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Tobacco Plants as a Production Platform for Biofuel Lee Greenawald

Learn how tobacco plants are being explored for biofuel production due to their potential to accumulate up to 40% of oil in seeds. By over-expressing specific genes, such as DGAT and LEC2, researchers have achieved significant increases in oil production, making tobacco a promising source of biofuels. This study explores how manipulating metabolic pathways in tobacco plants can lead to at least a twofold increase in oil accumulation, potentially yielding double the extractable fatty acids compared to conventional crops like soybean. With further research on gene overexpression and exploring alternative methods to boost oil accumulation, tobacco plants hold the promise of becoming a major player in sustainable biofuel production.

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Tobacco Plants as a Production Platform for Biofuel Lee Greenawald

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  1. Tobacco Plants as a Production Platform for BiofuelLee Greenawald

  2. How Does Biofuel Work? • Bxx • Like standard diesel fuel, biofuels burn as result of internal combustion in the engine (no major modification to cars) • Makes engines more efficient in converting/using energy • Combustion in biodiesel engines results from the heat of compression; glow plugs in engine increase the temperature • Produces heat required for combustion to occur and engine to work Photo courtesy Nebraska Soybean Board http://www.biodiesel.org/resources/users/images/Bio-Beetlesmall.jpg

  3. Why Tobacco is Being Looked At Closer • Crop not used for food; doesn’t interrupt leaf production • Grown in 100 countries • 170 tons of green tissues when grown for biomass production • Multiple harvests in a single year since can re-sprout after cutting • Can accumulate up to 40% of seed weight in oil http://climateprogress.org/wp-content/uploads/2008/04/corn.jpg

  4. Storage reserves for triacylglycerols

  5. Usta (2005) • Research proves tobacco seed oil successful for biodiesel • “In this study, 86% of the oil was converted to biodiesel using the transesterification process described. This is an acceptable yield for a crude oil [54].However, an ongoing research in our laboratory is being carried but to be able to increase this percentage.” (Usta 2005)

  6. Thomas Jefferson University, PA • Over-expressing genes in tobacco plant leads to increase in oil produced in two methods: • Arabidopsis thaliana gene diacylglycerol acyltransferase (DGAT) coding for a key enzyme in triacylglycerol (TAG) biosynthesis • Arabidopsis gene LEAFY COTYLEDON 2 (LEC2), a master regulator of seed maturation and seed oil storage under the control of an inducible Alc promoter 20-fold increase in TAG/twofold increase in fatty acids Increase of 6.8% per dry weight of total extracted FA

  7. TAG Biosynthesis Pathway Considered rate limiting step http://ejournal.vudat.msu.edu/index.php/mmg445/article/view/378/360

  8. 1. Over-expression of DGAT coding for enzyme in TAG biosynthesis • DGAT linked with C-terminal with c-myc tag assembled in T-DNA region of pBIN-Plus vector • Placed under control of a strong ribulose-biphosphate carboxylase small subunit (RbcS) promoter and terminator • Introduced into two tobacco cultivars: Nicotinana tabacum Wisconsin-38 and N. tabacum, NC-55 • Agrobacterium

  9. Overexpression of DGAT gene cont. • 50 independent transgenic lines of Wisconsin-38 • 9 independent transgenic lines of NC-55 generated by kanamycin selection • Presence of DGAT gene was confirmed by PCR • Protein expression determined by Western blot using c-myc-specific antibodies to detect c-myc tag fused to DGAT

  10. Overexpression of DGAT gene cont. 54 kDa band indicates transgenic line of DGAT DGAT WT

  11. Overexpression of DGAT gene cont. • Leaves with intense orange color were analyzed by LC-MS • Confirmed increase in TAG accumulation in transgenic plants • Over-expressing DGAT up to 20-fold • Transgenic lines with highest TAG expression levels also had highest total fatty acid content(1.5%-~25%) • Twofold increase in phospholipids

  12. 2. Over-expression of LEC2 for Fatty Acids • Regulates expression of many seed-specific genes in uniform manner + formation of oil bodies • Under control of Alc promoter system • Transgenic tobacco plants generated by Agrobacterium-mediated transformation

  13. Over-expression of LEC2 for Fatty Acids cont. • Transcription factor B3 encoded by LEC2 verified by expression of c-myc0-tagged protein • Expression of transcription factor stimulated by soil-drenching 6-8 week old plants with 0.1% or 1% acetaldehyde • Best responding plants found by kanamycin medium

  14. Over-expression of LEC2 for Fatty Acids cont. • Accumulation of FA examined by analyzing best responding plants over a 120 hour period • Result from low mobility of acetaldehyde from roots to leaves • LEC2 mRNA levels increased steadily=increase in FA level • 15 transformants confirmed by PCR were tested

  15. Over-expression of LEC2 for Fatty Acids cont. • Gas chromatography used for total FA content • Centrifugation of frozen plant tissues combined with chloroform fraction, evaporated to dryness under nitrogen gas flow and stored at -20 C until methylation • 0.1% acetaldehyde boosted FA content 5.5% • 1.0% acetaldehyde boosted FA content 2.9%-6.8%

  16. Conclusions • Overcoming rising oil prices and faulty crops needed for wide-scale biofuel production • Over-expressing genes demonstrates potential of manipulating metabolic pathways  at least a twofold increase in oil accumulation • 170 metric tons/ha harvest= 20 tons of dry biomass • Engineered plants achieve 6% increase in FA • Produce at least 2X as much bio-diesel as soybean

  17. Further Research • Over-expression of both genes in plant=double accumulation of extractable FA? • Other means of increasing oil accumulation: • Strong enhancers/promoters in combination with DGAT or other key enzymes influencing biosynthesis • Gene amplification technology • Blockage of lipid breakdown • Inhibition of pathways diverting energy and metabolite flow from oil biosynthesis • Selecting optimal tobacco plant tobacco-facts.net

  18. References • Andrianov, V.; Borisjuk, N.; Pogrebnyak, N.; Brinker, A.; Dixon, J.; Spitsin, S.; Flynn, J.; Matyszczuk, P.; Andryszak, K.; Laurelli, M.; Golovkin, M.; Koprowski, H. Tobacco as a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass. Plant Biotechnology Journal. 2010, 8,277-287. • Engineered tobacco plants have potential as biofuel feedstock; expressing oil in the leaves. http://www.greencarcongress.com/2009/12/tobacco-20091231/html. (accessed November 1, 2010). • Luo, K.; Duan, H.; Zhao, D.; Zheng, X.; Deng, W.; Chen, Y.; Stewart, N.; McAvoy, R.; Jiang, X.; Wu, Y.; He, A.; Pei, Y.; Li, Y. Plant Biotechnology Journal. 2007, 5, 263-274. • Stricklen, M. Plant genetic engineering to improve biomass characteristics for biofuels. Current Opinion in Biotechnology. 2006, 17, 315-319. • Usta, N. Use of tobacco seed oil methyl ester in a turbocharged indirect injection diesel engine. Biomass and Bioenergy. 2005, 28, 77-86. • Wu, S.; Chappel, J. Metabolic engineering of natural products in plants; tools of the trade and challenges for the future. Current Opinion in Biotechnology. 2008, 19, 145-152. http://www.inhabitat.com/wp-content/uploads/2010/01/smokingad-ed02.jpg

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