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@ARTICLE{Mller:908892,
      author       = {Müller, Carolin and Bakkes, Patrick J. and Lenz, Patrick
                      and Waffenschmidt, Vera and Helleckes, Laura M. and Jaeger,
                      Karl-Erich and Wiechert, Wolfgang and Knapp, Andreas and
                      Freudl, Roland and Oldiges, Marco},
      title        = {{A}ccelerated strain construction and characterization of
                      {C}. glutamicum protein secretion by laboratory automation},
      journal      = {Applied microbiology and biotechnology},
      volume       = {106},
      number       = {12},
      issn         = {0171-1741},
      address      = {New York},
      publisher    = {Springer},
      reportid     = {FZJ-2022-02899},
      pages        = {4481 - 4497},
      year         = {2022},
      abstract     = {Secretion of bacterial proteins into the culture medium
                      simplifies downstream processing by avoiding cell disruption
                      for target protein purification. However, a suitable signal
                      peptide for efficient secretion needs to be identified, and
                      currently, there are no tools available to predict optimal
                      combinations of signal peptides and target proteins. The
                      selection of such a combination is influenced by several
                      factors, including protein biosynthesis efficiency and
                      cultivation conditions, which both can have a significant
                      impact on secretion performance. As a result, a large number
                      of combinations must be tested. Therefore, we have developed
                      automated workflows allowing for targeted strain
                      construction and secretion screening using two platforms.
                      Key advantages of this experimental setup include lowered
                      hands-on time and increased throughput. In this study, the
                      automated workflows were established for the heterologous
                      production of Fusarium solani f. sp. pisi cutinase in
                      Corynebacterium glutamicum. The target protein was monitored
                      in culture supernatants via enzymatic activity and split GFP
                      assay. Varying spacer lengths between the Shine-Dalgarno
                      sequence and the start codon of Bacillus subtilis signal
                      peptides were tested. Consistent with previous work on the
                      secretory cutinase production in B. subtilis, a ribosome
                      binding site with extended spacer length to up to 12 nt,
                      which likely slows down translation initiation, does not
                      necessarily lead to poorer cutinase secretion by C.
                      glutamicum. The best performing signal peptides for cutinase
                      secretion with a standard spacer length were identified in a
                      signal peptide screening. Additional insights into the
                      secretion process were gained by monitoring secretion stress
                      using the C. glutamicum K9 biosensor strain.},
      cin          = {IBG-1 / IMET},
      ddc          = {570},
      cid          = {I:(DE-Juel1)IBG-1-20101118 / I:(DE-Juel1)IMET-20090612},
      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)16},
      pubmed       = {35759036},
      UT           = {WOS:000817028300003},
      doi          = {10.1007/s00253-022-12017-7},
      url          = {https://juser.fz-juelich.de/record/908892},
}