title: Engineering the CYP101 system for in vivo oxidation of unnatural substrates.
abstract: The protein engineering of CYP enzymes for structure-activity studies and the oxidation of unnatural substrates for biotechnological applications will be greatly facilitated by the availability of functional, whole-cell systems for substrate oxidation. We report the construction of a tricistronic plasmid that expresses the CYP101 monooxygenase from Pseudomonas putida, and its physiological electron transfer co-factor proteins putidaredoxin reductase and putidaredoxin in Escherichia coli, giving a functional in vivo catalytic system. Wild-type CYP101 expressed in this system efficiently transforms camphor to 5-exo-hydroxycamphor without further oxidation to 5-oxo-camphor until >95% of camphor has been consumed. CYP101 mutants with increased activity for the oxidation of diphenylmethane (the Y96F-I395G mutant), styrene and ethylbenzene (the Y96F-V247L mutant) have been engineered. In particular, the Y96F-V247L mutant shows coupling efficiency of approximately 60% for styrene and ethylbenzene oxidation, with substrate oxidation rates of approximately 100/min. Escherichia coli cells transformed with tricistronic plasmids expressing these mutants readily gave 100-mg quantities of 4-hydroxydiphenylmethane and 1-phenylethanol in 24-72 h. This new in vivo system can be used for preparative scale reactions for product characterization, and will greatly facilitate directed evolution of the CYP101 enzyme for enhanced activity and selectivity of substrate oxidation.
authors.affiliation: Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, UK.
authors.forename: S G
authors.initials: SG
authors.lastname: Bell
authors.forename: C F
authors.initials: CF
authors.lastname: Harford-Cross
authors.forename: L L
authors.initials: LL
authors.lastname: Wong
mesh_terms: D000473:Alkanes / Q000378:metabolism; D001555:Benzene Derivatives / Q000378:metabolism; D001559:Benzhydryl Compounds / Q000378:metabolism; D002164:Camphor / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000737:chemistry*; D003001:Cloning, Molecular; D004926:Escherichia coli; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids; D015202:Protein Engineering; D016958:Pseudomonas putida; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020058:Styrene / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000473:Alkanes; D001555:Benzene Derivatives; D001559:Benzhydryl Compounds; D005288:Ferredoxins; D011994:Recombinant Proteins; D020058:Styrene; C100137:putidaredoxin; D002164:Camphor; D019475:Camphor 5-Monooxygenase; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase; C007932:n-hexadecane; C010129:diphenylmethane; C004912:ethylbenzene