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Extending the substrate scope of a Baeyer-Villiger monooxygenase by multiple-site mutagenesis

Dudek, H. M., Fink, M. J., Shivange, A. V., Dennig, A., Mihovilovic, M. D., Schwaneberg, U. & Fraaije, M. W., May-2014, In : Applied Microbiology and Biotechnology. 98, 9, p. 4009-4020 12 p.

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  • Extending the substrate scope of a Baeyer-Villiger monooxygenase by

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DOI

  • Hanna M Dudek
  • Michael J Fink
  • Amol V Shivange
  • Alexander Dennig
  • Marko D Mihovilovic
  • Ulrich Schwaneberg
  • Marco W Fraaije

Baeyer-Villiger monooxygenase-catalysed reactions are attractive for industrial processes. Here we report on expanding the substrate scope of phenylacetone monooxygenase (PAMO). In order to introduce activity on alicyclic ketones in PAMO, we generated and screened a library of 1,500 mutants. Based on recently published structures of PAMO and its mutants, we selected previously uncharacterised positions as well as known hot-spots to be targeted by focused mutagenesis. We were able to mutate 11 positions in a single step by using the OmniChange method for the mutant library generation. Screening of the library using a phosphate-based activity detection method allowed identification of a quadruple mutant (P253F/G254A/R258M/L443F) active on cyclopentanone. The substrate scope of this mutant is extended to several aliphatic ketones while activity on aromatic compounds typical for PAMO was preserved. Moreover, the mutant is as thermostable as PAMO. Our results demonstrate the power of screening structure-inspired, focused mutant libraries for creating Baeyer-Villiger monooxygenases with new specificities.

Original languageEnglish
Pages (from-to)4009-4020
Number of pages12
JournalApplied Microbiology and Biotechnology
Volume98
Issue number9
Publication statusPublished - May-2014

    Keywords

  • Baeyer-Villiger monooxygenase, Oxygenation, OmniChange mutagenesis, Protein engineering, Biocatalysis, RNA DIFFERENTIAL DISPLAY, CYCLOHEXANONE MONOOXYGENASE, PHENYLACETONE-MONOOXYGENASE, SIMULTANEOUS IDENTIFICATION, DIRECTED EVOLUTION, THERMOBIFIDA-FUSCA, CLONING, SEQUENCE, GENE, BIOCATALYSTS

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