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@PHDTHESIS{Kbbing:801739,
      author       = {Köbbing, Sebastian},
      othercontributors = {Blank, Lars M. and Wierckx, Nick},
      title        = {{D}evelopment of synthetic biology tools for {P}seudomonas
                      putida; 1. {A}uflage},
      volume       = {23},
      school       = {RWTH Aachen University},
      type         = {Dissertation},
      address      = {Aachen},
      publisher    = {Apprimus Verlag},
      reportid     = {RWTH-2020-09053},
      isbn         = {978-3-86359-899-0},
      series       = {Applied microbiology},
      pages        = {1 Online-Ressource (XVI, 162 Seiten) : Illustrationen,
                      Diagramme},
      year         = {2020},
      note         = {Auch veröffentlicht auf dem Publikationsserver der RWTH
                      Aachen University; Dissertation, RWTH Aachen University,
                      2020},
      abstract     = {Biotechnological applications become a forward-looking
                      alternative to chemical production of fine and bulk
                      chemicals. The envisaged circular (bio)economy is aiming to
                      replace fossil resources by renewable biomass, CO2, and
                      other carbon-rich streams like plastic. Microbial hosts with
                      an expanded range of substrates are used to produce all
                      required products. Here, synthetic biology is used for the
                      tailoring of hyperproducing strains, starting with the
                      prominent chassis strain Pseudomonas putida. P. putida
                      KT2440 has a versatile metabolism and high resistance
                      towards oxidative stress. Biotechnological applications
                      become a forward-looking alternative to chemical production
                      of fine and bulk chemicals. The envisaged circular
                      (bio)economy is aiming to replace fossil resources by
                      renewable biomass, CO2, and other carbon-rich streams like
                      plastic. Microbial hosts with an expanded range of
                      substrates are used to produce all required products. Here,
                      synthetic biology is used for the tailoring of
                      hyperproducing strains, starting with the prominent chassis
                      strain Pseudomonas putida. P. putida KT2440 has a versatile
                      metabolism and high resistance towards oxidative stress.
                      This thesis aimed to increase the number of rationally
                      designed and well-characterized genetic tools for P. putida
                      KT2440 to foster synthetic biology. This thesis contributes
                      to the reliability of synthetic biology that often proved as
                      a major issue. We constructed and characterized several
                      sigma-70 factor dependent synthetic promoters and
                      combinations for heterologous gene expression. A library was
                      constructed with single nucleotide exchanges, which reveals
                      that the -35 and -10 regions are crucial for efficient
                      promoter activity. Combined promoters, so-called stacked
                      promoters, allowed, after detailed characterization of the
                      genetic context, prediction of the resulting expression
                      strength. Stable genomic integration is often used for
                      metabolic engineering, but characterized sites, so-called
                      landing pads, across the genome of P. putida KT2440 are
                      missing. We analyzed RNA-Seq data towards regions that are
                      equally expressed to identify suitable integration sites
                      across the genome of P. putida KT2440. These landing pads
                      enabled high heterologous expression with low variability.
                      With well-characterized promoters and landing pads, fine
                      tuning of gene expression can be conducted on two levels,
                      promoter and integration site. Additionally, the thesis
                      delivers tools for marker recycling and an alternative sigma
                      dependent promoter. In summary, this thesis contributes to
                      P. putida KT2440 synthetic biology. The increased number of
                      well-characterized tools will further support the many
                      efforts to establish this microbe as a workhorse in the
                      bioeconomy and biotechnological applications. Getting the
                      tough challenges, like the use of aromatic compounds from
                      plastic or lignin as substrates, will distinguish P. putida
                      KT2440 from the well-established microbes.},
      cin          = {161710 / 160000},
      ddc          = {570},
      cid          = {$I:(DE-82)161710_20140620$ / $I:(DE-82)160000_20140620$},
      pnm          = {P4SB - P4SB - From Plastic waste to Plastic value using
                      Pseudomonas putida Synthetic Biology (633962)},
      pid          = {G:(EU-Grant)633962},
      typ          = {PUB:(DE-HGF)11 / PUB:(DE-HGF)3},
      doi          = {10.18154/RWTH-2020-09053},
      url          = {https://publications.rwth-aachen.de/record/801739},
}