Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases

Journal of Biotechnology
Frank HannemannR Bernhardt

Abstract

The 15beta-hydroxylase (CYP106A2) from Bacillus megaterium, one of the few bacterial steroid hydroxylases, which has been isolated and characterized so far, catalyses the 15beta-hydroxylation of a variety of steroids. The enzyme can be supported in its activity with adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenals, supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. This three-component electron transfer chain was implemented in Escherichia coli by coexpression of the corresponding coding sequences from two plasmids, containing different selection markers and compatible origins of replication. The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin. The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach. The transformed bacteria show the inducible ability to efficiently convert 11-deoxycorticosterone (DOC) to 15beta-DOC at an average rate of 1 mM/d in culture volumes of 300 ml. The steroid conversion system was downscaled to the microtiter plate format and a robot set-up was developed for a fluorescence-based conver...Continue Reading

References

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Citations

Feb 27, 2010·Applied Microbiology and Biotechnology·Marco GirhardVlada B Urlacher
Jul 21, 2010·Applied Microbiology and Biotechnology·Yogan KhatriRita Bernhardt
Apr 25, 2012·Applied Microbiology and Biotechnology·Marco GirhardVlada B Urlacher
May 9, 2012·Applied Microbiology and Biotechnology·Marina V Donova, Olga V Egorova
Jul 7, 2009·Annual Review of Plant Biology·James Kirby, Jay D Keasling
Jul 26, 2011·Trends in Biotechnology·Vlada B Urlacher, Marco Girhard
Jun 12, 2010·Biochimica Et Biophysica Acta·Kerstin Maria EwenRita Bernhardt
Jul 29, 2009·Biochimica Et Biophysica Acta·Guillermina GoñiMilagros Medina
Jul 28, 2007·Journal of Biotechnology·Harald J RuijssenaarsJan A M de Bont
May 5, 2012·IUBMB Life·Kerstin Maria EwenRita Bernhardt
Apr 24, 2015·Applied Microbiology and Biotechnology·Flora Marta KissRita Bernhardt
Sep 19, 2006·Biochimica Et Biophysica Acta·Frank HannemannRita Bernhardt
Sep 17, 2015·Microbial Cell Factories·Simone Brixius-AnderkoRita Bernhardt
Feb 13, 2016·Chembiochem : a European Journal of Chemical Biology·Simon JanochaRita Bernhardt
Dec 17, 2014·Chembiochem : a European Journal of Chemical Biology·Alexander SchifrinRita Bernhardt
Feb 26, 2010·Chembiochem : a European Journal of Chemical Biology·Daniela ZehentgruberStephan Lütz
Jan 29, 2011·Chembiochem : a European Journal of Chemical Biology·Sabrina BleifRita Bernhardt
Jun 6, 2018·Frontiers in Microbiology·Lorena Fernández-CabezónJosé L García
Nov 1, 2006·Biochemical Society Transactions·C VirusR Bernhardt
Apr 26, 2012·Chembiochem : a European Journal of Chemical Biology·Kim Thoa NguyenRita Bernhardt
Jul 23, 2018·Applied Microbiology and Biotechnology·Maciej SzaleniecMarina Donova
Mar 5, 2021·Microbial Biotechnology·Carmen Felpeto-SanteroJosé Luis García
Aug 8, 2021·Microorganisms·Carmen Felpeto-SanteroJosé Luis García

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