Cargando…

Metabolic network capacity of Escherichia coli for Krebs cycle-dependent proline hydroxylation

BACKGROUND: Understanding the metabolism of the microbial host is essential for the development and optimization of whole-cell based biocatalytic processes, as it dictates production efficiency. This is especially true for redox biocatalysis where metabolically active cells are employed because of t...

Descripción completa

Detalles Bibliográficos
Autores principales: Theodosiou, Eleni, Frick, Oliver, Bühler, Bruno, Schmid, Andreas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517350/
https://www.ncbi.nlm.nih.gov/pubmed/26215086
http://dx.doi.org/10.1186/s12934-015-0298-1
_version_ 1782383172611211264
author Theodosiou, Eleni
Frick, Oliver
Bühler, Bruno
Schmid, Andreas
author_facet Theodosiou, Eleni
Frick, Oliver
Bühler, Bruno
Schmid, Andreas
author_sort Theodosiou, Eleni
collection PubMed
description BACKGROUND: Understanding the metabolism of the microbial host is essential for the development and optimization of whole-cell based biocatalytic processes, as it dictates production efficiency. This is especially true for redox biocatalysis where metabolically active cells are employed because of the cofactor/cosubstrate regenerative capacity endogenous in the host. Recombinant Escherichia coli was used for overproducing proline-4-hydroxylase (P4H), a dioxygenase catalyzing the hydroxylation of free l-proline into trans-4-hydroxy-l-proline with a-ketoglutarate (a-KG) as cosubstrate. In this whole-cell biocatalyst, central carbon metabolism provides the required cosubstrate a-KG, coupling P4H biocatalytic performance directly to carbon metabolism and metabolic activity. By applying both experimental and computational biology tools, such as metabolic engineering and (13)C-metabolic flux analysis ((13)C-MFA), we investigated and quantitatively described the physiological, metabolic, and bioenergetic response of the whole-cell biocatalyst to the targeted bioconversion and identified possible metabolic bottlenecks for further rational pathway engineering. RESULTS: A proline degradation-deficient E. coli strain was constructed by deleting the putA gene encoding proline dehydrogenase. Whole-cell biotransformations with this mutant strain led not only to quantitative proline hydroxylation but also to a doubling of the specific trans-4-l-hydroxyproline (hyp) formation rate, compared to the wild type. Analysis of carbon flux through central metabolism of the mutant strain revealed that the increased a-KG demand for P4H activity did not enhance the a-KG generating flux, indicating a tightly regulated TCA cycle operation under the conditions studied. In the wild type strain, P4H synthesis and catalysis caused a reduction in biomass yield. Interestingly, the ΔputA strain additionally compensated the associated ATP and NADH loss by reducing maintenance energy demands at comparably low glucose uptake rates, instead of increasing the TCA activity. CONCLUSIONS: The putA knockout in recombinant E. coli BL21(DE3)(pLysS) was found to be promising for productive P4H catalysis not only in terms of biotransformation yield, but also regarding the rates for biotransformation and proline uptake and the yield of hyp on the energy source. The results indicate that, upon a putA knockout, the coupling of the TCA-cycle to proline hydroxylation via the cosubstrate a-KG becomes a key factor constraining and a target to further improve the efficiency of a-KG-dependent biotransformations. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0298-1) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-4517350
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-45173502015-07-29 Metabolic network capacity of Escherichia coli for Krebs cycle-dependent proline hydroxylation Theodosiou, Eleni Frick, Oliver Bühler, Bruno Schmid, Andreas Microb Cell Fact Research BACKGROUND: Understanding the metabolism of the microbial host is essential for the development and optimization of whole-cell based biocatalytic processes, as it dictates production efficiency. This is especially true for redox biocatalysis where metabolically active cells are employed because of the cofactor/cosubstrate regenerative capacity endogenous in the host. Recombinant Escherichia coli was used for overproducing proline-4-hydroxylase (P4H), a dioxygenase catalyzing the hydroxylation of free l-proline into trans-4-hydroxy-l-proline with a-ketoglutarate (a-KG) as cosubstrate. In this whole-cell biocatalyst, central carbon metabolism provides the required cosubstrate a-KG, coupling P4H biocatalytic performance directly to carbon metabolism and metabolic activity. By applying both experimental and computational biology tools, such as metabolic engineering and (13)C-metabolic flux analysis ((13)C-MFA), we investigated and quantitatively described the physiological, metabolic, and bioenergetic response of the whole-cell biocatalyst to the targeted bioconversion and identified possible metabolic bottlenecks for further rational pathway engineering. RESULTS: A proline degradation-deficient E. coli strain was constructed by deleting the putA gene encoding proline dehydrogenase. Whole-cell biotransformations with this mutant strain led not only to quantitative proline hydroxylation but also to a doubling of the specific trans-4-l-hydroxyproline (hyp) formation rate, compared to the wild type. Analysis of carbon flux through central metabolism of the mutant strain revealed that the increased a-KG demand for P4H activity did not enhance the a-KG generating flux, indicating a tightly regulated TCA cycle operation under the conditions studied. In the wild type strain, P4H synthesis and catalysis caused a reduction in biomass yield. Interestingly, the ΔputA strain additionally compensated the associated ATP and NADH loss by reducing maintenance energy demands at comparably low glucose uptake rates, instead of increasing the TCA activity. CONCLUSIONS: The putA knockout in recombinant E. coli BL21(DE3)(pLysS) was found to be promising for productive P4H catalysis not only in terms of biotransformation yield, but also regarding the rates for biotransformation and proline uptake and the yield of hyp on the energy source. The results indicate that, upon a putA knockout, the coupling of the TCA-cycle to proline hydroxylation via the cosubstrate a-KG becomes a key factor constraining and a target to further improve the efficiency of a-KG-dependent biotransformations. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0298-1) contains supplementary material, which is available to authorized users. BioMed Central 2015-07-29 /pmc/articles/PMC4517350/ /pubmed/26215086 http://dx.doi.org/10.1186/s12934-015-0298-1 Text en © Theodosiou et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Theodosiou, Eleni
Frick, Oliver
Bühler, Bruno
Schmid, Andreas
Metabolic network capacity of Escherichia coli for Krebs cycle-dependent proline hydroxylation
title Metabolic network capacity of Escherichia coli for Krebs cycle-dependent proline hydroxylation
title_full Metabolic network capacity of Escherichia coli for Krebs cycle-dependent proline hydroxylation
title_fullStr Metabolic network capacity of Escherichia coli for Krebs cycle-dependent proline hydroxylation
title_full_unstemmed Metabolic network capacity of Escherichia coli for Krebs cycle-dependent proline hydroxylation
title_short Metabolic network capacity of Escherichia coli for Krebs cycle-dependent proline hydroxylation
title_sort metabolic network capacity of escherichia coli for krebs cycle-dependent proline hydroxylation
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517350/
https://www.ncbi.nlm.nih.gov/pubmed/26215086
http://dx.doi.org/10.1186/s12934-015-0298-1
work_keys_str_mv AT theodosioueleni metabolicnetworkcapacityofescherichiacoliforkrebscycledependentprolinehydroxylation
AT frickoliver metabolicnetworkcapacityofescherichiacoliforkrebscycledependentprolinehydroxylation
AT buhlerbruno metabolicnetworkcapacityofescherichiacoliforkrebscycledependentprolinehydroxylation
AT schmidandreas metabolicnetworkcapacityofescherichiacoliforkrebscycledependentprolinehydroxylation