Antonia Jekkel

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 9α-Hydroxylase in Mycobacterium smegmatis mc2155

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 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 9α-hydroxylation and carrying the transposon insertion and flanking DNA sequences, and used to isolate a chromosomal fragment encoding the 9α-hydroxylase. The open reading frame encodes the 383-amino-acid terminal oxygenase of 3-ketosteroid 9α-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 9α position.

Genetic Recombination by Spheroplast Fusion of Sterol-transforming Mycobacterium Strains

Wall-deficient forms of fast-growing mycobacteria were produced in growth medium containing vancomycin and glycine, and spheroplasts were prepared by lysozyme treatment of wall-deficient cells. Spheroplasts gave rise to recombinants with high frequency (2-6%) when they were fused using polyethylene glycol 6000. The results demonstrated that in vivo genetic recombination could be used to produce genetically modified Mycobacterium strains with applications in transformation of steroids. Useful intermediates of steroid drug synthesis and new degradation products were obtained from sterols by selected recombinant strains.

Microbial transformation of β-sitosterol and stigmasterol into 26-oxygenated derivatives

The genetically modified Mycobacterium sp. BCS 396 strain has been used to transform sterols with stigmastane side chain in order to obtain 26-oxidized metabolites. beta-Sitosterol (I) was transformed to 4-stigmasten-3-one (II), 26-hydroxy-4-stigmasten-3-one (III), and 3-oxo-4-stigmasten-26-oic acid (IV), while stigmasterol (V) was converted to 4,22-stigmastadien-3-one (VI), 6 beta-hydroxy-4,22-stigmastadien-3-one (VII), 26-hydroxy-4,22-stigmastadien-3-one (VIII), 3-oxo-4,22-stigmastadien-26-oic acid methyl ester (IX), and 3-oxo-1,4,22-stigmastatrien-26-oic acid methyl ester (X) with that strain. In both beta-sitosterol and stigmasterol, 26-oxidation generates the R-configuration on C-25.

Microbial hydroxylation of 13β-ethyl-4-gonene-3,17-dione

Abstract Among the microbiological transformations of steroids 15α-hydroxylation of 13β-ethyl-4-gonene-3,17-dione is an industrially important one [Von H. Hofmeister, K. Annen, H. Laurent, K. Petzoldt, R. Wiechert, Arzneim.-Forsch. 36 (1986) 781], since it results in an intermediate of the synthesis of Gestoden, a widely used contraceptive drug. The aim of our research was to select fungal strains for hydroxylation of 13β-ethyl-4-gonene-3,17-dione which produce the 15α-hydroxylated product in a high yield. According to our taxonomical studies, several species of Aspergillus, Fusarium, Mortierella, and Penicillium genera fulfill this requirement. It has been reported that the 15α-hydroxylating enzyme of Penicillium raistrickii is inducible by various steroidal compounds [S. Irrgang, D. Schlosser, H.-P. Schmauder, Biotechnol. Lett. 14 (1992) 33]. We found that the enzyme of Fusarium nivale (VJ-63 strain) is also advantageously induced by norethisterone, which significantly increased the economic efficiency of this biotransformation process [A. Jekkel, E. Ilkőy, J. Sutő, G. Ambrus, Gy. Horvath, I. Bősinger, I. Pallagi, I. Lang, E. Gyepessy, Hungarian Patent Appl. P-9602249 (1996)].

Novel 26-oxygenated products in microbial degradation of ergosterol

In order to investigate the effect of the different stereochemistry of C-24 on the microbial C-26 oxidation of sterol side-chain the genetically modified Mycobacterium sp. BCS 396 strain was used to transform erogsterol. Ergosterol was converted to 3-oxo-4,22-ergostadien-26-oic acid methyl ester, 3-oxo-1,4,22-ergostatrien-26-oic acid methyl ester, and 3-oxo-1,4,22-ergostatrien-26-oic acid, the structures of which have been determined by IR, 1H NMR, 13C NMR, and mass spectroscopy. The X-ray structure of 3-oxo-4,22-ergostadien-26-oic acid methyl ester revealed that oxidation at C-26 of the ergostane side-chain generates a chiral center with S-configuration at C-25 as a result of chiral induction of the C-24 center.

Novel intermediates of microbial side chain degradation of sitosterol

Abstract We describe the structure and stereochemistry of some new 26-oxygenated steroid derivatives obtained by microbial transformation of sitosterol.