Engineering fatty acid biosynthesis

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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.

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Engineering fatty acid biosynthesis

  1. 1. Engineering Fatty Acid Biosynthesis to Improve Biofuel Production in Higher Plants Vishnu Hembade
  2. 2. • Introduction • Biofuels • General pathways for Fatty Acid biosynthesis • Enzymes Modifications • Strategies for Genetic Engineering • Mechanism for Biodiesel Production • Case studies • Conclusion Contents
  3. 3. How Much do We have for Consume ?
  4. 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. 5. (Lu et al., 2011) Status of Vegetables oil
  6. 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. 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. 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. 9. What is Biodiesel? Fatty Acid Alcohol Glycerin Vegetable Oil BiodieselFA FAFA FA (Olofssonet al., 2008)
  10. 10. Why ?
  11. 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. 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. 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. 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. 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. 16. Biodiesel TAG Glycerol (Timothy et al..2008)
  17. 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. 18. Arild et. al…2005 Metabolism of Fatty Acid
  19. 19. Tom et al….2009 Overview of Lipid Synthesis
  20. 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. 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. 22. Qing Liu et al….2002 contd… Schematic diagram of the chimeric silencing constructs transformed into cotton
  23. 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. 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. 25. Thelen et.al…..2002
  26. 26. Case study 1
  27. 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. 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. 29. Western Blot 59 kDa Wisconsin 38 , using anti c- myc monoclonal antibodies Light micrograph Sudan Dye DGAT Expression
  30. 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. 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. 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. 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. 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. 35. Case Study 2
  36. 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. 37. 5 week old-DL 7 week old-SL Roles of DGAT1 and PDAT1 in TAG Synthesis during Leaf Development.
  38. 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. 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. 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. 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. 42. Fatt Acid composition of Membrane Lipids from leaves of PDAT1 Overexpressor
  43. 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. 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. 45. Conclusion
  46. 46. Discussion
  47. 47. Thank You

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