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@PHDTHESIS{Sonntag:886052,
      author       = {Sonntag, Christiane},
      title        = {{M}olecular tools for genome engineering of
                      {C}orynebacterium glutamicum},
      volume       = {230},
      school       = {Heinrich-Heine-Universität Düsseldorf},
      type         = {Dissertation},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
      reportid     = {FZJ-2020-04239},
      isbn         = {978-3-95806-532-1},
      series       = {Schriften des Forschungszentrums Jülich. Reihe
                      Schlüsseltechnologien / Key Technologies},
      pages        = {VIII, 111 S.},
      year         = {2021},
      note         = {Biotechnologie 1; Dissertation, Heinrich-Heine-Universität
                      Düsseldorf, 2020},
      abstract     = {Facing the demand for environmental friendly and
                      sustainable production processes, microorganisms are
                      engineered for the industrial biosynthesis of chemicals,
                      fuels, or food and feed additives from renewable resources.
                      However, microbial strain development is still laborious,
                      time consuming and expensive, which constricts the
                      transition to a more bio-based economy. Therefore,
                      development and consistent improvement of molecular tools
                      for genetic engineering as well as methods for the
                      high-throughput characterization of engineered strain
                      variants are of great importance. For this purpose, the
                      CRISPR/ Cas12a recombineering method for
                      $\textit{Corynebacterium glutamicum}$, a well-characterized
                      microorganism employed in the industrial amino acid
                      production, was refined by developing the flexible and easy
                      to assemble crRNA delivery vector pJYScr. Targeting and
                      editing efficiency of this new CRISPR/ Cas12a system was
                      systematically evaluated by inserting genetic mutations
                      proximal and distal to a selected PAM site in a genomic lacZ
                      gene encoding for $\beta$-galactosidase. Subsequently, this
                      improved method allowing for accelerated genome editing of
                      $\textit{C. glutamicum}$ was applied in a strain engineering
                      campaign aiming for improved L-glutamateefflux. For this
                      purpose single-stranded DNA oligonucleotides targeting
                      critical amino acid residuesin the mechanosensitive channel
                      MscCG of $\textit{C. glutamicum}$ were used for CRISPR/
                      Cas12 recombineering. Several generated strain variants were
                      characterized with regard to theirrespective L-glutamate
                      efflux identifying new gain-of-function mutations, which
                      improve L-glutamateexport in $\textit{C. glutamicum}$. To
                      the same extent as fast and reliable genetic engineering,
                      rapid identification of producing strain variants in larger
                      libraries is a crucial step in strain development. In this
                      respect, transcription factorbased, fluorescent biosensors
                      are valuable tools in metabolic engineering allowing for
                      semiquantitative determination of metabolites in single
                      cells. However, transcriptional biosensors are often limited
                      by intrinsic characteristics of the used native regulatory
                      circuit. Moreover, signal saturation at low inducer
                      concentrations typically limits their use in producer
                      strains at advanced engineering stages, and the application
                      of biosensors in heterologous host systems is often not
                      possible. Therefore, a unified biosensor design was
                      established, which allows fine-tuning of important sensor
                      parameters and ensures a sensor response in a heterologous
                      expression host. As a key feature of the design, the
                      regulator activity can be controlled through modulation of
                      the regulator gene expression level by using different
                      (synthetic) constitutive promoters. Several biosensors based
                      on transcriptional regulators LysG and PhdR and their
                      cognate promoters from $\textit{C. glutamicum}$ were
                      constructed for applications in the native host and in
                      $\textit{Escherichia coli}$. Detailed characterization of
                      these biosensors in liquid cultures and on the single-cell
                      level using flow cytometry showed that the sensor design
                      enables customization of important biosensor parameters as
                      well as application of these sensors in two different
                      bacterial species.},
      cin          = {IBG-1},
      cid          = {I:(DE-Juel1)IBG-1-20101118},
      pnm          = {2172 - Utilization of renewable carbon and energy sources
                      and engineering of ecosystem functions (POF4-217)},
      pid          = {G:(DE-HGF)POF4-2172},
      typ          = {PUB:(DE-HGF)3 / PUB:(DE-HGF)11},
      urn          = {urn:nbn:de:0001-2021051035},
      url          = {https://juser.fz-juelich.de/record/886052},
}