—An Emerging Oil Feedstock. Progress and Prospects for Biotechnological Improvement

Edgar Cahoon Center for Science Innovaon University of Nebraska Lincoln, Nebraska USA : Eat It or Burn It?

Food Uses: Frying oils, , salad oils

Nonfood Uses: , 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 consumpon 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 -type 592,627,000 substutes for crude oil: /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 Producon to the Great Plains? . Lower soil ferlity, lower rainfall than in producon regions . No significant oilseed producon currently in large porons of the Great Plains Is Camelina sava the best choice for an biofuel/industrial oilseed crop in the age of ? Camelina sava (false flax, gold of pleasure) Prior to World War II, was anestablished oilseed crop in Eastern Europe and now an emerging oilseed in the Great Plains and Pacific Northwest. *Producve on marginal land. *Not widely used in the U.S. for food. *Can use exisng equipment and infrastructure for harvesng and processing. *Can be grown as a rotaon or fallow crop *Super-easy to transform: amenable to metabolic engineering of novel traits. *Genecally similar to Arabidopsis: Good for translaon 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 .

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: Producon of Bio-Based Lubricants in a Dedicated Industrial Oilseed Crop

hp://camelinagene.org/

European Commission Seventh Framework Program: ICON, Industrial Producing Added Value Oils for Novel Chemicals

hp://icon.slu.se/ICON/

U.S. Department of Energy, Energy Froners Research Center: Center for Advanced (CABS)

hp://www.danforthcenter.org/cabs/

U.S. Department of Energy, ARPA-E Center for Enhanced (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 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 Mockais (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 content of the oil. >Increased content of

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 Polyunsaturaon 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 Fay Acid Composion 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 exisng vegetable oils, including camelina oil, are enriched in C16-C18 fay acids….too long for jet fuel.

Vegetable oils with short- and medium-chain fay 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 fay 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 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 for medium chain FA production Cuphea: A Rich Source of Short/Medium Chain Fay 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 , 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 -Producve on with low ferlity and limited rainfall and grown in rotaons -Can be easily transformed -Tools are in place for metabolic engineering of novel oil and protein traits: mul-gene vectors with mulple choices of selecon markers, seed-specific promoters, genomic informaon.

*Progress is being made in improving the anoxidant and fay acid composion 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 Aviaon 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