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@PHDTHESIS{Spielmann:872523,
      author       = {Spielmann, Alina},
      title        = {{NADPH}‐related studies performed with a {S}ox{R}‐based
                      biosensor in $\textit{{E}scherichia coli}$},
      volume       = {207},
      school       = {Universität Düsseldorf},
      type         = {Dr.},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
      reportid     = {FZJ-2020-00043},
      isbn         = {978-3-95806-438-6},
      series       = {Schriften des Forschungszentrums Jülich. Reihe
                      Schlüsseltechnologien / Key Technologies},
      pages        = {IV, 73 S.},
      year         = {2019},
      note         = {Universität Düsseldorf, 2019},
      abstract     = {The SoxRS regulatory system of $\textit{Escherichia coli}$
                      responds to NADPH, presumably due to the NADPH dependent
                      reduction of the transcriptional regulator SoxR, switching
                      it to the inactive state. In a previous study, this
                      NADPH-responsiveness was used to construct the genetically
                      encoded NADPH biosensor pSenSox, in which the SoxR-activated
                      soxS promoter controls expression of the reporter gene
                      $\textit{eyfp}$, allowing detection of SoxR activation at
                      the single cell level $\textit{via}$ eYFP fluorescence. The
                      biosensor was reported to sense intracellular NADPH
                      availability, because increased cellular NADPH demands
                      during the biotransformation of methyl acetoacetate (MAA) to
                      $\textit{R}$-methyl 3-hydroxybutyrate (MHB) by the strictly
                      NADPH-dependent alcohol dehydrogenase of
                      $\textit{Lactobacillus brevis}$ ($\textit{Lb}$ADH) led to
                      increased $\textit{eyfp}$ expression. Most importantly, the
                      specific eYFP fluorescence of $\textit{E. coli}$ cells
                      catalyzing MAA reduction to MHB correlated not only with the
                      amount of MAA reduced by the cells, and consequently with
                      the NADPH demand, but also with the specific
                      $\textit{Lb}$ADH activity when a fixed MAA concentration was
                      provided. The latter property enables high-throughput
                      screening of large NADPH-dependent enzyme libraries for
                      variants with improved activity using fluorescenceactivated
                      cell sorting (FACS). Based on the correlation of the
                      specific fluorescence of the biosensor with the cellular
                      NADPH demand and the activity of NADPH-dependent enzymes,
                      the pSenSox biosensor was used in this thesis to (1) study
                      NADPH-related processes in $\textit{E. coli}$ and to (2)
                      identify variants of the NADPH-dependent $\textit{Lb}$ADH
                      with improved catalytic properties. (1) NADPH plays a
                      crucial role in cellular metabolism for biosynthesis and
                      oxidative stress responses. Here, pSenSox was used to study
                      the influence of various NADPH-related parameters on the
                      soxRS response in $\textit{E. coli}$. Specifically, the
                      influence of different growth media, of the redox-cycling
                      drugs paraquat and menadione, of the SoxR-reducing system
                      RsxABCDGE and RseC, and of transhydrogenases SthA and PntAB
                      on the pSenSox signal was examined. Redox-cycling drugs
                      activated the NADPH biosensor. The absence of RsxABCDGE
                      and/or RseC caused an enhanced biosensor response, in
                      agreement with their function as a SoxRreducing system. The
                      absence of the membrane-bound transhydrogenase PntAB caused
                      an increased biosensor response, whereas the lack of the
                      soluble transhydrogenase SthA or of SthA and PntAB was
                      associated with a strongly decreased response. These data
                      support the opposing functions of PntAB in NADP$^{+}$
                      reduction and of SthA in NADPH oxidation. In conclusion, the
                      biosensor pSenSox was shown to be a useful tool for
                      analyzing environmental conditions and genes with respect to
                      their influence on the NADPH availability in the cell. (2)
                      The NADPH-dependent $\textit{Lb}$ADH is widely used in
                      industrial biotechnology for the biocatalytic production of
                      chiral alcohols. To find an optimized LbADH variant for the
                      substrate 2,5-hexanedione, the biosensor pSenSox was used
                      for FACS-based high-throughput screening of an
                      $\textit{Lb}$ADH library to isolate clones showing increased
                      fluorescence during the biotransformation of
                      2,5-hexanedione. Using this approach, the improved variant
                      $\textit{Lb}$ADH$^{K71E}$ was identified in which lysine-71
                      was replaced by glutamate, causing a charge reversal at the
                      surface of the protein. Kinetic measurements with purified
                      enzymes revealed that $\textit{Lb}$ADH$^{K71E}$ has a 16\%
                      higher affinity (K$_{M}$= 4.3 ± 0.5 mM) and a 17\% higher
                      activity (V$_{max}$= 173.3 ± 11.1 μmol
                      min$^{-1}$mg$^{-1}$) compared to the wild-type enzyme
                      (K$_{M}$= 5.1 ± 0.6 mM; V$_{max}$= 148.5 ± 12.3 μmol
                      min$^{-1}$mg$^{-1}$) with 2,5- hexanedione as substrate.
                      Moreover, the $\textit{Lb}$ADH$^{K71E}$ enzyme also showed
                      higher activity for the alternative substrates acetophenone,
                      acetylpyridine, 2-hexanone, 4-hydroxy-2-butanone, and MAA.
                      The isolation of the optimized variant
                      $^\textit{Lb}$ADH$^{K71E}$ demonstrates that the application
                      of the biosensor combined with high-throughput screening is
                      a powerful workflow for the identification of unexpected
                      beneficial mutations of NADPH-dependent ADHs.},
      cin          = {IBG-1},
      cid          = {I:(DE-Juel1)IBG-1-20101118},
      pnm          = {899 - ohne Topic (POF3-899)},
      pid          = {G:(DE-HGF)POF3-899},
      typ          = {PUB:(DE-HGF)3 / PUB:(DE-HGF)11},
      urn          = {urn:nbn:de:0001-2020012911},
      url          = {https://juser.fz-juelich.de/record/872523},
}