Transcript Synthetic Consordium for Cellulose Hydrolysis and Ethanol Production

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
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 in Hydrolysis 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.
Figure 1. Cost and Efficency of Consolidated
Bioprocessing
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.
Results
Figure 6. Digestion of pCEL15 and Dockerin
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.
Goal
Figure 3. Benefits of
consortium
Middle Lane: pCEL15 vector
Right Lane: Dockerin insert
Figure 7. Mini Prep of pCEL15/EG1
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
Future
Methods
The Basics of Molecular Cloning
I. PCR : Replication target insert (Endoglucanase 1 (EG1) or Dockerin)
II. Digestion : Cutting of insert and vector (pCEL15) to provide a common
conjunction
Figure 2. Plant Biomass Composition
Both Left Lanes: 1Kb DNA Ladder
Use X-Gal and ITPG for
Blue/White screening to
ensure the target vector is
produced.
Figure 8.
Blue/White
Screening
When insert is placed
between the Lac operon
cells are white.
If Lac operon is intact cells
are blue.
III. Gel Purification: Removal of digestion enzymes (Xho1, BglII, EcoR1, and
HindIII)
Vector pCEL15
IV. Ligation: Fusion of insert and vector
Figure 5. Shuttle vector
between E-coli and yeast
V. Transformation: Delivery of plasmid into the target cell
Dockerin inserted between XhoI and
BglII
EG1 inserted between EcoRI and
HindIII
•Cellulose ranges from 40-50% of plant biomass
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VI. Inoculation: Replication of the target cell
VII. Mini-prep: Purification of plasmid from cellular components for analysis
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.