Oilseeds As Crop Biofactories for Industrial Raw Materials

AOF Forum, 2004 Grain & chemical industry drivers

Oilseeds as crop ! Global competition increasing biofactories for ! Downward pressure on price and market share industrial raw ! Need to diversify away from commodities ! Need to capture maximum value materials

Allan Green ! Desirable to replace petroleum with renewable CSIRO Plant Industry sources of industrial raw materials ! Need for increased biodegradability of C O C O C O industrial products C C C C C C O C O C O C

Opportunities for industrial use Seed oils are triglycerides

! Current Australian non-food use of vegetable oils ! Seed oils are comprised almost entirely of is about 12,000 tonnes (~3% of total oil usage) triglycerides composed of fatty acids ! Long-term opportunities exist for:- ! Oils are deposited in the seed in oilbodies - direct use in lubricants and inks - bio-diesel fuels (e.g. rapeseed ME) - specialty oleochemicals - pure fatty acids (e.g. oleic acid) - fatty acid derivatives (e.g. erucamide) - alkyl units for polymers (e.g. nylon) - biodegradable plastics - pharmaceutical proteins

Fatty acids are like petrochemicals Oil composition can be changed

! Fatty acids are simply hydrocarbon chains ! Seed oils are needed only for energy of various lengths with a carboxyl group storage and release during germination (~COOH) at one end (i.e. no structural role) ! Fatty acids can have bond types or functional groups that allow them to be ! Fatty acid composition can therefore be cleaved or derivatised by chemical dramatically modified, provided that new processing fatty acids are:- - deposited in triglycerides (and excluded from ! Olechemical industry competes with membranes) petrochemical industry as source of raw - able to be broken down during germination materials for chemicals and plastics

1 Engineering novel oils Engineering novel oils

! Great potential to modify oils to produce ! Great potential to modify oils to produce new compounds of industrial importance new compounds of industrial importance • great diversity of fatty acids in nature • great diversity of fatty acids in nature • fatty acid biosynthetic pathways are well known • fatty acid biosynthetic pathways are well known • many genes in pathway are already cloned • many genes in pathway are already cloned • oilseeds can all be genetically transformed • oilseeds can all be genetically transformed ! Good progress in metabolic engineering • high-laurate rapeseed • wax-ester rapeseed • epoxy fatty acids • biodegradable plastics

Food oils have simple composition Unusual fatty acids for industrial use

Oilseeds developed for food use are Oilseeds developed for food use are composed of a limited range of fatty acids composed of a limited range of fatty acids of nutritional importance of nutritional importance

SATURATES MUFA PUFA 16:0 18:0 18:1 18:2 18:3 palmitic stearic oleic linoleic linolenic … but in nature there are thousands of unusual fatty acids containing a range of functional groups that are of interest as raw materials for chemical industry

Unusual fatty acids for industrial use Example: High-laurate rapeseed

erucic acid ricinoleic acid ! Coconut oil and palm kernel oils are rich in lauric (polymers, cosmetics, (lubricants, cosmetics inks, pharmaceuticals) pharmaceuticals) acid (C12:0) which is used in - detergents - confectionery fats

vernolic acid lauric acid (resins, coatings, ! All oilseeds produce laurate, but then convert it (detergents) plasticisers) all into longer chain fatty acids

! Rapeseed has been genetically engineered to produce high levels of laurate by removing it from conjugated fatty acids petroselenic acid (superior drying oils) (polymers, detergents) fatty acid synthesis before it can be elongated

2 Example: High-laurate rapeseed Example: High-laurate rapeseed

rapeseed oil rapeseed oil laurate thioesterase gene laurate thioesterase gene zero laurate from California bay tree zero laurate from California bay tree TE TE

40% laurate 40% laurate

C12-specific TE liberates more laurate than can be incorporated into triglycerides LPAAT LPAAT laurate acyltransferase laurate acyltransferase gene from coconut 70% laurate gene from coconut 70% laurate β-oxidation is induced, creating a FUTILE CYCLE of laurate synthesis, breakdown and resynthesis

Example: Wax ester rapeseed Example: Wax ester rapeseed

Unique physical properties make waxes HEAR Arabidopsis useful in cosmetics, food products and industrial lubricants

Coordinate expression of genes for 3 enzymes from jojoba required to produce waxes in rapeseed + + ++ [aldehyde] acyl reductase acyl reductase acyl elongase wax synthase acyl (jojoba) (jojoba) (Lunaria) (jojoba) reductase fatty acid long chain long chain precursors fatty acids 1o alcohols (e.g. C18:1) elongase (C20-C24) wax synthase

0.2% long 70% wax esters in seed lipids chain alcohols wax esters

Example: Epoxy fatty acids Example: Epoxy fatty acids

! Epoxy fatty acids are highly reactive and have Crepis palaestina seeds contain high value uses in glues, resins and surface high contents (70%) of vernolic acid a ∆12-epoxygenated C18 fatty acid coatings ! Currently obtained either as petrochemical or by chemical epoxidation of soybean or linseed oil

Cpal2

COOH 18:3, linolenic acid

O O O COOH tri-epoxy-18:3 CSIRO cloned ∆12-epoxygenase gene and is attempting to develop oilseeds with high levels of vernolic ! Highly epoxidised oils are 2-3 times the price of acids by transferring it into linseed commodity food oils

3 Example: Epoxy fatty acids Example: Epoxy fatty acids

Epoxy fatty acids in seed oil (%) Fatty acid epoxygenase 070! Next step is to introduce additional genes gene (Cpal2) cloned from Crepis palaestina from vernolic acid producing species (e.g. Crepis palaestina, Euphorbia lagascae) that may enable transgenic

+ Cpal2 What limits accumulation? plants to accumulate higher levels of vernolic acid + Cpal2 + Cpal2 + Cpdes

& silence competing enzymes ??

What is required to engineer high levels of epoxy fatty acids in oilseeds?

Tri-epoxy fatty acids Unusual epoxy fatty acids

! Vernolic acid has some specific high value

uses but is relatively low-volume market 12 9 Vernonia galamensis O COOH Vernolic acid Euphorbia lagascae

! Tri-epoxy is much larger market Crepis palaestina ! Requires epoxy groups to be introduced at O COOH Coronaric acid Sunflower both ∆9 & ∆15 positions of vernolic acid

9,10-epoxy O COOH Flax rust ! Research challenge is to clone genes for stearic acid these functions and to see if they can be

14,15-epoxy-11- O COOH Alchornea combined in a single molecule eicosenoic acid

Unusual hydroxy fatty acids Conjugated fatty acids

12 9 13 11 9 COOH Ricinoleic Castor COOH Eleostearic Tung OH

Dihydroxy Bushy Conjugated Ruminant COOH COOH stearic rock-cress Linoleic (CLA) bacteria HO OH

OH COOH Calendic Marigold COOH Coriolic Coriaria

COOH 2-hydroxy COOH Stellaheptaenoic Microalgae linolenic OH Thyme Moth Dodecadienoic COOH Nervonic COOH pheromone OH

4 Acetylenic fatty acids Combinatorial fatty acids

Can fatty acids be developed that contain multiple types of functional groups? 12 9 COOH Crepenynic Crepis alpina

OH Conjugated Coriaria hydroxy COOH COOH Oropheic Annonaceae

COOH Isanic Isano seed COOH Olacaceae

Conjugated acetylenic

COOH Dihydro-matricaric Soldier beetle COOH Asteraceae

Low accumulation in transgenics Low accumulation in transgenics

Initial expression of the gene for fatty acid synthesis Initial expression of the gene for fatty acid synthesis enzyme only results in low level accumulation of enzyme only results in low level accumulation of unusual fatty acid in transgenic seed oil unusual fatty acid in transgenic seed oil

Content in seed oils of Unusual fatty acid Source plant Transgenic plant Epoxy (vernolic) 70% 5% Hydroxy (ricinoleic) 90% 17% Background biosynthetic pathways need to be Acetylenic 80% 25% modified to ensure that unusual fatty acids are:- Conjugated 65% 17% Petroselenic 85% 10% • Moved out of phospholipids C16:1 (∆6) 80% 10% • Accumulated in triglycerides (TAG) Cyclopropenoic 40% 5% Omega-3 (EPA/DHA) 60% 5% • Released from TAG during germination

Example: PHB synthesis Example: PHB synthesis

C O C O C O CLS ketothiolase Chloroplast Localisation Signal C + C C C C C (CLS) added to target each CLS reductase O C O C HO C enzyme to chloroplasts β-keto Aceto-acetyl- CLS synthase thiolase CoA reductase reporter reporter

PHB is synthesised from acetyl-CoA PHB synthase by action of only three enzymes

C O C O C O C O C C C C C C C C O C O C O C O C normal vigour PHB = 14% (dry wt)

5 Example: PHB synthesis Chemical Plants

Biodegradable polymers based on a family of R-3 hydroxy-alkanoic acids C genes from wild plants C C C C C C O C O C O C O crops C C C C C C C C HO C HO C HO C HO C 3-OH-butyrate 3-OH-valerate 3-OH-caproate 3-OH-heptanoate renewable industrial raw C O C O C O C O materials C C C C C C C C “We can envisage processing factories being designed C C C C O O O O to specifically match up with the genetics in the crops” Polyhydroxybutyrate (PHB) Richard McConnell, Pioneer Hi-Bred, 1997

Crop Biofactories Initiative

! New CSIRO and GRDC initiative to develop novel ways to produce and isolate high value industrial raw materials from plants ! Identify new oil and biopolymer products exploiting novel plant biochemistries ! Make new plant products that enable new industrial chemistries ! Provide technology for a sustainable crop biofactory industry