Universität zu Köln

Goldbeck, Oliver ORCID: 0000-0002-5708-8600, Eck, Alexander W. and Seibold, Gerd M. (2018). Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP. Front. Microbiol., 9. LAUSANNE: FRONTIERS MEDIA SA. ISSN 1664-302X

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Abstract

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 Delta pgi, 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 Delta pgi was observed; as expected the internal NADPH concentration was significantly higher for C. glutamicum Delta pgi (0.31 mM) when compared to C. glutamicum WT (0.19 mM). Addition of paraquat to E. coil cells carrying mBFP led as expected to an immediate decrease of intracellular NADPH concentrations, showing the versatile use of mBFP as intracellular sensor.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Goldbeck, Oliver UNSPECIFIED orcid.org/0000-0002-5708-8600 UNSPECIFIED Eck, Alexander W. UNSPECIFIED UNSPECIFIED UNSPECIFIED Seibold, Gerd M. UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-169006
DOI:	10.3389/fmicb.2018.02564
Journal or Publication Title:	Front. Microbiol.
Volume:	9
Date:	2018
Publisher:	FRONTIERS MEDIA SA
Place of Publication:	LAUSANNE
ISSN:	1664-302X
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language PYRIDINE-NUCLEOTIDE TRANSHYDROGENASE; SHORT-CHAIN DEHYDROGENASE/REDUCTASE; CELLULAR FUNCTIONS; VIBRIO-VULNIFICUS; METABOLISM; EXPRESSION; CELLS; FLUORESCENCE; DEFICIENT; REVEALS Multiple languages Microbiology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/16900