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In Vitro Bioconversion of Pyruvate to n-Butanol with Minimized Cofactor Utilization

Due to enhanced energy content and reduced hygroscopicity compared with ethanol, n-butanol is flagged as the next generation biofuel and platform chemical. In addition to conventional cellular systems, n-butanol bioproduction by enzyme cascades is gaining momentum due to simplified process control....

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Autores principales: Reiße, Steven, Haack, Martina, Garbe, Daniel, Sommer, Bettina, Steffler, Fabian, Carsten, Jörg, Bohnen, Frank, Sieber, Volker, Brück, Thomas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066087/
https://www.ncbi.nlm.nih.gov/pubmed/27800475
http://dx.doi.org/10.3389/fbioe.2016.00074
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author Reiße, Steven
Haack, Martina
Garbe, Daniel
Sommer, Bettina
Steffler, Fabian
Carsten, Jörg
Bohnen, Frank
Sieber, Volker
Brück, Thomas
author_facet Reiße, Steven
Haack, Martina
Garbe, Daniel
Sommer, Bettina
Steffler, Fabian
Carsten, Jörg
Bohnen, Frank
Sieber, Volker
Brück, Thomas
author_sort Reiße, Steven
collection PubMed
description Due to enhanced energy content and reduced hygroscopicity compared with ethanol, n-butanol is flagged as the next generation biofuel and platform chemical. In addition to conventional cellular systems, n-butanol bioproduction by enzyme cascades is gaining momentum due to simplified process control. In contrast to other bio-based alcohols like ethanol and isobutanol, cell-free n-butanol biosynthesis from the central metabolic intermediate pyruvate involves cofactors [NAD(P)H, CoA] and acetyl-CoA-dependent intermediates, which complicates redox and energy balancing of the reaction system. We have devised a biochemical process for cell-free n-butanol production that only involves three enzyme activities, thereby eliminating the need for acetyl-CoA. Instead, the process utilizes only NADH as the sole redox mediator. Central to this new process is the amino acid catalyzed enamine–aldol condensation, which transforms acetaldehyde directly into crotonaldehyde. Subsequently, crotonaldehyde is reduced to n-butanol applying a 2-enoate reductase and an alcohol dehydrogenase, respectively. In essence, we achieved conversion of the platform intermediate pyruvate to n-butanol utilizing a biocatalytic cascade comprising only three enzyme activities and NADH as reducing equivalent. With reference to previously reported cell-free n-butanol reaction cascades, we have eliminated five enzyme activities and the requirement of CoA as cofactor. Our proof-of-concept demonstrates that n-butanol was synthesized at neutral pH and 50°C. This integrated reaction concept allowed GC detection of all reaction intermediates and n-butanol production of 148 mg L(−1) (2 mM), which compares well with other cell-free n-butanol production processes.
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spelling pubmed-50660872016-10-31 In Vitro Bioconversion of Pyruvate to n-Butanol with Minimized Cofactor Utilization Reiße, Steven Haack, Martina Garbe, Daniel Sommer, Bettina Steffler, Fabian Carsten, Jörg Bohnen, Frank Sieber, Volker Brück, Thomas Front Bioeng Biotechnol Bioengineering and Biotechnology Due to enhanced energy content and reduced hygroscopicity compared with ethanol, n-butanol is flagged as the next generation biofuel and platform chemical. In addition to conventional cellular systems, n-butanol bioproduction by enzyme cascades is gaining momentum due to simplified process control. In contrast to other bio-based alcohols like ethanol and isobutanol, cell-free n-butanol biosynthesis from the central metabolic intermediate pyruvate involves cofactors [NAD(P)H, CoA] and acetyl-CoA-dependent intermediates, which complicates redox and energy balancing of the reaction system. We have devised a biochemical process for cell-free n-butanol production that only involves three enzyme activities, thereby eliminating the need for acetyl-CoA. Instead, the process utilizes only NADH as the sole redox mediator. Central to this new process is the amino acid catalyzed enamine–aldol condensation, which transforms acetaldehyde directly into crotonaldehyde. Subsequently, crotonaldehyde is reduced to n-butanol applying a 2-enoate reductase and an alcohol dehydrogenase, respectively. In essence, we achieved conversion of the platform intermediate pyruvate to n-butanol utilizing a biocatalytic cascade comprising only three enzyme activities and NADH as reducing equivalent. With reference to previously reported cell-free n-butanol reaction cascades, we have eliminated five enzyme activities and the requirement of CoA as cofactor. Our proof-of-concept demonstrates that n-butanol was synthesized at neutral pH and 50°C. This integrated reaction concept allowed GC detection of all reaction intermediates and n-butanol production of 148 mg L(−1) (2 mM), which compares well with other cell-free n-butanol production processes. Frontiers Media S.A. 2016-10-17 /pmc/articles/PMC5066087/ /pubmed/27800475 http://dx.doi.org/10.3389/fbioe.2016.00074 Text en Copyright © 2016 Reiße, Haack, Garbe, Sommer, Steffler, Carsten, Bohnen, Sieber and Brück. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Reiße, Steven
Haack, Martina
Garbe, Daniel
Sommer, Bettina
Steffler, Fabian
Carsten, Jörg
Bohnen, Frank
Sieber, Volker
Brück, Thomas
In Vitro Bioconversion of Pyruvate to n-Butanol with Minimized Cofactor Utilization
title In Vitro Bioconversion of Pyruvate to n-Butanol with Minimized Cofactor Utilization
title_full In Vitro Bioconversion of Pyruvate to n-Butanol with Minimized Cofactor Utilization
title_fullStr In Vitro Bioconversion of Pyruvate to n-Butanol with Minimized Cofactor Utilization
title_full_unstemmed In Vitro Bioconversion of Pyruvate to n-Butanol with Minimized Cofactor Utilization
title_short In Vitro Bioconversion of Pyruvate to n-Butanol with Minimized Cofactor Utilization
title_sort in vitro bioconversion of pyruvate to n-butanol with minimized cofactor utilization
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066087/
https://www.ncbi.nlm.nih.gov/pubmed/27800475
http://dx.doi.org/10.3389/fbioe.2016.00074
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