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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
About NRC • 2012-13 budget: $774M
• Over 4,000 employees and
650 volunteer and independent visitors
• Wide variety of disciplines and broad array of services
and support to industry
IRAP
Research
facilities
3
Saskatoon
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
National Research
Natural Health Products
Algal Carbon Capture
Wheat
Bio-based Specialty Chemicals
Crop Platform Technologies
Biologics
Vaccines
Life Sciences
Aquatic and Crop Resource Development
Human Health
Therapeutics
Medical Devices
5 NATURAL HEALTH PRODUCTS
Oil Seed Crops at NRC
6
From Arabidopsis to Canola to Brassica carinata....
30+ years of R&D on industrial oils and their applications
Source: Wikipedia
24:1 15 (cis-tetracos-15- enoic acid) -9
Nervonic Acid
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 nutrition-
Pregnant/lactating women
• High-level training adults/athletes: enriched energy
supplements with neuro-protective effect
.
10
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
11
• 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)
12
Industrial Applications
2- Bioplastics and Polymers
Others Industrial Applications
• 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)
13
Source Globalgrind
Natural Sources for Nervonic Acid
Nervonic acid content (mg/100g)
Plant sources
Brassica oilseeds 69-83 Flaxseed 64 Sesame seeds 35 Macademia nuts 18 Tropaeolum speciosum 10 Lunaria (money plant) 8 Cardamine graeca 5.4 Acer (Purpleblow maple) 2.3 Borage Sinapis alba Hemp
Indigofera tinctoria (indigo)
Animal sources
King Salmon (Chinook) 140 Sockeye salmon 40 Cerebroside extracts Pseudaxinella cf. lunaecharta
14
Nervonic Acid Suppliers and Market Values Some Examples
15
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 nervonic acid powder 10-1000
Hangzhou Boduo Industrial Trade Co
nervonic acid capsules 1500-2000
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
Production of Nervonic Acid in Brassica
Cardamine graeca (bittercress)
The seed oil has 43 to 54% nervonic acid
15cm
1 cm
1 mm
Target KCS Gene Source
Expression of C. graeca KCS in
Brassica carinata
CG KCS NAPIN NOS
transform 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
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
% 18:1c9 % 20:1c11 % 22:1c13 % 24:1c15
Confined Field Trials (Saskatchewan)
CONTROLS Card KCS: B car Transgenics
MUFAs in B. carinata transformants
Wild Type Control Transgenic Control
Cardamine B. carinata Transgenic Line
18:1 20:1
22:1 24:1
Oil retains essential FAs: 25-30% -3 (18:3) + -6 (18:2)
45% 24:1
21
Mono-Unsaturated FAs Elongation
18:1 20:1 22:1 24:1 26:1 28:1 ?
erucic nervonic ximenic oleic eicosenoic
What we can do in a Brassica industrial platform crop
Ability to tailor oil profiles for specific industrial & health applications
Source: Teesdalia
NRC IP:
Crambe Cardamine Confidential
FA:
Patent Patent Patent PSIA
B carinata VLCFA OILS & APPLICATIONS
VLCFA JET FUELS EOR BIOPLASTICS NEURO HEALTH
20:1c11
22:1c13
24:1c15
26:1c17
Uses of Industrial Oilseeds
Future steps and collaborations
23
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
25
Nervonic
Nervonic
Nervonic
sn-1
sn-2
sn-3
Tri- nervonoyl TAG
26
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?
27
Mono-Unsaturated FAs Elongation
18:1 20:1 22:1 24:1
erucic nervonic eicosenoic
Source: Teesdalia
Cardamine
FA:
28
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 value-
added products
oForm strong collaboration and together take research from the lab
to the market
29 29
Thank you
Elizabeth-France Marillia David C. Taylor Laurel O’ Connor
Scientist Scientist Client Relationship Leader
Tel: 306-975-5282 Tel: 306-975-5270 Tel: 306-975-4573
[email protected] [email protected] Laurel.oconnor@nrc-cnrc. gc.ca
www.nrc-cnrc.gc.ca
