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

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

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

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

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

Processing of Bioethanol Focuses on the natural fermentation process: conversion of glucose into ethanol Current method (2 nd gen), two parts: 1)Obtain the cellulose source and process it with cellulases 2)Fermentation with yeast to produce ethanol (distillation) Figures left: (10), middle: (11), right: (12) glucose pyruvate ethanol

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

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

Costs: Consolidated Process Figure: (14)

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

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

The Cellulosome Reference: mentor Shen-long Tsai Cellulose Reducing Sugars

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

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

Engineering Cellulases **Can be specialized new cohesin/cellulase constructs Cloning Idea for New Constructs: -glucosidase: BglA + DocGf / exoglucanase: CelS + DocCc Secretion signal (with protein): MF1 + Gf protein Reference: mentor Shen-long Tsai C F

Method: Cloning 1.Identify the insert(s) and vector to use a)pET-24a b)protein: BglA c)cohesin: DocGf 2.Plan the order and restriction enzymes needed a)Restriction enzymes: cut a palindrome sequence of DNA b)Avoid restriction sites in inserts (destroys it)

Method: Cloning Figures left: (17), right: (18)

Method: Cloning References .TAGACCAAACGGTGAAAGACACGAAAAATACTCTTTGGCTCCAGTTGCTAAGGAATTGCAATCATTGTTGGGTAAGGATGTCACCTTCTTGAACGACTGTGTCGGTCCAGAAGTT GAAGCCGCTGTCAAGGCTTCTGCCCCAGGTTCCGTTATTTTGTTGGAAAACTTGTGTTACCACATCGAAGAAGAAGGTTCCAGAAAGGTCGATGGTCAAAAGGTCAAGGCTTCCAAG GAAGATGTTCAACAGTTCAGACACGAATTGAGCTCTTTGGCTGATGTTTACATCAACGATGCCTTCGGTACCGCTCACAGAGCTCACTCTTCTATGGTCGGTTTCGACTTGCCACAACG TGCTGCCGGTTTCTTGTTGGAAAAGGAATTGAAGTACTTCGGTAAGGCTTTGGAGAACCCAACCAGACCATTCTTGGCCGCGGCCGCAtgagatttccttcaatttttactgcagttttattcgcagca tcctccgcattagctgctccagtcaacactacaacagaagatgaaaccgcacaaattccggctgaagctgtcatcggttacttggatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttata aatactactattgccagcattgctgctaaagaagaaggggtatctttggataaaagagaggaaggcgaaccaGGATCCGGAACATATAACTATGGAGAAGCATTACAGAAATCAATAATGTTCTATGAATTCC AGCGTTCGGGAGATCTTCCGGCTGATAAACGTGACAACTGGAGAGACGATTCCGGTATGAAAGACGGTTCTGATGTAGGAGTTGATCTTACAGGAGGATGGTACGATGCAGGTGAC CATGTGAAATTTAATCTACCTATGTCATATACATCTGCAATGCTTGCATGGTCCTTATATGAGGATAAGGATGCTTATGATAAGAGCGGTCAGACAAAATATATAATGGACGGTATAA AATGGGCTAATGATTATTTTATTAAATGTAATCCGACACCCGGTGTATATTATTACCAAGTAGGAGACGGCGGAAAGGACCACTCTTGGTGGGGCCCTGCGGAAGTAATGCAGATGG AAAGACCGTCTTTTAAGGTTGACGCTTCTAAGCCCGGTTCTGCAGTATGTGCTTCCACTGCAGCTTCTCTGGCATCTGCAGCAGTAGTCTTTAAATCCAGTGATCCTACTTATGCAGAA AAGTGCATAAGCCATGCAAAGAACCTGTTTGATATGGCTGACAAAGCAAAGAGTGATGCTGGTTATACTGCGGCTTCAGGCTACTACAGCTCAAGCTCATTTTACGATGATCTCTCAT GGGCTGCAGTATGGTTATATCTTGCTACAAATGACAGTACATATTTAGACAAAGCAGAATCCTATGTACCGAATTGGGGTAAAGAACAGCAGACAGATATTATCGCCTACAAGTGGG GACAGTGCTGGGATGATGTTCATTATGGTGCTGAGCTTCTTCTTGCAAAGCTTACAAACAAACAATTGTATAAGGATAGTATAGAAATGAACCTTGACTTCTGGACAACTGGTGTTAA CGGAACACGTGTTTCTTACACGCCAAAGGGTTTGGCGTGGCTATTCCAATGGGGTTCATTAAGACATGCTACAACTCAGGCTTTTTTAGCCGGTGTTTATGCAGAGTGGGAAGGCTGT ACGCCATCCAAAGTATCTGTATATAAGGATTTCCTCAAGAGTCAAATTGATTATGCACTTGGCAGTACCGGAAGAAGTTTTGTTGTCGGATATGGAGTAAATCCTCCTCAACATCCTCA TCACAGAACTGCTCACGGTTCATGGACAGATCAAATGACTTCACCAACATACCACAGGCATACTATTTATGGTGCGTTGGTAGGAGGACCGGATAATGCAGATGGCTATACTGATGA AATAAACAATTATGTCAATAATGAAATAGCCTGCGATTATAATGCCGGATTTACAGGTGCACTTGCAAAAATGTACAAGCATTCTGGCGGAGATCCGATTCCAAACTTCAAGGCTATC GAAAAAATAACCAACGATGAAGTTATTATAAAGGCAGGTTTGAATTCAACTGGCCCTAACTACACTGAAATCAAGGCTGTTGTTTATAACCAGACAGGATGGCCTGCAAGAGTTACG GACAAGATATCATTTAAATATTTTATGGACTTGTCTGAAATTGTAGCAGCAGGAATTGATCCTTTAAGCCTTGTAACAAGTTCAAATTATTCTGAAGGTAAGAATACTAAGGTTTCCGG TGTGTTGCCATGGGATGTTTCAAATAATGTTTACTATGTAAATGTTGATTTGACAGGAGAAAATATCTACCCAGGCGGTCAGTCTGCGTGCAGACGAGAAGTTCAGTTCAGAATTGCC GCACCACAGGGAAGAAGATATTGGAATCCGAAAAATGATTTCTCATATGATGGATTACCAACCACCAGTACTGTAAATACGGTTACCAACATACCTGTTTATGATAACGGCGTAAAA GTATTTGGTAACGAACCCGCAGGTGGATCAGAACCCGGCACAAAGCTCGTTCCTACATGGGGCGATACAAACTGCGACGGCGTTGTAAATGTTGCTGACGTAGTAGTTCTTAACAGA TTCCTCAACGATCCTACATATTCTAACATTACTGATCAGGGTAAGGTTAACGCAGACGTTGTTGATCCTCAGGATAAGTCCGGCGCAGCAGTTGATCCTGCAGGCGTAAAGCTCACAG TAGCTGACTCTGAGGCAATCCTCAAGGCTATCGTTGAACTCATCACACTTCCTCAACTCGAGCACCACCACCACCACCACTGAGGATCCTCTAGAATCATGTAATTAGTTATGTCACGC TTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATA TTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCGGCCCTGCATTAATG AATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA GGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGCCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT..

Method: Cloning 3.Perform digestion of the insert and vector. 4.Perform ligation if successful. 5.Transformation of the DNA into a host cell.

Method: Cloning

6.Mini-preparation of colonies to test construct 7.Transformation into strain BL21.

Detection: SDS-Page Immunofluorescence Reducing Sugar Measurements

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

The Graph Figure: (14)

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

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

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

Thank you!

Questions?

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