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Engineering Bioactive Seed Oils

Randall J. Weselake

Professor and Canada Research Chair in Agricultural Lipid Biotechnology

Scientific Director of Alberta Innovates Phytola Centre

Department of Agricultural, Food and Nutritional Science University of Alberta

Edmonton, Alberta, Canada

Agriculture Biotechnology International Conference September 17, 2013

randall.weselake@ualberta.ca

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Images: ngm.nationalgeographic.com; Canola Council of Canada

Oilsands

Oilseeds

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Example of a Petrochemical

A component of gasoline

Seed Oil is Mainly Composed of Triacylglycerol (TAG)

Nelson DL, Cox MM (2005) Lehninger. Principles of Biochemistry, Fourth Edition, Freeman, New York

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Presentation Outline

• Production of plant oils for food, feed and industrial applications

• Introduction to the Alberta Innovates Phytola Centre • Seed oil formation

• Potential benefits of stearidonic acid (SDA)-enriched flax oil

• Towards producing pomegranate-like seed oil in an oil seed crop • Super enzymes for boosting seed oil content

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Nine Major Oil Crops Producing Multi-purpose Oils

Oil crop Global production 2009-2010 (MMT)

Coconut (Cocos nucifera) 3.7

Cotton seed (Gossypium hirsutum) 4.7

Olive (Olea europaea) 3.0

Palm (Elaeis guineensis) 45.0

Palm kernel (Elaeis guineensis) 5.5

Peanut (Arachis hypogea) 4.6

Oilseed rape (Brassica napus)a 22.1

Soybean (Glycine max) 37.9

Sunflower (Helianthus annuus) 11.1

TOTAL 138.6

a Predominantly low erucic acid/low glucosinolate cultivars

Taylor et al. (2011) In: Comprehensive Biotechnology 2nd Edition, Volume 4: Agricultural and Related Technologies; J Moo-Young (editor-in-chief); B Grodzinski, WA King, R Yada (volume editors); Elsevier Inc.; Oxford; pp 67-85; DOI: 10.101016/B978-0-08-088504-9.00256-7

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Plant Oils for Food and Feed

• Human lipid nutrition

• Frying and baking applications

• Supplements with nutraceutic properties

• Livestock lipid nutrition • Aqua-feed applications

• Pet food applications

Canola Council of Canada

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Examples of Major Non-food Uses of Plant Oils

• Fuel (e.g. biodiesel) • Lubricants (e.g. greases, fuel additives) • Hydraulic fluids • Surfactants • Printing inks • Surface coatings (paints, varnishes) • Solvents (e.g. paint removers) • Adhesives • Polymers (e.g. foams) • Plasticizers • Waxes and candles • Personal care products (e.g. lipstick) • Pharmaceuticals • Dust suppressants

Taylor et al. (2011) In: Comprehensive Biotechnology 2nd Edition, Volume 4: Agricultural and Related Technologies; J Moo-Young (editor-in-chief); B Grodzinski, WA King, R Yada (volume editors); Elsevier Inc.; Oxford; pp 67-85; DOI: 10.101016/B978-0-08-088504-9.00256-7

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Other Sources of Plant Oil • Numerous other oilseed crops (e.g., flax, castor, camelina) • By-products from the forest industry (e.g., tall oil)

• Microalgae and diatoms

– avoids competition with land used for food production • Conversion of starch-forming plants to oil-forming plants • Production of oil in vegetative and root tissue

Carlsson (2009) Biochimie 91: 665-670 Chapman et al. (2013) Plant Sci 207:128-134 Weselake et al. (2009) Biotech Adv 27:866-876

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Alberta Innovates Phytola Centre Vision and Mission

Vision: Phytola will be a leader in the development of novel oilseeds and specialty oils—solving market needs in collaboration with industry.

Mission: To provide market-responsive, oilseed biotechnology products & technology solutions in partnership with industry.

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Scientific Director: Randall Weselake, Ph.D.

Business Development Director: Joseph Boothe, Ph.D.

Centre Manager: Chris Kazala, M.Sc.

Management Advisory Board & Science Advisory Board

www.phytola.com

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Network Phytola has developed an extended network of collaborators within universities, research institutes and government organizations that extend its research and product development capabilities both upstream and downstream. Current key collaborations include: • Dr. Jie Chen (University of Alberta/NRCC): Nanobiotechnology - plant transformation

• Dr. Catherine Field (University of Alberta): Human nutrition and metabolism – the effects of lipids on immune system function and disease prevention

• Dr. Xiao Qiu (University of Saskatchewan): Lipid biosynthesis – isolation of genes for novel enzymes from plants and microbes

• Dr. Habibur Rahman (University of Alberta): Plant breeding - development of superior canola germplasm

• Dr. Saleh Shah (Alberta Innovates Technology Futures): Plant biotechnology – genetic engineering and plant transformation

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Bioactive oils

- Nutraceuticals

- Omega-3 eggs

- Aqua-feed

Increasing seed

oil content

Bio-industrial oils Platforms

- Enzyme engineering

- Plant transformation

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Recently Awarded a Major Infrastructure Grant

‘Instrumentation for Molecular Breeding for Specialty Oils’

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Oil Formation Occurs During Seed Development

Canola Council of Canada

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Photosynthesis

Sucrose

Carbon flow

Fatty acid biosynthesis & production of MUFA (18:1)

CYTOSOL

PLASTID

Acyl-CoA pool

ENDOPLASMIC RETICULUM (ER)

- Fatty acid elongation - Acyl-exchange with ER acyl chains

TAG

PUFA formation

TAG assembly

CoA

Bicarbonate Acetyl-CoA ATP

TAG

Overview of Seed Oil Formation

Weselake (2011) In: Canola: Description, Variety Development, Agronomy, Composition, and Utilization; JK Daun, D Hickling, NAM Eskin (editors); AOCS Press; Urbana, IL; pp 57-91

MUFA, monounsaturated fatty acid

PUFA, polyunsaturated fatty acid

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Production of Stearidonic Acid in Flax

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Flax (Linum usitatissimum) Production in Canada

Canada is the world’s largest producer and exporter of flax

~ 930,000 tonnes in 2009/2010

– Flax Council of Canada &

Flax Canada 2015

Flax oil is enriched in

alpha-linolenic acid (ALA)

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Δ6-desaturase

α-linolenic acid (ALA, C18:3)

stearidonic acid (SDA, C18:4)

eicosatetraenoic acid (ETA, C20:4)

eicosapentaenoic acid (EPA, C20:5)

docosahexaenoic acid (DHA, C22:6)

docosapentaenoic acid (DPA, C22:5)

The Omega-3 Route

From ALA to SDA, EPA and DHA

“Bottleneck”

• EPA, DHA & SDA have numerous

health benefits

Lorente-Cebrián et al. (2013) J Physiol Biochem 69:633-651

Siriwardhana et al. (2012) Adv Food Nutr Res 65:211-222

Swanson et al. (2012) Adv Nutr 3:1-7

Walker et al. (2013) Nutrition 29:363-369

Whelan (2009) J Nutr 139:5-10

www.livestrong.com

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Metabolic Engineering of Flax to Produce Seed Oil Enriched in Stearidonic Acid (SDA)

ALA

SDA

Fatty acid Control LO High SDA LO F06396B LO

16:0 5.0±0.1 6.4±0.2 5.4±0.1

18:0 3.4±0.1 2.7±0.1 2.2±0.1

Total SFA 8.7±0.2 9.1±0.3 7.6±0.2

18:1n-9 16.2±0.1 12.9±1.2 8.2±0.6

Total MUFA 16.2±0.1 12.9±1.2 8.2±0.6

18:2n-6 16.3±0.1 4.1±0.3 8.8±0.1

18:3n-6 ---- 15.2±0.2 ----

20:3n-6 ---- 0.8±0.1 ----

Total n-6 PUFA 16.3±0.1 20.1±0.6 8.8±0.1

18:3n-3 58.3±0.2 31.2±1.2 74.8±0.8

18:4n-3 (SDA) ---- 25.7±0.7 0.2±0.1

20:4n-3 ---- 0.7±0.1 ----

Total n-3 PUFA 58.3±0.2 57.7±2.0 75.0±0.9

Total PUFA 74.8±0.2 77.8±2.6 83.8±1.0

Values are means ± S.E.M.; n = 3 for control LO; n = 5 for high SDA

---- = Not detected

LO, linseed oil

MUFA, monounsaturated fatty acids

PUFA, polyunsaturated fatty acids

SFA, saturated fatty acids

• Delta-6 desaturase cloning: Xiao Qiu;

University of Saskatchewan

• Flax transformation: Saleh Shah;

Alberta Innovates Technology Futures

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Evaluation of SDA-enriched Flax Oil

• Anti-cancer effects (Catherine Field, University of Alberta)

• Omega-3 enrichment of eggs (Doug Korver, Mirko Betti;

University of Alberta)

• Omega-3 enrichment of trout tissue (Murray Drew; University of

Saskatchewan)

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SDA Treatment Reduces the Viability of the MDA-MB-231 Human Breast Cancer Cells

a a a

b b b b

Howe-Ming Yu & Catherine Field

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Mouse Studies (nu/nu mouse)

Tumour implanted (MDA-MB-231)

Tumour (50 mm)

Control diet

Control diet SDA flax oil diet

Diets isocaloric and nutritionally adequate

2 weeks

From Julia Ewaschuk & Catherine Field

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Effect of Diet Oil on Tumour and Body Weight for the Tumour-bearing Mice

P<0.07

n = 7 n = 6

SDA flax oil

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Effect of SDA-enriched Flax Oil on Production of n-3 Long Chain PUFA in Eggs

Egg yolks collected at 0, 7, 14 and 21 d, for lipid profile analysis.

Control

(4% Corn oil) Body Weight

Feed Intake

Egg Traits

Traditional

(4% Flax oil)

Novel

(4% SDA Flax oil)

Sandeep Nain, Robert Renema, Doug Korver & Mirko Betti

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P <.0001

0.35 0.06 0.00 0.06

0.59

4.93

0.12 0.16 0.13

1.14

3.74

0.53

0.24 0.29

1.33

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

LNA C 20:3 n-3 EPA DPA DHA

Pe

rce

nta

ge c

om

po

siti

on

(%

)

Control

Flax

SDA-Flax

b

a

b b b

b

a

a

a a c

c c

c

ETA

Egg: Total Yolk Various Long Chain n-3 PUFA

C18:3 C20:4 C20:5 C22:5 C22:6

ALA

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Production of Punicic Acid in Oil Crops

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Pomegranate (Punica granatum) Seed Oil • Anti-cancer properties (breast and prostate cancer) • Health and cosmetic products to postpone skin aging and improve skin elasticity

• Prevention of arteriosclerosis, hypertension and diabetes • Anti-inflammatory properties • Punicic acid (trichosanic acid) is about 65% of the fatty acids in the oil

Afaq et al. (2009) Exp Dermatol 18:553-561

Grossmann et al. (2010) Int J Oncol 36:421-426

Johanningsmeier & Harris (2011) Annu Rev Food Sci Technol 2:181-201

McFarlin et al. (2009) Br J Nutr 102:54-59

Applications:

• Cosmetics

• Nutraceuticals

• Functional food ingredients

Pomegranate seeds

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0

10

20

30

40

50

60

% F

A

18:1 18:2 Punicic acid

Engineering of the Model Plant, Arabidopsis,

to Produce About 20% Punicic Acid in the Seed Oil

Elzbieta Mietkiewska et al.

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Increasing Seed Oil Content

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Canola (Brassica napus) Production in Canada

• Farm gate value for sale of seed & oil is > $2.5 billion per year

generating > $15 billion in economic activity for Canada

• 10-12 million acres per year

• 7 million tonnes per year

• A 1% increase in seed oil content

will result in an additional $90 million

per year for the crushing and

processing industry

• 50-75% increase in canola oil production

required to meet the demand for seed oil

over the next 10 years

• Increasing seed oil content & oil yield per acre are top priorities

Canola Council of Canada

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$

Before

After

Widening the Bottleneck in the Flow of Carbon into

Seed Oil

DGAT

More

DGAT

activity Weselake et al. (2008) J Exp Bot 59:3523-3549

Weselake et al. (2009) Biotech Adv 27:866-878

Taylor et al. (2009) Botany 87:533-543

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Development of a “Super” DGAT

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Conclusions • Major oil crops could be used to produce alternatives to fish oil and

other bioactive oils for applications in food, feed and nutraceuticals.

SDA-enriched flax oil may be useful in cancer treatment.

• Major oil crops may be used to produce punicic acid-enriched oil for

nutraceutical applications including prevention of certain forms of cancer

and cardiovascular disease.

• Seed oil content can be increased through metabolic engineering. Further

increases are anticipated through the use of new “super enzymes”. Increased

seed oil content is important for both food and non-food applications.

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Acknowledgements

• Mirko Betti

• Disa Brownfield

• Anders Carlsson

• Gavin Chen

• Nancy Cranston

• Murray Drew

• John Dyer

• Julia Ewaschuk

• Catherine Field

• Tara L Furukawa-Stoffer

• Allen Good

• John Harwood

• Xiaohua He

• Chris Kazala

• Robert Renema

• Saleh Shah

• Rodrigo MP Siloto

• Crystal L Snyder

• Sten Stymne

• Mingguo Tang

• David C Taylor

• Martin Truksa

• Howe-Ming Yu

• Weiming Zhu

• Jitao Zou

• Doug Korver

• Arvind Kumar

• André Laroche

• Qin Liu

• Thomas McKeon

• Elzbieta Mietkiewska

• Maurice Moloney

• Sandeep Nain

• Cory Nykiforuk

• Xiao Qiu

• Patti A Quant

• Gerhard Rakow

• Phillip Raney

• Kyla Randall

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• Alberta Agricultural Research Institute

• Alberta Canola Producers Commission

• Alberta Crop Industry Development Fund

• Alberta Enterprise and Advanced Education

• Alberta Innovates Bio Solutions

• Alberta Innovates Technology Futures

• Agragen

• AVAC Ltd. (Wendy Lam, Senior Investment Manager)

• Biotechnology and Biological Sciences Research Council (UK)

• Canada Foundation for Innovation

• Canada Research Chairs Program

• Cargill

• Genome Alberta, Genome Prairie & Genome Canada

• National Research Council of Canada

• Natural Sciences and Engineering Research Council of Canada

• United States Department of Agriculture

• University of Alberta

• Advisors to the Bioactive Oils Program & the Alberta Innovates Phytola Centre

Acknowledgements

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