title: Directed evolution of P450cin for mediated electron transfer.
abstract: Directed evolution is a powerful method to optimize enzyme properties for application demands. Interesting targets are P450 monooxygenases which catalyze the stereo- and regiospecific hydroxylation of chemically inert C-H bonds. Synthesis employing P450s under cell-free reaction conditions is limited by low total turnover numbers, enzyme instability, low product yields and the requirement of the expensive co-factor NADPH. Bioelectrocatalysis is an alternative to replace NADPH in cell-free P450-catalyzed reactions. However, natural enzymes are often not suitable for using non-natural electron delivery systems. Here we report the directed evolution of a previously engineered P450 CinA-10aa-CinC fusion protein (named P450cin-ADD-CinC) to use zinc/cobalt(III)sepulchrate as electron delivery system for an increased hydroxylation activity of 1,8-cineole. Two rounds of Sequence Saturation Mutagenesis (SeSaM) each followed by one round of multiple site-saturation mutagenesis of the P450 CinA-10aa-CinC fusion protein generated a variant (Gln385His, Val386Ser, Thr77Asn, Leu88Arg; named KB8) with a 3.8-fold increase in catalytic efficiency (28 µM-1min-1) compared to P450cin-ADD-CinC (7 µM-1min-1). Furthermore, variant KB8 exhibited a 1.5-fold higher product formation (500 µM µM-1P450) compared to the equimolar mixture of CinA, CinC and Fpr using NADPH as co-factor (315 µM µM-1P450). In addition, electrochemical experiments with the electron delivery system platinum/cobalt(III)sepulchrate showed that the KB8 variant had a 4-fold higher product formation rate (0.16 nmol (nmol) P450-1min-1cm-2) than the P450cin-ADD-CinC (0.04 nmol (nmol) P450-1min-1cm-2). In summary, the current work shows prospects of using directed evolution to generate P450 enzymes suitable for use with alternative electron delivery systems.
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Ketaki D
authors.initials: KD
authors.lastname: Belsare
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Horn
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Anna Joëlle
authors.initials: AJ
authors.lastname: Ruff
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Ronny
authors.initials: R
authors.lastname: Martinez
authors.affiliation: Biochemical Engineering Group, DECHEMA Research Institute, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, Germany.
authors.forename: Anders
authors.initials: A
authors.lastname: Magnusson
authors.affiliation: Biochemical Engineering Group, DECHEMA Research Institute, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Holtmann
authors.affiliation: Biochemical Engineering Group, DECHEMA Research Institute, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Schrader
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany u.schwaneberg@biotec.rwth-aachen.de.|DWI-Leibniz-Institut für Interaktive Materialien e. V., Forckenbeckstraße 50, 52074 Aachen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D055162:Biocatalysis; D003511:Cyclohexanols / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D004563:Electrochemistry; D004579:Electron Transport; D000077591:Eucalyptol; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D039821:Monoterpenes / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D003511:Cyclohexanols; D039821:Monoterpenes; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; C465212:cytochrome P-450 176A; D000077591:Eucalyptol
keywords: P450cin; bioelectrocatalysis; directed Evolution; electron mediator; protein engineering