Application of a new versatile electron transfer system for cytochrome P450-based Escherichia coli whole-cell bioconversions
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Cytochromes P450 monooxygenases are highly interesting biocatalysts for biotechnological applications, since they perform a diversity of reactions on a broad range of organic molecules. Nevertheless, the application of cytochromes P450 is limited compared to other enzymes mainly because of the necessity of a functional redox chain to transfer electrons from NAD(P)H to the monooxygenase. In this study, we established a novel robust redox chain based on adrenodoxin, which can deliver electrons to mitochondrial, bacterial and microsomal P450s. The natural membrane-associated reductase of adrenodoxin was replaced by the soluble Escherichia coli reductase. We could demonstrate for the first time that this reductase can transfer electrons to adrenodoxin. In the first step, the electron transfer properties and the potential of this new system were investigated in vitro, and in the second step, an efficient E. coli whole-cell system using CYP264A1 from Sorangium cellulosum So ce56 was developed. It could be demonstrated that this novel redox chain leads to an initial conversion rate of 55 μM/h, which was 52 % higher compared to the 36 μM/h of the redox chain containing adrenodoxin reductase. Moreover, we optimized the whole-cell biotransformation system by a detailed investigation of the effects of different media. Finally, we are able to demonstrate that the new system is generally applicable to other cytochromes P450 by combining it with the biotechnologically important steroid hydroxylase CYP106A2 from Bacillus megaterium.
KeywordsCytochromes P450 Biotechnology Adrenodoxin Escherichia coli reductase Redox chain Whole-cell system CYP264A1 CYP106A1
This work was supported by a fellowship awarded by the Scholarship Program of the German Federal Environmental Foundation [Deutsche Bundesstiftung Umwelt (DBU)] to Michael Ringle. We are also thankful to Wolfgang Reinle for the expression and purification of Adx and AdR.
- Bleif S, Hannemann F, Zapp J, Hartmann D, Jauch J, Bernhardt R (2012) A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-beta-boswellic acid (KBA) based on a recombinant cytochrome P450 system. Appl Microbiol Biotechnol 93:1135–1146PubMedCrossRefGoogle Scholar
- Ewen KM, Hannemann F, Khatri Y, Perlova O, Kappl R, Krug D, Hüttermann J, Müller R, Bernhardt R (2009) Genome mining in Sorangium cellulosum So ce56—identification and characterization of the homologous electron transfer proteins of a myxobacterial cytochrome P450. J Biol Chem 284:28590–28598PubMedCrossRefGoogle Scholar
- Martinez-Gomez K, Flores N, Castaneda HM, Martinez-Batallar G, Hernandez-Chavez G, Ramirez OT, Gosset G, Encarnacion S, Bolivar F (2012) New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol. Microb Cell Fact 11:46PubMedCrossRefGoogle Scholar
- Werck-Reichhart D, Feyereisen R (2000) Cytochromes P450: a success story. Genome Biol 1:REVIEWS3003Google Scholar