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@ARTICLE{Fricke:897397,
      author       = {Fricke, Philipp Moritz and Lürkens, Martha and Hünnefeld,
                      Max and Sonntag, Christiane K. and Bott, Michael and Davari,
                      Mehdi D. and Polen, Tino},
      title        = {{H}ighly tunable {T}et{R}-dependent target gene expression
                      in the acetic acid bacterium {G}luconobacter oxydans},
      journal      = {Applied microbiology and biotechnology},
      volume       = {105},
      number       = {18},
      issn         = {1432-0614},
      address      = {New York},
      publisher    = {Springer},
      reportid     = {FZJ-2021-03763},
      pages        = {6835 - 6852},
      year         = {2021},
      note         = {Biotechnologie 1},
      abstract     = {For the acetic acid bacterium (AAB) Gluconobacter oxydans
                      only recently the first tight system for regulatable target
                      gene expression became available based on the heterologous
                      repressor-activator protein AraC from Escherichia coli and
                      the target promoter ParaBAD. In this study, we tested pure
                      repressor-based TetR- and LacI-dependent target gene
                      expression in G. oxydans by applying the same plasmid
                      backbone and construction principles that we have used
                      successfully for the araC-ParaBAD system. When using a
                      pBBR1MCS-5-based plasmid, the non-induced basal expression
                      of the Tn10-based TetR-dependent expression system was
                      extremely low. This allowed calculated induction ratios of
                      up to more than 3500-fold with the fluorescence reporter
                      protein mNeonGreen (mNG). The induction was highly
                      homogeneous and tunable by varying the anhydrotetracycline
                      concentration from 10 to 200 ng/mL. The already strong
                      reporter gene expression could be doubled by inserting the
                      ribosome binding site AGGAGA into the 3’ region of the
                      Ptet sequence upstream from mNG. Alternative plasmid
                      constructs used as controls revealed a strong influence of
                      transcription terminators and antibiotics resistance gene of
                      the plasmid backbone on the resulting expression
                      performance. In contrast to the TetR-Ptet-system,
                      pBBR1MCS-5-based LacI-dependent expression from PlacUV5
                      always exhibited some non-induced basal reporter expression
                      and was therefore tunable only up to 40-fold induction by
                      IPTG. The leakiness of PlacUV5 when not induced was
                      independent of potential read-through from the lacI
                      promoter. Protein-DNA binding simulations for pH 7, 6, 5,
                      and 4 by computational modeling of LacI, TetR, and AraC with
                      DNA suggested a decreased DNA binding of LacI when pH is
                      below 6, the latter possibly causing the leakiness of
                      LacI-dependent systems hitherto tested in AAB. In summary,
                      the expression performance of the pBBR1MCS-5-based TetR-Ptet
                      system makes this system highly suitable for applications in
                      G. oxydans and possibly in other AAB.},
      cin          = {IBG-1},
      ddc          = {570},
      cid          = {I:(DE-Juel1)IBG-1-20101118},
      pnm          = {2171 - Biological and environmental resources for
                      sustainable use (POF4-217)},
      pid          = {G:(DE-HGF)POF4-2171},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {34448898},
      UT           = {WOS:000690366300002},
      doi          = {10.1007/s00253-021-11473-x},
      url          = {https://juser.fz-juelich.de/record/897397},
}