Elzbieta Mietkiewska1, Saleh Shah1, Annette Scheick2, Ziliang Song1, Catherine J. Field1, Randall J. Weselake1*

1Alberta Innovates Bio Solutions Phytola Centre, Department of Agricultural, Food and Nutritional Science,

University of Alberta, Edmonton, Alberta, Canada

2Bioresource Technologies - Alberta Innovates Technology Futures, Vegreville, Alberta, Canada

Installing Attributes of Pomegranate Oil into Canola

* randall.weselake@ualberta.ca

Presentation Outline

• Nutraceutic benefits of pomegranate oil and punicic acid • Biosynthesis of seed oil containing punicic acid • Engineering punicic acid production in Arabidopsis thaliana (proof-of-concept using a model plant) • Engineering punicic acid production in canola type Brassica napus

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

Seeds from the fruit of pomegranate (a sub-tropical plant)

Fatty Acid Composition of Oil from the Mature P. granatum Seed

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16:0 18:0 18:1 18:2 18:3 c9,t11, c13

CLNAs

Fatty

aci

d (%

wt/w

t)

Punicic acid (18:3∆9cis,11trans,13cis)

Punicic Acid (PA) Decreases the Viability of Human Breast Cancer Cells

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MDA-MB-231 MCF-7 MCF-12A

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OAL

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Human Breast Cell Type

OALA (40/40 μM) 2.5 μM PA 5 μM PA 7.5 μM PA 10 μM PA

* *

OA, oleic acid (18:1∆9cis) LA, linoleic acid (18:2 ∆9cis,12cis)

Non-malignant cells Cancer cell lines

Field et al., unpublished data

Brassicaceae with Oil-forming Seeds

www3.uis.no/araperoxv1/ www.seedgenes.org

Canola Council of Canada

• Arabidopsis thaliana

• Family: Brassicaceae

• Model plant

• Canola type Brassica napus

• Family: Brassicaceae

• Canada’s major oil crop Developing zygotic embryos

Seed Oil is Mainly Composed of Triacylglycerol (TAG)

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

Why Should Canola be Engineered to Produce a Specialty Oil Enriched in Punicic Acid?

• Canola oil is already established as a healthy oil • Pomegranate oil enriched in punicic acid has proven nutraceutic benefits (e.g., anti-cancer properties) • If punicic acid-enriched oils are to be used on a larger scale, a more reliable, consistent and cheaper supply of high quality oil will be required • Opportunity for economic benefit?

Photosynthesis

Sucrose

Carbon flow

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

Cytosol

PLASTID Endoplasmic reticulum (ER)

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

TAG

PUFA formation

TAG assembly

CoA

Bicarbonate Acetyl-CoA ATP

TAG

TAG Biosynthesis in Embryos of Developing Seeds

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

Acyl-CoA pool

OH HO

P

FA HO

P

FA FA

P

FA 18:1

OH

FA FA

FA

TAG Assembly, Membrane-based FA Modification and Acyl-editing

FA-CoA pool

CoA, coenzyme A DAG, sn-1, 2-diacylglycerol DGAT, diacylglycerol acyltransferase FA, fatty acyl FAD, fatty acid desaturase G3P, sn-glycerol-3-phosphate PUFA, polyunsaturated fatty acid

phosphocholine

FA 18:1

Phosphatidylcholine

TAG DAG

DGAT

CoA CoA CoA

phosphocholine

FA PUFA

FADs

Acyl-exchange

Pi

Kennedy pathway (sn-glycerol-3-phosphate pathway)

G3P

Chapman & Ohlrogge (2012) J Biol Chem 287:2288-2294

Woodfield et al. (2015) 5 Inform 26:78-83

Chen et al. (2015) Lipids 50:1057-1068

Free PUFA FA elongation

Biosynthesis of Punicic Acid

• In previous studies, expression of the P. granatum FADX gene during seed development in Arabidopsis resulted in low accumulation of punicic acid in the seed oil (3.5%, w/w).

• This may be due to limited availability of sn-2-18:2∆9cis,12cis-PC (the FADX substrate). The expression of PgFADX may have resulted in down-regulation of endogenous FAD2. Indeed, the oleic acid (18:1) content of the seed oil of transgenic Arabidopsis was enhanced.

• Trichosanthes kirilowii FADX expressed in B. napus to produce oil with 2.5% punicic acid.

Hornung et al. (2002) Eur J Biochem 269:4852-4859

Iwabuchi et al. (2003) J Biol Chem 278:4603-4610

Koba et al. (2007) J Agric Food Chem 55:3741-3748

Mietkiewska et al. (2014) Biocatal Agric Biotechnol 3:44-48

16:0

18:3-Conj

phosphocholine

16:0

18:2

phosphocholine

FADX (Conjugase)

PC, phosphatidylcholine

16:0

18:1

phosphocholine

FAD2

FAD, fatty acid desaturase

sn-2 position

PgFADX: P. granatum conjugase

PgFAD2: P. granatum Δ12 desaturase Transformation platform:

• fad3/fae1 Arabidopsis – Smith et al. (2003) Planta 217:507-516

• High linoleic acid (18:2) background for use with molecular genetic constructs NCJ and NCJD

Transformation of the Model Plant

Napin PgFAD2 Nos Napin Nos PgFADX

Napin Nos PgFADX NCJ

NCJD

Fatty Acid Compositions of fad3/fae1 Arabidopsis T3 Seeds Expressing

the PgFADX Gene

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% F

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18:1 18:2 Punicic acid

Weselake & Mietkiewska. U.S. Patent Appl. No. 14/224,582; filed March 25, 2014 Mietkiewska et al. (2014) Planta 240:575-583

Transgenic lines

Fatty Acid Compositions of fad3/fae1 Arabidopsis T3 Seeds Co-expressing

PgFAD2 and PgFADX

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18:1 18:2 Punicic acid

Transgenic lines

NCJ-1-2-1 NCJ-19-2-1 NCJD-30-2-1 nt-fad3fae1

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sion

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Expression of PgFADX in fad3/fae1 Arabidopsis Suppresses AtFAD2 Expression

• Introduction of PgFAD2 activity during Arabidopsis seed development is required for enhancing the production of 18:2Δ9cis,12cis

NCJD: PgFADX+PgFAD2 SAF4: PgFADX+PgFAD2+PgDGAT2

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18:1 18:2 punicic acid

Weselake & Mietkiewska. U.S. Patent Appl. No. 14/224,582; filed March 25, 2014

Expression of PgFAD2, PgFADX and PgDGAT2 in fad3fae1 Arabidopsis Seeds Resulted in About 25% (w/w)

Punicic Acid in Some Transgenic Lines (SAF4 NCJD T4 Seed)

Transgenic lines

Production of Punicic Acid in T3 Seeds of Selected Lines Carrying Promoter/PgFADX Constructs

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7SL

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7SL

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3SL

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7SL

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11SL

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12SL

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Napin Phaseolin Linin Conlinin

Homozygous lines Heterozygous lines Null Segregants

Song et al., unpublished data

NCJD-3

NCJD-4

NCJD-5B

NCJD-7

NCJD-11

NCJD-13

NCJD-15A

NCJD-23

NCJD-26

NCJD-25

NCJD-27 WT

% p

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cid

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Each dot represents the data from a 2 seed sample

Production of Punicic Acid in T1 Canola Seeds Co-expressing PgFAD2 and PgFADX

B. napus DH12075

Canola Council of Canada

Production of Punicic Acid in T2 Canola Seeds Co-expressing PgFAD2 and PgFADX

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18:1 18:2 punicic acid

Null segregants

Mietkiewska et al., unpublished data

Transgenic lines

Pomegranate is Much More Efficient than Arabidopsis in Trafficking Punicic

Acid from PC to TAG

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P. granatum Arabidopsis with PgFADX+PgFAD2

Mietkiewska et al. (2014) Planta 240:575-583

Canola is More Efficient than Arabidopsis in Trafficking Punicic Acid from PC to TAG

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PC TAG

Arabidopsis T3 seed

Canola T2 seed

Mietkiewska et al., unpublished data

Closing Comments • Co-expression of pomegranate FAD2 and FADX in Arabidopsis or canola is an effective strategy for producing punicic acid in these temperate Brassicaceae

• Canola oil with about 10% punicic acid was produced

• Combined expression of pomegranate FAD2, FADX and DGAT2 (or genes encoding other oil assembly or acyl- trafficking enzymes) in canola may result in further increases in the punicic acid content of canola oil

• Use of the linin promoter (from flax) in molecular genetic constructs may further enhance punicic acid content

Other Phytola team members: Arabidopsis fad3/fae1:

Joseph Boothe Ljerka Kunst (University British Columbia) Guanqun Chen Michael S. Greer E. Chris Kazala Robin Miles Ariff Firman Sahibollah Aruna Wickramarathna Annie Wong

I000 Plants Project:

Michael Deyholos (University of Alberta)

B. napus DH12075:

Ginette Séguin-Swartz Gehard Rakow (Agriculture and Agri-Food Canada, Saskatoon)

Acknowledgements