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AuthorEck, Alexander
AuthorSeibold, Gerd
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AbstractAnalyses 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 (KD 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 1pgi, 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 1pgi was observed; as expected the internal NADPH concentration was significantly higher for C. glutamicum 1pgi (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.dc.description.abstract
PublisherUniversität Ulmdc.publisher
LicenseCC BY 4.0 Internationaldc.rights
Link to license text
Keywordredox statedc.subject
Keywordshort chain dehydrogenasedc.subject
Dewey Decimal GroupDDC 570 / Life sciencesdc.subject.ddc
Dewey Decimal GroupDDC 610 / Medicine & healthdc.subject.ddc
LCSHCorynebacterium glutamicumdc.subject.lcsh
LCSHEscherichia colidc.subject.lcsh
TitleReal time monitoring of NADPH concentrations in corynebacterium glutamicum and escherichia coli via the genetically encoded sensor mBFPdc.title
Resource typeWissenschaftlicher Artikeldc.type
FacultyFakultät für Naturwissenschaftenuulm.affiliationGeneral
InstitutionInstitut für Mikrobiologie und Biotechnologieuulm.affiliationSpecific
Peer reviewjauulm.peerReview
DCMI TypeTextuulm.typeDCMI
In cooperation withUniversität zu Kölnuulm.cooperation
Is Supplemented By
DOI of original publication10.3389/fmicb.2018.02564dc.relation1.doi
Source - Title of sourceFrontiers in Microbiologysource.title
Source - Place of publicationFrontiers Mediasource.publisher
Source - Volume9source.volume
Source - Year2018source.year
Source - Article number2564source.articleNumber
Source - eISSN1664-302Xsource.identifier.eissn
Fundinge:Bio initiative /BMBF [031A302D]uulm.funding
University Bibliographyjauulm.unibibliographie

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CC BY 4.0 International
Except where otherwise noted, this item's license is described as CC BY 4.0 International