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Dead Ligmen Tell No Tales:Development of Synthetic Yeast for Enzymatic Pretreatment of Lignocellulosic Biomass

2015 M. Aronson, B. Clark, H. Edmondson, H. Kubo, E. Leazer, A. Liu, K. Lowrey, H. Lysandrou, S. Magill, C. Martin, S. Mohan, J. Osterhus, A. Petrucianni, M. Robins, T. Tan, J. WelchPurdue University, West Lafayette

Background• Need to find more sources of renewable energy which

can alleviate problems in areas such as food production and water purification

• Ethanol biofuels are promising, but there are drawbacks of using food crops & fertilizers

• Lignocellulosic biofuels avoid food and other resource commit-ments, but conversion is inefficient due to recalcitrance of lignin to degradation and monomers poisoning crucial enzymes.

• Traditional approaches to circumvent lignin require pre-treating with high heat/energy or toxic/expensive chemicals

• Recently, a synthetic yeast reported to be capable of spatially separating the final biofuel conversion steps prevents the poisoning

• An opportunity arises for an enzymatic pretreatment to degrade lignin to increase conversion efficiency

Methodology

Results• The parts contained overlapping restriction sites• Successfully Gibson assembled 4 of 6 enzymes• Oxygen killswitch was completed

Human Practices • Toured Cardinal Ethanol Plant (Union City, IN)• Challenges facing cellulosic ethanol production:• Economic• Infrastructure• Implementation

• Outreach:• Synbio training for Girl

Scouts• Mentoring MASI high

school students• Host 4-H workshop for synbio, teach high school

students lab technique

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Special thanks to:Professor Jenna Rickus, Soo Ha, Sam Lee, Janie Brennan, Professor Michael Gribskov, Professor Michael Scharf, Professor Nathan Mosier, and the Cardinal Ethanol Plant.

Thanks to financial support from:IDT and the Purdue University: Office of the Provost; Office of the Vice President of Research; College of Agriculture; College of Science; College of Engineering; Department of Agricultural and Biological Engineering; Honors College; Learning Beyond the Classroom Grant; Summer Undergraduate Research Fellowship Program; Molecular Agriculture Summer Institutes Program; and Day of Giving.

References:Simmons, B.A.; Loqué, D.; & Ralph, J. “Advances in modifying lignin for enhanced biofuel production.” Current Opinion in Plant

Biology 13 (2010): 313-320.Sethi, A.; Slack, J.M.; Kovaleva, E.S.; Buchman, G.W.; Scharf, M.E. “Lignin-associated metagene expression in a lignocellulose-digest-

ing termite.” Biochem Mol Biol. 2013 Jan; 43(1):91-101. doi: 10.1016.

Future DirectionsExperiments• Bradford assay to measure protein expression levels• Peroxidase assay to measure activity of Lignin Peroxi-

dase, Versatile Peroxidase, and Manganese Peroxidase• DMP assay to measure laccase activity• Klason Procedure to determine levels of lignin degradation

Modeling• Use data to construct enzy-

matic reaction rate models for each enzyme

• Design of experiments to evaluate interaction effects of helper enzymes

• Statistical optimization to determine most effective en-zyme cocktail

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Conclusion• Our synthetic biology solution to biofuel conversion

from lingo-cellulosic biomass to ethanol did not reach its full potential. Overlapping restriction site errors and gBlock design errors blocked its progress. Better gene design would be necessary for future research. However, we still believe the idea is viable and shows potential for future progress, if supplied more time and resources.

• This project could have extensive applications if a low total cost for our yeast construct was achieved. An ideal standardized platform for bioreactors could be created in the future to allow for consistent testing.

Construct Design

Hexamer designed for PCR

Cut sites for EcoRI and XbaI

Promoter Region

Kozak Sequence N-Terminal Fluorescent Protein Tag

Linker Sequence

Enzyme Sequence Linker Sequence

C-Terminal Secretion Tag

Terminator Cut sites for SpaI and PstI

Hexamer designed for PCR

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Implementation1. Identify lignin degrading enzymes

from termites and fungi2. Insert genes into yeast for secre-

tion3. Add feedstock to pretreatment

with recombinant yeast4. Degraded lignocellulosic biomass

moves to biofuel conversion to produce ethanol

Figure 1: Lignin structure

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