title: 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 9alpha-hydroxylase in Mycobacterium smegmatis mc(2)155.
abstract: 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 mc(2)155 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 9alpha-hydroxylation and carrying the transposon insertion and flanking DNA sequences, and used to isolate a chromosomal fragment encoding the 9alpha-hydroxylase. The open reading frame encodes the 383-amino-acid terminal oxygenase of 3-ketosteroid 9alpha-hydroxylase in M. smegmatis mc(2)155 and has domains typically conserved in class IA terminal oxygenases. Escherichia coli containing the gene could hydroxylate the steroid ring at the 9alpha position.
authors.affiliation: Institute for Drug Research Ltd., PO Box 82, H-1325 Budapest, Hungary. attila.andor@idri.hu
authors.forename: Attila
authors.initials: A
authors.lastname: Andor
authors.forename: Antónia
authors.initials: A
authors.lastname: Jekkel
authors.forename: David A
authors.initials: DA
authors.lastname: Hopwood
authors.forename: Ferenc
authors.initials: F
authors.lastname: Jeanplong
authors.forename: Eva
authors.initials: E
authors.lastname: Ilkoy
authors.forename: Attila
authors.initials: A
authors.lastname: Kónya
authors.forename: István
authors.initials: I
authors.lastname: Kurucz
authors.forename: Gábor
authors.initials: G
authors.lastname: Ambrus
mesh_terms: D003001:Cloning, Molecular; D019976:Cloning, Organism; D015870:Gene Expression; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D013261:Sterols / Q000378:metabolism*
chemical_list: D013261:Sterols; D010105:Oxygenases