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@PHDTHESIS{Unthan:256301,
      author       = {Unthan, Simon},
      title        = {{R}obot-{A}ssisted {P}henotyping of {G}enome-{R}educed
                      ${C}orynebacterium$ $glutamicum$ {S}train {L}ibraries to
                      {D}raft a {C}hassis {O}rganism},
      volume       = {132},
      school       = {RWTH Aachen},
      type         = {Dr.},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
      reportid     = {FZJ-2015-06265},
      isbn         = {978-3-95806-169-9},
      series       = {Schriften des Forschungszentrums Jülich. Reihe
                      Schlüsseltechnologien / Key Technologies},
      pages        = {122 S.},
      year         = {2016},
      note         = {RWTH Aachen, Diss., 2015},
      abstract     = {In this work, concepts were developed and applied to guide
                      the construction of a $\textit{Corynebacterium glutamicum}$
                      chassis organism for synthetic biology approaches. The aim
                      was to delete irrelevant genes from the wild type strain in
                      order to obtain a chassis growing on defined CGXII medium
                      with D-glucose with unaltered biological fitness, which was
                      defined by the maximum specific growth rate and biomass
                      yield. Initially, workflows were developed on a robotic Mini
                      Pilot Plant (MPP), for example, to harvest cell-free
                      cultivation supernatants from BioLector cultivations in
                      response to individually defined triggers. Subsequently,
                      assays for amino acids and D-glucose were established in
                      384-well plate scale in order to quantify these metabolites
                      in cell-free culture supernatants in fully automated
                      workflows [1]. During initial reference experiments,
                      protocatechuic acid was identified as a hidden co-substrate
                      in the well-known defined CGXII medium. The additional TCA
                      feed via acetyl-CoA and succinyl-CoA, which are derived from
                      protocatechuic acid, elevates the growth rate by about 50 \%
                      in highly diluted cultures [2]. The first step toward a
                      chassis was the deletion of prophage elements contributing
                      to about 6.7 \% of the $\textit{C. glutamicum}$ genome. The
                      respective strain MB001 showed unaltered biological fitness
                      and an increased heterologous protein expression, caused by
                      the removal of a restriction-modification system in prophage
                      CGP3 [3]. As a next step, 36 strains with deletion of
                      non-essential gene clusters were tested thoroughly and 26
                      clusters were found irrelevant for the biological fitness of
                      $\textit{C. glutamicum}$ and offered the potential to reduce
                      the genome by about 22 \% [4]. Some clusters were also
                      deleted in the L-lysine model producer DM1933 and the
                      derived strain GRLP45 showed an 51 \% increased L-lysine
                      titer applying the automated MPP methods, what was finally
                      confirmed in lab-scale bioreactors [1]. During the final
                      combinatorial deletion of irrelevant gene clusters, some
                      interdependencies were observed resulting in a decreased of
                      biological fitness of the respective strains. One of those
                      strains was characterized in-depth and revealed the general
                      interplay of ribosome capacity and maximum growth rate of
                      $\textit{C. glutamicum}$. In the end, two pre-chassis,
                      namely W127 and W121, were obtained that displayed a total
                      genome reduction of 8.8 \% and 12.8 \%, respectively. Both
                      strains fulfilled the target criteria of unaltered
                      biological fitness on defined CGXII medium in BioLector
                      cultivations. Finally, the in-depth analysis of both
                      pre-chassis in bioreactors revealed a morphological
                      divergence of W121 which could be narrowed down to a single
                      cluster deletion. However, W127 did not show any drawback
                      compared to the wild type when tested under stress
                      conditions and on different cultivation scales. In fact,
                      this strain even grew faster on some C-sources, making it a
                      good basis for synthetic biology approaches.},
      cin          = {IBG-1},
      cid          = {I:(DE-Juel1)IBG-1-20101118},
      pnm          = {89581 - Biotechnology (POF2-89581)},
      pid          = {G:(DE-HGF)POF2-89581},
      typ          = {PUB:(DE-HGF)3 / PUB:(DE-HGF)11},
      urn          = {urn:nbn:de:0001-2017040716},
      url          = {https://juser.fz-juelich.de/record/256301},
}