Production of Nervonic acid in Brassica carinata for Industrial and

Production of Nervonic acid
in Brassica carinata
for Industrial and Health Applications
Elizabeth-France Marillia & David C.Taylor
ACRD-NRC
April 2014
Bio-Based Specialized Chemicals Program-ACRD
Overview
• NRC and its new RTO structure & mandate
• Life Science programs and industrial oils at NRC
• Nervonic acid: functions, uses and production
• Production in Brassica carinata: a Proof of Concept
• Future steps and collaboration with the industry
2
Saskatoon
About NRC
IRAP
• 2012-13 budget: $774M
Research
facilities
• Over 4,000 employees and
650 volunteer and independent visitors
• Wide variety of disciplines and broad array of services
and support to industry
3
New Research Technology
Organization (RTO) model
• Vision:
oTo be the most effective research and technology
organization in the world and stimulate sustainable
domestic prosperity
• Mission:
oWork with clients and partners to provide innovation
support, strategic research and technical services (fee for
services).
oBe dedicated to building economic competitiveness and
improving national productivity
4
NRC Life Sciences Research
Life Sciences
Aquatic and Crop
Resource Development
Human Health
Therapeutics
National Research
 Natural Health Products
 Algal Carbon Capture
 Wheat
 Bio-based Specialty Chemicals
 Crop Platform Technologies
 Biologics
 Vaccines
Medical Devices
NATURAL HEALTH PRODUCTS
5
Oil Seed Crops at NRC
From Arabidopsis to Canola to Brassica carinata....
30+ years of R&D on industrial oils and their applications
Source: Wikipedia
6
Nervonic Acid
24:1 15 (cis-tetracos-15- enoic acid) -9
Produced by elongation of erucic acid 22:1
Physiological Functions
• Physiological activity of nervonic acid discovered only 35 years ago
• Necessary for development and maintenance of brain tissue &
function:
o Component of myelin sheath. Serves to speed conduction of
nerve impulses
o Maintains integrity & regulates function of Ca2+ ion channeling in
cell membranes of nerve tissues
o Thought to play a major role in memory
o Role in postponing senility ?
Pharmacological Indications
Symptomatic treatment of:
• De-myelinating diseases: Multiple Sclerosis, Parkinson, ALD
• Schizophrenia: nervonic acid levels low in these patients
• Deficiencies associated w neurological disorders: Alzheimer’s
• HIV ? Nervonic acid inhibits HIV-1 Reverse Transcriptase
• Many applications in nutritional markets (senility, memory aid etc.)
• Treatments of arthritis, liver diseases and obesity
Dietary Supplements
• Babies/infants during the myelinating age. Baby foods/formulas
(Hangzhou Sweetant Biotech)
• Pre-term babies, no longer benefiting from maternal nutritionPregnant/lactating women
• High-level training adults/athletes: enriched energy
supplements with neuro-protective effect
.
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Industrial Applications
1- Biofuels & Surfactants
• Next generation of alternative fuels:
ARA Biofuels ISOCONVERSION: ReadiJet, Readi Diesel.
Test flight with NRC in 2012
• “Green Surfactants”:
Enhance Oil Recovery (EOR) for crude oil (Oil Chem
Technologies). VLCFAs feedstocks used to reduce water &
energy consumption during bitumen recovery & processing
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Industrial Applications
2- Bioplastics and Polymers
• Specialized polyamides, polyesters, polyols:
Feedstock for diacid production in the bioplastic
industry (Phytowelt Green Technologies; Mitsubishi)
• Paving bed polymers (low temp. stabilization):
Feedstock for moisture displacement additives in
asphalt paving sealers (SWEPCO)
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Others Industrial Applications
Source Globalgrind
• Cosmetics; hair dyeing; nail varnish (L’Oreal, Novance)
• Medical: biological diagnosis (Konica); improved vaccine
composition (Eurocine vaccines);
• Photosensitive semiconductors (Samsung); superconductive
wire (Hitachi)
• Gels and films (YOUTEC)
• Recording medium (HP)
• Coating oil (Nalso) and ink (JNC)
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Natural Sources for Nervonic Acid
Nervonic acid content (mg/100g)
Plant sources
Brassica oilseeds
Flaxseed
Sesame seeds
Macademia nuts
Tropaeolum speciosum
Lunaria (money plant)
Cardamine graeca
Acer (Purpleblow maple)
Borage
Sinapis alba
Hemp
69-83
64
35
18
10
8
5.4
2.3
Indigofera tinctoria (indigo)
Animal sources
King Salmon (Chinook)
Sockeye salmon
Cerebroside extracts
Pseudaxinella cf. lunaecharta
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140
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Nervonic Acid Suppliers and Market Values
Some Examples
Suppliers
Product
Market value
US$/Kg
Shanghi Soyoung
Biotechnology
nervonic acid food
additives
1-27
Shaanxi Pioneer Biotech Co natural nervonic acid
300-400
Wuxi Cima Science Co
10-1000
nervonic acid powder
Hangzhou Boduo Industrial
nervonic acid capsules
Trade Co
15
1500-2000
Production of Nervonic Acid in Brassica
Rationale:
High Erucic Acid (HEA) Brassica oilseeds contain traces of 24:1 in the
seed oil because of…... the absence of an elongase (KCS condensing
enzyme) specific for converting 22:1 to 24:1.
Goal:
Isolation & characterization of key gene(s) for the production of high
levels of nervonic acid in Brassica oilseed platform crops
Target KCS Gene Source
Cardamine graeca (bittercress)
15cm
1 cm
1 mm
The seed oil has 43 to 54% nervonic acid
Expression of C. graeca KCS in
Brassica carinata
NAPIN
CG KCS
transform
NOS
Brassica carinata
[ Ethiopian mustard ]
Why B. carinata?
● Excellent “vehicle crop” for HEA Brassica work;
22:1 ca 35-38%; 30% oil; Yields 2,500 kg / ha
● Highly transformable (up to 50%)
● Low out-crossing
● Highly resistant to fungal diseases; weed tolerant
● Suitable for growth in drier regions
Confined Field Trials (Saskatchewan)
% 18:1c9
% 20:1c11
% 22:1c13
% 24:1c15
45.00
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00
Card KCS: B car Transgenics
CONTROLS
MUFAs in B. carinata transformants
Wild Type Control
18:1
20:1
22:1
Transgenic Control
24:1
Cardamine B. carinata
Transgenic Line
45% 24:1
Oil retains essential FAs: 25-30% -3 (18:3) + -6 (18:2)
Mono-Unsaturated FAs Elongation
What we can do in a Brassica industrial platform crop
18:1
20:1
22:1
24:1
26:1
FA: oleic
eicosenoic
erucic
nervonic
ximenic
Source:
Teesdalia
Crambe
Cardamine
Confidential
NRC IP:
Patent
Patent
Patent
PSIA
28:1 ?
Ability to tailor oil profiles for specific industrial & health applications
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Uses of Industrial Oilseeds
B carinata VLCFA OILS & APPLICATIONS
VLCFA
20:1c11
22:1c13
24:1c15
26:1c17
JET FUELS
EOR
BIOPLASTICS
NEURO HEALTH
Future steps and collaborations
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Next generation:
Ultra-High Nervonic Carinata (UHN) oil
What do we have?
High-Nervonic carinata line: ~45% nervonic acid
and essential FA profile of WT
17 g/100g seed; > any other seed source (~0.08g)
Market demand ?
An improved UHN carinata line (>80% 24:1)
Why?
Less purification required for industrial
applications where high PUFAs not desired
How?
Produce seed oils (TAG) with nervonic moieties
at all 3 sn- positions on the glycerol backbone
Tri- nervonoyl TAG
sn-1
Nervonic
sn-2
Nervonic
sn-3
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Nervonic
Additional Genetic Modifications
to Achieve Commercial Success
•Over-express LPAT2 for esterification at the sn2 position
•Health applications: reduce residual erucic acid from 7% to
<2% ? or start with a low erucic B carinata?
•Industrial applications: silence endogenous B carinata KCS
and over-express KCS genes from Teesdalia (20:1) and
Cardamine (24:1) in tandem?
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Mono-Unsaturated FAs Elongation
18:1
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20:1
FA:
eicosenoic
Source:
Teesdalia
22:1
erucic
24:1
nervonic
Cardamine
Industrial Partnerships
What we need to achieve together:
•Identify & understand industry demand: needs, characteristics, trends
•Establish applied research collaborations:
oIdentify partners with complementary R&D for developing valueadded products
oForm strong collaboration and together take research from the lab
to the market
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Thank you
Elizabeth-France Marillia
Scientist
Tel: 306-975-5282
Elizabeth.marillia@nrc-cnrc.gc.ca
www.nrc-cnrc.gc.ca
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David C. Taylor
Laurel O’ Connor
Scientist
Client Relationship Leader
Tel: 306-975-5270
Tel: 306-975-4573
David.taylor@nrc-cnrc.gc.ca Laurel.oconnor@nrc-cnrc. gc.ca