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Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP
Analyses of intracellular NADPH concentrations are prerequisites for the design of microbial production strains and process optimization. mBFP was described as metagenomics derived, blue fluorescent protein showing NADPH-dependent fluorescence. Characterization of mBFP showed a high specificity for...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2018
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207642/ https://www.ncbi.nlm.nih.gov/pubmed/30405597 http://dx.doi.org/10.3389/fmicb.2018.02564 |
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author | Goldbeck, Oliver Eck, Alexander W. Seibold, Gerd M. |
author_facet | Goldbeck, Oliver Eck, Alexander W. Seibold, Gerd M. |
author_sort | Goldbeck, Oliver |
collection | PubMed |
description | Analyses of intracellular NADPH concentrations are prerequisites for the design of microbial production strains and process optimization. mBFP was described as metagenomics derived, blue fluorescent protein showing NADPH-dependent fluorescence. Characterization of mBFP showed a high specificity for binding of NADPH (K(D) 0.64 mM) and no binding of NADH, the protein exclusively amplified fluorescence of NADPH. mBFP catalyzed the NADPH-dependent reduction of benzaldehyde and further aldehydes, which fits to its classification as short chain dehydrogenase. For in vivo NADPH analyses a codon-optimized gene for mBFP was introduced into Corynebacterium glutamicum WT and the phosphoglucoisomerase-deficient strain C. glutamicum Δpgi, which accumulates high levels of NADPH. For determination of intracellular NADPH concentrations by mBFP a calibration method with permeabilized cells was developed. By this means an increase of intracellular NADPH concentrations within seconds after the addition of glucose to nutrient-starved cells of both C. glutamicum WT and C. glutamicum Δpgi was observed; as expected the internal NADPH concentration was significantly higher for C. glutamicum Δpgi (0.31 mM) when compared to C. glutamicum WT (0.19 mM). Addition of paraquat to E. coli cells carrying mBFP led as expected to an immediate decrease of intracellular NADPH concentrations, showing the versatile use of mBFP as intracellular sensor. |
format | Online Article Text |
id | pubmed-6207642 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-62076422018-11-07 Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP Goldbeck, Oliver Eck, Alexander W. Seibold, Gerd M. Front Microbiol Microbiology Analyses of intracellular NADPH concentrations are prerequisites for the design of microbial production strains and process optimization. mBFP was described as metagenomics derived, blue fluorescent protein showing NADPH-dependent fluorescence. Characterization of mBFP showed a high specificity for binding of NADPH (K(D) 0.64 mM) and no binding of NADH, the protein exclusively amplified fluorescence of NADPH. mBFP catalyzed the NADPH-dependent reduction of benzaldehyde and further aldehydes, which fits to its classification as short chain dehydrogenase. For in vivo NADPH analyses a codon-optimized gene for mBFP was introduced into Corynebacterium glutamicum WT and the phosphoglucoisomerase-deficient strain C. glutamicum Δpgi, which accumulates high levels of NADPH. For determination of intracellular NADPH concentrations by mBFP a calibration method with permeabilized cells was developed. By this means an increase of intracellular NADPH concentrations within seconds after the addition of glucose to nutrient-starved cells of both C. glutamicum WT and C. glutamicum Δpgi was observed; as expected the internal NADPH concentration was significantly higher for C. glutamicum Δpgi (0.31 mM) when compared to C. glutamicum WT (0.19 mM). Addition of paraquat to E. coli cells carrying mBFP led as expected to an immediate decrease of intracellular NADPH concentrations, showing the versatile use of mBFP as intracellular sensor. Frontiers Media S.A. 2018-10-24 /pmc/articles/PMC6207642/ /pubmed/30405597 http://dx.doi.org/10.3389/fmicb.2018.02564 Text en Copyright © 2018 Goldbeck, Eck and Seibold. 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) and the copyright owner(s) 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 | Microbiology Goldbeck, Oliver Eck, Alexander W. Seibold, Gerd M. Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP |
title | Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP |
title_full | Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP |
title_fullStr | Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP |
title_full_unstemmed | Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP |
title_short | Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP |
title_sort | real time monitoring of nadph concentrations in corynebacterium glutamicum and escherichia coli via the genetically encoded sensor mbfp |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207642/ https://www.ncbi.nlm.nih.gov/pubmed/30405597 http://dx.doi.org/10.3389/fmicb.2018.02564 |
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