Genetic engineering for the production of unusual fatty acids in seeds. Plants synthesize more than...

Genetic engineering for the production of unusual fatty acids in seeds.

• Plants synthesize more than 300 different fatty acids. The fatty acids found in glycerolipids (in membranes) resemble those found in animal cells. (But plants have different glycerolipids!)

• Unusual fatty acids accumulate only in seeds as triglycerides (TAGs) in oil bodies.

• Most plants store fatty acids of different length (C12 to C18) and fatty acids can have one (oleate), two (linoleate), three (linolenate) or four unsaturated bonds.

• Linoleic and linolenic acid are “essential” in the human diet• Some plants store unusual (e.g. hydroxylated) fatty acids that

are of interest to industry as feed stocks or lubricants. Plant oils have many industrial applications

What is the problem with the fats and oils in our diet?

1. Too many saturated fats (cheese, butter, meat)

2. Too many trans-fatty acids (TFAs) (margerine)

3. Not enough -3 fatty acids

4. Too much oxidized fat from deep fried foods

(Reactive oxygen species = ROS)

5. Too little exercise considering fat intake!

Too much fat and poor fatty acid profile cause obesity,

increased atherosclerosis, coronary heart disease and cancer.

The wrong-3/6 ratio can cause inflammation, asthma and other health problems.

QuickTime™ and aTIFF (Uncompressed) decompressor

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Fats, fatty acids and human health

French fries = papas fritas = everyone’s

favorite food Fry them in lard? Ugh!

Fry them in hydrogenated plant oil? Eat TFAs!Fry them in plant oil? Heat degrades and oxidizes the poly-

unsaturated fatty acids (PUFAs) creating free radicals. Fry them in tri-oleate fat (olive oil) would be good.

Don’t eat too many would be the best!





Early in the XVth Century, the Flemish painter Jan van Eyck combined linseed (or flax) oil

(Linum usitatissimum) with pigment particles and ground-up bones to

produce fast drying paint. This paint dries fast because linseed oil is high (58%) in linolenic acid (18:3) which

becomes oxidized. In this case oxidative instability of an oil is a

benefit. Other industrial applications also require fast-drying

oils.Soybeans transformed with FAD3 have 50 % 18:3, vs. 10 % in the

control.

Vegetable oils are used in industry

Vegetable oils are promising candidates as base fluid for eco-friendly lubricants because of their excellent lubricity, biodegradability, viscosity–temperature characteristics and low volatility. Their use, however, is restricted due to low thermo-oxidative stability (unsaturated bonds are oxidized) and poor cold flow behavior.

In animals and plants carbohydrates are converted to fatty acids via Acetyl-CoA!

**

Acetyl-CoA is central to all of metabolism. Glycolysis generates pyruvate which enters the mitochondria, is decarboxylatedto acetate and enters the TCA cycle as acetyl-CoA. Catabolism of acetyl-CoAgenerates energy for your workout in the gym.Acetate can also remain in the cytosol where it forms acetyl-CoA andis carboxylated to malonyl-CoA, the building block of fatty acids, which accumulate in your adipose cells!

High fructose corn syrup

Adipose tissue! The entire cell is filledwith fat (triglyceride)





C18 fatty acid structures

Stearic acid 18:0 Saturated. Present abundantly in butter and meat fat.

Oleic acid 18:1 One double bond, one kink Oleum (L) = oil

Linoleic acid 18:2 Two double bonds, two kinks. Linoleic acid is a polyunsaturated fatty acid or PUFA. Linum = flax

Linolenic acid 18:3 Three double bds; Also a PUFA, with three kinks (not shown) There are two forms, alpha and gamma,depending on the location of the ==

You need quite a bit of kinkiness in the fatty acids to make cell membranes dynamic!

If all the fatty acids were saturated they would be straight and closely packed and the membranes would have a high melting

temperature. Unsaturated bonds (kinked fatty acids) prevent close packing and increase membrane fluidity. Even the position of the

unsaturated bond (right) influences the melting temperature


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Melting temp of phosphatidyl cholinewith two similar C18 fatty acids

When poly-unsaturated fatty acids are hydrogenated at high temp (200 C) with a Ni catalyst, some of the cis hydrogen atoms are converted to trans hydrogens (without reducing the double bond), causing the formation of linear molecules that behave

like saturated fatty acids in lipid membranes. Nutritionally they also have the same effects: they increase total plasma cholesterol levels and LDL cholesterol.

We know that TFAs are detrimental to our health; manufacturers are now requiredto label the TFA content of foods.





C18:1 = Oleic acid

C18:1 = Elaidic acid = TFA

Double bond with trans hydrogen atoms

What are trans fatty acids (TFAs)?

cis

-3 and -6 fatty acids are members of the PUFA family. The terms are derived from the position of the double bond in

the carbon chain. The carbon atom furthest away from the carbonic acid

group is labeled “omega” () (it is at the end of the molecule). The numbers 3 and

6 denote the number of the C-atom, counted from the C-atom, at which the first double bond occurs. Linoleic acid,

- linolenic acid and arachidonic acid are -6 fatty acids, whereas linolenic acid,

EPA, and DHA are -3 fatty acids. Humans and mammals cannot synthesize -3 or -6 fatty acids, and linoleic acid,

and-linolenic acid, which can be converted to the various other -3 and -

6 fatty acids that humans need, are essential fatty acids.

Essential fatty acids: Omega-3 (-3) and omega-6 (-6) fatty acids

-6

-3

Myristic 14:0

Palmitic 16:0

Stearic 18:0

Oleic 18:1

Linoleic 18:2

Linolenic 18:3

Linolenic 18:3

Arachidonic 20:4

*

*

Eicosapentaenoic (EPA) 20:5

Docosohexaenoic (DHA) 22:6

-6

-3

Humans cannot introduce unsaturated bonds at the -3 and -6 positions because they lack the requisite enzymes. They do have desaturases for -9 and

carbons closer to the carboxyl end of the fatty acids

-3-6

-9

Oleic acid 18:1 -9Methyl-end

Carboxyl-end

= omega, means “the last”, or methyl carbon

Flax has the highest -linolenic acid ( -3); sunflower and safflowercorn, soy and cotton are high in linoleic acid, which readily oxidizes in cooking; olive and canola are high in oleic, a mono-unsaturate. Canolahas an excellent balance. Palm and coconut are too high in saturates.Add some flaxseed oil to the olive oil for salad dressing or use canola!

2:1

(-3)

(-6)

Fatty acids of the -3 and -6 series have multiple and different functions.

1. Major structural components of membrane phospholipids, esp. in the brain. C22:4 and C22:6 (DHA) make up 30 % of fatty acids in brain phospholipids. DHA accounts for 60 % of fatty acids in the outer rod segments. These -3 fatty acids are made from -linolenic acid (-3), but conversion in infants and old people may be too slow to meet demand. This may result in poor synapse formation, poor brain development and memory loss in the elderly.

• Eicosanoids are signaling molecules (prostaglandins). Discovered as active molecules in human seminal fluid (made by the prostate gland). There are -3 and -6 prostaglandins; prostaglandins have anti and pro-inflammatory functions. Since inflammation is both good and bad, we need both.

EPA 20:5

DHA 22:6

Humans make a number of molecules derived from the essential -3 and -6 fatty acids in our diet. To do this correctly we need

the right ratio of -3 and -6. In the US diet only 5 % of the PUFAs are -3 (-linolenic acid) and 95 % are -6

(linoleic acid). For an optimal diet we need 25 % to 33% of dietary

PUFAs as -3 (-linolenic acid). To change the ratio we should increase -3 and decrease -6.

We need -3 fatty acid (-linolenic acid) to synthesize EPA (eicosa-penta-enoic acid 20:5) and DHA (docosa-hexa-enoic acid 22:6), necessary for brain

development, and to promote cardiovascular health.

Omega-3 Deficiency Implicated in CVD

Ra

tio d

ieta

ry o

me

ga

-6:3 4:1

80:1

Japan

UK

US Diet

Canada

Recommendations are for a ratio of 4:1 omega-6 to omega-

3

CV deaths/100,00050 200

EPA and DHA are not found in the seeds of oil crops. They are synthesized by plankton and lower organisms and accumulate in thefood chain and are therefore found in fish oils. Eating more fish helps to increase -3 fatty acids in the diet. However farmed fish is low in -3 unless the fish diet has been fortified with fish meal, algae or with plants (e.g. flax seeds) that contain them.

Structure of DHA (22:6)

In a small vesicle, the inner lipid leaflethas more room for the hydrophobic fattyacid tails and the outer leaflet has moreroom for the polar headgroups of phospho-lipids, e.g. phosphoinositides.

The battle of the omega-3s: plants vs marine sources

www.chiro.org/nutrition/FULL/Marine_vs_Veggie_Omega-3.html

Cold water fish have oils rich in omega-3 (but some also have Hg!)

The micro-alga Crypthecodinium cohnii grown in fermentors yields an oil rich in omega-3 that is approved in the USA

Flax seed oil has 50% -linolenic and 18:4 stearidonic acid which are both -3 and are readily converted to EPA and DHA

The “essential” fatty acids in our diet are precursors needed for the synthesis of different prostagladins.

-6 family Arachidonic acid

18:2 -> 18:3 -> 20:3 -> 20:4 -> 22:4 -> 22:5

PGE1 PGE2

-3 family (EPA) (DHA) 18:3 -> 18:4 -> 20:4 -> 20:5 -> 22:5 -> 22:6

PGE3 PGs PGs

Biosynthesis in each pathway depends on the other one, because the -3 elongasesand desaturases are inhibitedby the -6 fatty acids. That iswhy you need to eat them inthe correct ratio.

Stearidonicacid

Liver enz.

Pro-inflammatory Anti-inflammatory

Prostaglandin synthesis pathway.

COX

POX

-6

-6

-3

The -6 family can give rise to pro and anti inflammatory prostaglandins.

Oils accumulate in seeds in special organelles called oil bodies or oleosomes. They have a single

phospholipid layer into which special proteins (oleosins) are embedded.

Fatty acid, triglyceride and phospholipid synthesis involve the

chloroplast and the ER.

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Oil body

oleosin

Oil body

Oil bodies are ER-derivedtriglyceride storage

compartments.

Suppression of oleosin synthesis creates giant oil bodies (Moloney laboratory)

Suppressed Control

Fatty acid synthesis is a multistep process.Step 1 takes place in the chloroplasts

Acetyl CoA carboxylase (ACCase) catalyzes the first committed step in fatty acid synthesis. It carboxylates Acetyl CoA with CO2 to give malonyl-CoA,

which is then transferred to acyl carrier protein (ACP). Multiple cycles elongate the fatty acid, two carbon atoms at a time, each time using one malonyl CoA.

The first condensation will produce butyryl CoA. The multiple cycles are catalyzed by three ketoacyl-ACP synthases (KAS I, II and III) that synthesize

(elongate) fatty acids of different lengths.

Three ketoacyl ACP synthases are required for fatty acid synthesis.

KAS III adds the first acetyl group from acetyl-CoA to malonyl-ACP, which afterreduction, dehydration and further reduction (by 3 different enzymes) becomes butyryl-ACP. Subsequent elongation steps by KAS III utilize malonyl-ACP and make keto acyl-ACP up to C10-C12.

KAS I prefers C10 - C12 as substrate for further elongation up to C16-ACP

KAS II elongates C16-ACP to C-18ACP.

The synthesis of a C18 fatty acids involves 48 steps carried out by 12 different proteins.It is likely that these proteins are organized in a supramolecular complex to provide metabolic channeling for more efficient synthesis.

KAS III

Reductase

Dehydratase

Reductase

KAS III

Acetyl CoA-carboxylase

Step 2: FAB1 and FAB2: Desaturation takes place in the chloroplasts and the ER

The first desaturation step takes place in the chloroplast and uses ACP-bound fatty acids; the substrate is ACP-18:0 (stearic) and the product is ACP-18:1 (oleic) for FAB2 and the substrate is ACP-

16:0 (palmitic acid) for FAB1. Other desaturation steps can occur in the chloroplast after incorporation of the FA into a

phospholipid. These fatty acids then stay in the chloroplasts.



Fatty acid synthesis in the chloroplast is terminated by the

activity of a fatty acid specific acyl-ACP-

thioesterase. The major thioesterase are shown.

The enzyme transfers the fatty acid from the Acyl Carrier Protein (ACP) to Coenzyme A and Acyl-CoA is exported to the

cytoplasm.



Found inSeeds of Cuphea orcoconut

Found inall species

Step 3: Detaching the fatty acyl goups from ACP and export to the ER

High laurate canola made by over-expression of 12:0 lauryl:ACP thioesterase in the chloroplasts.

Overexpression interrupts the elongation pathway.



Isolation of Arabidopsis mutants that are deficient in fatty acid desaturation led to the identification of desaturase genes and

the enzymes they encode.

1. FAB 1 and 2 are soluble chloroplast enzymes that act on ACP-16:0 and ACP-18:0. The desaturated fatty

acids are exported to the ER as CoA derivatives.2. FAD 2 and 3 are ER bound enzymes that use a

fatty acyl moiety that is part of phosphatidyl choline as their preferred substrates

3. FAD 4-8 are chloroplast enzymes that use different glycerolipids as their preferred substrates. These desaturated fatty acids remain in the chloroplasts.

FAB = fatty acid biosynthesis FAD = fatty acid desaturation.

Terminology confusion: two terminologies are in use to denote the placement of the unsaturated bond. counts from the methyl carbon, but counts from the carboxyl

carbon. So, oleic acid is both -9 and -9, because there are 18 carbons.

-3-6

-9

Oleic acid 18:1 -9 or -9Methyl-end

Carboxyl-end

= omega, means “the last”, or methyl carbon

-12

-15

Step 4: Chloroplasts export 16:0, 16:1, 18:0 and 18:1 as CoA derivatives to the ER, which imports 3-phosphoglycerate to make glycerolipids,

especially phosphatidyl choline (PC). It is at this stage that FAD 2 and FAD 3 introduce more double bonds.

PC with two 18:1 fatty acids is the substrate for FAD2 and this produces PC with one or two 18:2, which is the substrate for FAD3.

Fatty acid desaturation occurs in the chloroplast and the ER on different types of

substrates In the first step, 18:0-ACP is desaturated to

18:1-ACP9 (oleate) by a soluble chloroplast desaturase. Subsequent steps occur in the ER by membrane-bound enzymes after the fatty acid

has been incorporated into a phospholipid.The first step creates linoleate (9,12) and the

second step linolenate (9,12,15).

Summary of desaturation

Formation of oil bodies in developing oil seeds involves the synthesis of oleosins on the ER and synthesis of triacylglycerides

(TAGs) in the ER. Oil bodies bud off from the ER. TAGs are formed from a pool of acyl-CoA in the ER (after transfer from

the glycerolipid back to CoA!). The first step of TAG biosynthesis is

transfer of fatty acyl group to glycerol-3-phosphate to create lyso-phosphatidic acid and then in second step phosphatidic acid. Now the phosphate is removed to create

diacyl glycerol. A final transfer after possible elongation and desaturation

creates TAGs. All positions called sn1, sn2 and sn3 are not equivalent in their

reactivity.

Step 5: Biosynthesis of triacylglycerols (oils): fatty acids are transferred from Acyl-CoA moieties to acceptors.

All three final steps havebeen documented in plants

G3PAT

LPAAT

PAP

DAGAT

Glycerol-3-phosphate acyl transferase

Lysophospatidic acid acyl transferase

Phospatidic acid phosphatase

Diacylglycerol acyl transferase

TAG



Plants cells have two types of Acetyl-CoA-Carboxylases. The chloroplast enzyme elongates up to C18. The cytosolic enzyme supplies malonyl-CoA for many reactions, including

fatty acid elongation beyond C18 in the ER.









Goals of breeding/genetic engineering

More oilEliminate toxic oils (erucic acid) Lower saturates (more healthy)

Less PUFAs (more stable to heat)More -3 (stearidonic acid et al)

Breeding of canola eliminated erucic acidTraditional breeding eliminated erucic acid (22:1) from rape seed and the

resulting cultivars are called “canola”. Canola is the main oilseed crop in Europe and Canada. The seeds have 45 % oil.

HEAR = High erucic acid rape LEAR = Low erucic acid rapeWhether erucic acid is really toxic is not clear because rape seed oil is widely used

in India as a food source.



Overexpression of ACCase in the plastids (with a transit sequence) of canola with a seed specific promoter caused increase in oil content of the seeds from 35.5% to 37.5%.


are needed to see this picture.*

*

High laurate canola was produced by transformation with the gene for 12:0-ACP thioesterase, the chloroplast enzyme that cleaves the fatty acid from the ACP before

the fatty acid leaves the chloroplasts. In the transformants, the sn-1 and sn-3 positions of triglycerides had up to 60 % laurate, but the sn-2 position had only up

to 15 %. This shows that LPAAT of canola strongly discriminates against this medium chain fatty acid not found in canola. Some tropical oils (coconut) have

medium chain fatty acids at the sn-2 position. So, plants transformed with the ACP-thioesterase gene and the coconut LPAAT gene have up to 75 % laurate at the sn-2,

and have 67 % overall laurate. (Knutzon et al., (1999) Plant Phys 120, 739, Calgene)



sn-2

In canola transformed with the ACP thioesterase, 58 % of the fatty acids were laurate (C12). But, of the fatty acids initially

synthesized, 40 % were broken down because fatty acid degradation enzymes were induced in the glyoxysomes, setting up

a futile cycle.

Changing the level of desaturation and avoiding trans fatty acids (TFAs)

Schematic diagram of biosynthetic pathway for the major saturated, monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids in oilseeds and their key nutritional and functional properties.

Contain TFAs whenhydrogenated.

Engineering of High-Oleic Soybean Oil by Suppression of 18:1 Desaturase

(composition now similar to olive oil with more 18:1 and less 18:2 and 18:3; no need to hydrogenate)

010

2030

4050

60

7080

90

Mol %

'16:0 '18:1 '18:2 '18:3

Fatty Acid

ControlTS-Fad2

Manipulation of a single gene increases oleic, reduces saturates,reduces polyunsaturates. Similar results were obtained by screening 1000s of seeds for a FAD2 mutant. Data of Kinney et al., DuPont

Many of these products are based on genetically enhanced high oleic (HO) soybean oils, with unmatched stability, producing significant environmental

advantages over conventional industrial lubricants. ELM's products are derived from a renewable resource. They biodegrade quickly in the

environment, are less toxic than conventional industrial lubricants, require less energy in production and reduce dependence on imported oil.

Improved Soybean Oil Composition for Food

Standard Soybean

Low Lin

Linoleic18:2

Oleic18:1

Sats18:0 / 16:0

Linolenic18:3

Mid Oleic + Low Lin

Zero Sat + MO + LL

Eliminate Trans-fats

Breeding

Low LinolenicSoy Oil Improve Flavor & Shelf

Life with Oxidative Stability

Breeding

Mid Oleic and Low Lin Reduce Saturated Fat

and Improve Cardiovascular

Health

Transgenic

Low Saturates with Mid Oleic and Low Lin

New Monsanto Product Pipeline:

2006 2008 2011

Omega-3 Canola and Soybean oil

Scientific Progress Multi-gene expression Soybean transformation Omega-3 oil production

Omega-3 Deficiency Implicated in CVD

Mono-unsaturates

Saturates

Other poly-unsaturates

New bio- available Omega-3

0

10

20

30

40

50

60

70

80

90

100

Control Biotech Control Biotech

Canola Soybean

Fa

tty

Aci

d (

wt%

)

Steari-donic acid 18:4

Echium oil, derived from the seeds of Echium Plantagineum, is enriched in stearidonic acid (SDA; 18:4 -3), whichis the immediate product of -6desaturation of 18:3 -3 to 18:4

Non-transgenic -3

The -6 desaturase canadd a double bond in the -6 (= -12) position, 6

carbons from the carboxyl end. This converts 18:2

linoleic into 18:3 gamma linolenic in the -6 family.

The elongase adds two carbons to the carboxyl end to make 20:3 and shifting the

numbers by two . The -5 desaturase can now make a double bond 5 carbons from the carboxyl end to produce

20:4 (arachidonic acid)

Desaturation and elongation of -6 C18:2 and -3 C18:3 fatty acids in

lower organisms, mammals and some plants.

The -6 desaturase adds a double bond in the

-6 (= -12) position, 6 carbons from the carboxyl end. This converts 18:2

linoleic into 18:3 - linolenic in the -6 family.

The elongase adds two carbons to the carboxyl end to make 20:3 and shifting

the numbers by two . The -5 desaturase can now make a double bond

5 carbons from the carboxyl end to produce 20:4 (arachidonic acid).

A similar reaction sequence takes place for the -3 18:3 fatty acid and

generates first stearidonic acid (18:4) and then 20:5 EPA.

Schematic representation of the substrate-dichotomy bottleneck: desaturation and elongation use different fatty acyl substrates.

In plants, desaturation uses glycerolipid-linked substrates, whereas fatty acid elongation

requires acyl-CoA substrates inall organisms. The exchange of the

fatty acids between phospholipids and the acyl-CoA pool is an

enzyme-mediated process (via acyltransferases). Non-native fatty

acids (i.e., the products of transgenic VeryLongChain-PUFA activities) may not be efficiently

exchanged between these two metabolically active pools. This

complicates genetic engineering for VLC-PUFAs

Genetic engineering of seeds of tobacco and linseed for the

production of ARA (20:4) and EPA (20:5)

Abbadi et al, Biosynthesis of Very-Long-Chain Polyunsaturated Fatty Acids in Transgenic Oilseeds: Constraints on Their Accumulation. The Plant Cell 16:2734-2748 (2004)

Genes were selected from a wide variety of VLCPUFA-producing organisms such as fungi (Mortierella alpina), algae (Phaeodactylum tricornutum), mosses (Physcomitrella patens), plants (Borago officinalis) and lower animals (Caenorhabditis Elegans). Four different combinations of these sequences were placed in binary vectors and used for trans-formation of N. tabacum (high in linoleic acid, 18:2(9,12) and Linum usitatissimum (high in -linolenic acid, 18:3(9,12,15) to identify a useful combination and a suitable expression host.

In tobacco, 18:2 was converted to 18:3 quite

efficiently, but very little20:3 and 20:4 accumulated,

suggesting that the 6 desaturase was active but the next two enzymes had

minimal activity. In linseed, 18:3

accumulated as well as 18:4, but little of the higher

products (EPA = 20:5) was formed and no DHA (22:6)

Enzyme assays of seed extracts showed that all 3 enzymes were present and

equally active.

Is there sufficient C18:2 Acyl CoA in the cytosol to permit elongation to be carried out?

Profiles of Acyl-CoAs from Developing Wild-Type and Transgenic Linseed.

Immature seeds were harvested at 14, 24, and 35 DAF from wild-type (A) and transgenic (B) linseed expressing construct C and subjected to acyl-CoA extraction. The acyl-CoAs were converted to their etheno-derivatives and analyzed by HPLC.

The substrates for the elongase are not detectable in the cytosol, so they must be absent or present at very low levels.

Analysis of the fatty acids on phosphatidylcholine (PC) showed that PC was enriched inthe various higher fatty acids (more desaturated and longer) as was the TAG pool. This is best explained by channeling: once the acyl group is desaturated it is not necessarily detached by the LPCAT but channeled directly into TAGs by PDAT.

Genetic engineering of seeds of Brassica juncea for the

production of EPA (20:5) and DHA (22:6)

DHA

Adding more genes from different organisms permits

synthesis of very small amounts of DHA in Brassica juncea.

Nature Biotechnology 23, 1013 - 1017 (2005)Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acidsin plants. G Wu et al.

The two routes begin with linoleic acid (18:2n-6, LA) and -linolenic acid (18:3n-3, ALA), respectively, followed by sequential 6 desaturation, 6 elongation and 5 desaturation, leading to the synthesis of arachidonic acid (20:4n-6, AA) in the n-6 and eicosapentaenoic (20:5n-3, EPA) in the n-3 pathway. The two pathways can be interconnected by a omega3 desaturase that converts AA into EPA. Further Delta5 elongation and 4 desaturation reactions lead to the synthesis of docosapentaenoic (22:5n-3, DPA) and finally docosahexaenoic acid (22:6n-3, DHA).

The first construct (BJ3) introduced into Brassica juncea contained a 6 desaturase from Pythium irregulare, a 5 desaturase from Thraustochytrium sp. and a 6 fatty acid elongase from Physcomitrella patens. This represents the minimal set of transgenes required for the synthesis of AA and EPA from endogenous LA and ALA.

To increase LA and reduce the side-product 18:2n-9, we added a 12 desaturase gene from Calendula officinalis 15 to the triple construct (BJ4)

We attempted to enhance elongation by adding a second 6 elongase from Thraustochytrium sp. But the effect was minimal.

To use these n-6 fatty acids for the production of n-3 fatty acids, we included a omega3 desaturase from Phytophtora infestans in the construct BJ6. The omega3 desaturase also effectively converted AA into EPA in transgenic seeds. As a result, the EPA content increased significantly, from an average of 1.4% in BJ5 to 8.1% in BJ6 plants,

Three more genes were added to BJ6, creating the nine-gene construct BJ9. An elongase from Oncorhynchus mykiss that can elongate both 18- and 20-carbon fatty acids, a 4 desaturase from Thraustochytrium sp. The third gene, also from Thraustochytrium sp., represents a putative lysophosphatidic acid acyltransferase.

Eicosa-penta-enoic Acid and Docosa-hexa-enoic Acid (EPA and DHA) These two difficult -to-pronounce fatty acids are responsible for t he beneficial effects of fish oils. EPA produces eicosanoids that have many beneficial effects in the body. Research demonstrates that fish oils conta ining EPA and DHA have therapeutic benefits in areas inclu ding: rheumatoid arthritis, high blood triglycerides, high blood pressure, cardiac arrhythmia (irregular heart beat), infant brain development, and cancer. Arachidonic Acid (AA) AA is necessary for the infant brain development and small amounts are required for overall fetal development. However, it is not generally deemed a "good" fat, because, in excess, AA may have som e harmful effects. AA is produced in the body from linoleic acid. It is also f ound in meat, eggs, and some shellfish. The body uses AA to pr oduce a class of eicosanoids that are strongly pro-inflammatory, constrict our blood vessels, and increase the possibil ity of b lood clotting. These compounds are very useful when you accidentally cut your skin while peeling p otatoes - without them you would bleed t o death. But once you have an excessive amount of these eicosanoids, the blood can clot in places you don't really want it to – for example, arteries.

Linoleic Acid (LA) Omega 6,9 Referred to as omega-6LA is found in processed foods, margarine, and vegetable oils. LA helps improve skin conditions. It may also be partially converted to GLA in the body (see more on GLA below). The typical North American diet includes an excess of LA.

Alpha Linolenic Acid (ALA) omega 3, 6, 9 Referred to as omega-3ALA is found primarily in flax seed oil. The positive effects of ALA have been documented in areas including: high blood cholesterol, high blood pressure, immune system function, male infertility and cancer. The body also converts a portion of ALA into two other fatty acids, EPA and DHA (see below).

Gamma Linolenic Acid (GLA) omega 6, 9, 12A healthy body may derive some GLA from LA (see above).The richest natural source of GLA is borage oil. The body uses GLA to produce eicosanoids that are highly anti-inflammatory, dilate blood vessels, and reduce blood clotting. GLA is popularly used by women suffering from PMS. However, GLA has been clinically indicated to have therapeutic benefits in many other health conditions including: rheumatoid arthritis, cardiovascular disease, diabetic neuropathy, cancer, and skin diseases such as eczema and psoriasis.

Genetic engineering for the production of unusual fatty acids in seeds. Plants synthesize more than...

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