Camelina—An Emerging Biofuel Oil Feedstock. Progress and Prospects for Biotechnological Improvement
Edgar Cahoon Center for Plant Science Innova on University of Nebraska Lincoln, Nebraska USA Vegetable Oil: Eat It or Burn It?
Food Uses: Frying oils, margarine, salad oils
Nonfood Uses: Biodiesel, bio-materials
Soybean oil: 80% Food use/20% Non-food use (2011)
Is there enough vegetable oil for both food and fuel/biomaterial uses? Vegetable Oil-Derived Biodiesel is a More Significant Biofuel than Ethanol in Europe
Biodiesel has 1.4X more specific energy density than ethanol. Strong World Demand and Limited Supply Has Resulted in Increased Prices of Major Vegetable Oils
Lu et al. (2011) Current Op. Biotechnol.
Total world vegetable oil consump on Major vegetable oil prices
World vegetable oil consumption have nearly doubled over the past decade and will double again by 2030
How do we meet this demand? Alternative sources of vegetable oils Increased yields per hectare 2006 US Petroleum Product Usage (Barrels/yr) Finished Motor Gasoline 3,369,922,000 Vegetable oils offer renewable Distillate Fuel Oil 1,522,922,000 Kerosene-type Jet Fuel 592,627,000 subs tutes for crude oil: Propane/Propylene 439,086,000 Still Gas 258,613,000 fuels, chemical feedstocks, Ethane/Ethylene 257,814,000 Petrochemical Feedstocks (i.e. Naptha and lubricants & other oils) 250,553,000 Residual Fuel Oil 248,581,000 Lubes 41,862,000 Uses of Crude Oil
Product Yield From a Barrel of Crude Oil 88% Fuels
7% Source: Energy Information Administration Petrochemicals & Lubricants Expanding Oilseed Produc on to the Great Plains? . Lower soil fer lity, lower rainfall than in soybean produc on regions . No significant oilseed crop produc on currently in large por ons of the Great Plains Is Camelina sa va the best choice for an biofuel/industrial oilseed crop in the age of biotechnology? Camelina sa va (false flax, gold of pleasure) Brassicaceae Prior to World War II, was anestablished oilseed crop in Eastern Europe and now an emerging oilseed in the Great Plains and Pacific Northwest. *Produc ve on marginal land. *Not widely used in the U.S. for food. *Can use exis ng equipment and infrastructure for harves ng and processing. *Can be grown as a rota on or fallow crop *Super-easy to transform: amenable to metabolic engineering of novel traits. *Gene cally similar to Arabidopsis: Good for transla on of lab findings from a model plant to a crop plant. Camelina seed composition:
Oil: 30 to 40% of seed weight Protein: 25% to 30% of seed weight Relatively low in glucosinolates
Arabidopsis Camelina Brassica napus 0.02 to 0.03 mg/seed ~1 mg/seed 3 to 5 mg/seed Nebraska: Excellent crop land in the east/marginal land in the west
Very productive for corn and soybeans.
Marginal crop land: Niche for camelina? Camelina: Requires about 1/3 of the fertility as canola Productive with limited rainfall and irrigation Field trials in the Nebraska Panhandle with 12 inches of irrigation: 2,385 lbs/acre (52 bushels per acre) of camelina versus 2,903 lbs/acre of canola Camelina: Maturity 20 days earlier than canola. *Alexander Pavlista & Gary Hergert—University of Nebraska Camelina Metabolic Engineering-Based Projects
USDA-AFRI: Produc on of Bio-Based Lubricants in a Dedicated Industrial Oilseed Crop
h p://camelinagene.org/
European Commission Seventh Framework Program: ICON, Industrial Crops Producing Added Value Oils for Novel Chemicals
h p://icon.slu.se/ICON/
U.S. Department of Energy, Energy Fron ers Research Center: Center for Advanced Biofuels (CABS)
h p://www.danforthcenter.org/cabs/
U.S. Department of Energy, ARPA-E Center for Enhanced Camelina Oil (CECO) Development of a Metabolic Engineering Tool Box for Camelina
*Simple, non-labor intensive Agrobacterium-based transformation system.
*Construction of binary vectors for multiple genes with different selection markers for complex traits.
*Preparation of gene expression cassettes with a range of seed-specific promoters.
*Fluorescent protein markers for easy selection of transgenic seeds and maintenance of transgenic lines.
*Development of genomic resources for camelina. Camelina is a Dream Crop for Metabolic Engineers Camelina can be transformed by floral vacuum infiltration of agrobacterium…similar to Arabidopsis Soybean
Somatic Selection Multiplication & Regeneration/ Embryogenesis Maturation Plant Growth
Biolistic Phenotypic Transformation Analysis
Camelina Timeline of Transformation: Soybean versus Camelina
Agrobacterium Infiltration 0 10 Months Binary vector used for seed specific expression of candidate genes
Gene of Gene of Gene of interest 1 interest 2 interest 3 Seed Seed Seed specific specific specific promoter 3’ promoter 3’ UTR promoter 3’ UTR UTR (terminator) (terminator) (terminator) T-DNA T-DNA LB RB
Gene Gene Gene CMV NOS cassette cassette cassette promoter terminator DsRed • Other binaries with kanamycin, Basta, and hygromycin selection markers for trait stacking • Different seed specific promoter/terminator cassettes can be easily cloned into binary vectors Use of DsRed Fluorescent Protein Marker Facilitates Camelina Metabolic Engineering
*Transgenic seeds can be detected with a green LED flashlight and red camera filter.
*T1 seeds from the vacuum infiltrated plants can be analyzed for desired seed composition trait.
Development of Genomic Resources for Camelina
*Generation of ESTs from developing camelina seeds
*454 sequencing of developing seeds 789 Mb of total sequence Average read lengths of two runs: 353 bp & 433bp
Wenyu Yang Brian Scheffler (USDA-ARS) Keithanne Mockai s (Indiana U) Lipid Gene Database from Camelina 454 Data
Jason Macrander Camelina EST Analysis: Comparison of Nucleotide Sequence Identity With Arabidopsis and Brassica napus % Identity w/ % Identity w/ Gene At ID Arabidopsis Brassica napus
a-tubulin At1g04820 93 (564 bp) 91 (562 bp) FAD2 At3g12120 93 (542 bp) 84 (542 bp) PEP At2g42600 93 (482 bp) 84 (482 bp) carboxylase Stearoyl-ACP At2g43710 95 (650 bp) 89 (650 bp) desaturase FABI (KASII) At1g74960 93 (611 bp) 88 (611 bp)
Sphingolipid At2g46210 92 (592 bp) 84 (593 bp) D8 desaturase BCCP At5g16390 91 (430 bp) 84 (593 bp) LEC1 At1g21970 86 (716 bp) 79 (662 bp)
*Arabidopsis sequences are suitable for use for RNAi experiments in camelina. Significant Infrastructure for Biotech Camelina in Nebraska
Soil-bed greenhouse
Scottsbluff Sidney Mead Camelina Variety Testing Biotech Fields
Oil Analysis Capability Engineering Camelina Oil for Improved Biofuel and Biolubricant Properties
*Improved lubricant/biodiesel functionality of camelina vegetable oil
→Enhanced oxidative stability >Reduced polyunsaturation/increased oleic acid content of the oil. >Increased content of vitamin E antioxidants
In progress
*Novel fatty acids: conjugated, hydroxy, epoxy fatty acids.
*Novel high temperature lubricants: wax esters.
*Modify both oil and protein traits for improved industrial functionality of the complete seed. Petroleum Diesel Versus Biodiesel
Palmitate Stearate Oleate Linoleate Linolenate Carbons 16 18 18 18 18 Double 0 0 1 2 3 bonds Cold flowa Worse Worse Similar ND ND Fuel Good Good Satisfactory Poor Poor stabilityb NO x Lower Lower Similar Higher Higher emissionsc Ignition Higher Higher Higher Similar Lowere qualityd
Durrett et al. Plant J. 2008 May;54(4):593-607 A high oleic acid vegetable oil is best for biodiesel Goal 1: Reduce the Polyunsatura on of Camelina Oil
40 35 Wild-type AtFAD2-RNAi/ 30 AtFAE1-RNAi 25 20 15 10 % of total fatty acids totalfatty %of % of total fatty acids totalfatty %of 5 wt wt 0 16:0 18:0 18:1 18:2 18:3 20:0 20:1 22:1 16:0 18:0 18:1 18:2 18:3 20:0 20:1 22:1
AtFAD3-RNAi/ CsFAE1-RNAi Basta selection marker used in order to stack DsRed-linked trait genes. % of total fatty acids totalfatty %of wt
16:0 18:0 18:1 18:2 18:3 20:0 20:1 22:1 Tailoring Fa y Acid Composi on of Vegetable Oils for Specific Fuel Markets
Example: Jet fuel ~6% of total crude oil usage Jet A - Composition
Aromatics C8-C9 aliphatic 18% hydrocarbons 9% C15-C17 aliphatic hydrocarbons 7%
C10-C14 aliphatic hydrocarbons 66%
Source: Chevron Jet A (Kerosene and paraffin oil-based fuel) comprises C8-C16 hydrocarbons
Problem/Challenge: Most exis ng vegetable oils, including camelina oil, are enriched in C16-C18 fa y acids….too long for jet fuel.
Vegetable oils with short- and medium-chain fa y acids can be generated using variant FatB acyl-ACP thioesterases from plants such as Cuphea, California bay laurel, and elm. Production of Vegetable Oils Enriched in Short/ Medium Chain Length Fatty Acids by Expression of Specialized Acyl-ACP Thioesterases
45% 40% 35% 30% 25% 16:0 20% 14:0 15% 12:0 10%
% of total fa y acids 10:0 5% 8:0 Mol 0%
Homozygous transgenic camelina lines Dr. Jill Silva To achieve very high levels of jet-fuel type short- and medium-chain fatty acids in camelina seed oil requires placement of these fatty acids at all three positions of triacylglycerol
Typical oilseeds do not introduce short- and medium-chain fatty acids at the sn-2 position of TAG:
Need specialized acyltransferases Engineering short/medium chain fatty acid biosynthesis in camelina
Approach: Use genes known to be involved in medium chain fatty acid biosynthesis and new target genes from Cuphea 454 sequencing to transform Camelina sativa for medium chain FA production Cuphea: A Rich Source of Short/Medium Chain Fa y Acids
•C. hookeriana accumulates up to 75% 8:0 and 10:0 in its seed oil •* C. pulcherrima ~95% 8:0 •* C. viscosissima accumulates 25% 8:0, 50-70% 10:0 •C. lanceolata >80% 10:0 *used for 454 sequencing Cuphea Seed Transcriptomic Analysis
Cuphea viscosissima (25% 8:0, >50% 10:0 ): • 554 Mb of sequence was obtained, with an average read length of 393 bases
Cuphea pulcherrima (~95% 8:0): • More than 624 Mb of sequence was obtained, with an average read length of 425.6 bases
Now testing Cuphea genes in camelina for improved short- and medium-chain fatty acid accumulation
Challenges of Development of Camelina as a Biofuel Crop
In contrast to soybean and rapeseed, camelina has received little breeding effort for improvement of agronomic and oil traits.
Example: Elite rapeseed germplasm—40 to 50% seed oil content Camelina germplasm—30 to 40% seed oil content
CECO Goal: Speed up improvement of camelina for biofuel production by stacking of ~12 oil enhancement-related transgenes.
Principal Investigator: Jan Jaworski (DDPSC) Co-PI- Sam Wang (DDPSC/UMSL) Co-PI- Ed Cahoon (UNL) Co-PI- Dick Sayre (NMC/LANL) Co-PI- Chaofu Lu (Montana State) Co-PI- Doug Allen (DDPSC/USDA) Co-PI- Dave Kramer (MSU) Consultant- J. Alan Weber Consultant- Duane Johnson
CECO: Simultaneously targeting multiple pathways for metabolic engineering of camelina seed oil content and quality Don’t Forget the Protein Meal: Camelina Seeds Have ~25-30% Protein
At Cs Bn kDa
70 55 35 12S 25 a b 15 2S 10 L Tam Nguyen S Seed storage protein profile of camelina is similar to that of Arabidopsis and Brassica napus: 12S globulins and 2S albumins Can we alter the seed storage protein composition of camelina?
RNAi silencing of 2S seed storage proteins (napin)
-72 -55 -36 -28
-17
Suppression of 2S proteins -10
Future: Combine industrial protein and oil traits to have a completely industrial camelina Conclusions: *Camelina holds considerable promise as a biotech industrial oilseed -Not widely used as a food crop in the US -Produc ve on with low fer lity and limited rainfall and grown in rota ons -Can be easily transformed -Tools are in place for metabolic engineering of novel oil and protein traits: mul -gene vectors with mul ple choices of selec on markers, seed-specific promoters, genomic informa on.
*Progress is being made in improving the an oxidant and fa y acid composi on of camelina for lubricant and biodiesel uses.
Camelina in the news headlines:
“Japan Airlines biofuels flight test a success; camelina, algae, jatropha used in B50 biofuel mix; fuel economy higher than Jet-A” February 2009
“Camelina Acreage for Avia on Biofuel in US to More Than Double in 2010” January 2010 Acknowledgments
Lab Contributors Contributors
Chunyu Zhang Chaofu Lu (Montana St.) Wenyu Yang Tom Clemente (UNL) Rebecca Cahoon Johnathan Napier (Rothamsted) Tara Nazarenus Basil Nikolau (Iowa St.) Jill Silva Jeong-Won Nam (Danforth Center) Tam Nguyen Jan Jaworski (Danforth Center) Anjireddy Konda Brian Scheffler (USDA) Sten Stymne (Swedish Agricultural U.) Ljerka Kunst (UBC) Jay Shockey (USDA) John Dyer (USDA) Keithanne Mockaitis (Indiana U.)
Funding: USDA, US Department of Energy, National Science Foundation