Triacylglycerols produced by plants are one of the most energy-rich and abundant forms of reduced carbon available from nature. Given their chemical similarities, plant oils represent a logical substitute for conventional diesel, a non-renewable energy source. However, as plant oils are too viscous for use in modern diesel engines, they are converted to fatty acid esters. Apart from seed oil vegetative tissue is potential source as bio mass for biofuel production, taking 15 tonnes per hectare as an average dry matter yield for a perennial grass, an oil content of 20– 25% by weight will produce about 3400 l of biodiesel (Heaton et al., 2004). There is growing interest in engineering green biomass to expand the production of plant oils as feed and biofuels. Here, we show that PHOSPHOLIPID: DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is a critical enzyme involved in triacylglycerol (TAG) synthesis in leaves. Overexpression of PDAT1 increases leaf TAG accumulation, leading to oil droplet overexpansion through fusion. Ectopic expression of oleosin promotes the clustering of small oil droplets. Coexpression of PDAT1 with oleosin boosts leaf TAG content by up to 6.4% of the dry weight without affecting membrane lipid composition and plant growth. PDAT1 overexpression stimulates fatty acid synthesis (FAS) and increases fatty acid flux toward the prokaryotic glycerolipid pathway (Julian at al..2013). First, an Arabidopsis thaliana gene diacylglycerol acyltransferase (DGAT) coding for a key enzyme in triacylglycerol (TAG) biosynthesis, was expressed in tobacco under the control of a strong ribulose-biphosphate carboxylase small subunit promoter. This modification led to up to a 20-fold increase in TAG accumulation in tobacco leaves and translated into an overall of about a twofold increase in extracted fatty acids (FA) up to 5.8% of dry biomass in Nicotiana tabacum cv Wisconsin, and up to 6% in high-sugar tobacco variety NC-55 ( Andrianovet al 2010). Therefore Biotechnology has important and perhaps critical part to play in large-scale development of Biodiesel.

1. Engineering Fatty Acid Biosynthesis to
Improve Biofuel Production in Higher
Plants
Vishnu Hembade

2. • Introduction
• Biofuels
• General pathways for Fatty Acid biosynthesis
• Enzymes Modifications
• Strategies for Genetic Engineering
• Mechanism for Biodiesel Production
• Case studies
• Conclusion
Contents

3. How Much do We have for Consume ?

4.  Seed oils of plants
 Renewable sources for food
applications(frying, baking, processed foods)
 Fuel (Biodiesel)
 industrial raw material (soaps, detergents, paints, lubricants)
 Vegetable oils account for ~85% of the world’s edible fat and oil
production
 Oil palm, soybeans, rapeseed and sunflower, which together
account for ≈ 79% of the total production.
Introduction

5. (Lu et al., 2011)
Status of Vegetables oil

6. o Fatty acids are carboxylic acids with a long hydrocarbon chain attached.
-Saturated fatty acids
- Monounsaturated fatty acids
- Polyunsaturated fatty acids
o Triacylglycerols
Triacylglycerols are contained primarily in seeds but also in vegatative
part,Leaves, fruits such as olives or avocados.
Triacylglycerols containing three fatty acids are of a nonpolar nature.
(Slater et al.,2011)
Fatty acids and Storage lipid or oil

7. Common name Chemical structure Δx C:D
Lauric acid 12:0
Myristic acid 14:0
Myristoleic acid CH3(CH2)3CH=CH(CH2)7COOH cis-Δ9 14:1
Palmitic acid 16:0
Palmitoleic acid CH3(CH2)5CH=CH(CH2)7COOH cis-Δ9 16:1
Sapienic acid CH3(CH2)8CH=CH(CH2)4COOH cis-Δ6 16:1
Stearic acid 18:0
Oleic acid CH3(CH2)7CH=CH(CH2)7COOH cis-Δ9 18:1
Linoleic acid CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH cis,cis-Δ9,Δ12 18:2
α-Linolenic acid CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH cis,cis,cis-Δ9,Δ12,Δ15 18:3
Arachidonic acid
CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH
2)3COOH
cis,cis,cis,cis-Δ5Δ8,Δ11,Δ14 20:4
Eicosapentaenoic
acid
CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2C
H=CH(CH2)3COOH
cis,cis,cis,cis,cisΔ5,Δ8,Δ11,Δ14,
Δ17
20:5
Erucic acid CH3(CH2)7CH=CH(CH2)11COOH cis-Δ13 22:1
Docosahexaenoic
acid
CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2C
H=CHCH2CH=CH(CH2)2COOH
cis,cis,cis,cis,cis,cis-
Δ4,Δ7,Δ10,Δ13,Δ16,Δ19
22:6
Nomenclature of FAs

8.  A biofuel is a fuel that contains energy from geologically recent carbon
fixation. These fuels are produced from living organisms.
 Examples of this carbon fixation occur in plants and microalgae. These fuels
are made by a biomass conversion (biomass refers to recently living
organisms, most often referring to plants or plant-derived materials).
 Bioethanol:.
 Biodiesel:
Biodiesel refers to a vegetable oil- or animal fat-based diesel fuel consisting
of long-chain alkyl (methyl, ethyl, or propyl) esters. Biodiesel is typically
made by chemically reacting lipids (e.g., vegetable oil, animal fat (with an
alcohol producing fatty acid esters.
Biofuel

9. What is Biodiesel?
Fatty Acid
Alcohol
Glycerin
Vegetable Oil
BiodieselFA
FAFA
FA
(Olofssonet al., 2008)

10. Why
?

11. • Pure Biodiesel (B100) or blended with petroleum diesel
(B20, BXX).
• Rudolf Diesel: peanut oil.
• Use existing fuel distribution network.
• Available now.
Biodiesel can be used in existing Diesel Engines

12. • Burning fossil fuels increases atmospheric levels of carbon dioxide
• Fossil fuels are a
finite resource
Graph taken from USF Oceanography webpage
Biodiesel’s Closed
Carbon Cycle
30% Increase
Environmental Issues

13. 0 20 40 60 80 100 120 140 160
Gasoline
CNG
LPG
Diesel
Ethanol 85%
B20
Diesel Hybrid
Electric
B100
Data from “A Fresh Look at CNG: A Comparison of Alternative
Fuels”, Alternative Fuel Vehicle Program, 8/13/2001
B100 = 100% Biodiesel
B20 = 20% BD + 80% PD
Relative Greenhouse Gas Emissions

14.  Limited use
 Problem with fuel Characteristics.
-Poor cold temp. performance
-Higher Oxidation
-More Emmission of NOx
 Cost and Supply limitations
(Timothy et al..2008)
Limitations

15.  Cold Temperature Flow Characteristics
CP: Cloud Point-: -16 (soy Biodiesel: 0 C)
PP:Pouring Point: -27 (soy Biodisel: -2 C) (Timothy et al..2008)
 Indeed, the CP and PP of FAMEs derived from low-palmitate soybean oil are -7C
and -9C respectively, at least 5C lower than for FAMEs from normal soybean oil
(Lee et al., 1995)
 Oxidation
 The influence of molecular structure on the rate of oxidation of biodiesel is
greater than the influence of environmental conditions such as air, light and the
presence of metal (Knothe and Dunn, 2003)
 Tocopherols(vitamin E) present naturally in soybean oil can reduce the rate of
biodiesel oxidation by more than a factor of 10(Knothe et al., 2005)
 NOx Emission
Strategies for Improving Biofuel Properties

16. Biodiesel
TAG
Glycerol
(Timothy et al..2008)

17.  Strategies to produce oil in vegetative tissue.
 Because of their very high biomass yields and low fertilizer or other inputs, perennial
grasses are projected to be a major future source of biofuels (Heaton et al., 2004).
 Taking 15 tonnes per hectare as an average dry matter yield for a perennial grass, an oil
content of 20– 25% by weight will produce about 3400 l of biodiesel (Heaton et
al., 2004).
 The extraction of oil and conversion to biodiesel requires less energy than
lignocellulose hydrolysis, fermentation to ethanol and distillation, the net energy
balance and greenhouse gas benefits for biodiesel are even more favorable (Hill et
al., 2006).
 Miscanthus Grass.
contd…

18. Arild et. al…2005
Metabolism of Fatty Acid

19. Tom et al….2009
Overview of Lipid Synthesis

20. Enzymes to be manipulated
• Fatty acid synthase:- KASI, KASII, KASIII
• Thioesterases - produce medium chain FAs by removing acyl
group.
• Elongases - produce 20:1 and 22:1 FAs from oleate
• Desaturases- introduce double bonds into FA chain.
• Stearoyl-ACP Δ9-desaturase:- in the plastid stroma that
converts stearate into oleate.
• Δ12-desaturase, Δ15-desaturase
• Acyltransferases - incorporate FAs into DAG and TAG.
• Hydroxylases - incorporate hydroxyl groups in the FA chain.

21. Sixth largest source of vegetable oil
26% palmitic acid (C16:0),
3% Stearic acid (C18:0)
15% oleic acid (C18:1),
58% linoleic acid (C18:2)
Stearoyl-acyl-carrier protein (ACP) Δ9-
desaturase
oleoyl-phosphatidylcholine (PC)
ω6-desaturase
40%
77%
In addition, palmitic acid was significantly
lowered in both high-stearic and high-oleic
lines.
Qing Liu et al….2002
High-Stearic and High-Oleic Cottonseed Oils Produced by Hairpin RNA-
Mediated Post-Transcriptional Gene Silencing

22. Qing Liu et al….2002
contd…
Schematic diagram of the chimeric
silencing constructs transformed into cotton

23. Thelen et.al…..2002
1) Acyl-ACP thioesterase
2) Acyl-ACP thioesterase
3) Acyl-ACP thioesterase
4) Acyl-ACP thioesterase
5)Stearoyl-ACP D-9 desat
6) Palmitoyl-ACP D-4 desa
Fatty acid synthesis, modification, and assembly into
triacylglycerols in plants.

24. 7) Oleoyl-D-12 desaturase
8) b-Ketoacyl-CoA synthase (
9) acyl-CoA desaturase
10) Oleate-12-hydroxylase
11) Acetylenase
12) b-ketoacyl synthase
13) acyl-CoA reductase
14) wax synthase
contd…

25. Thelen et.al…..2002

26. Case study 1

27.  Generation of DGAT and LEC2 expression cassettes and plant
transformation
 Recombinant DNA and protein analysis
 Light microscopy
 Induction of recombinant LEC2 expression
 Quantitative reverse transcriptase-polymerase chain reaction
analysis of LEC2 expression
 Lipid extraction and analysis
Materials and methods

28.  Two Cultivars
1) Nicotiana tabacum, cv. Wisconsin-38
2)high-sugar variety N. tabacum, NC-55
 DGAT
 LEC2
 Shift in lipid composition in transgenic tobacco plants
expressing DGAT
 Overexpression of DGAT in transgenic tobacco leads to
increased biosynthesis and accumulation of plant lipids.
 Inducible expression of LEC2 affects FA accumulation and
composition
Results

29. Western Blot
59
kDa
Wisconsin 38 , using anti c- myc monoclonal antibodies
Light micrograph
Sudan Dye
DGAT Expression

30. TAG amounts are given in
equivalents
(mean ± SD) of
trinonanoin used as a
quantitative standard in
LC-MS analysis.
FA by Gas
Chromatography
5.8
6
Quantitative analysis of triacylglycerol (TAG) and FA in transgenic
tobacco expressing diacylglycerol acyltransferase

31. DGAT-Wisconsin
Chromatogram:
TAG Fraction
Ln-Linolenic
L-Linoeic
O-Oleic
P-Palmitic
O=1.5-25%
Ln=67-35%
Ln=20-12%
O=18-44%
Calculated by peak Integration
Changes in fatty acid (FA) composition in transgenic
tobacco expressing diacylglycerol acyltransferase (DGAT).

32. Detection of LEC2 protein of
expected MW by anti-c-myc
MAb .
(+), recombinant protein with
c-myc tag used as a
positive control.
42 KDa
Analysis of LEAFY COTYLEDON 2 (LEC2) expression in
tobacco plants.Changes in fatty acid (FA) composition in transgenic
tobacco expressing diacylglycerol acyltransferase (DGAT).

33. Correlated accumulation of fatty acids
mRNA values are given as copy
number increase compared with
mRNA copy number for housekeeping
gene actin.
Accumulation of lipids in transgenic tobacco expressing
LEAFY COTYLEDON 2 (LEC2).

34.  Quantitative changes in fatty acid accumulation because of
overexpression of DGAT and LEC2 in transgenic tobacco
 Increased oil accumulation triggers a shift in fatty acid oil
composition of DGAT and LEC2 in transgenic tobacco.
 Potential of tobacco for biofuel production.
Conclusion of Article

35. Case Study 2

36.  The Relative Contribution of PDAT1 and DGAT1 to TAG
Synthesis in Leaves.
 Overexpression of PDAT1 Enhances TAG
Accumulation, Leading to OD Overexpansion in Leaves.
 Ectopic Expression of Oleosin Promotes the Clustering of
Small ODs and Boosts Oil Accumulation in PDAT1 Transgenic
Plants.
 Rate of FA Synthesis Is Enhanced in PDAT1 Transgenic Plants
Results

37. 5 week old-DL
7 week old-SL
Roles of DGAT1 and PDAT1 in TAG Synthesis during Leaf
Development.

38. TAG Accumulation in Leaves of
Transgenic Plants Overexpressing
PDAT1
Values are means and SD of
three biological replicates.
7 week old, independent
transgenic lines
OD Accumulation in Leaves of
Transgenic Plants Overexpressing
PDAT1.
7 week old A,B,C,D –stained with Nile
Red , C&D- TEM, (A&C WT)

39. • Ectopic Expression of Oleosin Promotes the Clustering of Small ODs and
Boosts Oil Accumulation in PDAT1 Transgenic Plants.
• Oleosins are known to play a key role in preventing ODs from coalescing in
oilseeds (Siloto et al., 2006; Shimada et al., 2008).
• To test the functional role of oleosins in TAG accumulation in vegetative
tissues, OELOSIN1 (OLE1), the most abundant seed OD-specificprotein of
Arabidopsis was C-terminally tagged with green fluorescent protein (GFP),
and this fusion gene was expressed in Arabidopsis wild-type plants under
the control of the 35S cauliflower mosaic virus promoter.
Oleosin Promoter

40. A) TEM imaging of ODs in leaves of the
OLE1-GFP–overexpressing line 1.
(B) Confocal microscopy of OD clusters
in leaves of the OLE1-GFP–
overexpressing line 1.
Ectopic Expression of OLE1 Promotes the Clustering of Small
ODs.

41. A)Fat Red dye.
B) 74 fold increase-7 week
old
C) Stained wih Nile Red
D) confocal microscopy
TAG Accumulation in Transgenic Lines Coexpressing PDAT1
and OLE1.

42. Fatt Acid composition of Membrane Lipids from leaves of
PDAT1 Overexpressor

43. o Study shows that PDAT1 has a dual role in enhancing FAS and
directing FAs from membrane lipids to TAG in Arabidopsis leaves.
o They showed that the combined expression of PDAT1 and OLE1
increases leaf TAG to 6.4% per DW in the wild type and 8.6% per
DW in tgd1 without major negative growth consequences.
o Given the growing recognition of the potential benefits of
maximizing TAG content in vegetative tissues of crops this
clarification of the role of PDAT1 in plants may enable new
strategies for future genetic engineering efforts aimed at enhancing
oil accumulation in biomass crops used for biofuel production.
Conclusion of Article

44.  Jatropha
 d1 mohan bio oils ltd
 In May 2005, Chief Minister Raman Singh became the first
head of a state government to use jatropha diesel for his
official vehicle.
 Railway
Thanjavur to Nagore section
Tiruchirapalli to Lalgudi, Dindigul and Karur sections.
Altenburg, Tilman (2009). Biodiesel in India :
value chain organisation and policy options for
rural development.
Biodiesel in India

45. Conclusion

46. Discussion

47. Thank You