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Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli

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Abstract

The major limitation in the synthetic application of two-component Baeyer–Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.

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Acknowledgments

We thank the European Research Council (ERC AdG 247014), the Swedish Research Council, the Deutsche Forschungsgemeinschaft (Grant Bo1862/6-1) and the Deutsche Bundesstiftung Umwelt (AZ13234 and AZ20013/231) for financial support.

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Authors and Affiliations

  1. Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, Svante Arrhenius Väg 16C, 106 91, Stockholm, Sweden

    Maria Kadow & J.-E. Bäckvall

  2. Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany

    Maria Kadow, Kathleen Balke & Uwe T. Bornscheuer

  3. Curnow Consultancies, Trewithen House, Ashton, Helston, TR13 9PQ, UK

    Andrew Willetts

  4. Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Strasse 4, 17487, Greifswald, Germany

    Uwe T. Bornscheuer

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  1. Maria Kadow
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  2. Kathleen Balke
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Correspondence to Uwe T. Bornscheuer.

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Kadow, M., Balke, K., Willetts, A. et al. Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli . Appl Microbiol Biotechnol 98, 3975–3986 (2014). https://doi.org/10.1007/s00253-013-5338-3

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