The most important oilseed crops are Oil palm, Soybeans, Rapeseed and Sunflower,which together account for ≈ 79% of the total production of oils.Oils that are low in palmitic acid and rich in either oleic acid or stearic acid are novel oils. Selective breeding utilizing natural variants or induced mutations has been used to develop a range of improved oils. The vegetable oil is used for different applications as renewable sources of food used for frying, baking, processed foods,Fuel (Biodiesel),medicine and can be used as industrial raw material for preparation of soaps, detergents, paints, lubricants etc. The recommended ratio of omega6/omega3 fatty acids in the human diet is approximately 2:1 to 6:1 (Simopoulos., 2000; Wijendran and Hayes., 2004) and the much higher ratio of omega 6 fatty acids in the typical Western diet (approximately 20:1) is thought to be a major contributor to cardiovascular disease (Simopoulos., 2000). For metabolic engineering of oil quality improvement fatty acid composition and enzymes involved are very important so we can reduce expression of endogenous enzymes by adding new enzyme ,overexpressing existing enzyme and by using antisense RNA. It proved that genes for membrane-bound fatty acid-modifying enzymes not only from plants but also from bacterial,animal,yeast have been shown to function in transgenic plants.The enzymes such as Fatty acid synthase ,Thioesterases ,Elongases ,Desaturases ,Stearoyl-ACP desaturase ,Δ12-desaturase, , Δ15-Desaturase ,Acyltransferases and Hydroxylases are important in fatty acid manipulation.Suppression of the oleate D12-desaturase gene (which normally converts 18:1 to 18:2) in soybean, sunflower, cotton and canola has resulted in the production of oils with a high oleic acid content, which have greater oxidative stability and improved performance in high-temperature cooking applications. (Metzger and Bornscheuer., 2006).

1. Metabolic engineering for oil quality
improvement
Pusadkar Pratik Prabodh

2. • Introduction
• Overview
• Enzymes used
• General pathways for oil biosynthesis
• Oil quality factors
• Applications
• Petroselinic acid production
• Case studies

3. Introduction
 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.

4. contd…
 Oilseed crops occupy next to cereals both irrigated and rainfed
conditions.
 Vegetable oils - principally of energy-dense triacylglycerols
- Calories in human and animal diets
- Preparation of margarines, salad oils, and fried foods .
- Sensory characteristics of numerous processed food
 Plant kingdom opens - bio-based industrial formulations
Ex. Lubricants and drying oils
(Lu et al., 2011)

5. Growth rate for primary oilseeds

6. Status of Vegetables oil
limitation of land for cropping
- Limitation petroleum
- Price of oils (Almost double )
growing reliance on biodiesel for liquid transportation fuel
- Now 7Mtns (Lu et al., 2011)

7. Limitations
• Poor spread of high yielding varieties and hybrids for yield
enhancement
• Non-availability of quality seed.
• Composition of oils, proteins, and carbohydrates in seeds of
Need
oil.
• Enhance the nutritional quality of plant oils
• Improve oil stability and long-term storage
• Production of novel oils in plants (designer oilseed)
• Increase oil content and reduce production costs
• Alter the nutritional or functional properties of the harvested
plant for use in foods, animal feeds, or industrial products.

8. Why Plant Oils Are Attractive Targets for
Metabolic Engineering

9. Oil Metabolic engineering
Main objectives are
• Increase content of ‘‘healthy’’ fatty acids and reduce
unhealthy’’ fatty acids.
• Improve oil stability to expand applications and reduce the
need for hydrogenation.
• Expand the repertoire of fatty acids through exploitation of
genetic diversity and enzyme engineering.

10. Nomenclature of FAs
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
CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2
Arachidonic acid )3COOH
cis,cis,cis,cis-Δ5Δ8,Δ11,Δ14 20:4
Eicosapentaenoic CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH cis,cis,cis,cis,cisΔ5,Δ8,Δ11,Δ14,
=CH(CH2)3COOH
20:5
acid Δ17
Erucic acid CH3(CH2)7CH=CH(CH2)11COOH cis-Δ13 22:1
Docosahexaenoic CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH cis,cis,cis,cis,cis,cis-
=CHCH2CH=CH(CH2)2COOH
22:6
acid Δ4,Δ7,Δ10,Δ13,Δ16,Δ19

11. Storage lipid or oil
• Triacylglycerols
• Triacylglycerols are contained primarily in seeds but also in
some fruits such as olives or avocados
• Fats and oils are an essential part of the human diet, on
average 25 kg per person per year, mostly (80%) from plant
sources.
Triacylglycerols containing three fatty acids are of a nonpolar nature.
(Slater et al.,2011)

12. Overview of Fatty Acid and Triacylglycerol
Biosynthesis in Plants

14. Fatty acid composition of oils from major oil crops
(Dyer et al.,2008)

15. Strategies for metabolic engineering in plants
• The production of more of a specific desired compound.
• The production of less of a specific unwanted compound.
• The production of a novel compound (i.e. a molecule that is
produced in nature.

16. Strategies for metabolic engineering
•Engineering of single step in a pathway to increase or decrease
metabolic flux to target compounds.
•To block competitive pathways or to introduce short cuts that
divert metabolic flux in a particular way.
•Metabolic engineering has been also used in an attempt to
increase starch yields.

17. 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.

18. General Overview

19. Production of medium chain fatty acids

20. Biochemical pathway for storage oils

21. Improvement of properties of oils
• Cooking oils generally contain a higher proportion of mono-
unsaturated FAs (oleic acid).
• Margarines and spreads are often rich in saturated fatty acids (e.g.
palmitic and stearic acids).
• Other oils, such as salad oils, contain more polyunsaturated FAs
(e.g. linoleic and α-linolenic acids).
• Production of oils for specific applications has been achieved by
mixing of various plant oils.
• Partial hydrogenation
(Ascherio.,2006).

22. Seed oil quality
• Fatty acid composition of storage triacylglycerols (TAGs).
• The number of double bonds.
• Degree of unsaturation in the fatty acyl moieties is the prime
determinant of melting point.
• Oxidative stability.
• Crystallization properties.
• Nutritional characteristics.
• Chain length.
• Presence of additional functional groups

23. Improvement of oil quality
• The production of oils with a high lauric acid (12:0) content in
Arabidopsis and rapeseed.
• Transgenic expression of a laurate-specific acyl-ACP
thioesterase gene from the California bay tree (Umbellularia
californica).
• Firstly in Arabidopsis and subsequently in rapeseed led to the
accumulation of over 50% laurate in the seed TAG.
• Analysis of triglycerides in the transgenic rapeseed oils,
however, revealed that laurate was present in high amounts.
• Poorly incorporated at sn-2. To overcome this limitation, the
gene for a laurate-specific LPAAT obtained from coconut.
(Wiberg et al., 2000)

24. Erucic acid improvement
• Breeding strategies applied to increase the content of
linolenic acid (18:3) from around 45% to over 65% in flax and
erucic acid (22:1) in rapeseed.
• Erucic acid in rapeseed is not efficiently esterified to the
center (sn-2) position.erucic acid in this species would
therefore be only 67% of total seed fatty acids.
• By overexpressing the condensing enzyme responsible for
erucic acid synthesis with an acyltransferase enzyme capable
of catalyzing the incorporation of erucic acid into the sn-2
position.
(Han et al., 2001)

25. VLC-PUFA
• (VLC-PUFAs) - arachidonic acid (20:4D5,8,11,14), eicosapentaenoic acid
(20:5D5,8,11,14,17) and docosahexaenoic acid (22:6D4,7,10,13,16,19).
• VLCPUFAs confer flexibility, fluidity and selective permeability to
cellular membranes, and may also be metabolized to produce lipid
signalling molecules such as eicosanoids.
• Respective VLC-PUFA derivatives referred to as omega-6 and omega-3
fatty acids as they contain double bonds located six or three carbons
from the methyl (omega) end of the fatty acids.
• The recommended ratio of omega6/omega3 fatty acids in the human
diet is approximately 2:1 to 6:1.
(Simopoulos, 2000; Wijendran and Hayes, 2004),

26. VLC-PUFAs synthesis

27. Applications of VLC-PUFAs
• VLC-PUFAs are found in many food applications, including infant
formulas, adult dietary supplements, animal feed and food
additives, and are used as precursors for the production of
pharmaceuticals.
• The world wholesale market for infant formula alone is estimated
to be valued at $10 billion per year (Ward and Singh, 2005).
• GLA is used in the treatment of skin conditions such as atopic
eczema as well as having possible anti-viral and anti-cancer
properties.
• Oral health supplement.
(Napier.,2000)

28. Metabolic role
Alonso et al.,2000

29. Fish oil
• Some mosses have been reported to contain AA and EPA ( Kaewsuwan et
al., 2006), but the main organisms responsible for producing the EPA and
DHA present in the human diet are marine microalgae (Carlsson et
al.,2007).
• The food chain to accumulate in fish oils
• An increased intake of this food has been recommended as a way towards
a more balanced ratio of omega6 to omega3 fatty acids. (Sargent and
Tacon, 1999)
• But various problems associated with commercial fish farming (Naylor et
al., 2000).
• Generation of plants that produce high amounts of ‘fish oil’-type fatty
acids

30. Engineering of Castor oil
• The low oxidative stability of vegetable oils can be improved
through chemical modification of plant oils such as castor bean
oil (Ricinus communis).
• This speciality oil for high-temperature applications (Schneider,
2006).
• Demand for castor oil is high, however cultivation of this crop is
restricted due to the presence of a toxin (ricin) and allergenic
proteins
• Cost of castor oil is relatively high.

31. Transgenic Rapeseed
• The first transgenic crop with a modified seed composition to
be approved for unrestrictive commercial cultivation in the
USA was a lauric oil rapeseed grown in 1995.
• Rapeseed, Brassica napus, is a species that is relatively
amenable to transformation and regeneration.
• Secondly, the metabolic pathways involved in storage oil
biosynthesis appeared at first to be well defined and
potentially straightforward to manipulate via single gene
insertions.
• Traditional rapeseed oil accumulates high amounts of erucic
acid (C22:1) comprising 45–50% of the total fatty acids.

32. Transgenic plants
• Suppression of the oleate D12-desaturase gene (which
normally converts 18:1 to 18:2) in soybean, sunflower, cotton
and canola has resulted in the production of oils with a high
oleic acid content, which have greater oxidative stability and
improved performance in high-temperature cooking
applications.
(Metzger and Bornscheuer, 2006).

33. Some genetically modified oils

34. Non-food applications
•It is estimated that about 14% of the fats and oils are used
chemically and 6% as feed material (Patel et al., 2006).
•In Europe where biodiesel is already a major fuel derived from
oils such as rapeseed, sunflower or palm Durrett et al. (2008).
•Industrial usage of plant oils is ‘soy ink’, which is produced from
soybean oil that is blended with pigments, resins and waxes to
make environmentally friendly printing inks (Erhan et al., 1992).

35. Non-food applications
• The market for lauric acid alone is estimated to be worth
more than $1.4 billion annually.
• Erucic acid is used to produce erucamide, which is used as a
slipping agent for production of extruded polyethylene and
propylene films such as shopping or refuse bags (Wang et al.,
2003).
• Global demand for erucic acid and the related behenic acid
(22:0) is expected to continue to increase, rising from 18 and
15 million tonnes in 1990 to 35 and 46 million tonnes,
respectively, by 2010 (Jadhav et al., 2005).

36. Derivatives Uses,Applications
Fatty acids and derivatives Metallic soaps,detergents,soaps,cosmatics,paints,
textile,leather and paper industries,rubber,
lubricants.
Fattty acid methyl esters Biodiesel,cosmetics,solvents,intermediates in the
production of alcohols.
Glycerol and derivatives Cosmetics,toothpaste,pharmaceuticals,food,paints,p
lastics,synthetic resins,tobacco,explosives,cellulose
processsing
Fatty alcohols and derivatives Detergents,cosmatics,textile,leather and paper
industries,duplicator stencils,petrolium additives.
Fatty amines and derivatives Surfectants,fabric softners,mining,road building,
biocides,textile and fiber industries,petrolium
additives
Drying oils Paints,varnish,linoleum
Castor oil,ricinoleic acid Polyamide 11,alkyd resins
(Patel et al.,2006)

37. Petroselinic acid
• Useful industrial raw material for polymer and detergent
manufacture.
• Hardening agent for margarines .
• Dietary studies in rats however, indicate that petroselinic acid
ingestion is associated with liver abnormalities and inhibition
of arachidonic acid biosynthesis.
• Indicates that such transgenic oil crops may be better
targeted initially to produce industrial, rather than edible
products.

38. • The species such as coriander produce a high percentage of
petroselinic acid upto 80% in their seed oils.
• A cDNA has been isolated from coriander that codes for an
acyl-ACP desaturase involved in petroselinic acid biosynthesis.
• Three enzymes involved in Biosynthesis.
• Acyl-ACP desaturase act on Palmitoyl-ACP
• 3-Ketoacyl-ACP synthase for elongation
• Acyl-ACP thioesterase for release.

39. Petroselinic acid structure and biosynthesis

40. • The production of GLA in Borage and Evening primose crops is more
than other oilseed crops.
• D6 fatty acid desaturase enzyme having conserved histidine boxes
essential for enzyme function.
• This gene driven by constitutive promotor cloned and introduced in
tobacco and in sunflower resulting in increase GLA content in oil.

41. PUFA Synthesis

43. Two approches
• A novel FatB thioesterase from Diploknema butyracea was
engineered into the B. juncea crop, driven by the seed-
specific napin promoter.
• The B. juncea fatty acid elongase was restricted at the genetic
level by incorporation of hair-pin RNA known to cause post-
transcriptional gene silencing.

44. T-DNA region and Binary vector pCAMBIA-1300

45. Pathway for Erucic acid

46. Results

48. Paper contd…
• Technique of hairpin RNA-mediated gene silencing to down-
regulate the seed expression of two key fatty acid desaturase
genes, ghSAD-1-encoding stearoyl-acyl-carrier protein 9
desaturase and ghFAD2-1-encoding oleoyl
phosphatidylcholine 6-desaturase.
• Hairpin RNA-encoding gene constructs (HP) targeted against
either ghSAD-1 or ghFAD2-1 were transformed into cotton
(Gossypium hirsutum cv Coker 315).

49. High-Stearic and High-Oleic Cottonseed Oils Produced by Hairpin RNA-Mediated Post-
Transcriptional Gene Silencing
Sixth largest source of vegetable oil
26% palmitic acid (C16:0),
3% Stearic acid (C18:0) 40%
15% oleic acid (C18:1), 77%
58% linoleic acid (C18:2)
Stearoyl-acyl-carrier protein (ACP) Δ9-
desaturase
oleoyl-phosphatidylcholine (PC)
ω6-desaturase
In addition, palmitic acid was significantly
lowered in both high-stearic and high-oleic
lines.
Sometimes hydrogenated to achieve the very
high stability required in deepfrying food

50. • Down-regulation of the ghSAD-1 gene substantially increased
stearic acid from the normal levels of 2% to 3% up to as high
as 40%
• Silencing of the ghFAD2-1 gene resulted in greatly elevated
oleic acid content, up to 77% compared with about 15% in
seeds of untransformed plants.
• By intercrossing the high-stearic and high-oleic genotypes, it
was possible to simultaneously down-regulate both ghSAD-1
and ghFAD2-1 to the same degree as observed in the
individually silenced parental lines.