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@PHDTHESIS{Kortmann:888102,
      author       = {Kortmann, Maike},
      title        = {{S}train and tool development for the production of
                      industrially relevant compounds with
                      $\textit{{C}orynebacterium glutamicum}$},
      volume       = {227},
      school       = {Heinrich-Heine-Universität Düsseldorf},
      type         = {Dissertation},
      publisher    = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
      reportid     = {FZJ-2020-04679},
      isbn         = {978-3-95806-522-2},
      series       = {Schriften des Forschungszentrums Jülich. Reihe
                      Schlüsseltechnologien / Key Technologies},
      pages        = {II, 138 S.},
      year         = {2021},
      note         = {Biotechnologie 1; Dissertation, Heinrich-Heine-Universität
                      Düsseldorf, 2020},
      abstract     = {$\textit{Corynebacterium glutamicum}$ is one of the most
                      important model organisms in white biotechnology for the
                      industrial production of numerous amino acids, most notably
                      L-glutamate and L-lysine, but also of other metabolites and
                      proteins. In order to further expand possible applications
                      of this organism for the conversion of renewable feedstocks
                      into industrially relevant compounds, two different
                      approaches of strain engineering of $\textit{C. glutamicum}$
                      were addressed in the present thesis. In the first part of
                      this work, a new expression system based on a chromosomally
                      encoded T7 RNA polymerase and a plasmid-based T7 promoter
                      was established and characterized in $\textit{C.
                      glutamicum}$. For this purpose, the T7 RNA polymerase (gene
                      1) was integrated into the prophage-free strain $\textit{C.
                      glutamicum}$ MB001 under the control of the IPTG-inducible
                      lacUV5 promoter, resulting in strain MB001(DE3). In
                      addition, the expression plasmids pMKEx1 and pMKEx2 were
                      constructed into which a target gen can be cloned under
                      control of the T7 promoter and, if necessary, can be fused
                      to a polyhistidine-tag or a Strep-tag. The T7 expression
                      system was evaluated with the reporter gene $\textit{eyfp}$
                      and compared to the well-established pEKEx2 system, which is
                      based on the $\textit{tac}$ promotor. The basal expression
                      of the T7 system was lower than that of the pEKEx2 system.
                      The specific fluorescence of eYFP increased 450-fold after
                      maximal induction of the T7 system with 250 μM IPTG and was
                      3.5-fold higher than the specific eYFP fluorescence obtained
                      after maximal $\textit{eyfp}$ expression with the pEKEx2
                      system. Furthermore, it was shown that proteins such as
                      pyruvate kinase or secretory GFP proteins can also be
                      successfully produced in higher amounts with the T7 system
                      than with the pEKEx2 system. In the second part of this
                      work, a genetically encoded biosensor for the cytoplasmic
                      lysine concentration was used to screen for mutated variants
                      of pyruvate carboxylase (PCx), which enabled an improved
                      Llysine production. PCx catalyzes the ATP-dependent
                      carboxylation from pyruvate to oxaloacetate and plays an
                      important role as anaplerotic enzyme. It replenishes the
                      tricarboxylic acid cycle during growth on sugars when
                      intermediates have been removed from the cycle by branching
                      metabolic pathways. This reaction is for example of great
                      importance for L-lysine production and it has already been
                      shown that overexpression of the $\textit{pyc}$ gene as well
                      as the mutation P458S in PCx lead to an increased L-lysine
                      yield in $\textit{C. glutamicum}$. In order to find further
                      advantageous mutations for lysine production, a plasmidbased
                      library with mutated $\textit{pyc}$ variants was generated
                      in this work using error-prone PCR. Out of this library, two
                      PCx variants could be isolated with the help of the lysine
                      biosensor pSenLys-Spec and FACS-based high-throughput
                      screening, which showed an increased lysine formation
                      compared to the strain with wild-type PCx. The variants
                      PCx$^{T343A, I1012S}$ and PCx$^{T132A}$ enabled an increase
                      of the lysine titer by 9\% and 19\%, respectively, after
                      plasmid-based overexpression of the respective genes in the
                      strain DM1868Δ$\textit{pyc}$/pSenLys-Spec. When the
                      mutations were introduced one by one into the genome of the
                      lysine-producing strain DM1868, the variant PCx$^{T132A}$
                      produced 7\% more lysine and PCx$^{T343A}$ 15\% more lysine
                      compared to strain DM1868 encoding wild-type PCx. In
                      previous studies, PCx activity of $\textit{C. glutamicum}$
                      could only be measured with permeabilized cells. For a more
                      detailed characterization, conditions were established that
                      enabled the measurement of PCx activity in cell-free
                      extracts and the isolation of the enzyme in active form. For
                      purified PCx, K$_{m}$ values of 3.8 mM for pyruvate, 0.6 mM
                      for ATP, and 13.3 mM for bicarbonate were determined. ADP
                      and aspartate inhibited PCx activity with $\textit{K}_{i}$
                      values of 1.5 mM and 9.3 mM, respectively. Initial
                      characterization of the variants PCx$^{T132A}$ and
                      PCx$^{T343A}$ revealed that their activity was not inhibited
                      up to aspartate concentrations of approx. 7.5 mM. The
                      $\textit{K}_{i}$ values of 13.2 mM for PCx$^{T132A}$ and
                      10.8 mM for PCx$^{T343A}$ were higher than for wild-type PCx
                      and provide an explanation for the increased lysine
                      production obtained with these variants in $\textit{C.
                      glutamicum}$.},
      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:0001-2021051022},
      url          = {https://juser.fz-juelich.de/record/888102},
}