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

Edgar  Cahoon  Center  for  Plant  Science  Innova5on  

University  of  Nebraska  Lincoln,  Nebraska  USA  

Vegetable  Oil:  Eat  It  or  Burn  It?  

Food  Uses:  Frying  oils,    margarine,  salad  oils  

Is  there  enough  vegetable  oil  for  both  food  and  fuel/biomaterial  uses?  

Nonfood  Uses:  Biodiesel,  bio-­‐materials  

Soybean  oil:  80%  Food  use/20%  Non-­‐food  use  (2011)  

Vegetable Oil-Derived Biodiesel is a More Significant Biofuel than Ethanol in Europe

Biodiesel has 1.4X more specific energy density than ethanol.

Lu  et  al.  (2011)  Current  Op.  Biotechnol.  

Strong  World  Demand  and  Limited  Supply  Has  Resulted  in  Increased  Prices  of  Major  Vegetable  Oils  

Total  world  vegetable  oil  consumpRon   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

Finished Motor Gasoline 3,369,922,000Distillate Fuel Oil 1,522,922,000Kerosene-type Jet Fuel 592,627,000Propane/Propylene 439,086,000Still Gas 258,613,000Ethane/Ethylene 257,814,000Petrochemical Feedstocks (i.e. Naptha & other oils) 250,553,000Residual Fuel Oil 248,581,000Lubes 41,862,000

2006 US Petroleum Product Usage (Barrels/yr)

Product Yield From a Barrel of Crude Oil

Source: Energy Information Administration

88%  

7%  

Fuels  

Uses  of  Crude  Oil  

Petrochemicals  &  Lubricants  

Vegetable  oils  offer  renewable    subs5tutes  for  crude  oil:  fuels,  chemical  feedstocks,  

and  lubricants  

Expanding  Oilseed  ProducRon  to  the  Great  Plains?    Lower  soil  ferRlity,  lower  rainfall  than  in  soybean  producRon  regions    No  significant  oilseed  crop  producRon  currently  in  large  porRons                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.  

*ProducRve  on  marginal  land.  *Not  widely  used  in  the  U.S.  for  food.  *Can  use  exisRng  equipment  and        infrastructure  for  harvesRng  and  processing.  *Can  be  grown  as  a  rotaRon  or  fallow  crop  *Super-­‐easy  to  transform:  amenable      to  metabolic  engineering  of  novel  traits.  *GeneRcally  similar  to  Arabidopsis:      Good  for  translaRon  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 0.02 to 0.03 mg/seed

Camelina ~1 mg/seed

Brassica napus 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

h^p://www.danforthcenter.org/cabs/  

h^p://icon.slu.se/ICON/  

h^p://camelinagene.org/  

Camelina  Metabolic  Engineering-­‐Based  Projects  

USDA-­‐AFRI:  ProducRon  of  Bio-­‐Based  Lubricants  in  a  Dedicated    Industrial  Oilseed  Crop  

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

U.S.  Department  of  Energy,  Energy  FronRers  Research  Center:  Center  for  Advanced  Biofuels  (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

Somatic Embryogenesis

Biolistic Transformation

Selection Multiplication & Maturation

Regeneration/ Plant Growth

Phenotypic Analysis

0 10 Months

Soybean

Agrobacterium Infiltration

Camelina Timeline of Transformation: Soybean versus Camelina

Binary vector used for seed specific expression of candidate genes

T-DNA LB

T-DNA RB

Seed specific

promoter

Gene of interest 1

3’ UTR

(terminator)

Seed specific

promoter

Gene of interest 2

3’ UTR (terminator)

Seed specific

promoter

Gene of interest 3

3’ UTR (terminator)

Gene cassette

Gene cassette

Gene cassette

NOS terminator

CMV promoter

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  MockaiRs  (Indiana  U)  

Lipid  Gene  Database  from  Camelina  454  Data  

Jason  Macrander  

Gene At ID % Identity w/ Arabidopsis

% Identity w/ Brassica napus

a-tubulin At1g04820 93 (564 bp) 91 (562 bp) FAD2 At3g12120 93 (542 bp) 84 (542 bp)

At2g42600 93 (482 bp) 84 (482 bp) PEP carboxylase

At2g43710 95 (650 bp) 89 (650 bp) Stearoyl-ACP desaturase

At1g74960 93 (611 bp) 88 (611 bp) FABI (KASII)

At2g46210 92 (592 bp) 84 (593 bp) Sphingolipid D8 desaturase

At5g16390 91 (430 bp) 84 (593 bp) BCCP

At1g21970 86 (716 bp) 79 (662 bp) LEC1

Camelina EST Analysis: Comparison of Nucleotide Sequence Identity With Arabidopsis and Brassica napus

*Arabidopsis sequences are suitable for use for RNAi experiments in camelina.

Significant Infrastructure for Biotech Camelina in Nebraska

Scottsbluff Camelina Variety

Testing

Mead Sidney

Biotech Fields

Oil Analysis Capability

Soil-bed greenhouse

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.

Carbons 16 18 18 18 18 Double bonds 0 0 1 2 3

Cold flowa Worse Worse Similar ND ND Fuel stabilityb Good Good Satisfactory Poor Poor

NOx emissionsc Lower Lower Similar Higher Higher

Ignition qualityd Higher Higher Higher Similar Lowere

Palmitate Stearate Oleate Linoleate Linolenate

Petroleum Diesel Versus Biodiesel

A high oleic acid vegetable oil is best for biodiesel

Durrett et al. Plant J. 2008 May;54(4):593-607

05

10152025303540

16:0 18:0 18:1 18:2 18:3

wt%

of t

otal

fatty

aci

ds

20:0 20:1 22:1 16:0 18:0 18:1 18:2 18:3

wt%

of t

otal

fatty

aci

ds

20:0 20:1 22:1

16:0 18:0 18:1 18:2 18:3

wt%

of t

otal

fatty

aci

ds

20:0 20:1 22:1

Wild-type AtFAD2-RNAi/ AtFAE1-RNAi

AtFAD3-RNAi/ CsFAE1-RNAi

Goal  1:  Reduce  the  PolyunsaturaRon  of  Camelina  Oil  

Basta selection marker used in order to stack DsRed-linked trait genes.

C10-C14 aliphatic

hydrocarbons66%

C15-C17 aliphatic

hydrocarbons7%

Aromatics18%

C8-C9 aliphatic hydrocarbons

9%

Jet A - Composition

Source: Chevron

Tailoring  Fa^y  Acid  ComposiRon  of  Vegetable  Oils  for  Specific  Fuel  Markets  

Example:  Jet  fuel  ~6%  of  total  crude  oil  usage  

Jet  A  (Kerosene  and  paraffin  oil-­‐based  fuel)    comprises  C8-­‐C16  hydrocarbons  

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.  

Problem/Challenge:    Most  exisRng  vegetable  oils,  including  camelina  oil,  are  enriched  in  C16-­‐C18  fa^y  acids….too  long  for  jet  fuel.    

Production of Vegetable Oils Enriched in Short/ Medium Chain Length Fatty Acids by Expression of

Specialized Acyl-ACP Thioesterases

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

Mol%  of  total  fa^y

 acids  

Homozygous transgenic camelina lines

16:0  14:0  12:0  10:0  8:0  

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

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

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.

CECO: Simultaneously targeting multiple pathways for metabolic engineering of camelina seed oil content and quality

70 55

35 25

15

10

kDa

12S

2S

a

b

L S

At Cs Bn

Don’t Forget the Protein Meal: Camelina Seeds Have ~25-30% Protein

Seed storage protein profile of camelina is similar to that of Arabidopsis and Brassica napus: 12S globulins and 2S albumins

Tam Nguyen

-10

-17

-28 -36

-55 -72

Can we alter the seed storage protein composition of camelina?

RNAi silencing of 2S seed storage proteins (napin)

Suppression of 2S proteins

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          -­‐ProducRve  on  with  low  ferRlity  and  limited  rainfall  and  grown  in    rotaRons          -­‐Can  be  easily  transformed          -­‐Tools  are  in  place  for  metabolic  engineering  of  novel  oil  and                protein  traits:    mulR-­‐gene  vectors  with  mulRple  choices  of                  selecRon  markers,  seed-­‐specific  promoters,  genomic  informaRon.    *Progress  is  being  made  in  improving  the  anRoxidant  and  fa^y  acid      composiRon  of  camelina  for  lubricant  and  biodiesel  uses.    

“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  AviaRon  Biofuel  in  US  to  More  Than  Double  in  2010”    January  2010  

Camelina  in  the  news  headlines:  

Acknowledgments

Contributors

Chaofu Lu (Montana St.) Tom Clemente (UNL) Johnathan Napier (Rothamsted) Basil Nikolau (Iowa St.) Jeong-Won Nam (Danforth Center) Jan Jaworski (Danforth Center) Brian Scheffler (USDA) Sten Stymne (Swedish Agricultural U.) Ljerka Kunst (UBC) Jay Shockey (USDA) John Dyer (USDA) Keithanne Mockaitis (Indiana U.)

Lab Contributors

Chunyu Zhang Wenyu Yang Rebecca Cahoon Tara Nazarenus Jill Silva Tam Nguyen Anjireddy Konda

Funding: USDA, US Department of Energy, National Science Foundation