Transcript Over-expression of LEC2 for Fatty Acids cont.

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
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/wpcontent/uploads/2008/04/corn.jpg
Storage reserves for triacylglycerols
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)
Thomas Jefferson University, PA
 Over-expressing genes in tobacco plant
leads to increase in oil produced in two
methods:
1. Arabidopsis thaliana gene diacylglycerol
acyltransferase (DGAT) coding for a key
enzyme in triacylglycerol (TAG)
biosynthesis
2. 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
TAG Biosynthesis Pathway
Considered rate limiting
step
http://ejournal.vudat.msu.edu/index.php/mmg445/articl
e/view/378/360
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
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
Overexpression of DGAT gene cont.
54 kDa band
indicates
transgenic line
of DGAT
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
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
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
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
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%
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
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
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.
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