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Title: Chemical genomic guided engineering of gamma-valerolactone tolerant yeast

Abstract

Gamma valerolactone (GVL) treatment of lignocellulosic bomass is a promising technology for degradation of biomass for biofuel production; however, GVL is toxic to fermentative microbes. Using a combination of chemical genomics with the yeast ( Saccharomyces cerevisiae) deletion collection to identify sensitive and resistant mutants, and chemical proteomics to monitor protein abundance in the presence of GVL, we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast. Chemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate cinnamic acid and its derivatives to vinyl forms, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were moremore » abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain. We found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol accumulation in aerobically grown cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates.« less

Authors:
 [1];  [2];  [3];  [3];  [4];  [3];  [3];  [3];  [3];  [5];  [6];  [3];  [7]
  1. Univ. of Wisconsin-Madison, Madison, WI (United States); Technische Univ. Munchen, Straubing (Germany)
  2. Univ. of Wisconsin-Madison, Madison, WI (United States); Georgia Inst. of Technology, Atlanta, GA (United States)
  3. Univ. of Wisconsin-Madison, Madison, WI (United States)
  4. Univ. of Wisconsin Biotechnology Center, Madison, WI (United States)
  5. Univ. of Wisconsin-Madison, Madison, WI (United States); Morgridge Inst. for Research, Madison, WI (United States); Genome Center of Wisconsin, Madison, WI (United States)
  6. Univ. of Minnesota-Twin Cities, Minneapolis, MN (United States)
  7. Univ. of Wisconsin-Madison, Madison, WI (United States); Yumanity Therapeutics, Cambridge, MA (United States)
Publication Date:
Research Org.:
Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1510822
Grant/Contract Number:  
FC02-07ER64494
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Microbial Cell Factories
Additional Journal Information:
Journal Volume: 17; Journal Issue: 1; Journal ID: ISSN 1475-2859
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Chemical genomics; Gamma-valerolactone; Lignocellulosic; Biofuel; Biocatalysts; Saccharomyces cerevisiae

Citation Formats

Bottoms, Scott, Dickinson, Quinn, McGee, Mick, Hinchman, Li, Higbee, Alan, Hebert, Alex, Serate, Jose, Xie, Dan, Zhang, Yaoping, Coon, Joshua J., Myers, Chad L., Landick, Robert, and Piotrowski, Jeff S. Chemical genomic guided engineering of gamma-valerolactone tolerant yeast. United States: N. p., 2018. Web. doi:10.1186/s12934-017-0848-9.
Bottoms, Scott, Dickinson, Quinn, McGee, Mick, Hinchman, Li, Higbee, Alan, Hebert, Alex, Serate, Jose, Xie, Dan, Zhang, Yaoping, Coon, Joshua J., Myers, Chad L., Landick, Robert, & Piotrowski, Jeff S. Chemical genomic guided engineering of gamma-valerolactone tolerant yeast. United States. doi:10.1186/s12934-017-0848-9.
Bottoms, Scott, Dickinson, Quinn, McGee, Mick, Hinchman, Li, Higbee, Alan, Hebert, Alex, Serate, Jose, Xie, Dan, Zhang, Yaoping, Coon, Joshua J., Myers, Chad L., Landick, Robert, and Piotrowski, Jeff S. Fri . "Chemical genomic guided engineering of gamma-valerolactone tolerant yeast". United States. doi:10.1186/s12934-017-0848-9. https://www.osti.gov/servlets/purl/1510822.
@article{osti_1510822,
title = {Chemical genomic guided engineering of gamma-valerolactone tolerant yeast},
author = {Bottoms, Scott and Dickinson, Quinn and McGee, Mick and Hinchman, Li and Higbee, Alan and Hebert, Alex and Serate, Jose and Xie, Dan and Zhang, Yaoping and Coon, Joshua J. and Myers, Chad L. and Landick, Robert and Piotrowski, Jeff S.},
abstractNote = {Gamma valerolactone (GVL) treatment of lignocellulosic bomass is a promising technology for degradation of biomass for biofuel production; however, GVL is toxic to fermentative microbes. Using a combination of chemical genomics with the yeast (Saccharomyces cerevisiae) deletion collection to identify sensitive and resistant mutants, and chemical proteomics to monitor protein abundance in the presence of GVL, we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast. Chemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate cinnamic acid and its derivatives to vinyl forms, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain. We found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol accumulation in aerobically grown cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates.},
doi = {10.1186/s12934-017-0848-9},
journal = {Microbial Cell Factories},
issn = {1475-2859},
number = 1,
volume = 17,
place = {United States},
year = {2018},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
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Cited by: 2 works
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Figures / Tables:

Fig. 1 Fig. 1: Production of GVL hydrolysates and toxicity. The half-maximal inhibitory concentration ( IC50) of GVL against our control strain in rich media was estimated using an 8 pt dose curve (Mean ± SE, n = 3)

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    Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.