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.
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.
Andor
Attila
A
Institute for Drug Research Ltd., PO Box 82, H-1325 Budapest, Hungary. attila.andor@idri.hu
Jekkel
Antónia
A
Hopwood
David A
DA
Jeanplong
Ferenc
F
Ilkoy
Eva
E
Kónya
Attila
A
Kurucz
István
I
Ambrus
Gábor
G
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*
D013261:Sterols; D010105:Oxygenases