Development of a imidazolium ionic liquid tolerant, xylose-fermenting yeast via

chemical genomics guided biodesign

Quinn Dickinson, Scott Bottoms Li Hinchman, Trey Sato, Robert Landick, Jeff Piotrowski

Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI

The vinyl derivative of coumaric acid (4-vinylphenol) is significantly more toxic than the acid form

Exponential Phase

of hydrolysate

Background

Chemical genomics predicts ionic liquids target mitochondria

Chemical genomics uses barcoded

deletion and overexpression collection to

determine the genome-wide response of

an organism to a toxic compound2.

Multiplexed, next-generation sequence is

used to assess mutant performance

following exposure of the mutants to a

compound relative to a control. The

resulting chemical genomic profile gives

functional insight into the compound‘s

mode-of-action and cellular target.

Ionic liquid treatment damages mitochondria

Deletion of the serine kinase PTK2 dramatically improves ionic liquid tolerance

of hydrolysate

Proposed model of PTK2 in ionic liquid tolerance

Summary and next steps

Acknowledgements

This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494).

Decreasing mitochondrial membrane potential

Control

EMIM-Cl

Antimycin Valinomycin

WaterWater

EMIM-ClEMIM-Cl EMIM-Cl

EMIM-ClEMIM-Cl

EMIM-Cl

Glucose Glycerol

Ionic liquids (ILs) are a promising means of chemical hydrolysis and pre-

treatment of lignocellulosic hydrolysates

Following ionic liquid pre-treatemt of biomass, these compound may persist at

residual levels in the hydrolysates at levels up to 1%

Ionic liquids are toxic to fermentative microbes at levels below 1%, and this

require IL tolerant microbes to ensure viability of this means of biofuel production

The mode of action by which ILs are toxic remains poorly characterized

This study was designed with 2 main goals:

1. Determine the mechanism of action of IL toxicity

2. Determine what gene modifications can be made to improved IL tolerance

in a xylose-fermenting yeast

We describe the first genome-wide screen of imidazolium ionic liquids, which has

led to a proposed mechanism of action and development of a xylose-fermenting

yeast tailored for general tolerance of imidazolium ionic liquids

Chemical genomic profiling identified 220

mutants significantly resistant to EMIM-Cl. Of

these, a deletion mutant of PTK2 was the most

significantly resistant, and deletion mutant of

SKY1 was the second most significantly resistant.

We found significant enrichment (p<0.01) for

mitochondria genes among the most sensitive

We confirmed the individual sensitivity

and resistant to the top 2 most

responsive strains. The top resistant

mutants (PTK2 and SKY1) had

considerable tolerance to high levels of

EMIM-Cl

Overexpression of the essential

proton pump Pma1p, which is

regulated by Ptk2p, significantly

reduced EMIM-Cl tolerance;

however, overexpression of PTK2

also increased EMIM-Cl

sensitivity, but not significantly

Deletion of PTK2 in the xylose-fermenting yeast strain Y133

conferred significantly greater tolerance of EMIM-Cl, BMIM-Cl,

and EMIM-Ac (p<0.01). BMIM-Cl was the most toxic of the ILs

tested

The Y133 ptk2∆ mutant grew,

consumed sugars, and produced ethanol

at greater levels than Y133 in the

presence of 1 % EMIM-Cl

Because we observed many

mitochondrial genes mutants

were highly sensitive to ILs, we

predicted that they may exert

toxicity on mitochondria. Further

the chemical genomic profile of

EMIM-Cl had highest correlation

with that of valinomycin, an

ionophore that damages

mitochondrial membrane potential

The toxicity of EMIM-Cl is greater

when yeast are grown on a non-

fermentable carbon source

(glycerol)

Yeast grown in the presence

of EMIM-Cl show a dose

dependent decrease in

mitochondrial structureSimilar to other mitochondrial drugs,

IL treatment causes a rapid decrease

in mitochondrial membrane potential

Together these data allow us to make the following predictions for the model of how a PTK2 deletion confers

IL resistance:

1. PTK2 activates the H+ efflux pump Pma1p, and H+ efflux is coupled with influx of the toxic imidazolium

cation which is toxic to the mitochondria

2. In the absence of PTK2, H+ efflux is reduced, as a result less of the imidazolium cation enters the cell

3. Toxicity is reduced under anaerobic conditions, where mitochondrial function is reduced

4. Toxicity is reduced at lower pH, where H+ efflux is reduced

Fermentation data in the presence of BMIM-Cl support this model. To note, while toxicity is reduced at lower

pH and under anaerobic conditions, deletion of the PTK2 mutations improves xylose-fermentation even at

these optimal conditions

pH 6.5

anaerobic

pH 6.5

aerobic

pH 5.0

anaerobic

pH 5.0

aerobic

The effect of the PTK2 modification on IL

tolerance is pH dependent. At neutral pH, ILs

have greater toxicity, but this is reduced at a

lower pH

The effect of the PTK2 modification on IL

tolerance is pH dependent. At neutral pH, ILs

have greater toxicity, but this is reduced at a

lower pH133

133 ptk2∆

Basic model for how a

PTK2 deletion confers

IL tolerance