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@ARTICLE{Bakkes:902122,
      author       = {Bakkes, Patrick J. and Lenz, Patrick and Müller, Carolin
                      and Bida, Astrid and Dohmen-Olma, Doris and Knapp, Andreas
                      and Oldiges, Marco and Jaeger, Karl-Erich and Freudl,
                      Roland},
      title        = {{B}iosensor-{B}ased {O}ptimization of {C}utinase
                      {S}ecretion by {C}orynebacterium glutamicum},
      journal      = {Frontiers in microbiology},
      volume       = {12},
      issn         = {1664-302X},
      address      = {Lausanne},
      publisher    = {Frontiers Media},
      reportid     = {FZJ-2021-04052},
      pages        = {750150},
      year         = {2021},
      abstract     = {The industrial microbe Corynebacterium glutamicum is
                      gaining substantial importance as a platform host for
                      recombinant protein secretion. We recently developed a
                      fluorescence-based (eYFP) C. glutamicum reporter strain for
                      the quantification of Sec-dependent protein secretion by
                      monitoring the secretion-related stress response and now
                      demonstrate its applicability in optimizing the secretion of
                      the heterologous enzyme cutinase from Fusarium solani pisi.
                      To drive secretion, either the poor-performing PelSP or the
                      potent NprESP Sec signal peptide from Bacillus subtilis was
                      used. To enable easy detection and quantification of the
                      secreted cutinase we implemented the split green fluorescent
                      protein (GFP) assay, which relies on the GFP11-tag fused to
                      the C-terminus of the cutinase, which can complement a
                      truncated GFP thereby reconstituting its fluorescence. The
                      reporter strain was transformed with different mutant
                      libraries created by error-prone PCR, which covered the
                      region of the signal peptide and the N-terminus of the
                      cutinase. Fluorescence-activated cell sorting (FACS) was
                      performed to isolate cells that show increased fluorescence
                      in response to increased protein secretion stress. Five
                      PelSP variants were identified that showed a 4- to 6-fold
                      increase in the amount and activity of the secreted cutinase
                      (up to 4,100 U/L), whereas two improved NprESP variants were
                      identified that showed a $∼35\%$ increase in secretion,
                      achieving ∼5,500 U/L. Most of the isolated variants
                      carried mutations in the h-region of the signal peptide that
                      increased its overall hydrophobicity. Using site-directed
                      mutagenesis it was shown that the combined mutations F11I
                      and P16S within the hydrophobic core of the PelSP are
                      sufficient to boost cutinase secretion in batch cultivations
                      to the same level as achieved by the NprESP. Screening of a
                      PelSP mutant library in addition resulted in the
                      identification of a cutinase variant with an increased
                      specific activity, which was attributed to the mutation A85V
                      located within the substrate-binding region. Taken together
                      the biosensor-based optimization approach resulted in a
                      substantial improvement of cutinase secretion by C.
                      glutamicum, and therefore represents a valuable tool that
                      can be applied to any secretory protein of interest.},
      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       = {34777299},
      UT           = {WOS:000717705900001},
      doi          = {10.3389/fmicb.2021.750150},
      url          = {https://juser.fz-juelich.de/record/902122},
}