Dead Ligmen Tell No Tales Design of a Microbial Pretreatment for

Lignin Degradation using S. cerevisiae

Purdue iGEM 2015

Part One Motivation and Background

Initial Questions

Project Development

Water Sanitation Energy Building Agriculture

What are

Biodigesters?

• Definition: systems which decompose biological matter

using bacteria in an anaerobic environment in order to

produce a fuel source

• Four steps:

• Work best if separated

• One chamber bioreacters

Pretreatment Digestion Processing

And Filtration

Reuse and Disposal of

Waste

Why Biodigesters?

• Broad application

• Renewable energy

• Transportation

• Electricity generation

• Areas for improvement

Types of

Biodigestion

First Generation

Food plants

Second Generation

Fuel Crops

Third Generation

Algae

Fourth Generation

GM plants with less lignin

Lignin

• Nonrepeating

structure

• Protects plants

• Requires

pretreatment

Pretreatment

Thermal Mechanical

Chemical Enzymatic

Containment

• Usually rely on things from an industry setting

• But in less contained environments?

• Need a reliable, effective, affordable killswitch

Part Two Problem and Approach

Initial Problem

How can we design a simplified biological system

that covers all four stages of biodigestion?

Pretreatment

Disposal Processing

Digestion

J4 yeast Our system

Refined Problem

How can we use synthetic biology to

create a lower energy alternative to

thermal pretreatment?

How can we contain such a system in different

settings including third world application?

Solution

Cellulose

Lignin

Enzyme Cocktail

Our System

J4 Yeast

Enzyme Selection

Aldo-Keto Reductase Lignin Peroxidase Laccase

Tyrosinase Manganese Peroxidase Versatile Peroxidase

Genetic Construct

Design

Biobrick prefix

Kozack Sequence

Biobrick suffix

Manganese Peroxidase Fusion Terminator

yEGFP

Constitutive Promoter

Linker Linker

Manganese Peroxidase Secretion Tag

Killswitch

Considerations

• Several promoters considered

• Robust killswitch

• Ethanol based spill kit

Biobrick prefix

Kozack Sequence

Biobrick suffix

Lambda Holin Coding Sequence Terminator Negative Promoter

Plasmid Optimization

Codon

Optimization

Refinement

for blocks

gBlock

Format

Restriction

Site Check

Part Three Methods and Results

One Project,

Two Teams

Enzyme Team

• Suraj, Jill, Erich, and Melissa

• Construction of enzyme constructs and ligation into yeast

vector

Killswitch Team

• Bowman, Kate, and Alexa

• Construction of oxygen based killswitch

Enzyme and Killswitch Track

Methodology

Enzyme Track

Progress

Enzyme Track

Problems

• Gibson was not able to:

• Combine the G-blocks at

proper site

• Produce DNA at ample

concentrations for

transformation

• Attempted Overlap PCR to

alleviate issues

• Limited success with

Manganese Peroxidase

LiP VP AKR MnP

Assays

• Completed

• MTT Assay • Yeast Growth/Death Curves

• Future Experiments

• Bradford Protein Assay • Protein concentration measurement

• Peroxidase Assay • Lignin, Manganese, and Versatile Peroxidase activity

• DMP Assay • Laccase activity assay

• Klason Procedure • Lignin Degradation Assay

Overall Progress

Enzyme PCR Amp

Purify/Extract

Gibson E.coli Vector Insert

E.coli Transformation

Miniprep Sequencing Yeast Transformation

Lignin Peroxidase

Laccase

Manganese Peroxidase

AKR

Versatile Peroxidase

Tyrosinase

Completed Stopped

Enzyme Analysis

• Since enzymes are exported from cells, only supernatant needs to be

analyzed

• Enzymes would then be tested in multiple combinations

• Linear regression model used to calculate lignin breakdown of each

enzyme

• Statistical optimization used to eliminate combinations with little effect and

determine amount of enzymes needed for bioreactor

Conclusions

• Unable to reach proof of concept point

• Errors:

• Overlapping restriction site error

• block design errors

• Still believe the idea is viable

• Better gene design in the future

Part Four Human Practices and Beyond

Changing Human

Practices Focus

• Started broad

• Shift to pretreatment

• Biofuel application

• Broad potential

Enzymatic Pretreatment B

road

Bro

ad

Biofuels

Cardinal Ethanol

Plant

• Full time ethanol plant

• 100 million gal / year

• Ethanol is sold to gasoline companies

Nonscientific Issues

Surrounding Biofuels

• Lignocellulosic biofuels only

• Transportation infrastructure

• Cost

• All biofuels

• Cannot be shipped in current pipelines

• Cannot be used in most current vehicles

• Current gasoline market

Viability

So is any of this even worth it?

We don’t know yet.

But…

Assay data would have helped build a model.

Model would have been used to predict overall costs and

evaluate how much is being saved.

Final Thoughts

• Lowered cost opens doors

• Standardized platform

• Wide spectrum of application

Summary

• Examined problems facing biodigesters

• Surveyed range of lignin-degrading enzymes

• Selected candidate enzymes based on design criteria

• Designed genetic constructs for expression

• Had constructs synthesized and assembled four of the devices

• Designed oxygen-dependent kill switch

• Constructed kill switch using registry parts

• Visited Cardinal Ethanol Plant

• Used real world perspective to develop standards of feasibility

Attributions

• Advisors

– Professor Jenna Rickus

– Soo Ha

– Sam Lee

– Janie Brennan

– Professor Michael Gribskov

• Project consultation

– Professor Michael Scharf

– Professor Nathan Mosier

• Collaboration

– Cardinal Ethanol Plant

– Purdue Society of Women

Engineers

Funding

• Purdue University Office of the Provost

• Purdue University Office of the Vice

President of Research

• Purdue University College of Agriculture

• Purdue University College of Science

• Purdue University College of Engineering

• Purdue University Honors College

• Purdue University Department of Agricultural

and Biological Engineering

• Purdue University Learning Beyond the

Classroom Grant

• Purdue University Summer Undergraduate

Research Fellowship Program

• Purdue University Molecular Agriculture

Summer Institutes Program

• Purdue University Day of Giving

Thank You Questions?

Background

&

Motivation

Problem

&

Approach

Methods

&

Results

Human Practices

&

Beyond