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Title: Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes

Abstract

n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. In this paper, a synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70%more » conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Finally and thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures.« less

Authors:
 [1];  [2];  [2];  [3];  [3];  [2]
  1. North Carolina State Univ., Raleigh, NC (United States). Dept. of Chemical and Biomolecular Engineering
  2. North Carolina State Univ., Raleigh, NC (United States). Dept. of Chemical and Biomolecular Engineering
  3. Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology
Publication Date:
Research Org.:
North Carolina State Univ., Raleigh, NC (United States); Univ. of Georgia, Athens, GA (United States)
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E); National Science Foundation (NSF); National Inst. of Health (NIH) (United States)
OSTI Identifier:
1470144
Grant/Contract Number:  
AR0000081; CBET-1264052; CBET-1264053; 2T32GM008776
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 81; Journal Issue: 20; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Loder, Andrew J., Zeldes, Benjamin M., Garrison, G. Dale, Lipscomb, Gina L., Adams, Michael W. W., and Kelly, Robert M. Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes. United States: N. p., 2015. Web. doi:10.1128/AEM.02028-15.
Loder, Andrew J., Zeldes, Benjamin M., Garrison, G. Dale, Lipscomb, Gina L., Adams, Michael W. W., & Kelly, Robert M. Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes. United States. https://doi.org/10.1128/AEM.02028-15
Loder, Andrew J., Zeldes, Benjamin M., Garrison, G. Dale, Lipscomb, Gina L., Adams, Michael W. W., and Kelly, Robert M. 2015. "Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes". United States. https://doi.org/10.1128/AEM.02028-15. https://www.osti.gov/servlets/purl/1470144.
@article{osti_1470144,
title = {Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes},
author = {Loder, Andrew J. and Zeldes, Benjamin M. and Garrison, G. Dale and Lipscomb, Gina L. and Adams, Michael W. W. and Kelly, Robert M.},
abstractNote = {n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. In this paper, a synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Finally and thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures.},
doi = {10.1128/AEM.02028-15},
url = {https://www.osti.gov/biblio/1470144}, journal = {Applied and Environmental Microbiology},
issn = {0099-2240},
number = 20,
volume = 81,
place = {United States},
year = {2015},
month = {8}
}

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Cited by: 7 works
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Works referencing / citing this record:

Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor
journal, December 2018


Ethanol production by the hyperthermophilic archaeon Pyrococcus furiosus by expression of bacterial bifunctional alcohol dehydrogenases
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