1. 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
Water
Water
EMIM-Cl
EMIM-Cl EMIM-Cl
EMIM-Cl
EMIM-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 structure
Similar 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