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Title: An iterative computational design approach to increase the thermal endurance of a mesophilic enzyme

Abstract

Strategies for maximizing the microbial production of bio-based chemicals and fuels include eliminating branched points to streamline metabolic pathways. While this is often achieved by removing key enzymes, the introduction of nonnative enzymes can provide metabolic shortcuts, bypassing branched points to decrease the production of undesired side-products. Pyruvate decarboxylase (PDC) can provide such a shortcut in industrially promising thermophilic organisms; yet to date, this enzyme has not been found in any thermophilic organism. Incorporating nonnative enzymes into host organisms can be challenging in cases such as this, where the enzyme has evolved in a very different environment from that of the host. Here, we use computational protein design to engineer the Zymomonas mobilis PDC to resist thermal denaturation at the growth temperature of a thermophilic host. We generate thirteen PDC variants using the Rosetta protein design software. We measure thermal stability of the wild-type PDC and PDC variants using circular dichroism. We then measure and compare enzyme endurance for wild-type PDC with the PDC variants at an elevated temperature of 60º (thermal endurance) using differential interference contrast imaging. We find that increases in melting temperature (T m) do not directly correlate with increases in thermal endurance at 60º. We alsomore » do not find evidence that any individual mutation or design approach is the major contributor to the most thermostable PDC variant. Rather, remarkable cooperativity among sixteen thermostabilizing mutations is key to rationally designing a PDC with significantly enhanced thermal endurance. These results suggest a generalizable iterative computational protein design approach to improve thermal stability and endurance of target enzymes.« less

Authors:
 [1];  [2];  [1];  [1];  [1];  [1];  [1];  [2];  [1];  [1];  [3];  [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States). Biosciences Center
  2. Univ. of Colorado, Boulder, CO (United States). Dept. of Chemistry and Biochemistry and the BioFrontiers Inst.
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Institutes of Health (NIH)
OSTI Identifier:
1472243
Report Number(s):
NREL/JA-5100-71815
Journal ID: ISSN 1754-6834
Grant/Contract Number:  
AC36-08GO28308; R01GM103843; F30CA180249
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 97 MATHEMATICS AND COMPUTING; computational protein design; biofuels; thermal stability; pyruvate decarboxylase

Citation Formats

Sammond, Deanne W., Kastelowitz, Noah, Donohoe, Bryon S., Alahuhta, Markus, Lunin, Vladimir V., Chung, Daehwan, Sarai, Nicholas S., Yin, Hang, Mittal, Ashutosh, Himmel, Michael E., Guss, Adam M., and Bomble, Yannick J. An iterative computational design approach to increase the thermal endurance of a mesophilic enzyme. United States: N. p., 2018. Web. doi:10.1186/s13068-018-1178-9.
Sammond, Deanne W., Kastelowitz, Noah, Donohoe, Bryon S., Alahuhta, Markus, Lunin, Vladimir V., Chung, Daehwan, Sarai, Nicholas S., Yin, Hang, Mittal, Ashutosh, Himmel, Michael E., Guss, Adam M., & Bomble, Yannick J. An iterative computational design approach to increase the thermal endurance of a mesophilic enzyme. United States. doi:10.1186/s13068-018-1178-9.
Sammond, Deanne W., Kastelowitz, Noah, Donohoe, Bryon S., Alahuhta, Markus, Lunin, Vladimir V., Chung, Daehwan, Sarai, Nicholas S., Yin, Hang, Mittal, Ashutosh, Himmel, Michael E., Guss, Adam M., and Bomble, Yannick J. Mon . "An iterative computational design approach to increase the thermal endurance of a mesophilic enzyme". United States. doi:10.1186/s13068-018-1178-9. https://www.osti.gov/servlets/purl/1472243.
@article{osti_1472243,
title = {An iterative computational design approach to increase the thermal endurance of a mesophilic enzyme},
author = {Sammond, Deanne W. and Kastelowitz, Noah and Donohoe, Bryon S. and Alahuhta, Markus and Lunin, Vladimir V. and Chung, Daehwan and Sarai, Nicholas S. and Yin, Hang and Mittal, Ashutosh and Himmel, Michael E. and Guss, Adam M. and Bomble, Yannick J.},
abstractNote = {Strategies for maximizing the microbial production of bio-based chemicals and fuels include eliminating branched points to streamline metabolic pathways. While this is often achieved by removing key enzymes, the introduction of nonnative enzymes can provide metabolic shortcuts, bypassing branched points to decrease the production of undesired side-products. Pyruvate decarboxylase (PDC) can provide such a shortcut in industrially promising thermophilic organisms; yet to date, this enzyme has not been found in any thermophilic organism. Incorporating nonnative enzymes into host organisms can be challenging in cases such as this, where the enzyme has evolved in a very different environment from that of the host. Here, we use computational protein design to engineer the Zymomonas mobilis PDC to resist thermal denaturation at the growth temperature of a thermophilic host. We generate thirteen PDC variants using the Rosetta protein design software. We measure thermal stability of the wild-type PDC and PDC variants using circular dichroism. We then measure and compare enzyme endurance for wild-type PDC with the PDC variants at an elevated temperature of 60º (thermal endurance) using differential interference contrast imaging. We find that increases in melting temperature (Tm) do not directly correlate with increases in thermal endurance at 60º. We also do not find evidence that any individual mutation or design approach is the major contributor to the most thermostable PDC variant. Rather, remarkable cooperativity among sixteen thermostabilizing mutations is key to rationally designing a PDC with significantly enhanced thermal endurance. These results suggest a generalizable iterative computational protein design approach to improve thermal stability and endurance of target enzymes.},
doi = {10.1186/s13068-018-1178-9},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 11,
place = {United States},
year = {Mon Jul 09 00:00:00 EDT 2018},
month = {Mon Jul 09 00:00:00 EDT 2018}
}

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