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Engineering Yeast to Drive Cars The Road for the Cellulosome

Engineering Yeast to Drive Cars The Road for the Cellulosome. BRITE 2008 Presentation. Jeffrey Chen August 21, 2008 University of Texas Lab: Wilfred Chen. Cars and Gasoline. Demand and prices for oil have increased Supply of oil is limited. Figures – left: (1), right: (2). Biofuels.

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Engineering Yeast to Drive Cars The Road for the Cellulosome

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  1. Engineering Yeast to Drive Cars The Road for the Cellulosome BRITE 2008 Presentation Jeffrey Chen August 21, 2008 University of Texas Lab: Wilfred Chen

  2. Cars and Gasoline • Demand and prices for oil have increased • Supply of oil is limited Figures – left: (1), right: (2)

  3. Biofuels Reference: (3) • Definition: liquid or gaseous fuel that is produced predominately from biomass • Advantages: • Cleaner, more natural fuel • Readily available source, sustainable • Alternative security to petroleum

  4. Bioethanol Figure: (4) Reference: (5) • Ethanol produced as fuel • Wide-accepted use worldwide currently;popular fuel • Biomass obtained from any plantsource with cellulosic material(originally sugar cane / maize)

  5. Processing of Bioethanol glucose pyruvate ethanol Figures – left: (10), middle: (11), right: (12) • Focuses on the natural fermentation process:conversion of glucose into ethanol • Current method (2nd gen), two parts: • Obtain the cellulose source and process it with cellulases • Fermentation with yeast to produce ethanol (distillation)

  6. Disadvantages of Bioethanol Figures -- left: (6), right: (7) References: (8), (9) • Changes in bioethanol have shifted tocellulosic ethanol production, but still not very efficient • Not competitive with gasoline yet • Food crisis/famine currently

  7. Improvements for Bioethanol Reference: (13) • More emphasis on “cellulosic” ethanol; use of biomass • Increasing efficiency of cellulase digestion • Consolidating the two processes**

  8. Costs: Consolidated Process Figure: (14)

  9. Increasing Efficiency:The Cellulosome Reference: mentor Shen-long Tsai • Cellulosome: complex of enzymes that forms outsidethe cell for digestion • Not free-floating! • Organized complex • Can be specialized** • Only found in certain bacteria naturally

  10. The Cellulosome Cellulase Cohesin Dockerin Scaffoldin Reference: mentor Shen-long Tsai

  11. The Cellulosome Cellulose ReducingSugars Reference: mentor Shen-long Tsai

  12. The Cellulases Endoglucanase Exoglucanase Figures – top/middle: (15), bottom: (16)

  13. The Cellulases Endoglucanase Exoglucanase β-glucosidase Figures – top/middle: (15), bottom: (16)

  14. Engineering Cellulases C C F F Reference: mentor Shen-long Tsai • **Can be specialized – new cohesin/cellulase constructs • Cloning Idea for New Constructs: • β-glucosidase: BglA + DocGf / exoglucanase: CelS + DocCc • Secretion signal (with protein): MFα1 + Gf protein

  15. Method: Cloning • Identify the insert(s) and vector to use • pET-24a • protein: BglA • cohesin: DocGf • Plan the order and restriction enzymes needed • Restriction enzymes: cut a palindrome sequence of DNA • Avoid restriction sites in inserts (destroys it)

  16. Method: Cloning Figures – left: (17), right: (18)

  17. Method: Cloning …….TAGACCAAACGGTGAAAGACACGAAAAATACTCTTTGGCTCCAGTTGCTAAGGAATTGCAATCATTGTTGGGTAAGGATGTCACCTTCTTGAACGACTGTGTCGGTCCAGAAGTTGAAGCCGCTGTCAAGGCTTCTGCCCCAGGTTCCGTTATTTTGTTGGAAAACTTGTGTTACCACATCGAAGAAGAAGGTTCCAGAAAGGTCGATGGTCAAAAGGTCAAGGCTTCCAAGGAAGATGTTCAACAGTTCAGACACGAATTGAGCTCTTTGGCTGATGTTTACATCAACGATGCCTTCGGTACCGCTCACAGAGCTCACTCTTCTATGGTCGGTTTCGACTTGCCACAACGTGCTGCCGGTTTCTTGTTGGAAAAGGAATTGAAGTACTTCGGTAAGGCTTTGGAGAACCCAACCAGACCATTCTTGGCCGCGGCCGCAtgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaaccgcacaaattccggctgaagctgtcatcggttacttggatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctttggataaaagagaggaaggcgaaccaGGATCCGGAACATATAACTATGGAGAAGCATTACAGAAATCAATAATGTTCTATGAATTCCAGCGTTCGGGAGATCTTCCGGCTGATAAACGTGACAACTGGAGAGACGATTCCGGTATGAAAGACGGTTCTGATGTAGGAGTTGATCTTACAGGAGGATGGTACGATGCAGGTGACCATGTGAAATTTAATCTACCTATGTCATATACATCTGCAATGCTTGCATGGTCCTTATATGAGGATAAGGATGCTTATGATAAGAGCGGTCAGACAAAATATATAATGGACGGTATAAAATGGGCTAATGATTATTTTATTAAATGTAATCCGACACCCGGTGTATATTATTACCAAGTAGGAGACGGCGGAAAGGACCACTCTTGGTGGGGCCCTGCGGAAGTAATGCAGATGGAAAGACCGTCTTTTAAGGTTGACGCTTCTAAGCCCGGTTCTGCAGTATGTGCTTCCACTGCAGCTTCTCTGGCATCTGCAGCAGTAGTCTTTAAATCCAGTGATCCTACTTATGCAGAAAAGTGCATAAGCCATGCAAAGAACCTGTTTGATATGGCTGACAAAGCAAAGAGTGATGCTGGTTATACTGCGGCTTCAGGCTACTACAGCTCAAGCTCATTTTACGATGATCTCTCATGGGCTGCAGTATGGTTATATCTTGCTACAAATGACAGTACATATTTAGACAAAGCAGAATCCTATGTACCGAATTGGGGTAAAGAACAGCAGACAGATATTATCGCCTACAAGTGGGGACAGTGCTGGGATGATGTTCATTATGGTGCTGAGCTTCTTCTTGCAAAGCTTACAAACAAACAATTGTATAAGGATAGTATAGAAATGAACCTTGACTTCTGGACAACTGGTGTTAACGGAACACGTGTTTCTTACACGCCAAAGGGTTTGGCGTGGCTATTCCAATGGGGTTCATTAAGACATGCTACAACTCAGGCTTTTTTAGCCGGTGTTTATGCAGAGTGGGAAGGCTGTACGCCATCCAAAGTATCTGTATATAAGGATTTCCTCAAGAGTCAAATTGATTATGCACTTGGCAGTACCGGAAGAAGTTTTGTTGTCGGATATGGAGTAAATCCTCCTCAACATCCTCATCACAGAACTGCTCACGGTTCATGGACAGATCAAATGACTTCACCAACATACCACAGGCATACTATTTATGGTGCGTTGGTAGGAGGACCGGATAATGCAGATGGCTATACTGATGAAATAAACAATTATGTCAATAATGAAATAGCCTGCGATTATAATGCCGGATTTACAGGTGCACTTGCAAAAATGTACAAGCATTCTGGCGGAGATCCGATTCCAAACTTCAAGGCTATCGAAAAAATAACCAACGATGAAGTTATTATAAAGGCAGGTTTGAATTCAACTGGCCCTAACTACACTGAAATCAAGGCTGTTGTTTATAACCAGACAGGATGGCCTGCAAGAGTTACGGACAAGATATCATTTAAATATTTTATGGACTTGTCTGAAATTGTAGCAGCAGGAATTGATCCTTTAAGCCTTGTAACAAGTTCAAATTATTCTGAAGGTAAGAATACTAAGGTTTCCGGTGTGTTGCCATGGGATGTTTCAAATAATGTTTACTATGTAAATGTTGATTTGACAGGAGAAAATATCTACCCAGGCGGTCAGTCTGCGTGCAGACGAGAAGTTCAGTTCAGAATTGCCGCACCACAGGGAAGAAGATATTGGAATCCGAAAAATGATTTCTCATATGATGGATTACCAACCACCAGTACTGTAAATACGGTTACCAACATACCTGTTTATGATAACGGCGTAAAAGTATTTGGTAACGAACCCGCAGGTGGATCAGAACCCGGCACAAAGCTCGTTCCTACATGGGGCGATACAAACTGCGACGGCGTTGTAAATGTTGCTGACGTAGTAGTTCTTAACAGATTCCTCAACGATCCTACATATTCTAACATTACTGATCAGGGTAAGGTTAACGCAGACGTTGTTGATCCTCAGGATAAGTCCGGCGCAGCAGTTGATCCTGCAGGCGTAAAGCTCACAGTAGCTGACTCTGAGGCAATCCTCAAGGCTATCGTTGAACTCATCACACTTCCTCAACTCGAGCACCACCACCACCACCACTGAGGATCCTCTAGAATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCGGCCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGCCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT…….. References

  18. Method: Cloning Perform digestion of the insert and vector. Perform ligation if successful. Transformation of the DNA into a host cell.

  19. Method: Cloning

  20. Method: Cloning Mini-preparation of colonies to test construct Transformation into strain BL21.

  21. Detection: • SDS-Page • Immunofluorescence • Reducing Sugar Measurements

  22. Reducing Sugar Prediction BglA + Ec + At Ec + At Ec + BglA Ec + BglA control Glucose (OD) Time

  23. The Graph Figure: (14)

  24. Future Work • Run more varied conditions to express the Gf construct • Finish constructs; if successful, create new constructs • Perform growth curve, reducing sugar, and glucose level experiments/measurements

  25. References 1. Flickr. $2.299 [picture]. (August 2008) Available at http://flickr.com/photos/69282447@N00/241986026 2. Crazy-jokes. "Gas Pump #2" [picture]. (August 2008) Available at http://blogs.e-rockford.com/applesauce/category/michael-bloomberg/ 3. Demirbas, Ayhan (2006). Progress and recent trends in biofuels. Progress in Energy and Combustion Science, 33:1, 1-18. doi:10.1016/j.pecs.2006.06.001 4. my-biodiesel.org. "Corn Fuel" [picture] (August 2008) Available at http://my-biodiesel.org/web/ 5. Wikipedia (2008). Bioethanol. http://en.wikipedia.org/wiki/Biofuel 6. Experiments using urine and humus dervied from ecological toilets. Experimental maize field under investigation [picture]. (August 2008) Available at http://aquamor.tripod.com/KYOTO.htm 7. Wikipedia. Cornfield in South Africa2 [picture]. (August 2008) Available at http://en.wikipedia.org/wiki/Image:Cornfield_in_South_Africa2.jpg 8. Wikipedia (2008). Ethanol fuel. http://en.wikipedia.org/wiki/Bioethanol 9. Rosegrant, Mark (2008). Biofuels and Grain Prices: Impact and Policy Responses. International Food Policy Research Institute. http://www.ifpri.org/pubs/testimony/rosegrant20080507.pdf 10. Masters Athlete Physiology & Performance. Lactate Threshold [picture]. (August 2008) Available at http://home.hia.no/~stephens/lacthres.htm 11. Wikipedia. "Pyruvic acid 2D skeletal" [picture]. (August 2008) Available at http://en.wikipedia.org/wiki/Image:Pyruvic-acid-2D-skeletal.png 12. Wikipedia. "Ethanol 2D skeletal" [picture]. (August 2008) Available at http://en.wikipedia.org/wiki/Image:Ethanol-2D-skeletal.svg 13. Wikipedia (2008). Cellulosic ethanol. http://en.wikipedia.org/wiki/Cellulosic_ethanol 14. Lynd, Lee et al (2005). Consolidated bioprocessing of cellulosic biomass: an update. Current Opinion in Biotechnology, 16:5, 577-583. doi:10.1016/j.copbio.2005.08.009 15. Colebrook, Michael. Life Chemistry. “Cellulose” [picture] (August 2008) Available at http://www.greenspirit.org.uk/resources/LifeChemistry.htm 16. Sci-toys. “Lactose” [picture]. (August 2008) Available at http://sci-toys.com/ingredients/lactose.html 17. Novagen. "pET-24a" [picture]. (August 2008) Available at http://www.merckbiosciences.com/docs/docs/PROT/TB070.pdf 18. Queens University of Charlotte. Recombinant DNA Technology [picture]. (August 2008) Available at http://campus.queens.edu/faculty/jannr/bio103/helpPages/C16DNAtech.htm

  26. Acknowledgements • The BRITE program (NSF), Jun Wang, and UCR • Ph.D. candidate Shen-long Tsai • Professor Wilfred Chen • All of the people in the Chen Lab • All of the professors at UCR • Others…

  27. Thank you!

  28. Questions?

  29. Engineering Yeast to Drive Cars The Road for the Cellulosome BRITE 2008 Presentation Jeffrey Chen August 21, 2008 University of Texas Lab: Wilfred Chen

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