Examining Biofuel Precursors Via Increased SorbitolFlux & Lignin Peroxidase Expression in Escherichia coli

Wisconsin iGEM 2008 ~ University of Wisconsin ~ Madison, WI 53706 USA

Team Wisconsin used Escherichia coli to produce biofuel precursors in an effort to find alternative fuel sources. One project focused on using E. coli to efficiently produce sorbitol, a biofuel precursor. Using computer modeling, we determined a way to funnel gly-colysis’ intermediates towards the production of sorbitol via sorbitol-6-phosphate dehydrogenase. Wisconsin’s second aim was to isolate high-energy precursors from the plant cell wall through E. coli mediated breakdown of cell wall lignin. This was achieved by inserting the gene encoding lignin peroxidase, found in the white rot fungus, Phanerochaete chrysosporium, into genetically modi-fied E. coli, capable of producing and exporting the enzyme. Both projects improve current methods in the production of alterna-tive fuels via two unique routes, and have the potential to progress biofuel research.

Abstract & Biofuel Life Cycle

Sorbitol Dehydrogenase Lignin Peroxidase

CO2H2O

Biomass

Algae

Combustion

Distribution

Refining

Purification

BiomassPretreatment

Hydrolysis &Saccharification

Pyrolysis

Fermentation

Catalysis CrudeBiofuels

BiomassHarvesting CO, H2

HexosePentose

Overview, Goal & ApproachGOAL: Improve biofuel synthesis by: -Increasing Overall Energy Efficiency (OEE) -Allowing for hydrocarbon fuel precursor synthesis via solid catalysis -Sorbitol is water soluble but creates an organic phase when reduced -Allows for easy harvesting of precursors post catalytic reaction

-Examine the E. coli metabolic pathway to optimize sorbitol dehydrogenase synthesis in vivo -Knockout phosphofructokinase A -Knockout triose phosphate isomerase-Develop an assay to determine which cells have the highest sorbitol yield

Genetic Alteration - creating an srlD vectorCloning process for making a vector with srlD in MG1655 E. coli, including PCR amplification of chro-mosomal DNA, di-gestion of destination vector, ligation and transformation.

Overview, Goal & ApproachGOAL: Improve biofuel synthesis by: -Inserting genes from fungal decomposers into a bacterial system -Allowing greater access to plant cellulose -Attainable by degrading lignin from plant cell walls -Permitting the use of an entire plant for biofuel synthesis

-Examine the function of lignin peroxidase from the white rot fungus, Phanerochaete chrysosporium -Optimize the expression of lignin peroxidase (lip) in E. coli -Develop Lip export tag fusion protein for Lip harvesting-Develop an assay to determine the rate of lignin degradation

Plant Biomass, Lignin & Lignin Peroxidase Structures

Metabolic Mapping

Sorbitol

From sugar to biofuel precursor to biofuelA) X-ray crystal struc-ture - sorbitol dehy-drogenase. B) Pro-duced from fructose, sorbitol, a reduced sugar, can be further reduced to hexane and other fuel pre-cursors.A

B

Reduction by solid catalyst

Hexane

1. Plant Cell Wall Complex. Ceres: the energy crop company. <http://www.ceres.net/AboutUs/AboutUs-Biofuels-Carbo.html>.2.ProposedLigninStructure.TheUniversityofKentucky. <http://www.research.uky.edu/odyssey/winter07/green_energy.html>.3.Background-SeeWisconsiniGEMWiki2008 -<http://2008.igem.org/Team:Wisconsin/Project>

ReferencesAndyBraaschCharlieBurnsBenCoxMattiasGyllborgJackHoYash Jhala

The Team:JiaLuoSeanMcMasterTannerPeelenPeter Vander VeldenJoeYuen

Dr.AseemAnsariDr.FrancoCerrinaDr.BrianPflegerDr.JennieReed

Faculty & AdvisorsDr.MikeSussmanDr.DougWeibelBasudebBhattacharyya

GreatLakesBioenergyResearchCenter(DOE)MaterialsResearchScience&EngineeringCenterNanoscaleScience&EngineeringCenterUWCollegeofAgricultural&LifeSciencesChancellorJohnWiley

SponsorsUWCollegeofEngineeringClontech/TakaraBiosciencesLucigenCorporationPromegaCorporation

SRM 2008

A) Structure of lignin, formed by a radical reaction. B) X-ray crystal structure-lignin peroxidase breaks down lig-nin into aromatic compo-nents. C) Components of biomass. Lignin acts as an integral structural compo-nent and protective layer.

B C

Through computer modeling a gram per gram prediction of sorbitol pro-duction to sugar input was established, telling us the gene to knockout.

Modeling of glycolytic knockouts

00.20.40.60.8

11.21.41.61.8

Glucose Glycerol Pyruvate Acetate Δ tpiA:Glycerol

Δ pfkA:Glycerol

Minimal Media

Sorb

itol

Out

put

(gra

m

sorb

itol

/gra

m s

ugar

inpu

t)

FBA Model Predictions: Sorbitol Production

Absorption change in NAD+ due to sorbitol dehydrogenase activity. This assay is used to measure sorbitol produced in a given culture of cells.

Sorbitol Dehydrogenase Assay

Growth curves for RL257, srlD, tpiA and tpiA+srlD grown in C media. Absorbance values measured at 600nm at given time intervals.

Knockout Growth Curves

62.5 mL-1

0.170.180.190.200.210.220.230.240.25

0 50 100 150 200Time (Seconds)

Abs

orba

nce

(340

nm

)

Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5

Glycolysis in E. coli results in the forma-tion of pyruvate, which can be shuttled into different metabolic pathways. By blocking components of glycolysis, we can force intermediates into the syn-thesis of sorbitol. Logically, by knock-

ing-out phosphofructokinase A (pfkA) (red X), we can force fructose-6-phosphate into the sorbitol synthesis pathway. Computer modeling indicated that the same results can be achieved by knocking-out triose phos-phate isomerase (blue X); however, it would appear that the pfkA knockouts would yield higher efficiencies of sorbitol output per sugar input because less glycolytic by-products would be produced.

X

X

Lignin Peroxidase Expression

E. coli Inner Membrane Export Pathways

Sec TAT

A) Sec and TAT export pathways of E. coli. The Sec pathway in-volves unfolding, exporting and refolding the protein. The TAT pathway is simpler, capable of exporting entire folded proteins at once. In both cases, the final product is a folded periplasmic protein. We chose native E. coli export tags from torA, ycbK, ycdO, ycdB genes which work via TAT/Sec pathways.

1 2 3 4 5

B) An agarose gel showing the four synthesized E. coli export tags. Due to their small size (~100-180 bp), we were able to PCR synthesize these tags using six overlap-ping primers for each tag of interest.

Gel Lanes:1) 100bp Ladder2) ycdO (Sec tag)3) ycbK (TAT tag)4) ycdB (TAT/Sec tag)5) torA (TAT)

BA

SDS PAGE gel stained with Coomassie Blue. Cells induced with IPTG showed a darker band of protein, relative to the rest of the cell lysate, at ~45 KDa (red box) than the unin-duced cells. This band is consistent with our transcribed protein: lipD fused to a His tag and a T7 tag. Therefore, we conclude that we have successfully transformed and ex-pressed lignin peroxidase in E. coli. This experiment vali-dates a proof of concept wherein the additional nine lignin peroxidase genes could be cloned out of the white rot fun-gus, Phanerochaete chrysosporium.1) Uninduced culture2) Fermentas page ruler unstained protein ladder3) Induced pLysS cells with lipD in pET28a1 2 3

A) Structure of Azure B, a chemical dye. B) At a con-centration of 0.015 IU, lignin peroxidase affects lignin concentration in the solution over time, as measured by Azure B oxidation. The results closely emulate those from Archibald (1992), inferring the assay’s effectiveness in measuring lignin peroxidase cellular concentrations.

Lignin Peroxidase Assay

A

Absorption of Azure B Oxidation by Lignin Peroxidase

0.1

0.3

0.5

0.7

0.9

1.1

1.3

550 600 650 700Wavelength (nm)

Abso

rban

ce

0 min 3.33 min 6.67 min 10 min 13.33 min

16.67 min 20 min 23.33 min 26.67 min

B

Growth of RL257

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 1 2 3 4 5 6Time (hours)

log

(A60

0)

RL257 RL257+srlD RL257+tpiA RL257+tpiA+srlD

A