installing attributes of pomegranate oil into canola · • canola oil is already established as a...
TRANSCRIPT
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
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
0
10
20
30
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60
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80
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
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
MDA-MB-231 MCF-7 MCF-12A
Cell
viab
ility
com
pare
d to
OAL
A co
ntro
l
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Field et al., unpublished data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
0
10
20
30
40
50
60
% F
A
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
0
10
20
30
40
50
60
% F
A
18:1 18:2 Punicic acid
Transgenic lines
NCJ-1-2-1 NCJ-19-2-1 NCJD-30-2-1 nt-fad3fae1
Rel
ativ
e Ex
pres
sion
0.0
0.2
1.0
1.2
1.4
1.6
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
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
% F
A
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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SL2-
28-2
SL2-
28-3
SL2-
28-4
SL2-
28-8
SL2-
28-1
7SL
2-61
-14
SL2-
61-1
7SL
4-9-
3SL
4-9-
7SL
4-9-
11SL
4-9-
12SL
4-14
-14
SL4-
14-1
6SL
6-8-
1SL
6-8-
4SL
6-8-
7SL
6-8-
17SL
7-12
-4
SL2-
28-1
SL2-
28-7
SL2-
61-1
SL2-
61-4
SL4-
9-2
SL4-
14-1
SL4-
14-6
SL6-
8-2
SL6-
8-3
SL7-
12-1
1
SL2-
28-5
SL2-
28-6
SL2-
61-2
SL2-
61-3
SL4-
9-1
SL4-
9-4
SL4-
14-2
SL4-
14-3
SL6-
8-6
SL6-
8-9
SL7-
12-6
SL7-
12-1
0
% P
unic
ic a
cid
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
unic
ic a
cid
0
2
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10
12
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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10
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60
70
% F
A
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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PC TAG
Puni
cic
acid
con
tent
(%
of t
otal
FA
)
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PC TAGPu
nici
c ac
id c
onte
nt
(% o
f tot
al F
A)
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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2
4
6
8
10
12
14
% p
unci
c ac
id
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