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Applied and Environmental Microbiology, October 2006, p. 6554-6559, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00941-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Generation of Useful Insertionally Blocked Sterol Degradation Pathway Mutants of Fast-Growing Mycobacteria and Cloning, Characterization, and Expression of the Terminal Oxygenase of the 3-Ketosteroid 9{alpha}-Hydroxylase in Mycobacterium smegmatis mc2155

Attila Andor,1* Antónia Jekkel,1,{dagger} David A. Hopwood,2 Ferenc Jeanplong,1 Éva Ilkoy,1 Attila Kónya,1 István Kurucz,1 and Gábor Ambrus1

Institute for Drug Research Ltd., 47-49 Berlini St., H-1045 Budapest, Hungary,1 John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom2

Received 20 April 2006/ Accepted 22 July 2006

Integration of the pCG79 temperature-sensitive plasmid carrying Tn611 was used to generate libraries of mutants with blocked sterol-transforming ability of the sterol-utilizing strains Mycobacterium smegmatis mc2155 and Mycobacterium phlei M51-Ept. Of the 10,000 insertional mutants screened from each library, 4 strains with altered activity of the sterol-degrading enzymes were identified. A blocked 4-androstene-3,17-dione-producing M. phlei mutant transformed sitosterol to 23,24-dinorcholane derivatives that are useful starting materials for corticosteroid syntheses. A recombinant plasmid, pFJ92, was constructed from the genomic DNA of one of the insertional mutants of M. smegmatis, 10A12, which was blocked in 3-ketosteroid 9{alpha}-hydroxylation and carrying the transposon insertion and flanking DNA sequences, and used to isolate a chromosomal fragment encoding the 9{alpha}-hydroxylase. The open reading frame encodes the 383-amino-acid terminal oxygenase of 3-ketosteroid 9{alpha}-hydroxylase in M. smegmatis mc2155 and has domains typically conserved in class IA terminal oxygenases. Escherichia coli containing the gene could hydroxylate the steroid ring at the 9{alpha} position.

* Corresponding author. Mailing address: Institute for Drug Research Ltd., P.O. Box. 82, H-1325 Budapest, Hungary. Phone: (361) 399 3300. Fax: (361) 399 3356. E-mail: attila.andor{at}idri.hu.

{dagger} Deceased.

Applied and Environmental Microbiology, October 2006, p. 6554-6559, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00941-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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