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Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli

In clostridia, n-butanol production from carbohydrates at yields of up to 76% of the theoretical maximum and at titers of up to 13 g/L has been reported. However, in Escherichia coli, several groups have reported butyric acid or butanol production from recombinant expression of clostridial genes, at...

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Detalles Bibliográficos
Autores principales: Fischer, Curt R., Tseng, Hsien-Chung, Tai, Mitchell, Prather, Kristala L. J., Stephanopoulos, Gregory
Formato: Texto
Lenguaje:English
Publicado: Springer-Verlag 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2921503/
https://www.ncbi.nlm.nih.gov/pubmed/20625717
http://dx.doi.org/10.1007/s00253-010-2749-2
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author Fischer, Curt R.
Tseng, Hsien-Chung
Tai, Mitchell
Prather, Kristala L. J.
Stephanopoulos, Gregory
author_facet Fischer, Curt R.
Tseng, Hsien-Chung
Tai, Mitchell
Prather, Kristala L. J.
Stephanopoulos, Gregory
author_sort Fischer, Curt R.
collection PubMed
description In clostridia, n-butanol production from carbohydrates at yields of up to 76% of the theoretical maximum and at titers of up to 13 g/L has been reported. However, in Escherichia coli, several groups have reported butyric acid or butanol production from recombinant expression of clostridial genes, at much lower titers and yields. To pinpoint deficient steps in the recombinant pathway, we developed an analytical procedure for the determination of intracellular pools of key pathway intermediates and applied the technique to the analysis of three sets of E. coli strains expressing various combinations of butyrate biosynthesis genes. Low expression levels of the hbd-encoded S-3-hydroxybutyryl-CoA dehydrogenase were insufficient to convert acetyl-CoA to 3-hydroxybutyryl-CoA, indicating that hbd was a rate-limiting step in the production of butyryl-CoA. Increasing hbd expression alleviated this bottleneck, but in resulting strains, our pool size measurements and thermodynamic analysis showed that the reaction step catalyzed by the bcd-encoded butyryl-CoA dehydrogenase was rate-limiting. E. coli strains expressing both hbd and ptb-buk produced crotonic acid as a byproduct, but this byproduct was not observed with expression of related genes from non-clostridial organisms. Our thermodynamic interpretation of pool size measurements is applicable to the analysis of other metabolic pathways. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-010-2749-2) contains supplementary material, which is available to authorized users.
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spelling pubmed-29215032010-08-20 Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli Fischer, Curt R. Tseng, Hsien-Chung Tai, Mitchell Prather, Kristala L. J. Stephanopoulos, Gregory Appl Microbiol Biotechnol Applied Microbial and Cell Physiology In clostridia, n-butanol production from carbohydrates at yields of up to 76% of the theoretical maximum and at titers of up to 13 g/L has been reported. However, in Escherichia coli, several groups have reported butyric acid or butanol production from recombinant expression of clostridial genes, at much lower titers and yields. To pinpoint deficient steps in the recombinant pathway, we developed an analytical procedure for the determination of intracellular pools of key pathway intermediates and applied the technique to the analysis of three sets of E. coli strains expressing various combinations of butyrate biosynthesis genes. Low expression levels of the hbd-encoded S-3-hydroxybutyryl-CoA dehydrogenase were insufficient to convert acetyl-CoA to 3-hydroxybutyryl-CoA, indicating that hbd was a rate-limiting step in the production of butyryl-CoA. Increasing hbd expression alleviated this bottleneck, but in resulting strains, our pool size measurements and thermodynamic analysis showed that the reaction step catalyzed by the bcd-encoded butyryl-CoA dehydrogenase was rate-limiting. E. coli strains expressing both hbd and ptb-buk produced crotonic acid as a byproduct, but this byproduct was not observed with expression of related genes from non-clostridial organisms. Our thermodynamic interpretation of pool size measurements is applicable to the analysis of other metabolic pathways. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-010-2749-2) contains supplementary material, which is available to authorized users. Springer-Verlag 2010-07-13 2010 /pmc/articles/PMC2921503/ /pubmed/20625717 http://dx.doi.org/10.1007/s00253-010-2749-2 Text en © The Author(s) 2010 https://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
spellingShingle Applied Microbial and Cell Physiology
Fischer, Curt R.
Tseng, Hsien-Chung
Tai, Mitchell
Prather, Kristala L. J.
Stephanopoulos, Gregory
Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli
title Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli
title_full Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli
title_fullStr Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli
title_full_unstemmed Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli
title_short Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli
title_sort assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant escherichia coli
topic Applied Microbial and Cell Physiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2921503/
https://www.ncbi.nlm.nih.gov/pubmed/20625717
http://dx.doi.org/10.1007/s00253-010-2749-2
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