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. 2006 Oct;72(10):6554-9.
doi: 10.1128/AEM.00941-06.

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

Attila Andor  1 , Antónia Jekkel, David A Hopwood, Ferenc Jeanplong, Eva Ilkoy, Attila Kónya, István Kurucz, Gábor Ambrus
Affiliations

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

Attila Andor et al. Appl Environ Microbiol. 2006 Oct.

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.

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Figures

FIG. 1.
FIG. 1.
Microbial transformations of β-sitosterol in fast-growing mycobacteria.
FIG. 2.
FIG. 2.
Replicative transposition of Tn611 from pCG79 (top) and recovery of pFJ92 from the chromosome of M. smegmatis 10A12 (M.s.) (bottom). Solid bars indicate the PstI-PstI and EcoRI-EcoRI hybridization probes.
FIG. 3.
FIG. 3.
Identification of a Rieske [2Fe-2S] domain and a nonheme Fe(II) domain in the terminal oxygenase of the following 3-ketosteroid 9α-hydroxylases: terminal oxygenase of 3-ketosteroid 9α-hydroxylase in M. smegmatis mc2155 (DDBJ/EMBL/GenBank accession no. DQ357196) (M.s.9α-hydroxylaseA); putative terminal oxygenase of 3-ketosteroid 9α-hydroxylase in M. tuberculosis H37Rv (DDBJ/EMBL/GenBank accession no. CAB05051) (Rv 3526); and terminal oxygenase of 3-ketosteroid 9α-hydroxylase in R. erythropolis SQ1 (DDBJ/EMBL/GenBank accession no. AY083508) (KshA).
FIG. 4.
FIG. 4.
SDS-PAGE analysis of the terminal oxygenase of 3-ketosteroid 9α-hydroxylase from M. smegmatis mc2155 expressed in E. coli BL21/pOX17. Lane 1, soluble fraction of noninduced cell lysate; lane 2, soluble fraction of 0.4 mM IPTG-induced cell lysate; lane 3, inclusion body fraction of a noninduced cell lysate; lane 4, inclusion body fraction of a 0.4 mM IPTG-induced cell lysate; lane 5, Precision Plus protein standard (250 kDa, 150 kDa, 100 kDa, 75 kDa, 50 kDa, and 37 kDa; Bio-Rad).
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