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Metabolic Engineering for Healthy Oil Traits: From Lab-to-Field-to-Functional Evaluation

Metabolic Engineering for Healthy Oil Traits: From Lab-to-Field-to-Functional Evaluation. Ed Cahoon Center for Plant Science Innovation & Department of Biochemistry UNL. Soybean is the #1 Oilseed Crop in the United States and ~85% of U.S. Soybean Oil is Used for

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Metabolic Engineering for Healthy Oil Traits: From Lab-to-Field-to-Functional Evaluation

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  1. Metabolic Engineering for Healthy Oil Traits: From Lab-to-Field-to-Functional Evaluation Ed Cahoon Center for Plant Science Innovation & Department of Biochemistry UNL

  2. Soybean is the #1 Oilseed Crop in the United States and ~85% of U.S. Soybean Oil is Used for Food Products and Food Preparation Nebraska’s 2009 soybean crop was valued at $2.4 billion

  3. Cleaning Cracking Dehulling Full Fat Flakes Conditioning and Flaking Soybean Meal 48% Protein Solvent extraction Crude Oil Defatted Flakes Solvent removal Grinding & Sizing Refining Bleaching Carbohydrate removal Protein extraction 70 & 90 PDI Soybean Flour Deodorization Hydrogenation Protein precipitation Drying Deodorization RBD Soy Oil Conditioning Soy Protein Concentrate RBHD Soy Oil Drying Soy Protein Isolate Major Economic Components of Soybeans: Oil and Meal One Bushel of Soybeans: 48 lbs. of Meal & 11 lbs. of Oil

  4. Soybean Oil Consists Principally of Triacylglycerols But Also Contains Other Lipid Soluble Compounds that Contribute to the Nutritional and Functional Qualities of the Oil Triacylglycerols • >95% of vegetable oil • Fatty acid components are • important for: • *Human/animal nutrition • *Functional properties of oil • for food processing & • industrial uses • *Meat quality Vitamin E • ~0.2% of vegetable oil • Importance: *Human/animal nutrition *Oxidative stability of oil for food processing & industrial uses *Meat quality

  5. Soybean Oil is Composed of Five Fatty Acids Palmitic Acid (16:0) 10% Stearic Acid (18:0) 5% Oleic Acid (18:1) 24% Linoleic Acid (18:2) 54% Linolenic Acid (18:3) 7% Saturated Fatty Acids Monounsaturated Fatty Acids Polyunsaturated Fatty Acids

  6. Towards a Better Soybean Oil Approaches *Improved nutritional properties: High oleic Low palmitic High long chain omega-3 polyunsaturated fatty acids *Improved functional/bioactive properties: High oleic (oxidative stability) Low linolenic (oxidative stability) High stearic/high oleic (soft spread margarine) High vitamin E (oxidative stability, meat quality) Conjugated linoleic/linolenic (reduced fat/reduced obesity)

  7. What’s On the Market or Will Soon Be On the Market Low Linolenic Soybeans (not a biotech trait) High Oleic Soybeans Stearidonic Soybeans J. Whelan (2009) J. Nutrition 139: 5-10

  8. Towards New Oil Traits…. Starting from the lab: *Up- or down-regulation of genes native to soybean to shift flux toward the synthesis of desired fatty acid profile. *Identification and use of genes from other sources to redirect soybean lipid metabolism toward the production of a desired fatty acid or other lipidic compound.

  9. Enrichment or Removal of a Fatty Acid Component Can Typically Be Achieved by Altering the Expression of One or More Fatty Acid Desaturase or Thioesterase Genes D15 Desaturase/ FAD3 High/Low Linolenic Acid D12 Desaturase/ FAD2 High Oleic Acid Palmitoyl-ACP Thioesterase FATB High/Low Palmitic Acid D9 Desaturase High Stearic Acid

  10. PUFA Production: Example of gene discovery for oil modification

  11. Next step: Getting the transgenes into soybean b-Conglycinin Promoter Barley HGGT Phaseolin 3’UTR Hygromycin Selection Marker

  12. Soybean Somatic Embryogenesis Selection Multiplication & Maturation Regeneration/ Plant Growth Phenotypic Analysis Biolistic Transformation Camelina Timeline of Transformation: Soybean versus Camelina Agrobacterium Infiltration 0 10 Months …and then another two to three generations in the greenhouse to get homozygous lines and to bulk up seeds for the field

  13. Engineered Soybeans with New Oil Traits Can Be Taken to the Field Dedicated Field Site (app. 25 acres with Irrigation) Soybean Release Biotech fields are located in Mead and North Platte

  14. Insta Pro 2000 Extruder Insta Pro 1500 Horizontal Oil Press Downstream Processing

  15. 18:0 Stearic Acid 9 ∆ Desaturase 18:1 Oleic Acid 12 ∆ Desaturase 18:2 Linoleic Acid (LA) 15 6 ∆ ∆ Desaturase Desaturase 18:3 Linolenic Acid (ALA) 18:3 Linolenic Acid (GLA) Omega-6 -  - 6 ∆ Desaturase 18:4 Stearidonic acid (STA) Omega-3 Fatty Acid (FA) biosynthesis pathway

  16. 11.9% 3.0% 11.4% 3.6% 25.8% 6.4% 38% Event 535-9 Field 2005 (T4 Generation) Palmitic Acid Stearic Acid Oleic Acid Linoleic Acid Linolenic Acid GLA STA Borage (∆6desaturase) Arabidopsis (∆15desaturase)

  17. Displacing fishmeal and fish oil in aquaculture feeds with soy-based protein and lipids: Kona Kampachi™ and Steelhead Trout

  18. Steelhead trout at harvest Soy diet Control diet

  19. What’s Next? Stearic acid (18:0) Plant cell ∆9 desaturase Oleic acid (18:1) ∆12 desaturase ∆15 desaturase Linolenic acid (18:3) Linoleic acid (18:2) ∆6 desaturase EPA: omega-3 fatty acid Stearidonic acid (18:4) Linolenic acid (18:3) elongase ∆5 desaturase Arachidonic acid (20:4) Eicosapentaenoic acid (20:5) -6 pathway -3 pathway

  20. Structure of CLA isomers Linoleic (cis-9,cis-12) 12 9 11 9 Cis-9,trans-11 CLA 12 10 Trans-10,cis12 CLA

  21. CLA fat reduction in epididymal fat pads of mice fed a normal calorie diet + 0.5% CLA for 2 weeks Control CLA Treated

  22. CLA has limited effects in humans • Human trials show modest to no effects • Primary human adipocytes show strong effects just like mouse adipocytes • The mouse dose is much higher than the human dose. • Our Goal: Find mechanism(s) and nutraceuticals, possibly in combination with drugs, that facilitate fat loss in humans

  23. O HO Eleostearic Acid 18:3D9c,11t,13t Goal: Produce CLA-like compounds in soybean seeds. Approach: Isolate genes for the synthesis of conjugated fatty acids and introduce into soybeans. Potential Sources of Genes: Calendula officinalis Pot Marigold Momordica charantia Bitter Gourd O HO Calendic Acid 18:3D8t,10t,12c O HO a-Parinaric Acid 18:4D9c,11t,13t,15c Impatiens balsamina

  24. O PC “Fatty acid conjugases” convert an existing double bond into two conjugated double bonds. O O PC PC Oleic Acid 18:1D9c Linoleic Acid 18:2D9c,12c “Normal” FAD2 FAD2- Type “Conjugase” O PC Eleostearic Acid (18:3D9c,11t,13t) Linoleic Acid 18:2D9c,12c

  25. Calendic Acid Content of 20 to 25% of the Total Fatty Acids Is Achievable in Soybean Seeds 18:2 Soybean 18:1 Calendic (20%) 16:0 18:3 18:0 Arabidopsis (FAD3/FAE1 mutant) 18:1 18:2 Calendic (15%) 16:0 18:0 18:3 Seed-Specific Expression of the Calendula Conjugase cDNA Under Control of the Soybeana’-b-Conglycinin Promoter

  26. Vitamin E Antioxidant Biofortification of Soybean Result: Step 2: Transfer of Gene to Soybean and Expression in Seed Step 1: Isolation of a Tocotrienol Biosynthetic Gene From Barley Non- transformed +Barley Gene Six-Fold Enhancement of the Vitamin E Content of Soybean Seeds Tocotrienols: Improved meat quality, cholesterol-lowering compounds

  27. Can soybean be engineered to produce astaxanthin for farmed salmon? Brevundimonas sp. Maize

  28. Production of astaxanthin in soybean +crtZ/crtW/ phytoene synthase non-transformed

  29. Production of Astaxanthin and β-Carotene in Soybean Seeds non-transformed Detector Response (Absorbance 455nm) 5 10 15 20 25 min +psy/crtW/crtZ * 5 10 15 20 25 min astaxanthin std. 5 10 15 20 25 min β-carotene std. 5 10 15 20 25 min

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