Hydroxylation of steroids by <Emphasis Type="Italic">Curvularia lunata</Emphasis> mycelium in the presence of methyl-β-cyclodextrine

Transformation of 16 Δ⁵-3β-hydroxy- and Δ⁴-3-ketosteroids of androstane and pregnane classes was carried out using Curvularia lunata mycelium suspended in phosphate buffer with methyl-β-cyclodextrine (MCD). As the result, 20 monohydroxy- and dihydroxy-metabolites, whose structure was determined using spectra of proton magnetic resonance and mass-spectra, have been isolated. Hydroxylation of Δ⁵-3β-hydroxy-steroids occurred mostly in the C-7α position whereas hydroxylation of Δ⁴-3-ketosteroids was in the C-11β position. Only androst-4-en-3,17-dione, 9α-hydroxy-androstenedione, and androsta-1,4-diene-3,17-dione were hydroxylated at C-14α position. Besides main 11β-derivatives, the 6β- and 7β-hydroxy-derivatives with yield 10 and 30%, respectively, were isolated during transformation of progesterone and hydroxymethyl pregnadienone. The ratio of MCD to transforming steroid was 1: 1 (mol/mol). Hydrocortisone and 7α-hydroxyandrostenolone with the yield 55 and 77%, respectively, were obtained at the maximal concentrations of cortexolone 20 g/l and androstenolone acetate 10 g/l in the presence of MCD. Absorption of steroids on mycelium, lower speed of their transformation, low concentrations of modifying substrates, and low yield of hydroxyderivatives have been observed in the absence of MCD.     

Metabolism of androst-4-en-3,17-dione by the filamentous fungus Neurospora crassa

Microbial transformation of androst-4-en-3,17-dione (AD; I) using Neurospora crassa afforded six metabolites; 6beta,14alpha-dihydroxyandrost-4-en-3,17-dione (II), 6beta,9alpha-dihydroxyandrost-4-en-3,17-dione (III), 7alpha-hydroxyandrost-4-en-3,17-dione (IV), 9alpha-hydroxyandrost-4-en-3,17-dione (V), 14alpha-hydroxyandrost-4-en-3,17-dione (VI), and androst-4,6-dien-3,17-dione (VII). The steroid products were assigned by interpretation of their spectral data such as (1)H NMR, (13)C NMR, FTIR, and mass spectroscopy. The characteristic transformations observed were C-6beta, C-7alpha, C-9alpha, C-14alpha hydroxylations, and C6-C7 dehydrogenation. The best fermentation condition was found to be 6-day incubation at 25 degrees C and pH value of 5.0-6.5 according to TLC profiles. Time course study showed the accumulation of V and VI from the third day and IV from the fourth day of the fermentation. Optimum concentration of the substrate, which gave maximum bioconversion efficiency, was 3.5mM in one batch. Biotransformation was completely inhibited in a concentration above 7.0mM.     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria Mycobacterium neoaurum, Pimelobacter simplex, and Rhodococcus erythropolis

Abstract-Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9alpha-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5-15 nm) or the transformation in the presence of randomly methylated beta-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The technical product ADD was obtained in 75% yield, based on phytosterol. It contained as impurity 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment-the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD-no by products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5 AD was combined. The yield of 9-OH-AD (m.p. 218-220 degrees C) based on transformed phytosterol was 56%.     

Microbial transformation of 3-hydroxy-5,6-cyclopropanocholestanes--an alternative route to 6-methylsteroids

Incubation of 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholestane (4), 3beta-hydroxy-5,6beta-cyclopropano-5beta-cholestane (5), and 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholest-7-e ne (6) with Mycobacterium sp. (NRRL B-3805) gave a mixture of side chain cleaved 17-keto steroids as the major products in 52, 57, and 69% yields, respectively. Among these 17-keto steroids, the cyclopropyl ring eliminated product, androst-4-ene-3,17-dione (9), was isolated in 6, 4, and 8% yields, respectively. A cyclopropyl ring migration product, 6alpha,7alpha-cyclopropanoandrost-4-ene-3,17-dione (16), was isolated from the incubation mixture of 6 in 4% yield, also 10% yield of 16 was obtained when 5, 6alpha-cyclopropano-5alpha-androst-7-ene-3,17-dione (12) was incubated. The cyclopropyl ring opening and subsequent reduction followed by oxidation of the two major biotransformation products, 5, 6beta-cyclopropano-5beta-androsta-3,17-dione (10) and 5, 6alpha-cyclopropano-5alpha-androsta-3,17-dione (7), gave 6beta- and 6alpha-methylandrost-4-ene-3,17-dione in 60, and 45% yields, respectively.     

The chemistry of 9 alpha-hydroxysteroids. 1. Preparation of 9 alpha,17 beta-dihydroxy-17 alpha-ethynylandrost-4-en-3-one

9 alpha-Hydroxyandrost-4-ene-3,17-dione 1, when allowed to react with dipotassium acetylide in tetrahydrofuran, resulted, after chromatographic separation, in 4-methyl-19-norandrosta-4,9-diene-1,17-dione 2, 4 xi-methyl-19-norandrosta-5(10),9(11)-diene-1,17-dione 3, 4-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-4,9-dien-1-one 4, 4 xi-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-5(10),9(11)-dien- 1-one 5, and 17 alpha-ethynyl-17 beta-hydroxy-9,10-secoandrost-4-ene-3,9-dione 6. Selective protection of delta 4-3-ketone of 9 alpha-hydroxyandrost-4-ene-3,17-dione 1 as its dienol methyl ether 7, and subsequent reaction with lithium acetylide-ethylenediamine followed by acidic hydrolysis, afforded 9 alpha,17 beta-dihydroxy-17 alpha- ethynylandrost-4-en-3-one 8.     

Microbial transformation of mesterolone

The microbial transformation of mesterolone (= (1alpha,5alpha,17beta)-17-hydroxy-1-methylandrostan-3-one; 1), by a number of fungi yielded (1alpha,5alpha)-1-methylandrostane-3,17-dione (2), (1alpha,3beta,5alpha,17beta)-1-methylandrostane-3,17-diol (3), (5alpha)-1-methylandrost-1-ene-3,17-dione (4), (1alpha,5alpha,15alpha)-15-hydroxy-1-methylandrostane-3,17-dione (5), (1alpha,5alpha,6alpha,17beta)-6,17-dihydroxy-1-methylandrostan-3-one (6), (1alpha,5alpha,7alpha,17beta)-7,17-dihydroxy-1-methylandrostan-3-one (7), (1alpha,5alpha,11alpha,17beta)-11,17-dihydroxy-1-methylandrostan-3-one (8), (1alpha,5alpha,15alpha, 17beta)15,17-dihydroxy-1-methylandrostan-3-one (9), and (5alpha,15alpha,17beta)-15,17-dihydroxy-1-methylandrost-1-en-3-one (10). Metabolites 5-10 were found to be new compounds. All metabolites, except 2, 3, 6, and 7, exhibited potent anti-inflammatory activity. The structures of these metabolites were characterized on the basis of spectroscopic studies, and the structure of 5 was also determined by single-crystal X-ray-diffraction analysis.     

Progesterone side-chain degradation by some species ofAspergillus flavus group

Seventy isolates belonging to 6 species and one variety of A. flavus group were shown to degrade the progesterone side-chain to yield delta 4-androstene-3,17-dione and testosterone. The isolates of five species (A. flavo-furcatis, A. flavus, A. oryzae, A. parasiticus and A. tamarii) possessed enzyme systems catalyzing the opening of ring D and formed testololactone as final steroid metabolite in addition to their ability to produce the above mentioned two products. 11 beta-Hydroxy-delta 4-androstene-3,17-dione was formed by only A. flavus and A. tamarii while 11 beta-hydroxytestosterone was produced by A. flavo-furcatis, A. parasiticus and A. subolivaceus. The chromatographic resolution of the mixture products obtained (when the selective isolate of each species reacted with 1 g of progesterone) revealed that 60-75% of progesterone was converted into delta 4-androstene-3,17-dione (8-30%), testosterone (7-33%), testololactone (14-37%) and other products (3-40%). The most bioconversion activity was exhibited by A. oryzae, followed by A. parasiticus. The highest values of delta 4-androstene-3,17-dione (30% of added progesterone) and testosterone (33%) were formed by A. flavus var. columnaris while those of testololactone (37%) were produced by A. oryzae. A systematic variation could be observed between the different tested species of A. flavus group with respect to the transformation reactions of progesterone. Comparative biotransformation results showed that essential differences exist between the tested species in this group; this biochemical differentiation may supplement the morphological and other physiological criteria used in the identification of the different species in the A. flavus group.     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590.

The microbial degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 was studied, and two major products were isolated and identified as 7 alpha, 12 beta-dihydroxyandrosta-1,4-diene-3,17-dione and 7 alpha, 12 alpha-dihydroxy-3-oxopregna-1,4-diene-20-carboxylic acid. Four minor products were isolated and evidence is given for the following structures: 7 alpha, 12 alpha-dihydroxyandrosta-1,4-diene-3,17-dione, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 7 alpha, 12 beta, 17 beta-trihydroxyandrosta-1,4-dien-3-one and 7 alpha, 12 alpha-dihydroxy-3-oxopregn-4-ene-20-carboxylic acid. The significance of the production of the steroid products is discussed, along with the possible enzymic mechanisms responsible for their production.     

Microbial transformations of steroids--VI. Transformation of testosterone and androstenedione by Botryosphaerica obtusa

The 7 beta progesterone-hydroxylating microorganism Botryosphaerica obtusa was tested for its ability to hydroxylate at this site the C-19 androstene-based compounds, androstenedione (androst-4-ene-3,17-dione) and testosterone (17 beta-hydroxyandrost-4-en-3-one). Only very limited 7 beta hydroxylation of both substrates was observed. The products included traces of 7 beta-monohydroxytestosterone and 6 beta,7 beta-dihydroxyandrostenedione from testosterone, and of 6 beta,7 beta-dihydroxyandrostenedione from androstenedione. 6 beta,7 beta-Dihydroxyandrostenedione does not appear to have been reported previously as a microbial transformation product. Both substrates were monohydroxylated in significant amounts at the isomeric 7 alpha site and at the 6 beta site. Testosterone was also significantly monohydroxylated at the 15 alpha site and in minor amounts at the 11 alpha and 12 beta sites. Some monohydroxytestosterones had also been oxidised at their 17-OH group, converting them into the corresponding monohydroxy androstenediones. The 7 alpha-hydroxy metabolites and 15 alpha-hydroxytestosterone being chemically demanding to synthesis are valuable microbial transformation products.     

Microbial transformation ofβ-sitosterol byNocardia sp. M 29

Summary The degradation and conversion of -sitosterol to C-17-ketosteroids byNocardia sp. M 29 was studied. Maximal enzymatic activity was found after 24 h of incubation. Although the key enzymes involved in the decomposition of -sitosterol were inducible, no separate induction of side chain hydroxylase or 9-hydroxylase was possible. Inhibition of the steroid ring cleaving enzyme by ,-dipyridyl resulted in low yields of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione (total yield 22%). Addition of lipophilic organic adsorbents (Amberlite XAD-2 and XAD-4) stimulated the 1,4-androstadiene-3,17-dione formation (maximal 50% within 120 h of incubation). Furthermore, 3-oxo-23,24-dinor-1,4-choladienic acid and 3-oxo-23,24-dinor-1,4-choladienic acid methyl ester were accumulated in the presence of Amberlite XAD-2 in moderate yields (total 11%).     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 under anaerobic conditions.

The bacterial degradation of cholic acid under anaerobic conditions by Pseudomonas sp. N.C.I.B. 10590 was studied. The major unsaturated neutral compound was identified as 12 beta-hydroxyandrosta-4,6-diene-3,17-dione, and the major unsaturated acidic metabolite was identified as 12 alpha-hydroxy-3-oxochola-4,6-dien-24-oic acid. Eight minor unsaturated metabolites were isolated and evidence is given for the following structures: 12 alpha-hydroxyandrosta-4,6-diene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-4,6-dien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, 3,12-dioxochola-4,6-dien-24-oic acid and 12 alpha-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid. In addition, a major saturated neutral compound was isolated and identified as 3 beta,12 beta-dihydroxy-5 beta-androstan-17-one, and the only saturated acidic metabolite was 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholan-24-oic acid. Nine minor saturated neutral compounds were also isolated, and evidence is presented for the following structures: 12 beta-hydroxy-5 beta-androstane-3,17-dione, 12 alpha-hydroxy-5 beta-androstane-3,17-dione, 3 beta,12 alpha-dihydroxy-5 beta-androstan-17-one, 3 alpha,12 beta-androstan-17-one, 3 alpha,12 alpha-dihydroxy-5 beta-androstan-17-one, 5 beta-androstane-3 beta,12 beta,17 beta-triol, 5 beta-androstane-3 beta,12 alpha,17 beta-triol, 5 beta-androstane-3 alpha,12 beta,17 beta-triol and 5 beta-androstane-3 alpha,12 alpha,17 beta-triol. The induction of 7 alpha-dehydroxylase and 12 alpha-dehydroxylase enzymes is discussed, together with the significance of dehydrogenation and ring fission under anaerobic conditions.     

Conversion of sterols and triterpenes by mycobacteria. II. Transformation of 7-oxygenated sterols into androstane derivatives via a 7-deoxygenation

The bioconversion of 7-oxygenated sterols by Mycobacterium aurum was studied in a preliminary investigation of the microbial conversion of wool wax. 7-Oxocholesterol was found to be transformed mainly into 3,17-dioxygenated androstane derivatives. 7 xi-Hydroxylated sterols were formed in an initial reduction step, and the C-7 hydroxyl group was then eliminated in a dehydration reaction. This was thought to take place during the isomerisation of cholest-4-en-3-one to cholest-5-en-3-one. Deuterium labelling experiments showed that this elimination proceeded faster for the C-7 alpha isomer, although it was not stereospecific. The C-7 alpha and C-7 beta-hydroxy isomers were weakly interconverted via the 7-oxo derivatives. Cholest-4-en-3-one, cholest-1,4-dien-3-one and cholest-4,6-dien-3-one all lost their side chains following a hydrogenation/dehydrogenation reaction. The resulting 3,17-dioxoandrostene or 3,17-androstadiene derivatives were mainly hydrogenated into 5 alpha-androstane-3,17-dione and 5 alpha-androstane-3 beta-ol-17-one. Elimination of the 3 beta-hydroxyl groups giving cholesta-3,5-dien-7-one, and subsequent microbial degradation of the side chain was not observed to any significant extent. The convergence of the bioconversion pathways of cholesterol and the 7-oxygenated cholesterols enabled crude, partially auto-oxidised cholesterol to be used as a substrate for the production of 3,17-dioxygenated androstane derivatives by M. aurum.     

The facile bioconversion of testosterone by alginate-immobilised filamentous fungi

The potential for biotransformation of the substrate 17β-hydroxyandrost-4-en-3-one (testosterone) by six filamentous fungi, namely, Rhizopus oryzae ATCC 11145, Mucor plumbeus ATCC 4740, Cunninghamella echinulata var. elegans ATCC 8688a, Aspergillus niger ATCC 9142, Phanerochaete chrysosporium ATCC 24725 and Whetzelinia sclerotiorum ATCC 18687, was investigated. In this study both free cells and macerated mycelia immobilised in calcium alginate were utilised and the results (products, % yields, % transformation) were compared. In general the encapsulated cells of the microorganisms effectively generated products similar to those found using free cells. However, with immobilised macerated mycelia, isolation of the transformation products was expedited by the simple work up procedure, and their purification was facilitated by the absence of fungal secondary metabolites. Twenty seven analogues of testosterone were generated, wherein the androstane skeleton was functionalised at C-1β, -2β, -6β, -7α, -11α, -14, -15α, -15β and -16β by the moulds. Redox chemistry was also observed. Seven of the analogues, 6β,11α,17β-trihydroxyandrost-4-en-3-one, 6β,14α,17β-trihydroxyandrost-4-en-3-one, 2,6β-dihydroxyandrosta-1,4-diene-3,17-dione, 2β,16β-dihydroxyandrost-4-ene-3,17-dione, 2β,6β-dihydroxyandrost-4-ene-3,17-dione, 2β,15β,17β-trihydroxyandrost-4-en-3-one and 2β,3α,17β-trihydroxyandrost-4-ene, were novel compounds. Five others, namely, 7α,17β-dihydroxyandrost-4-en-3-one, 6β,14α-dihydroxyandrost-4-ene-3,17-dione, 15α,17β-dihydroxyandrost-4-en-3-one, 16β,17α-dihydroxyandrost-4-en-3-one and 2β,16β,17β-trihydroxyandrost-4-en-3-one, were fully characterised for the first time.     

Microbial oxidation of dehydroepiandrosterone and related compounds.

The oxidation of dehydroepiandrosterone (DHEA), 4-androstene-3, 17-dione, and estrone with Streptomyces roseochromogenes NRRL B-1233 was studied. The oxidation products were isolated and identified as as 16alpha-hydroxy-DHEA, 16alpha-hydroxy-4-androstene-3,17-dione and 16alpha-hydroxyestrone. The yields of these three products were 85%, 41% and 18%, respectively. This indicates the substrate stereospecificity of 16alpha-hydroxylase of the organism. An interrelationship between cell growth and the formation of 16alpha-hydroxylated steroid was observed in any case. For formation of 16alpha-hydroxy-DHEA, 16alpha-hydroxylase showed good activity at DHEA concentration of 3.47 x 10(-4)M. In the case of DHEA, 16alpha-hydroxy-4-androstene-3,17-dione and 5-androstene-3beta, 16alpha, 17beta-triol were obtained after the yield of 16alpha-hydroxy-DHEA reached the maximum yield for about 30 hr. The oxidation pathway of DHEA is discussed.     

The metabolism of steroids in the fatty liver induced by orotic acid feeding.

1. The metabolism of 4-[4-14C]androstene-3,17-dione, 4-[4-14C]pregnene-3,20-dione, 5alpha-[4-14C]androstane-3alpha,17beta-diol, [4-14C]cholesterol, 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one, 5beta-[7beta-3H]cholestane-3alpha,7alpha-diol and [3H]lithocholic acid was studied in the microsomal fraction of livers from control and orotic acid-treated male rats. 2. As a result of the treatment the orotic acid-fed rats had fatty livers and subnormal concentrations of cholesterol and triglycerides in serum. 3. The 6beta- and 7alpha-hydroxylation of 4-androstene3,17-dione, and the 2alpha-, 2beta- and 18-hydroxylation of 5alpha-androstane-3alpha,17beta-diol, and the 5alpha-reduction of 4-androstene-3,17-dione and 4-pregnene-3,20-dione were decreased by 40--50% in orotic acid-fed rats. Other oxidative and reductive reactions of the steroid hormones were not significantly affected. 4. The 12alpha-hydroxylation of 7alpha-hydroxy-4-cholesten-3-one was decreased by about 50%, whereas the 7alpha-hydroxylation of cholesterol and the 26-hydroxylation of 5beta-cholestane-3alpha,7alpha-diol were not significantly decreased. The 6beta-hydroxylation of lithocholic acid was stimulated by 40%. 5. The results are discussed in relation to present knowledge of the heapatic drug-metabolizing enzymes and to the recent findings of an abnormal bile acid metabolism in liver disease.     

HPLC analysis of PAMAM dendrimer based multifunctional devices

6-OXO, a new nutritional supplement commercially available on the internet, is sold as an aromatase-inhibitor and contains androst-4-ene-3,6,17-trione as active ingredient. This anabolic steroid is a prohibited substance in sports. Androst-4-ene-3,6,17-trione is metabolised to androst-4-ene-6alpha-ol-3,17-dione and androst-4-ene-6alpha,17beta-diol-3-one. A fast, sensitive and accurate LC/MS method was developed and validated for the quantification of androst-4-ene-3,6,17-trione and its metabolites in urine. The method is capable of determining the stereochemical position of the hydroxy-group at C-6 of the metabolites and consists of a liquid-liquid extraction step with diethylether after enzymatic hydrolysis, followed by separation on a reversed phase column. Ionisation of the analytes is carried out using atmospheric pressure chemical ionisation. The limit of quantification of the method was 5 ng/mL for all compounds. The accuracy ranged from 14.8 to 1.3% for androst-4-ene-3,6,17-trione, 9.4 to 1.6% for androst-4-ene-6alpha-ol-3,17-dione and 4.1 to 3.2% for androst-4-ene-6alpha,17beta-diol-3-one in the range of 5-1000 ng/mL. Using this method androst-4-ene-6alpha-ol-3,17-dione was identified as a major urinary metabolite, whereas androst-4-ene-6alpha,17beta-diol-3-one as a minor metabolite. While the parent compound is predominantly excreted in conjugated form, both metabolites are solely excreted as conjugates.     

Flexibility of the endogenous progesterone lactonisation pathway in Aspergillus tamarii KITA: transformation of a series of cortical steroid analogues

A range of cortical steroids have been transformed by the fungus Aspergillus tamarii, which has the ability to convert progesterone to testololactone in high yield through a four step enzymatic pathway. 16alpha,17alpha-Epoxyprogesterone underwent a rare epoxide opening resulting in a unique inversion of stereochemistry to give 16beta-hydroxy-17alpha-oxa-D-homo-androst-4-en-3,17-dione. The metabolism of deoxycorticosterone resulted in relatively efficient transformation to testololactone with no other products isolated. Transformation of 17alpha-hydroxyprogesterone yielded 17alpha-oxa-D-homo-androst-1,4-dien-3,17-dione, a lactone not previously isolated from A. tamarii. Cortexolone was transformed to the 20(R)-alcohol with no further transformation observed. Evidence is also presented for the presence of a highly flexible but stereospecific keto-reductase. All metabolites were isolated by column chromatography and were identified by 1H, 13C NMR, DEPT analysis and other spectroscopic data.     

Metabolism of 11-deoxycortisol by a Bacillus species

Microbial transformation by a Bacillus species was employed for the preparation of potentially important derivatives of 11-deoxycortisol. Each microbial metabolite was characterised by the application of various spectroscopic methods. The five metabolites of 11-deoxycortisol were characterised as 4-androstene-3,17-dione (2), 14-hydroxy-4-androstene-3,17-dione (3), 14,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (4), 6 beta,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (5) and 15 alpha,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (6). The availability of the metabolites enabled complete elucidation of their [13C]NMR spectra.     

Biotransformations of steroid compounds by Chaetomium sp. KCH 6651

Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14α-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6β-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6β position. Progesterone was transformed to a single product—6β,14α-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7α-hydroxy derivative, which was subsequently converted to 7α-hydroxyandrost-4-en-3,17-dione.     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria <Emphasis Type="Italic">Mycobacterium neoaurum,Pimelobacter simplex</Emphasis>, and <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5–15 μm) or the transformation in the presence of randomly methylated β-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The crude product ADD was obtained in 75% yield, based on phytosterol. It contained as by-products 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment—the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD—no by-products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5% AD was obtained. The yield of 9-OH-AD (m.p. 218–220°C) based on transformed phytosterol was 56%.