Engineering Fatty Acid Biosynthesis to Improve Biofuel Production in Higher

Plants

Vishnu Hembade

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

Contents

How Much do We have for Consume ?

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

(Lu et al., 2011)

Status of Vegetables oil

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

Common name Chemical structure Δx C:D

Lauric acid 12:0Myristic 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 acidCH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2

)3COOH cis,cis,cis,cis-Δ5Δ8,Δ11,Δ14 20:4

Eicosapentaenoic acid

CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH

=CH(CH2)3COOHcis,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=CHCH2CH

=CHCH2CH=CH(CH2)2COOHcis,cis,cis,cis,cis,cis-Δ4,Δ7,Δ10,Δ13,Δ16,Δ19

22:6

Nomenclature of FAs

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

What is Biodiesel?

Fatty AcidAlcoholGlycerin

Vegetable Oil

BiodieselFA

FAFA

FA

(Olofssonet al., 2008)

Why

?

• 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

• 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

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% BiodieselB20 = 20% BD + 80% PD

Relative Greenhouse Gas Emissions

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

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

BiodieselTAG

Glycerol

(Timothy et al..2008)

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…

Arild et. al…2005

Metabolism of Fatty Acid

Tom et al….2009

Overview of Lipid Synthesis

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.

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

Qing Liu et al….2002

contd…

Schematic diagram of the chimericsilencing constructs transformed into cotton

Thelen et.al…..2002

1) Acyl-ACP thioesterase2) Acyl-ACP thioesterase 3) Acyl-ACP thioesterase4) Acyl-ACP thioesterase5)Stearoyl-ACP D-9 desat6) Palmitoyl-ACP D-4 desa

Fatty acid synthesis, modification, and assembly into triacylglycerols in plants.

7) Oleoyl-D-12 desaturase8) b-Ketoacyl-CoA synthase (9) acyl-CoA desaturase10) Oleate-12-hydroxylase11) Acetylenase12) b-ketoacyl synthase13) acyl-CoA reductase14) wax synthase

contd…

Thelen et.al…..2002

Case study 1

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

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

Western Blot

59 kDa

Wisconsin 38 , using anti c- myc monoclonal antibodies

Light micrographSudan Dye

DGAT Expression

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 transgenictobacco expressing diacylglycerol acyltransferase

DGAT-Wisconsin

Chromatogram:TAG FractionLn-LinolenicL-LinoeicO-OleicP-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 transgenictobacco expressing diacylglycerol acyltransferase (DGAT).

Detection of LEC2 protein of expected MW by anti-c-myc MAb . (+), recombinant protein with c-myc tag used as apositive control.

42 KDa

Analysis of LEAFY COTYLEDON 2 (LEC2) expression intobacco plants.Changes in fatty acid (FA) composition in transgenic

tobacco expressing diacylglycerol acyltransferase (DGAT).

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 expressingLEAFY COTYLEDON 2 (LEC2).

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

Case Study 2

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

5 week old-DL7 week old-SL

Roles of DGAT1 and PDAT1 in TAG Synthesis during LeafDevelopment.

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)

• 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

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.

A)Fat Red dye. B) 74 fold increase-7 week

oldC) Stained wih Nile Red D) confocal microscopy

TAG Accumulation in Transgenic Lines Coexpressing PDAT1and OLE1.

Fatt Acid composition of Membrane Lipids from leaves of PDAT1 Overexpressor

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

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

Conclusion

Discussion

Thank You