Synthetic Consortium for Cellulose Hydrolysis and Ethanol Production

1. Synthetic Consortium for Cellulose Hydrolysis and Ethanol Production David Pham, Shen-Long Tsai, Anjali Mulchandani, and Dr. Wilfred Chen Department of Chemical and Environmental Engineering at University of California Riverside, CA 92521 Introduction Abstract Results Two of the world’s major problems in this era, Solid Municipal Waste (SMW) buildup and Gasoline depletion, can be solved with the process of converting cellulose into ethanol. In perfecting this technique, the high cellulose content of SMW, can be used as a renewable ethanol reserve to replace standard traditional gasoline. The conversion of amorphous cellulose into ethanol by yeast has already been achieved by Riaan Den Haan, et al inHydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae. However, Riann Den Haan’s method is inefficient and expensive due to the extra steps necessary to create a suitable environment for the conversion process: cellulase production as well as simultaneous saccharification and fermentation (SSCF). To alleviate problem we plan to create a synthetic consortium, or group, of yeast cells that includes populations that express the different genes needed for the digestion of cellulose. We expect this consortium to produce the necessary enzymes to hydrolyze cellulose and make ethanol without the additional environmental stability preparation steps needed to stabilize the enzymes, in a process called consolidated bioprocessing (CBP). As a result, the process will gain increased cost efficiency. Our goal is to create an engineered yeast consortium consisting of four intermingling yeast types to achieve the conversion of cellulose into ethanol. Each specialized yeast cell will each produce the one of the four major components needed for cellulose digestion into glucose, where in afterwards the yeast can naturally converts glucose into ethanol. The specific components secreted or displayed by the yeast are three cellulose digesting hydrolyzing enzymes: Endoglucanase, Exoglucanase, β-glucosidase, and the scaffolding to hold all three enzymes together. The three cellulase-secreting yeast secrete all the enzymes made out into the common medium while the scaffolding latches on to the cell membrane as a surface display glycoprotein. To create the four yeast cells, need we rely on the molecular cloning of E-coli to mass produce a vector with the right gene sequence that produce the target molecules. We use the pCEL15 plasmid as a shuttle vector between E-coli and yeast due to its dual compatibility with both organisms. The end should result in a yeast cell with the capabilities to express our desired gene sequence. Figure 6. Digestion of pCEL15 and Dockerin Both Left Lanes: 1Kb DNA Ladder Middle Lane: pCEL15 vector Right Lane: Dockerin insert Figure 7. Mini Prep of pCEL15/EG1 Goal Figure 3. Benefits of consortium Figure 4. Cellulose Hydrolyzing Synthetic Consortium • 12 wells with digested pCEL15/EG1 vector. The middle band suggests a contamination of a foreign vector while the outside bands show the digested pCEL15/EG1 vector ● Can perform multiple tasks without exhausting the cell. ● Better resilience to environmental changes ●Readily changed to fit production parameters Figure 1. Cost and Efficency of Consolidated Bioprocessing Future Figure 8. Blue/White Screening Use X-Gal and ITPG for Blue/White screening to ensure the target vector is produced. When insert is placed between the Lac operon cells are white. If Lac operon is intact cells are blue. Methods • The Basics of Molecular Cloning • PCR : Replication target insert (Endoglucanase 1 (EG1) or Dockerin) • Digestion : Cutting of insert and vector (pCEL15) to provide a common conjunction • Gel Purification: Removal of digestion enzymes (Xho1, BglII, EcoR1, and HindIII) • Ligation: Fusion of insert and vector • Transformation: Delivery of plasmid into the target cell • Inoculation: Replication of the target cell • Mini-prep: Purification of plasmid from cellular components for analysis Figure 2. Plant Biomass Composition Vector pCEL15 Figure 5. Shuttle vector between E-coli and yeast Dockerininserted between XhoI and BglII EG1 inserted between EcoRI and HindIII Acknowledgements We would like to thank the National Science foundation funding our program as well as the Chen lab for teaching us the lab techniques needed to perform this experiment. Lastly, we would like to thank Jun Wang for organizing and directing the BRITE program. • Cellulose ranges from 40-50% of plant biomass