metabolic engineering for healthy oil traits: from lab-to-field-to-functional evaluation
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Metabolic Engineering for Healthy Oil Traits:From Lab-to-Field-to-Functional Evaluation
Ed CahoonCenter for Plant Science Innovation &
Department of BiochemistryUNL
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
Soybean Meal 48% Protein
70 & 90 PDI Soybean
Flour
Conditioningand Flaking
Refining
Bleaching
Deodorization
RBHD Soy Oil
RBD Soy Oil
Full FatFlakes
Crude Oil
DefattedFlakes
Soy Protein Concentrat
e
Soy ProteinIsolate
Cleaning CrackingDehulling
Solventextracti
on
Solvent
removal
Grinding &
Sizing
Protein extraction
Carbohydrate
removal
Protein precipitati
onDrying
Conditioning
Drying
Hydrogenation
Deodorization
Major Economic Components of Soybeans: Oil and Meal
One Bushel of Soybeans: 48 lbs. of Meal & 11 lbs. of Oil
Vitamin E~0.2% of vegetable oil Importance: *Human/animal nutrition *Oxidative stability of oil for food processing & industrial uses *Meat quality
Triacylglycerols >95% of vegetable oilFatty acid components are important for: *Human/animal nutrition *Functional properties of oil for food processing & industrial uses *Meat quality
Soybean Oil Consists Principally of Triacylglycerols But AlsoContains Other Lipid Soluble Compounds that Contribute
to the Nutritional and Functional Qualities of the Oil
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 AcidsMonounsaturated Fatty AcidsPolyunsaturated Fatty Acids
Towards a Better Soybean Oil
Approaches
*Improved nutritional properties: High oleic
Low palmiticHigh 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)
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
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.
Towards New Oil Traits….
D9 DesaturaseHigh Stearic Acid
D12 Desaturase/FAD2
High Oleic Acid
Palmitoyl-ACPThioesterase
FATBHigh/Low Palmitic
Acid
D15 Desaturase/FAD3
High/Low Linolenic Acid
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
O
HO
HO
O
PUFA Production: Example of gene discovery for oil modification
b-ConglycininPromoter
BarleyHGGT
Phaseolin3’UTR
Hygromycin Selection Marker
Next step: Getting the transgenes into soybean
Somatic Embryogenesis
BiolisticTransformation
Selection Multiplication &Maturation
Regeneration/Plant Growth
PhenotypicAnalysis
0 10Months
Soybean
AgrobacteriumInfiltration
CamelinaTimeline of Transformation:Soybean versus Camelina
…and then another two to three generations in the greenhouse to get homozygouslines and to bulk up seeds for the field
Dedicated Field Site (app. 25 acres with Irrigation)
Soybean Release
Biotech fields are located in Mead and North Platte
Engineered Soybeans with New Oil Traits Can Be Taken to the Field
Insta Pro 1500 Horizontal Oil Press
Insta Pro 2000 Extruder
Downstream Processing
18:0 Stearic Acid
∆9 Desaturase
18:1 Oleic Acid
∆12 Desaturase
18:2 Linoleic Acid (LA)
∆15 Desaturase
18:3 - Linolenic Acid (ALA) 18:3 - Linolenic Acid (GLA) Omega-6
∆6 Desaturase
18:4 Stearidonic acid (STA) Omega-3
∆6 Desaturase
Fatty Acid (FA) biosynthesis pathway
Borage (∆6desaturase)
11.9% 3.0% 11.4% 3.6% 25.8% 6.4% 38%
Palmitic Acid Stearic Acid Oleic Acid Linoleic Acid Linolenic Acid GLA STA
Event 535-9 Field 2005 (T4 Generation)
Arabidopsis (∆15desaturase)
Displacing fishmeal and fish oil in aquaculture feeds with soy-based protein and lipids:Kona Kampachi™ and Steelhead Trout
Soy diet Control diet
Steelhead trout at harvest
Stearic acid (18:0)∆9 desaturase
Oleic acid (18:1)∆12 desaturase
Linoleic acid (18:2)∆15 desaturase
Linolenic acid (18:3)
Plant cell
∆6 desaturase
Linolenic acid (18:3) Stearidonic acid (18:4)elongase
Arachidonic acid (20:4) Eicosapentaenoic acid (20:5)
-6 pathway -3 pathway
∆5 desaturase
EPA: omega-3 fatty acid
What’s Next?
Structure of CLA isomers
Linoleic (cis-9,cis-12)
Cis-9,trans-11 CLA
Trans-10,cis12 CLA
9
9
10
12
11
12
CLA fat reduction in epididymal fat pads of mice fed a normal calorie diet + 0.5% CLA
for 2 weeksControl
CLA Treated
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
HO Calendic Acid18:3D8t,10t,12c
O
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 officinalisPot Marigold
Momordica charantiaBitter Gourd
Impatiens balsamina
O
HO-Parinaric Acid18:4D9c,11t,13t,15c
O
HO Eleostearic Acid18:3D9c,11t,13t
O
O
PC
PC
Oleic Acid 18:1D9c
Linoleic Acid 18:2D9c,12c
“Fatty acid conjugases” convert an existing double bond into two conjugated double bonds.
O
PC
Linoleic Acid 18:2D9c,12c“Normal”
FAD2FAD2-Type
“Conjugase”O
PC
Eleostearic Acid (18:3D9c,11t,13t)
16:0 18:0
18:1
18:3
18:2
16:018:0
18:1
18:3
18:2
Calendic(20%)
Calendic(15%)
Soybean
Arabidopsis(FAD3/FAE1 mutant)
Seed-Specific Expression of the Calendula Conjugase cDNAUnder Control of the Soybean ’-b-Conglycinin Promoter
Calendic Acid Content of 20 to 25% of the Total Fatty AcidsIs Achievable in Soybean Seeds
Step 1: Isolation of aTocotrienol Biosynthetic
Gene From Barley
Step 2: Transfer ofGene to Soybean and
Expression in Seed
Result:
Vitamin E Antioxidant Biofortification of Soybean
Six-Fold Enhancement of the Vitamin E Content
of Soybean Seeds
0
400
800
1200
1600
2000
2400
2800
Vita
min
E C
onte
nt (m
g/kg
see
d w
t.)
+BarleyGene
Non-transformed
Tocotrienols: Improved meat quality, cholesterol-lowering compounds
Brevundimonas sp.
Maize
Can soybean be engineered to produceastaxanthin for farmed salmon?
non-transformed+crtZ/crtW/
phytoene synthase
Production of astaxanthin in soybean
min5 10 15 20 25
min5 10 15 20 25
min5 10 15 20 25
min5 10 15 20 25
*
Det
ecto
r Res
pons
e (A
bsor
banc
e 45
5nm
)non-transformed
+psy/crtW/crtZ
astaxanthin std.
β-carotene std.
Production of Astaxanthin and β-Carotene in Soybean Seeds
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