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@ARTICLE{Skoczinski:841984,
      author       = {Skoczinski, Pia and Volkenborn, Kristina and Fulton,
                      Alexander and Bhadauriya, Anuseema and Nutschel, Christina
                      and Gohlke, Holger and Knapp, Andreas and Jaeger,
                      Karl-Erich},
      title        = {{C}ontribution of single amino acid and codon substitutions
                      to the production and secretion of a lipase by {B}acillus
                      subtilis},
      journal      = {Microbial cell factories},
      volume       = {16},
      number       = {1},
      issn         = {1475-2859},
      address      = {London},
      publisher    = {Biomed Central},
      reportid     = {FZJ-2018-00271},
      pages        = {160},
      year         = {2017},
      abstract     = {Background: Bacillus subtilis produces and secretes
                      proteins in amounts of up to 20 g/l under optimal
                      conditions. However, protein production can be challenging
                      if transcription and cotranslational secretion are
                      negatively affected, or the target protein is degraded by
                      extracellular proteases. This study aims at elucidating the
                      influence of a target protein on its own production by a
                      systematic mutational analysis of the homologous B. subtilis
                      model protein lipase A (LipA). We have covered the full
                      natural diversity of single amino acid substitutions at 155
                      positions of LipA by site saturation mutagenesis excluding
                      only highly conserved residues and qualitatively and
                      quantitatively screened about 30,000 clones for
                      extracellular LipA production. Identified variants with
                      beneficial effects on production were sequenced and analyzed
                      regarding B. subtilis growth behavior, extracellular lipase
                      activity and amount as well as changes in lipase transcript
                      levels. Results: In total, 26 LipA variants were identified
                      showing an up to twofold increase in either amount or
                      activity of extracellular lipase. These variants harbor
                      single amino acid or codon substitutions that did not
                      substantially affect B. subtilis growth. Subsequent
                      exemplary combination of beneficial single amino acid
                      substitutions revealed an additive effect solely at the
                      level of extracellular lipase amount; however, lipase amount
                      and activity could not be increased simultaneously.
                      Conclusions: Single amino acid and codon substitutions can
                      affect LipA secretion and production by B. subtilis. Several
                      codon-related effects were observed that either enhance lipA
                      transcription or promote a more efficient folding of LipA.
                      Single amino acid substitutions could improve LipA
                      production by increasing its secretion or stability in the
                      culture supernatant. Our findings indicate that optimization
                      of the expression system is not sufficient for efficient
                      protein production in B. subtilis. The sequence of the
                      target protein should also be considered as an optimization
                      target for successful protein production. Our results
                      further suggest that variants with improved properties might
                      be identified much faster and easier if mutagenesis is
                      prioritized towards elements that contribute to enzymatic
                      activity or structural integrity.},
      cin          = {JSC / NIC / ICS-6 / IBG-1 / IMET},
      ddc          = {610},
      cid          = {I:(DE-Juel1)JSC-20090406 / I:(DE-Juel1)NIC-20090406 /
                      I:(DE-Juel1)ICS-6-20110106 / I:(DE-Juel1)IBG-1-20101118 /
                      I:(DE-Juel1)IMET-20090612},
      pnm          = {581 - Biotechnology (POF3-581) / 574 - Theory, modelling
                      and simulation (POF3-574) / 511 - Computational Science and
                      Mathematical Methods (POF3-511) / BioSC - Bioeconomy Science
                      Center (BioSC)},
      pid          = {G:(DE-HGF)POF3-581 / G:(DE-HGF)POF3-574 /
                      G:(DE-HGF)POF3-511 / G:(DE-Juel1)BioSC},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:28946879},
      UT           = {WOS:000411760300001},
      doi          = {10.1186/s12934-017-0772-z},
      url          = {https://juser.fz-juelich.de/record/841984},
}