Selection and characterization of new microorganisms for the manufacture of 9-OH-AD from sterols.

Using a special selection procedure, several mutants of Mycobacterium vaccae were isolated which were capable of converting sterols to 9 alpha-hydroxyandrost-4-ene-3,17-dion (9-OH-AD). Two mutants, Mycobacterium vaccae ZIMET 11052 and 11053, respectively, were further investigated. Strains of the species Mycobacterium fortuitum are mainly used for commercially obtaining 9-OH-AD from sterols. In contrast to the species Mycobacterium fortuitum the species Mycobacterium vaccae has not been reported to contain pathogenic strains. This seems an advantage for industrial application. Mutants with the ability of converting sterols to 9 alpha-hydroxysteroids have a defect in the steroid-1-dehydrogenase activity which is, however, only a partial one. The remaining activity may cause an undesirable degradation of the steroid nucleus. The steroid-1-dehydrogenase activity was tested using an assay developed by ATRAT (1986). We confirmed two apparently distinct steroid-1-dehydrogenases in Mycobacterium fortuitum NRRL B-8119 as reported by WOVCHA et al. (1979). One of them has an activity on androst-4-ene-3,17-dion (AD). The activity is increased by induction with sitosterol. The other one is active on 9-OH-AD. But Mycobacterium vaccae does not possess steroid-1-dehydrogenase activity on 9-OH-AD, and the AD specific steroid-1-dehydrogenase is not effected by sitosterol. The consequence is a high level of protection against steroid nucleus degradation yielding an effective accumulation of 9-OH-AD in fermentations with Mycobacterium vaccae mutants.

Microbial degradation of 2 alpha, 3 alpha-dihydroxy-5 alpha-cholestan-6-one by Mycobacterium vaccae.

2 alpha,3 alpha-Dihydroxy-5 alpha-cholestan-6-one (3), which had the substitution pattern of brassinosteroids in the A/B-ring moiety, was transformed by Mycobacterium vaccae to give 2 alpha,3 alpha,6 alpha-trihydroxy-5 alpha-androstan-17-one (4) and 2 alpha-hydroxyandrost-4-ene-3,17-dione (5). The structures of these compounds were determined by spectroscopic methods, especially 1H nuclear magnetic resonance studies.

Formation of progesterone and 1-dehydroprogesterone from cholesterol in fermentation cultures of Mycobacterium aurum.

The formation of progesterone and 1-dehydroprogesterone from cholesterol in fermentation cultures of Mycobacterium aurum ATCC 25790 was studied with the aim of clarifying the microbial pathway. The C22-intermediate (20S)-20-carboxy-1,4-pregnadien-3-one was microbiologically converted via the undetectable corresponding aldehyde into the C22-alcohol. However in the fermentation broth without microorganisms, but containing 2,2'-bipyridyl and copper ions, synthetically prepared C22-aldehyde was oxidized to the corresponding C21-compound 1-dehydroprogesterone, suggesting that the enzymatically originated C22-aldehydes may be immediately chemically oxidized to the corresponding C21-ketones.

Influence of 1-double bond and 11 beta-hydroxy group on stereospecific microbial reductions of 4-en-3-oxo-steroids.

The yeast Rhodotorula glutinis and the anaerobic bacterium Clostridium paraputrificum were used for stereospecific reductions of 4-chloro-11 beta-hydroxy-17 alpha-methyl-testosterone and the corresponding 1-dehydro compound to prepare 5 alpha- and 5 beta-H derivatives, respectively. C. paraputrificum was able to 5 beta-reduce both substances, whereas the 5 alpha-reduction by R. glutinis was inhibited by the structure elements 1-en and 11 beta-OH so that the substrate with both structure elements was not 5 alpha-reduced. The microbial conversion of the four steroids with and without 1-en and 11 beta-OH was compared in semiquantitative experiments. A number of new substances are described, 11 beta-hydroxy and 11-oxo derivatives of 5 alpha- and 5 beta-dihydro-4-chloro-17 alpha-methyltestosterone including some 3-OH compounds and characterized by NMR, mass spectrometric and further data.

Studies on biotransformation of STS 557.

Transformation of STS 557 (17 alpha-cyanomethyl-17 beta-hydroxy-estra-4, 9-dien-3-one) by female rat liver microsomes demonstrates a lower transformation rate in comparison with the analogous compound without 9-double bond: 17 alpha-cyanomethyl-19-nortestosterone, and the basic substance: 19-nortestosterone. 17 alpha-Cyanomethyl-estra-1, 3, 5(10), 9(11)-tetraene-3, 17 beta-diol, 17 alpha-cyanomethyl-11 beta, 17 beta-dihydroxy-estra-4, 9-dien-3-one, and tentatively 17 alpha-cyanomethyl-6 alpha, 17 beta-dihydroxy-estra-4, 9-dien-3-one were identified as metabolites. Microbial model investigations with species known to hydrogenate the 4-double bond in 4-en-3-oxo steroids stereospecifically to 5 alpha H- or 5 beta H-metabolites indicate 5 alpha-hydrogenation to be prevented in STS 557 by the 9-double bond, whereas 5 beta-hydrogenation is not affected. Isolation and characterization of metabolites from beagle dog and rat urine following administration of 3H-STS 557 revealed the following pathways of biotransformation: Hydroxylation in different positions of the steroid molecule, aromatization of ring A, hydrogenation of a double bond, simultaneous hydroxylation and hydrogenation, and alteration of the 17 alpha-side chain with loss of nitrogen.

Microbial transformation of 17 alpha-cyanomethyl-17-hydroxy-4,9-estradien-3-one (STS 557) and 17 alpha-cyanomethyl-19-nortestosterone by Mycobacterium smegmatis.

Microbial transformation of the new progestagen STS 557 (17 alpha-cyanomethyl-17-hydroxy-4,9-estradien-3-one) by Mycobacterium smegmatis yielded predominantly ring A-aromatized compounds: 17 alpha-cyanomethyl-1,3,5(10),9(11)-estratetraene-3, 17-diol, 17 alpha-cyanomethyl-1,3,5(10)-estratriene-3,17-diol and the corresponding 3-methyl ethers. The analogous compound without the 9(10) double bond, 17 alpha-cyanomethyl-19-nortestosterone, was transformed mainly to 5 alpha-hydrogenated metabolites: 17 alpha-cyanomethyl-17-hydroxy-5 alpha-estran-3-one, 17 alpha-cyanomethyl-17-hydroxy-5 alpha-1-estren-3-one, 17 alpha-cyanomethyl-5 alpha-estrane-3 alpha, 17-diol, and 17 alpha-cyanomethyl-5 alpha-estrane-3 beta, 17-diol. From these results, it is concluded that 4,9-dien-3-oxo compounds are not substrates for enzymatic 5 alpha-hydrogenation.

[Steroid transformation with immobilized microorganisms. III. Degradation of the side chain of cholesterol derivatives with immobilized Mycobaterium phlei and M. smegmatis cells]. / Steroidtransformation mit immobilisierten Mikroorganismen. III. Abbau der Seitenkette von Cholesterolderivaten mit immobilisierten Zellen von Mycobacterium phlei und M. smegmatis.

The cholesterol derivatives 3,3-ethylendioxycholest-5-en I and 4-cholesten-3-(O-carboxymethyl)oxime II were transformed into the corresponding androstenedione derivatives by selective cleavage of the sterol side chain with immobilized preparations of Mycobacterium phlei IMET SG 1026 and M. smegmatis IMET H 124. The influence of three immobilization methods on fermentation activity and stability was investigated. The immobilized cells of M. phlei IMET SG 1026 were found to be suitable to transform the structure modified sterol II in a periodic process over a long period into the androstenedione in a yield of 50--70%. Immobilized cells of M. phlei have higher transformation stability in comparison with free intact cells.

[Steroid transformation using immobilized microorganisms. II. Degradation of the sidechain of cholesterol by immobilized cells of Nocardia erythropolis]. / Steroid transformation mit immobilisierten Mikroorganismen. II. Abbau der Seitenkette von Cholesterol mit immobilisierten Zellen von Nocardia erythropolis.

The degradation of cholesterol side chain was studied by immobilized cells of Nocardia erythropolis in the presence of an inhibitor of the microbial steroid-skeleton-degradation. Different immobilized preparations such as DEAE-cellulose adsorbates, ionic cross-linked polymer gels, i.e., Al/Coalginates and polyacrylates, respectively, and polyethylene maleic acid anhydride supports have shown a high transformation activity yielding the C22- and C19-degradation products. No side chain degradation activity could be observed after immobilization of the cells in polyacrylamide gel under usual conditions caused by the toxic effect of the acrylamide monomer on the Nocardia cells.

[Steroid transforming enzymes in microorganisms. XIII. Relationship between steroid structure and induction of 4-ene-3-oxosteroid: (acceptor)-1-ene-oxidoreductase in Nocardia opaca]. / Steroidumwandelnde Enzyme aus Mikroorganismen. XIII. Bezichungen zwischen Steroidstruktur und Induktion der 4-En-3-oxosteroid: (Akzeptor)-1-en-oxidoreduktase in Nocardia opaca.

The induction of the 4-ene-3-oxosteroid: (acceptor)-1-ene-oxidoreductase in Nocardia opaca has been shown to be dependent on both hydrophilic and hydrophobic structural features of the steroid molecule, i.g. the nature of the oxygen function in positions 11 and 20 and the presence of the methyl group in position 10 influence the induction of the enzyme drastically. In this connection the metabolism of the steroid inductors being degradated during the induction process has been considered.

[Degradation of steroids. XVI. Microbial side chain degradation of structurally modified sterols]. / Abbau von Steroiden. XVI. Mikrobieller Seitenkettenabbau von strukturmodifizierten Sterolen.

Different types of sterol derivatives substituted in position 3 have been investigated to be suitable substrates for microorganisms capable of degradating the side chain of sterols. Sterolesters were found to be either unattacked or hydrolyzed followed by complete degradation of the molecule. Derivatives of the alkylether - and ketal type were transformed to the corresponding C19-steroids by fermentation with strains of the genus Mycobacteria. With respect to stability, solubility and transformation rate the sterol-3-carboxymethyloxime-derivatives were shown to be most suitable.

Degradation of steroids by microorganisms. XVIII. The reversibility of steroid-1-dehydrogenation during microbial side chain degradation of sterols by Nocardia.

The aerobic side chain degradation of sterols often leads to mixtures of 1,4-diene and 4-ene compounds. By an additional anaerobic step a practically complete reverse reaction of the steroid-1-dehydrogenation occurs yielding only the corresponding 4-ene compound.

[Steroid transformation with immobilized microorganisms. I. Transformation of cholesterol to cholestenone in organic solvents]. / Steroidtransformation mit immobilisierten Mikroorganismen. I. Transformation von Cholesterin zu Cholestenon in organischen Lösungsmitteln.

The transformation of cholesterol to cholest-4-ene-3-one has been investigated in the presence of toluene and carbontetrachloride using cells of Nocardia erythropolis (IMET 7185) immobilized by different methods. The adsorption on DEAE-cellulose has been observed to be the most effective method. The stability of immobilized cells relating to cholesteroloxydase activity, transformation capacity, and the influence of water have been investigated.

[Steroid-transforming enzymes from microorganisms. X. Enrichment of a 4-en-3-oxosteroid-5 alpha-reductase from Mycobacterium smegmatis as well as separation and enrichment of the apoenzyme by means of affinity chromatography]. / Steroidumwandelnde Enzyme aus Mikroorganismen. X. Anreicherung einer 4-En-3-oxosteroid-5 alpha-Reduktase aus Mycobacterium smegmatis sowie Abtrennung und Anreicherung des Apoenzyms mit Hilfe der Affinitätschromatographie.

The 4-en-3-oxosteroid-5 alpha-reductase from Mycobacterium smegmatis was bound biospecifically on the affinant containing an immobilized testosterone ligand. The enzyme obtained by elution with ethylene glycol and urea in a 32 fold purity has a S. A. of 8.73 X 10(-3) microM androstenedione min-1 mg-1. The coenzyme (FAD) could be separated from the immobilized enzyme substrate complex on the affinity matrix, in the presence of (NH4)2SO4 at pH 3.0. After elution of the apoenzyme 97% of the initial enzyme activity was obtained by incubation with FAD. The reactivated enzyme results in a 40-fold enrichment.

[Steroid-transforming enzymes from microorganisms. XII. Induction characteristics of the 4-en-3-oxosteroid: (acceptor)-1-en-oxidoreductase in Nocardia opaca]. / Steroidumwandelnde Enzyme aus Mikroorganismen. XII. Merkmale der Induktion der 4-En-3-oxosteroid: (Akzeptor)-1-en-oxidoreduktase in Nocardia opaca.

17 alpha-Methyltestosterone and the corresponding 1(2)-dehydrocompound (Dianabol) are efficient inducers of the 4-en-3-oxosteroid: (acceptor)-1-en-oxidoreductase from Nocardia opaca. After a lag period of 4 hours the enzyme activity increases rapidly. During the induction the steroids are completely metabolized causing a drastical drop of specific enzyme activity. Using a fixed induction time the optimal steroid concentration and the temperature characteristic were found out. The influence of the concentration of the steroid water suspension on the induction effect is discussed to be dependent on the velocity of the dissolving of the steroid particles. Chloramphenicol and streptomycin are powerful inhibitors of the induction process.

[Steroid-transforming enzymes from microorganisms. IX. Affinity chromatographic preparation and studies of the apoenzyme from a 4-en-3-oxosteroid: (acceptor)-1-en-oxidoreductase from Nocardia opaca]. / Steroidumwandelnde Enzyme aus Mikroorganismen. IX. Affinitätschromatographische Präparation und Untersuchung des Apoenzyms einer 4-En-3-oxosteroid: (Akzeptor)-1-en-oxidoreduktase aus Nocardia opaca.

From the flavoenzyme, 4-en-3-oxosteroid: (acceptor)-1-en-oxidoreductase of Nocardia opaca, prosthetic group and apoenzyme were separated quantitatively by means of affinity chromatography in the presence of 2 M (NH4)2 at pH 3.0. Subsequently the apoenzyme was eluted from affinity matrix by 0.01 M phosphate buffer, pH 8.0, whereas under these conditions the intact enzyme could not be eluted. The whole enzyme activity applied could be restored by incubation of the eluted apoenzyme with FAD. The binding strength of the apoenzyme to the immobilized steroid ligand is highly decreased in comparison to the native enzyme and can be interpreted by the action of rest hydrophobicity. That indicates the essential character of FAD for both ligand binding and transformation.

[Steroid-transforming enzymes from microorganisms. XI. Reversibility of the dehydrogenation reaction of the steroid-1-dehydrogenase from Nocardia opaca]. / Steroidumwandelnde Enzyme aus Mikroorganismen. XI. Reversibilität der Dehydrierungsreaktion der Steroid-1-Dehydrogenase aus Nocardia opaca.

Highly purified preparations of the 4-en-3-oxosteroid: (acceptor)-1-en-oxidoreductase from Nocardia opaca have been investigated in both types of reactions: 1.2-dehydrogenation of the 4-en-3-oxo-derivative and 1.2-hydrogenation of the 1.4-dien-3-oxo-derivative. It was not possible to separate the hydrogenating activity from the dehydrogenating activity by affinity chromatography, disc electrophoresis, SDS-electrophoresis, and isoelectric focusing techniques. The pure enzyme preparation is discussed as only one FAD depending protein acting in response to the system as a dehydrogenase as well as a reductase.