Metabolic engineering for oil quality improvement

Pusadkar Pratik Prabodh

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

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.

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)

Growth rate for primary oilseeds

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)

Limitations

• 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.

• 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

oil. Need

Why Plant Oils Are Attractive Targets for Metabolic Engineering

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.

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 acidCH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2

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

Eicosapentaenoic acid

CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH

=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=CHCH2CH

=CHCH2CH=CH(CH2)2COOH

cis,cis,cis,cis,cis,cis-Δ4,Δ7,Δ10,Δ13,Δ16,Δ19

22:6

Nomenclature of FAs

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)

Overview of Fatty Acid and Triacylglycerol Biosynthesis in Plants

Fatty acid composition of oils from major oil crops

(Dyer et al.,2008)

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.

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.

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.

General Overview

Production of medium chain fatty acids

Biochemical pathway for storage oils

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).

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

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)

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)

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),

VLC-PUFAs synthesis

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)

Metabolic role

Alonso et al.,2000

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

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.

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.

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).

Some genetically modified oils

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).

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).

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,plastics,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)

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.

• 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.

Petroselinic acid structure and biosynthesis

• 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.

PUFA Synthesis

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.

T-DNA region and Binary vector pCAMBIA-1300

Pathway for Erucic acid

Results

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).

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) 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. Sometimes hydrogenated to achieve the very high stability required in deepfrying food

• 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.