Production of Nervonic acid in Brassica carinatakinkibio.com/news/NATIONAL RESEARCH COUNCIL.pdfProduction of Nervonic acid in Brassica carinata for Industrial and Health Applications

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

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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

.

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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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• 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)

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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

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Nervonic Acid Suppliers and Market Values Some Examples

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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

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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

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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

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Nervonic

Nervonic

Nervonic

sn-1

sn-2

sn-3

Tri- nervonoyl TAG

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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 20:1 22:1 24:1

erucic nervonic eicosenoic

Source: Teesdalia

Cardamine

FA:

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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