
2017
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24. Reduced triacylglycerol mobilization during seed germination and early seedling growth in Arabidopsis containing nutritionally important polyunsaturated fatty acids

Author

Shrestha, Pushkar, Callahan, Damien, Singh, Surinder P, Petrie, James R, Zhou, Xue-Rong, Shrestha, Pushkar, Callahan, Damien, Singh, Surinder P, Petrie, James R, and Zhou, Xue-Rong

Abstract

There are now several examples of plant species engineered to synthesize and accumulate nutritionally important polyunsaturated fatty acids in their seed triacylglycerols (TAG). The utilization of TAG in germinating seeds of such transgenic plants was unknown. In this study, we examined the TAG utilization efficiency during seed germination in transgenic Arabidopsis seeds containing several examples of these fatty acids. Seed TAG species with native fatty acids had higher utilization rate than the TAG species containing transgenically produced polyunsaturated fatty acids. Conversely, quantification of the fatty acid components remaining in the total TAG after early stages of seed germination revealed that the undigested TAGs tended to contain elevated levels of the engineered polyunsaturated fatty acids (PUFA). LC-MS analysis further revealed asymmetrical mobilization rates for the individual TAG species. TAGs which contained multiple PUFA fatty acids were mobilized slower than the species containing single PUFA. The mobilized engineered fatty acids were used in de novo membrane lipid synthesis during seedling development.

Published
2016

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

Author

Liu, Qing, primary, Guo, Qigao, additional, Akbar, Sehrish, additional, Zhi, Yao, additional, El Tahchy, Anna, additional, Mitchell, Madeline, additional, Li, Zhongyi, additional, Shrestha, Pushkar, additional, Vanhercke, Thomas, additional, Ral, Jean-Philippe, additional, Liang, Guolu, additional, Wang, Ming-Bo, additional, White, Rosemary, additional, Larkin, Philip, additional, Singh, Surinder, additional, and Petrie, James, additional

Published
2016
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33. Metabolic engineering of biomass for high energy density: Oilseed-like triacylglycerol yields from plant leaves

Author

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.

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.

Published
2014

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

Author

Liu, Qing, Guo, Qigao, Akbar, Sehrish, Zhi, Yao, El Tahchy, Anna, Mitchell, Madeline, Li, Zhongyi, Shrestha, Pushkar, Vanhercke, Thomas, Ral, Jean‐Philippe, Liang, Guolu, Wang, Ming‐Bo, White, Rosemary, Larkin, Philip, Singh, Surinder, and Petrie, James

Subjects
POTATOES, FOOD crops, TRIGLYCERIDES, TRANSGENES, LIPID synthesis
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 (Ca MV-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. [ABSTRACT FROM AUTHOR]

Published
2017
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35. Genetic enhancement of palmitic acid accumulation in cotton seed oil through RNAi down-regulation of gh KAS2 encoding β-ketoacyl- ACP synthase II ( KASII).

Author

Liu, Qing, Wu, Man, Zhang, Baolong, Shrestha, Pushkar, Petrie, James, Green, Allan G., and Singh, Surinder P.

Subjects
PALMITIC acid, COTTONSEED oil, BETA-ketoacyl-acyl-carrier-protein synthases, MOLECULAR structure of RNA, GENOTYPES
Abstract

Palmitic acid (C16:0) already makes up approximately 25% of the total fatty acids in the conventional cotton seed oil. However, further enhancements in palmitic acid content at the expense of the predominant unsaturated fatty acids would provide increased oxidative stability of cotton seed oil and also impart the high melting point required for making margarine, shortening and confectionary products free of trans fatty acids. Seed-specific RNAi-mediated down-regulation of β-ketoacyl- ACP synthase II ( KASII) catalysing the elongation of palmitoyl- ACP to stearoyl- ACP has succeeded in dramatically increasing the C16 fatty acid content of cotton seed oil to well beyond its natural limits, reaching up to 65% of total fatty acids. The elevated C16 levels were comprised of predominantly palmitic acid (C16:0, 51%) and to a lesser extent palmitoleic acid (C16:1, 11%) and hexadecadienoic acid (C16:2, 3%), and were stably inherited. Despite of the dramatic alteration of fatty acid composition and a slight yet significant reduction in oil content in these high-palmitic ( HP) lines, seed germination remained unaffected. Regiochemical analysis of triacylglycerols ( TAG) showed that the increased levels of palmitic acid mainly occurred at the outer positions, while C16:1 and C16:2 were predominantly found in the sn-2 position in both TAG and phosphatidylcholine. Crossing the HP line with previously created high-oleic ( HO) and high-stearic ( HS) genotypes demonstrated that HP and HO traits could be achieved simultaneously; however, elevation of stearic acid was hindered in the presence of high level of palmitic acid. [ABSTRACT FROM AUTHOR]

Published
2017
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38. Metabolic Engineering Camelina sativa with Fish Oil-Like Levels of DHA

Author

Petrie, James R., primary, Shrestha, Pushkar, additional, Belide, Srinivas, additional, Kennedy, Yoko, additional, Lester, Geraldine, additional, Liu, Qing, additional, Divi, Uday K., additional, Mulder, Roger J., additional, Mansour, Maged P., additional, Nichols, Peter D., additional, and Singh, Surinder P., additional

Published
2014
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39. Metabolic engineering of biomass for high energy density: oilseed‐like triacylglycerol yields from plant leaves

Author

Vanhercke, Thomas, primary, El Tahchy, Anna, additional, Liu, Qing, additional, Zhou, Xue‐Rong, additional, Shrestha, Pushkar, additional, Divi, Uday K., additional, Ral, Jean‐Philippe, additional, Mansour, Maged P., additional, Nichols, Peter D., additional, James, Christopher N., additional, Horn, Patrick J., additional, Chapman, Kent D., additional, Beaudoin, Frederic, additional, Ruiz‐López, Noemi, additional, Larkin, Philip J., additional, Feyter, Robert C., additional, Singh, Surinder P., additional, and Petrie, James R., additional

Published
2013
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40. Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway.

Author

Naim, Fatima, Shrestha, Pushkar, Singh, Surinder P., Waterhouse, Peter M., and Wood, Craig C.

Subjects
*PLANT gene silencing, *GENE expression in plants, *SUPPRESSOR mutation, *PLANT metabolism, *TRANSGENIC plants, *PLANTS
Abstract

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s) RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins ( VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous micro RNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid ( AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP- AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the si RNA amplification step that is not part of the mi RNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application. [ABSTRACT FROM AUTHOR]

Published
2016
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