Application of a new versatile electron transfer system for cytochrome P450-based Escherichia coli whole-cell bioconversions

Abstract

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.

Conversions of Tricyclic Antidepressants and Antipsychotics with Selected P450s fromSorangium cellulosumSo ce56

“…For the past few years, we have been engaged in the study of novel P450s from myxobacteria, and some P450s from S. cellulosum So ce56 have shown a potential for industrial and biotechnological applications (Khatri et al, 2010a(Khatri et al, ,b, 2013(Khatri et al, , 2014Ly et al, 2012;Ringle et al, 2013). We were also able to develop an efficient E. coli-based whole-cell bioconversion system for some myxobacterial P450s .…”

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

“…Therefore, we first identified efficient autologous redox partners to transfer electrons to the CYP267 family. Although the autologous redox system Fdx8/FdR_B from S. cellulosum So ce56 has already been shown to transfer electrons to myxobacterial CYP109D1, CYP260A1, and CYP264A1 Ringle et al, 2013), the heterologous bovine Adx 4-108 with AdR or the E. coli Fpr has been shown to be more efficient Ringle et al, 2013). However, in this study, the substitution of Adx 4-108 /Fpr by Fdx8/FdR_B showed a significant increase of product yields for drug molecules (Table 1) when using the members of the CYP267 family.…”

Characterization of the Gene Cluster CYP264B1‐geoA from Sorangium cellulosum So ce56: Biosynthesis of (+)‐Eremophilene and Its Hydroxylation

“…strain CL190 were co-expressed. For in vivo sesquiterpene oxidation by CYP264B1, the heterologous electron-transfer system consisting of Fpr and Adx [57] was used for the co-expression.…”

“…The CYP264B1 expression vector pET22b_ CYP264B1 was obtained by cloning the sequence-optimized CYP264B1 gene flanked by restriction sites NdeI and HindIII (Life Technologies) at the same restriction sites into pET-22b (Invitrogen). The vector pETC4oFA was constructed from three fragments: NdeI/HindIII-flanked CYP264B1, EcoRI/HindIII-digested pET_MR8, [57] and NdeI/EcoRI-digested pET-17b.…”