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@PHDTHESIS{Flachbart:865921,
      author       = {Flachbart, Lion},
      title        = {{D}evelopment of a transcriptional biosensor and
                      reengineering of its ligand specificity using
                      fluorescence-activated cell sorting},
      volume       = {226},
      school       = {Heinrich-Heine-Universität Düsseldorf},
      type         = {Dr.},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
      reportid     = {FZJ-2019-05198},
      isbn         = {978-3-95806-515-4},
      series       = {Schriften des Forschungszentrums Jülich. Reihe
                      Schlüsseltechnologien / Key Technologies},
      pages        = {VIII, 102 S.},
      year         = {2020},
      note         = {Biotechnologie 1; Heinrich-Heine-Universität Düsseldorf,
                      Diss., 2019},
      abstract     = {Important chemical compounds of our daily life such as
                      amino acids, antibiotics or vitamins are produced by
                      microorganisms at large‐scale. Also, there is growing
                      interest in the microbial synthesis of many other compounds
                      including pharmaceutically interesting secondary metabolites
                      from plants. However, development and improvement of the
                      microbial producer strains is time‐consuming and
                      cost‐intensive. In this context,biosensor‐based
                      fluorescence‐active cell sorting (FACS) to identify
                      suitable production strain variants representsa promising
                      approach to tackle these challenges. In this dissertation,
                      the application of transcription factor‐based biosensors
                      in combination with FACS for high-throughput screening of
                      enzyme libraries was investigated in $\textit{Escherichia
                      coli}$. Furthermore, the construction of biosensors with
                      modified ligand spectrum from an existing biosensor was
                      pursued to expand the repertoire of biosensor‐detectable
                      substances. Initially, the transcription factor‐based
                      biosensor pSenCA which can be used to convert cytosolic
                      concentrations of the phenylpropanoid trans‐cinnamic acid
                      (CA) to a fluorescence output signal, was constructed and
                      characterized. The biosensor is composed of the
                      transcriptional regulator HcaR from $\textit{Escherichia
                      coli}$ and its target promoter P$_{hcaE}$, transcriptionally
                      fused with the $\textit{eyfp}$ gene encoding an
                      autofluorescent protein. This biosensor was subsequently
                      used to optimize an L‐phenylalanine/L‐tyrosine ammonia
                      lyase from $\textit{Trichosporon cutaneum}$ (Xal$_{Tc}$), by
                      a directed evolution approach. Aromatic amino acid ammonia
                      lyases represent the key enzyme in many plant polyphenol
                      biosynthetic pathways. The use of an expression system with
                      titratable expression strength of the ammonia lyase gene as
                      well as a significant reduction of the initial cell density
                      priorto screening were prerequisites for an effective
                      isolation of CA producers from mixed cultures with
                      nonproducers. The established screening method was
                      subsequently used to screen a randomly mutagenized ammonia
                      lyase library of 2.4×10$^{6}$ variants for improved
                      fluorescence. All 182 clones isolated by FACS were CA
                      producers, 138 produced at least 10 \% more CA compared to
                      the parent strain. The best strain showed a 60 \% increase
                      in CA production. Seven Xal$_{Tc}$ variants investigated
                      $\textit{in vitro}$ exhibited up to 12 \% increased specific
                      activity and up to 20 \% increased substrate affinity. In
                      the second project, 15 amino acids in the ligand binding
                      site of the regulator protein HcaR, which were identified by
                      $\textit{in silico}$ structure analysis, were randomized by
                      site saturation mutagenesis. The resulting HcaR biosensor
                      libraries were screened for variants with increased
                      specificity for 3,5‐dihydroxyphenylpropionate using FACS.
                      These experiments resulted in the isolation of pSenGeneral,
                      a sensor variant with a significantly broadenedligand
                      spectrum. In a second round of biosensor evolution,
                      additional libraries based on pSenGeneral were constructed
                      and screened for variants with specificity for various
                      compounds of biotechnological interest. As a result,
                      biosensor variants for the detection of 4‐hydroxybenzoic
                      acid, 6‐methylsalicylate, $\textit{p}$‐coumaric acid, or
                      5‐bromoferulic acid could be isolated. In the future,
                      these newly designed biosensors for small aromatic compounds
                      could find an application during microbial strain
                      development and might represent a good starting point for
                      the development of additional biosensors for other aromatic
                      molecules of biotechnological interest.},
      cin          = {IBG-1},
      cid          = {I:(DE-Juel1)IBG-1-20101118},
      pnm          = {581 - Biotechnology (POF3-581)},
      pid          = {G:(DE-HGF)POF3-581},
      typ          = {PUB:(DE-HGF)3 / PUB:(DE-HGF)11},
      urn          = {urn:nbn:de:0001-2021080228},
      url          = {https://juser.fz-juelich.de/record/865921},
}