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Testosterone 15β-hydroxylation by solvent tolerant Pseudomonas putida S12

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Author: Ruijssenaars, H.J. · Sperling, E.M.G.M. · Wiegerinck, P.H.G. · Brands, F.T.L. · Wery, J. · Bont, J.A.M.de
Type:article
Date:2007
Institution: TNO Kwaliteit van Leven
Source:Journal of Biotechnology, 2, 131, 205-208
Identifier: 240153
doi: doi:10.1016/j.jbiotec.2007.06.007
Keywords: Biology · Biotechnology · CYP106A2 · Fer · Pseudomonas putida · Solvent tolerance · Steroid synthesis · Whole cell biocatalyst · Biocatalysts · Enzyme kinetics · Hydroxylation · Optimization · Solvents · Pseudomonas putida · Solvent tolerance · Steroid synthesis · Whole cell biocatalysts · Drug products · 15 beta hydroxylase · bacterial protein · cytochrome P450 · organic solvent · steroid monooxygenase · testosterone · threonine · valine · 15beta hydroxylase CYP106A2, Bacillus megaterium · 15beta-hydroxylase CYP106A2, Bacillus megaterium · solvent · article · Bacillus subtilis · biocatalyst · biotransformation · chemical structure · controlled study · high performance liquid chromatography · nonhuman · nuclear magnetic resonance · priority journal · protein expression · Pseudomonas putida · steroid hydroxylation · steroidogenesis · biological model · enzyme specificity · evaluation · gene expression regulation · genetics · hydroxylation · metabolism · site directed mutagenesis · transgenic organism · Bacillus megaterium · Bacillus subtilis · Pseudomonas putida · Bacterial Proteins · Cytochrome P-450 Enzyme System · Gene Expression Regulation, Bacterial · Hydroxylation · Models, Biological · Mutagenesis, Site-Directed · Organisms, Genetically Modified · Pseudomonas putida · Solvents · Substrate Specificity · Testosterone

Abstract

A steroid 15β-hydroxylating whole-cell solvent tolerant biocatalyst was constructed by expressing the Bacillus megaterium steroid hydroxylase CYP106A2 in the solvent tolerant Pseudomonas putida S12. Testosterone hydroxylation was improved by a factor 16 by co-expressing Fer, a putative Fe-S protein from Bacillus subtilis. In addition, the specificity for 15β-hydroxylation was improved by mutating threonine residue 248 of CYP106A2 into valine. These new insights provide the basis for an optimized whole-cell steroid-hydroxylating biocatalyst that can be applied with an organic solvent phase. © 2007 Elsevier B.V. All rights reserved.