title: A simplified process design for P450 driven hydroxylation based on surface displayed enzymes.
abstract: New production routes for fine and bulk chemicals are important to establish further sustainable processes in industry. Besides the identification of new biocatalysts and new production routes the optimization of existing processes in regard to an improved utilization of the catalysts are needed. In this paper we describe the successful expression of P450BM3 on the surface of E. coli cells with the Autodisplay system. The successful hydroxylation of palmitic acid by using surface-displayed P450BM3 was shown. Besides optimization of surface protein expression, several cofactor regeneration systems were compared and evaluated. Afterwards, the development of a suitable process for the biocatalytic hydroxylation of fatty acids based on the re-use of the catalysts after a simple centrifugation was investigated. It was shown that the catalyst can be used for several times without any loss in activity. By using surface-displayed P450s in combination with an enzymatic cofactor regeneration system a total turnover number of up to 54,700 could be reached, to the knowledge of the authors the highest value reported for a P450 monooxygenase to date. Further optimizations of the described reaction system can have an enormous impact on the process design for more sustainable bioprocesses. Biotechnol. Bioeng. 2016;113: 1225-1233. © 2015 Wiley Periodicals, Inc.
authors.affiliation: DECHEMA-Forschungsinstitut, Biochemical Engineering, Frankfurt am Main, Hessen, 60486, Germany.
authors.forename: Frank W
authors.initials: FW
authors.lastname: Ströhle
authors.affiliation: Autodisplay Biotech GmbH, Düsseldorf, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Kranen
authors.affiliation: DECHEMA-Forschungsinstitut, Biochemical Engineering, Frankfurt am Main, Hessen, 60486, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Schrader
authors.affiliation: Autodisplay Biotech GmbH, Düsseldorf, Germany.
authors.forename: Ruth
authors.initials: R
authors.lastname: Maas
authors.affiliation: DECHEMA-Forschungsinstitut, Biochemical Engineering, Frankfurt am Main, Hessen, 60486, Germany. holtmann@dechema.de.
authors.forename: Dirk
authors.initials: D
authors.lastname: Holtmann
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D006868:Hydrolysis; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases
keywords: P450 monooxygenases; cofactor regeneration; process design; surface display; total turnover numbers