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