h1

% IMPORTANT: The following is UTF-8 encoded.  This means that in the presence
% of non-ASCII characters, it will not work with BibTeX 0.99 or older.
% Instead, you should use an up-to-date BibTeX implementation like “bibtex8” or
% “biber”.

@PHDTHESIS{Unthan:670366,
      author       = {Unthan, Simon},
      othercontributors = {Wiechert, Wolfgang and Büchs, Jochen},
      title        = {{R}obot-assisted phenotyping of genome-reduced
                      {C}orynebacterium glutamicum strain libraries to draft a
                      chassis organism},
      volume       = {132},
      school       = {RWTH Aachen University},
      type         = {Dissertation},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH, Zentralbibliothek},
      reportid     = {RWTH-2016-07483},
      series       = {Schriften des Forschungszentrums Jülich. Reihe
                      Schlüsseltechnologien / Key Technologies},
      pages        = {1 Online-Ressource (122 Seiten) : Illustrationen,
                      Diagramme},
      year         = {2016},
      note         = {Druckausgabe: 2016. - Onlineausgabe: 2016. - Auch
                      veröffentlicht auf dem Publikationsserver der RWTH Aachen
                      University; Dissertation, RWTH Aachen University, 2015},
      abstract     = {In this work, concepts were developed and applied to guide
                      the construction of a 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 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
                      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 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          = {420410},
      ddc          = {620},
      cid          = {$I:(DE-82)420410_20140620$},
      typ          = {PUB:(DE-HGF)11 / PUB:(DE-HGF)3},
      urn          = {urn:nbn:de:hbz:82-rwth-2016-074839},
      url          = {https://publications.rwth-aachen.de/record/670366},
}