This study used chemical genomics to determine the mechanism of toxicity and develop tolerance to gamma valerolactone (GVL), a promising solvent used to generate fermentable hydrolysates. The results showed: 1. GVL damages cellular membranes, similar to ethanol. Deletion mutants involved in membrane transport were sensitive to GVL. 2. Deletion of the decarboxylases PAD1 and FDC1, which convert phenolic acids to more toxic vinyl derivatives, conferred increased GVL tolerance. 3. An engineered xylose-fermenting yeast with the PAD1 and FDC1 deletions showed improved growth and sugar consumption in the presence of GVL compared to the control

1. Understanding the microbial response to gamma valerolactone: Mode of action and
chemical genomics guided biodesign of a GVL tolerant, xylose fermenting yeast
Scott Bottoms, Quinn Dickinson, Li Hinchman, Ali Motagamwala, James Dumesic, Robert Landick, Jeff Piotrowski
Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI
Among deletion mutants significantly resistant to GVL, we saw significant enrichment for genes in phenylpropanoid metabolism (p<0.001), driven by the
mutants of the decarboxylases Pad1p and Fdc1p (A). We confirmed that individual mutants in these genes were more tolerant of GVL
The vinyl derivative of coumaric acid (4-vinylphenol) is significantly more toxic than the acid form
of hydrolysate
Background
Chemical genomics predicts GVL targets cellular membranes
GVL is a promising, new chemical hydrolysis
technology developed at the GLBRC
The advantage of GVL is that is a renewable solvent
than can be used to generate fermentable
hydrolysates with very high sugar concentrations
The disadvantage is that residual GVL (and other
compounds generated in the process) can be
inhibitory to fermentative microbes
GVL is the most abundant inhibitor found in GVL
produced hydrolysates, yet the mechanism of GVL
toxicity to microbes is unclear
This study was designed to:
1. Determine the mechanism of GVL toxicity
2. Identify points of rational engineering to create a
GVL tolerant derivative of the GLBRC’s xylose
fermenting yeast
We used genome-wide chemical genomic profiling
to predict the mode of toxicity and identify genes for
modification
Toxicity of major inhibitors found in GVL
hydrolysates
GVL, levulinic acid, and HMF are
the major inhibitors found n GVL
hydrolysates. While HMF and
levulinic are more toxic, GVL is far
more abundant, so we focused
specifically on this inhibitor
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.
Chemical genomic profiling revealed a significant enrichment for genes involved
in late endosome to vacuole transport (p<0.01) among the top gene mutants
sensitive to GVL. This process is sensitive to chemical agents that affect
membrane integrity. These 3 mutants are similarly sensitive to ethanol, which
damages cellular membranes. Single mutant validations of these individual
mutants confirmed they were significantly more sensitive to GVL
GVL rapidly damages cellular membrane integrity
Cells treated with GVL for 4h had a
significant increase in the number
of cells with a propidium iodide
signal as determined by FACS
analysis
Chemical genomic profiling predicted that GVL may have particular toxicity towards cellular
membranes. We tested the effects of GVL on membrane integrity using a dye (propidium
iodide) that is only taken up by cells with damaged membranes
GVL treatment compromised
membrane integrity in a dose
dependent manner, similar to
ethanol but of a greater
magnitude
GVL is synergistic
with ethanol toxicity,
which could further
inhibit final ethanol
production from GVL
hydrolysates
Deletion of the decarboxylases Pad1p and Fdc1P confers GVL tolerance
GVL alters membrane fatty acid composition
Among deletion mutants
significantly resistant to GVL, we
saw significant enrichment for
genes in phenylpropanoid
metabolism (p<0.001), driven by the
mutants of the decarboxylases
Pad1p and Fdc1p (A). We
confirmed that individual mutants in
these genes were more tolerant of
GVL
Overexpression profiling using
MoBY-ORF transformed Y133
demonstrated that overexpression of
PAD1 conferred significant GVL
sensitivity, supporting the deletion
mutant results . Increased
expression of PAD1 significantly
reduced GVL tolerance in single
mutant cultures
of hydrolysate
Vinyl products of Pad1p decarboxylation are synergistic with GVL
Deletion of PAD1 and FDC1 improves GVL tolerance in a xylose fermenting yeast
Summary and next steps
Acknowledgements
The decarboxylase Pad1p
converts phenolic acids to a
vinyl form
The vinyl derivative
of coumaric acid (4-
vinylphenol) is
significantly more
toxic than the acid
form
4-vinylphenol is
significantly synergistic
with GVL, and chemical
genomic profiling of
this compound similarly
predicts it targets
cellular membranes
We used a two-step PCR approach to
simultaneously delete PAD1 and
FDC1 in Y133, which are adjacent on
chromosome IV. The Y133 pad1∆
fdc1∆ mutant had significantly greater
(p<0.01) tolerance of GVL
Using a chemical genomics approach, we have defined the mode of action and relevant synergisms of GVL
We reveled deletions in the decarboxylases PAD1 and FDC1 can confer GVL tolerance, and we have
introduced these modification into a xylose-fermenting yeast strain to create a new yeast specifically
tailored to GVL hydrolysates
The process chemistry of GVL and other hydrolysis production methods is still developing, and new inhibitor
challenges may arise, which we can approach with similar tools
As GVL hydrolysate production is scaled up, we will be first to test our engineered stains in larger-scale
fermentations to evaluate performance
This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494).
Time (h)
0 10 20 30 40 50
Ratiooffattyacids
0.5
1.0
1.5
2.0
2.5
3.0
3.5
133 16:0/18:0
133 16:1/18:1
133 GVL 16:0/18:0
133 GVL 16:1/18:1
Cells grow in the presence of GVL (2.5%) show and increase
in the ratio of steric acid (18:0) to oleic acid (18:1), as well as
palmitic acid (16:0) to palmitoleic acid (16:1). Oleic acid
content in cells has been shown as a major contributor to
ethanol tolerance of yeast (Man you et al 2003) through it’s
effect on membrane fluidity. We documented a similar effect
with GVL, supporting the idea that these two solvent have a
similar mode of action via membrane disruption. This also
suggests that modifications to total oleic acid content could
be a means of enhancing GVL tolerance
Using a lab media and synthetic mimic of GVL hydrolysates, we tested if our
engineered strain had better fermentation in the presence of GVL. In aerobic rich
media, the engineered strain consumed sugars (glucose and xylose) and produced
ethanol faster then the control background strain. In synthetic media under anaerobic
conditions, the difference was stark. The control strain was unable to grow in the
presence of GVL, whereas the modified strain grew and fermented
Aerobic fermentation in rich media+ 2.5 % GVL
Anaerobic fermentation in Synthetic GVLH+1% GVL