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@PHDTHESIS{Frster:771961,
      author       = {Förster, Jan},
      othercontributors = {Blank, Lars M. and Oldiges, Marco},
      title        = {{I}nvestigating the metabolism of {P}ichia pastoris for
                      improved recombinant protein production; 1. {A}uflage},
      volume       = {15},
      school       = {RWTH Aachen University},
      type         = {Dissertation},
      address      = {Aachen},
      publisher    = {Apprimus Verlag},
      reportid     = {RWTH-2019-10507},
      series       = {Applied microbiology},
      pages        = {1 Online-Ressource (XIII, 172 Seiten) : Illustrationen,
                      Diagramme},
      year         = {2019},
      note         = {Auch veröffentlicht auf dem Publikationsserver der RWTH
                      Aachen University; Dissertation, RWTH Aachen University,
                      2019},
      abstract     = {With the ever-increasing demand for proteins, the
                      production host, including its metabolism, moves into focus.
                      Applications as different as additives in washing detergents
                      and biopharmaceuticals demand on the one hand low production
                      prices, and on the other hand high reproducibility and
                      comparability of post-translational modifications,
                      respectively. Next to bacteria and mammalian cells, yeasts
                      are well established. One such yeast is Pichia pastoris,
                      increasingly used in industry for recombinant protein
                      production. In this thesis, contributions for increasing the
                      knowledge space of P. pastoris, as well as tools for the
                      protein production pipeline were in focus. As stated, for
                      optimization, one requires knowledge, here, detailed
                      knowledge on the amino acid biosynthesis network.
                      Specifically, the amino acid leucine was chosen as prime
                      example, as it is a multi-enzyme compartmented pathway, also
                      encoded by duplicate genes in baker´s yeast. However, no
                      information in any other hemiascomycetes was available. A
                      combination of bioinformatics analysis and protein
                      localization studies highlighted that baker´s yeast cannot
                      be used as a general blueprint for every yeast amino acid
                      pathway. Finally, the structure of the leucine biosynthesis
                      pathway in P. pastoris could be determined. With this
                      information in hand, the idea of amino acid availability
                      increase by deregulation of synthesis pathways was
                      exemplified using leucine. Feedback inhibition, prominent in
                      many amino acid pathways, was deleted through mutagenesis of
                      the alpha-isopropylmalate synthase gene (LEU4) by the
                      non-metabolizable leucine analogon trifluoroleucine. Stable
                      isotope experiments with 13C-labelled glucose enabled the
                      measurement of de novo synthesized amino acids. Indeed, the
                      mutagenized leucine pathway resulted in increased leucine
                      biosynthesis. This approach enables the construction of
                      strains that are tailored towards the overproduction of
                      defined amino acids and can then be used for the production
                      of recombinant proteins with a bias towards that amino acid.
                      With P. pastoris, the challenge is to find the hyper protein
                      producer after transformation. Here, the yeast was modified
                      to become magnetic. A knockout of the vacuolar transporter
                      protein Ccc1p leads to accumulation of iron if ferric
                      citrate is present. While it was possible to move single
                      yeast, the magnetic attraction was low, resulting in no
                      shorter separation times as 6 h. An improved design is
                      discussed. For many applications glycosylation is key.
                      However, the metabolic impact on glycosylation is rarely
                      quantified. Here, special attention was given to the
                      glycosylation chain length in regard to the glycosylation
                      site number. The more glycosylation sites were present on
                      the protein variant, the shorter the chains got. The results
                      indicate a limitation in cytosolic GDP-mannose availability
                      and glycosyl transferase capacity. The results indicate that
                      metabolism not only impacts the rate of protein synthesis,
                      but also the quality of the protein of choice. Here, both
                      aspects were investigated in detail, contributing to the
                      ever increasing knowledge base of P. pastoris.},
      cin          = {161710 / 160000},
      ddc          = {570},
      cid          = {$I:(DE-82)161710_20140620$ / $I:(DE-82)160000_20140620$},
      typ          = {PUB:(DE-HGF)11 / PUB:(DE-HGF)3},
      doi          = {10.18154/RWTH-2019-10507},
      url          = {https://publications.rwth-aachen.de/record/771961},
}