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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!)

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

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    1. 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

    2. Fats, fatty acids and human health What is the problem with the fats and oils in our diet? Too many saturated fats (cheese, butter, meat) Too many trans-fatty acids (TFAs) (margerine) Not enough w-3 fatty acids 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w-3/w6 ratio can cause inflammation, asthma and other health problems.

    3. French fries = papas fritas = everyone’s favorite foodFry 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!

    4. 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.

    5. 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.

    6. 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 decarboxylated to acetate and enters the TCA cycle as acetyl-CoA. Catabolism of acetyl-CoA generates energy for your workout in the gym. Acetate can also remain in the cytosol where it forms acetyl-CoA and is 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 filled with fat (triglyceride)

    7. C18 fatty acid structuresStearic acid 18:0 Saturated. Present abundantly in butter and meat fat.Oleic acid 18:1 One double bond, one kink Oleum (L) = oilLinoleic acid 18:2 Two double bonds, two kinks. Linoleic acid is a polyunsaturated fatty acid or PUFA. Linum = flaxLinolenic 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!

    8. 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 Melting temp of phosphatidyl choline with two similar C18 fatty acids

    9. What are trans fatty acids (TFAs)? 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. C18:1 = Oleic acid We know that TFAs are detrimental to our health; manufacturers are now required to label the TFA content of foods. cis Double bond with trans hydrogen atoms C18:1 = Elaidic acid = TFA

    10. Essential fatty acids: Omega-3 (w-3) and omega-6 (w-6) fatty acids w-3 and w-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” (w) (it is at the end of the molecule). The numbers 3 and 6 denote the number of the C-atom, counted from the w C-atom, at which the first double bond occurs. Linoleic acid, - linolenic acid and arachidonic acid are w-6 fatty acids, whereas linolenic acid, EPA, and DHA are w-3 fatty acids. Humans and mammals cannot synthesize w-3 or w-6 fatty acids, and linoleic acid, and a-linolenic acid, which can be converted to the various other w-3 and w-6 fatty acids that humans need, are essential fatty acids. Myristic 14:0 Palmitic 16:0 Stearic 18:0 Oleic 18:1 w-6 Linoleic 18:2 * w-6 g  Linolenic 18:3 * Arachidonic 20:4 w-3 a  Linolenic 18:3 Eicosapentaenoic (EPA) 20:5 w-3 Docosohexaenoic (DHA) 22:6

    11. 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  = omega, means “the last”, or methyl carbon -9 -6 -3 Carboxyl-end Methyl-end Oleic acid 18:1 -9

    12. 2:1 (-6) (-3) Flax has the highest a-linolenic acid ( -3); sunflower and safflower corn, soy and cotton are high in linoleic acid, which readily oxidizes in cooking; olive and canola are high in oleic, a mono-unsaturate. Canola has 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!

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

    14. Omega-3 Deficiency Implicated in CVD Japan Recommendations are for a ratio of 4:1 omega-6 to omega-3 4:1 80:1 Canada UK Ratio dietary omega-6:3 US Diet 50 200 CV deaths/100,000 Humans make a number of molecules derived from the essential w-3 and w-6 fatty acids in our diet. To do this correctly we need the right ratio of w-3 and w-6. In the US diet only 5 % of the PUFAs are w-3 (a-linolenic acid) and 95 % are w-6 (linoleic acid). For an optimal diet we need 25 % to 33% of dietary PUFAs as w-3 (a-linolenic acid).To change the ratio we should increase w-3 and decrease w-6.We need w-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. EPA and DHA are not found in the seeds of oil crops. They are synthesized by plankton and lower organisms and accumulate in the food chain and are therefore found in fish oils. Eating more fish helps to increase w-3 fatty acids in the diet. However farmed fish is low in w-3 unless the fish diet has been fortified with fish meal, algae or with plants (e.g. flax seeds) that contain them.

    15. Structure of DHA (22:6) In a small vesicle, the inner lipid leaflet has more room for the hydrophobic fatty acid tails and the outer leaflet has more room for the polar headgroups of phospho- lipids, e.g. phosphoinositides.

    16. The battle of the omega-3s: plants vs marine sources 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 w-3and are readily converted to EPA and DHA

    17. The “essential” fatty acids in our diet are precursors needed for the synthesis of different prostagladins. -6 familyArachidonic acid 18:2 -> 18:3 -> 20:3 -> 20:4 -> 22:4 -> 22:5 PGE1PGE2 Liver enz. Biosynthesis in each pathway depends on the other one, because the -3 elongases and desaturases are inhibited by the -6 fatty acids. That is why you need to eat them in the correct ratio. -3 family (EPA) (DHA) 18:3 -> 18:4 -> 20:4 -> 20:5 -> 22:5 -> 22:6 PGE3 PGs PGs Stearidonic acid Pro-inflammatoryAnti-inflammatory

    18. Prostaglandin synthesis pathway. COX POX

    19. w-6 w-6 w-3

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

    21. Oil bodies are ER-derived triglyceride storage compartments. 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. Oil body Oil body oleosin

    22. Suppression of oleosin synthesis creates giant oil bodies (Moloney laboratory) Suppressed Control

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

    24. Three ketoacyl ACP synthases are required for fatty acid synthesis. KAS III adds the first acetyl group from acetyl-CoA to malonyl-ACP, which after reduction, 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.

    25. Acetyl CoA-carboxylase KAS III KAS III Reductase Reductase Dehydratase

    26. Step 2: FAB1 and FAB2: Desaturation takes place in the chloroplasts andthe ERThe 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.

    27. Step 3: Detaching the fatty acyl goups from ACP and export to the ER 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 in Seeds of Cuphea or coconut Found in all species

    28. High laurate canola made by over-expression of 12:0 lauryl:ACP thioesterase in the chloroplasts.Overexpression interrupts the elongation pathway.

    29. 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 substrates3. 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.

    30. 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.  = omega, means “the last”, or methyl carbon -9 -12 -6 -15 -3 Carboxyl-end Methyl-end Oleic acid 18:1 -9 or -9

    31. 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.

    32. Fatty acid desaturation occurs in the chloroplast and the ER on different types of substratesIn the first step, 18:0-ACP is desaturated to 18:1-ACPD9 (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 (D9,12) and the second step linolenate (D9,12,15). Summary of desaturation

    33. 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.

    34. Step 5: Biosynthesis of triacylglycerols (oils): fatty acids are transferred from Acyl-CoA moieties to acceptors. G3PAT Glycerol-3-phosphate acyl transferase LPAAT Lysophospatidic acid acyl transferase PAP Phospatidic acid phosphatase All three final steps have been documented in plants DAGAT Diacylglycerol acyl transferase TAG

    35. 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.

    36. Goals of breeding/genetic engineeringMore oilEliminate toxic oils (erucic acid) Lower saturates (more healthy)Less PUFAs (more stable to heat)More w-3 (stearidonic acid et al)

    37. 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.

    38. 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%. * *

    39. 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

    40. 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.

    41. 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 when hydrogenated.

    42. 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) 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

    43. 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.

    44. Low Saturates with Mid Oleic and Low Lin New Monsanto Product Pipeline: Mid Oleic and Low Lin Reduce Saturated Fat and Improve Cardiovascular Health Transgenic Low Linolenic Soy Oil Improve Flavor & Shelf Life with Oxidative Stability Breeding Eliminate Trans-fats Breeding 2011 2008 2006 Linolenic 18:3 Linoleic 18:2 Sats 18:0 / 16:0 Oleic 18:1 Standard Soybean Low Lin Mid Oleic + Low Lin Zero Sat + MO + LL Improved Soybean Oil Composition for Food

    45. Canola Soybean 100 New bio- available Omega-3 90 80 70 Other poly-unsaturates 60 Fatty Acid (wt%) 50 40 Mono-unsaturates 30 20 Saturates 10 0 Control Biotech Control Biotech Omega-3 Deficiency Implicated in CVD Omega-3 Canola and Soybean oil • Scientific Progress • Multi-gene expression • Soybean transformation • Omega-3 oil production Steari-donic acid 18:4 Non-transgenic -3 Echium oil, derived from the seeds of Echium Plantagineum, is enriched in stearidonic acid (SDA; 18:4 -3), which is the immediate product of -6 desaturation of 18:3 -3 to 18:4

    46. 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)

    47. 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.

    48. 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

    49. Genetic engineering of seeds of tobacco and linseed for the production of ARA (20:4) and EPA (20:5) 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. 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)

    50. 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.