Hydroxylation of Testosterone by Bacterial Cytochromes P450 Using theEscherichia coliExpression System

Abstract

Two hundred thirteen cytochrome P450 (P450) genes were collected from bacteria and expressed based on an Escherichia coli expression system to test their hydroxylation ability to testosterone. Twenty-four P450s stereoselectively monohydroxylated testosterone at the 2alpha-, 2beta-, 6beta-, 7beta-, 11beta-, 12beta-, 15beta-, 16alpha-, and 17-positions (17-hydroxylation yields 17-ketoproduct). The hydroxylation site usage of the P450s is not the same as that of human P450s, while the 2alpha-, 2beta-, 6beta-, 11beta-, 15beta-, 16alpha-, and 17-hydroxylation are reactions common to both human and bacterial P450s. Most of the testosterone hydroxylation catalyzed by bacterial P450s is on the beta face.

Cloning and Characterization of the Biosynthetic Gene Cluster of 16‐Membered Macrolide Antibiotic FD‐891: Involvement of a Dual Functional Cytochrome P450 Monooxygenase Catalyzing Epoxidation and Hydroxylation

“…1 H and 13 C NMR spectra were taken by DRX-500 (Bruker) or ECX500 (JEOL) spectrometer, and FAB-MS spectra were measured by JMS-700 (JEOL). The assignments of and SpeI, and the resulting DNA fragment was inserted into the corresponding site of pCYP-camAB [44] to obtain pGfsF-camAB. The pGfsF-camAB was introduced into E. coli BL21(DE3) by a standard chemical transformation.…”

Directed Evolution of the Actinomycete Cytochrome P450 MoxA (CYP105) for Enhanced Activity

“…The amplicon was digested with NdeI and SacI for moxA and SacI and SpeI for moxB. Equal amounts of the fragments were mixed and cloned into the NdeISpeI sites of pT7NS-camAB 11,13) to yield expression plasmid pMox100. This expression plasmid contains ''redox partners,'' viz., ferredoxin (moxB) from N. recticatena and putidaredoxin (pdx) and putidaredoxin (pdR) reductase both from P. putida, facilitating the coexpression of all these components under the control of the T7 promoter.…”

“…This expression plasmid contains ''redox partners,'' viz., ferredoxin (moxB) from N. recticatena and putidaredoxin (pdx) and putidaredoxin (pdR) reductase both from P. putida, facilitating the coexpression of all these components under the control of the T7 promoter. 11,13) Both pdx and pdR were inserted downstream of moxB to support electron transport in E. coli.…”

“…9,10) Recently, we identified P450 MoxA (P450moxA), a class I P450, from the actinomycete Nonomuraea recticatena NBRC 14525, which catalyzes the hydroxylation of fatty acids, steroids, and aromatic compounds. [11][12][13] Many of these reactions were human P450-like, indicating that the P450moxA-catalyzed transformation system would be applicable in the preparation of human or actinomycete-specific P450 metabolites from a diverse set of pharmaceuticals in the early stages of drug development. The broad substrate specificity of the enzyme is highly attractive for medicinal applications.…”

Generation of New Benanomicin Analogues by Biotransformation Using Escherichia coli Expressing Actinomycete Cytochrome P450

“…Based on the system, we created the bacterial P450 expression library which serves the rapid biotransformation screening with help of co-expressed Pseudomonas redox partner genes camAB [15]. In our attempt to obtain benanomicin derivatives with improved biological activities, we took an approach to test P450-catalyzed transformation of benanomicin A by the strain of the bacterial P450 expressed E. coli, since the P450s are able to catalyze monooxygenation (principally hydroxylation) to a diverse set of organic compounds.…”