Testosterone as biotransformation product in steroid conversion byMycobacterium sp.

Summary Testosterone production byMyc. sp. NRRL B-3683 is discussed. The unexpected finding that testosterone is not formed by single reduction of 17-keto group of 4-androstene-3,17-dione (AD) but by a double reduction of both 17-keto group and 1–2 doble bound of 1,4-androstadiene-3,17-dione (ADD) is presented.     

1-Ene-steroid reductase of Mycobacterium sp. NRRL B-3805

The microbial enzymatic reduction of 1,4-androstadiene-3,17-dione (ADD) to 4-androstene-3,17-dione (AD), testosterone and 1-dehydrotestosterone (DHT) is described. Two reducing activities observed in washed cell suspensions and cell free extracts of Mycobacterium sp. NRRL B-3805 were found to account for these bioconversions. One was a 1-ene-steroid reductase and the other a 17-keto steroid reductase. The first reducing activity was found to appear in the soluble cell fraction whereas the latter could be precipitated by centrifugation. Maximum 1-ene-steroid reductase specific activity was achieved during the exponential growth phase of the organism and significantly increased upon induction with ADD. The 1-ene-steroid reductase was partially purified (30-fold) by ammonium sulfate fractionation, gel-filtration and ion-exchange chromatography, and was eluted from a Sephacryl S-300 column with an Mr = 115,000. The 1-ene-steroid reductase activity was NADPH-dependent and had specificity towards steroid compounds containing C-1,2 double bond with an apparent Km for ADD of 2.2 X 10(-5) M. The reverse reaction catalyzing C-1,2 dehydrogenation could not be detected in our preparations. The results suggest that in Mycobacterium sp NRRL B-3805 and B-3683 the steroid C-1,2 dehydrogenation and 1-ene reduction are two separable activities.     

Production of androsta-1,4-diene-3,17-dione from cholesterol using immobilized growing cells of Mycobacterium sp. NRRL B-3683 adsorbed on solid carriers

Summary Living cells of Mycobacterium sp. NRRL B-3683 were immobilized by adsorption on different types of solid carriers in order to produce androsta-1,4-diene-3,17-dione (ADD) from cholesterol. Activated alumina proved to be the most preferred carrier for long-term operation when glucose and peptone were added to the reaction medium. In a repeated-batch process, the maximum productivity of ADD was about 0.19 g/l per day with a molar conversion rate of 77% when 1.0 g/l of cholesterol was added to the reaction medium. The half-life of the immobilized cells was more than 45 days and the system could be reactivated by incubating the immobilized cells in a cell growth medium.     

Production of androsta-1,4-diene-3,17-dione from cholesterol using two-step microbial transformation

A novel two-step transformation process for the production of androsta-l by microorganisms-diene-3,17-dione (ADD) from a high concetration of cholesterol by microorganisms is proposed. Cholesterol (20 g/l) was initially converted to cholest-4-en-3-one (cholestenone) by an inducible cholesterol oxidase-producing bacterium, Arthrobacter simplex U-S-A-18. The maximum productivity of cholestenone was 8 g/l per day and the molar conversion rate was 80%. Subsequently, a fine suspension of cholestenone (50 g/l), which was prepared directly from the fermentation broth of A. simplex, was converted to ADD by Mycobacterium sp. NRRL B-3683 in the presence of an androstenone adsorbent, Amberlite XAD-7. The maximum productivity of ADD was 0.91 g/l per day and the molar conversion rate was 35%. Correspondence to: W.-H. Liu     

Spectrophotometric estimation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153

A spectrophotometric method for simultaneously estimating 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) in a binary mixture has been developed using sulphuric acid chromogens. The method has been used to estimate both AD and ADD during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153.The authors are with the School of Life Sciences, Devi Ahilya Vishwavidyalaya. Vigyan Bhawan, Khandwa Road, Indore-452 001, India.     

Conversion of liposomal 4-androsten-3,17-dione by A. simplex immobilized cells in calcium pectate

Arthrobacter simplex ATCC 6946 free and immobilized cells were assayed for their ability to convert 4-androsten-3,17-dione (AD) to 1,4-androstadien-3,17-dione (ADD) in aqueous and liposomal media. Bioconversions were carried out in a 100 ml flask containing 25 ml of AD liposomal or aqueous medium for 3h, and AD concentrations ranging from 0.3 to 1.0 mM were tested. AD/ADD ratios in samples were determined by HPLC. Biotransformation of substrate entrapped in multilamellar vesicles (MLV) was demonstrated to be better than the corresponding free form. In the former case, 2h were necessary to completely bioconvert 1 mM AD. By contrast, 3h were needed to reach 50% bioconversion in (4%) ethanol medium containing 0.63 mM AD. The liposomal medium allows us to perform steroid conversions at high concentrations of AD, reusing immobilized cells in suitable conditions which are non-toxic for microorganisms.     

Isolation of a biodegradable sterol-rich fraction from industrial wastes

Several industrial waste materials were screened for their sterol content. The possibility of using these industrial by-products as sterol sources for the microbiological production of 4-androsten-3,17-dione (AD) and 1,4-androsta-diene-3,17-dione (ADD) was investigated. Two methods of obtaining the sterol fraction from wastes were developed. Sterol-rich (96-98%) fractions were isolated in a yield above 70%, from a tall-oil effluent of a paper pulp industry and from edible-oil deodorizates. These fractions were subsequently used as a substrate for microbial degradation by a Mycobacterium sp. strain and proved to be easily converted to AD and ADD.     

Nostoc muscorum: a regioselective biocatalyst for 17-carbonyl reduction of androst-4-en-3,17-dione and androst-1,4-dien-3,17-dione

Nostoc muscorum PTCC 1636 was examined for its ability to convert androst-4-en-3,17-dione (AD) and androst-1,4-dien-3,17-dione (ADD) to their 17-hydroxy related derivatives in BG-11 medium. Bioconversion procedures were carried out at 25 °C without shaking. The metabolites obtained were purified using chromatographic methods and characterized as testosterone and 1-dehydrotestosterone on the basis of their spectroscopic features. In both cases, the bioreaction characteristics observed were 17-carbonyl reduction.     

Molecular and functional characterization of kshA and kshB,encoding two components of 3-ketosteroid 9alpha-hydroxylase,a class IA monooxygenase,in Rhodococcus erythropolis strain SQ1

9 alpha-Hydroxylation of 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) is catalysed by 3-ketosteroid 9 alpha-hydroxylase (KSH), a key enzyme in microbial steroid catabolism. Very limited knowledge is presently available on the KSH enzyme. Here, we report for the first time the identification and molecular characterization of genes encoding KSH activity. The kshA and kshB genes, encoding KSH in Rhodococcus erythropolis strain SQ1, were cloned by functional complementation of mutant strains blocked in AD(D) 9 alpha-hydroxylation. Analysis of the deduced amino acid sequences of kshA and kshB showed that they contain domains typically conserved in class IA terminal oxygenases and class IA oxygenase reductases respectively. By definition, class IA oxygenases are made up of two components, thus classifying the KSH enzyme system in R. erythropolis strain SQ1 as a two-component class IA monooxygenase composed of KshA and KshB. Unmarked in frame gene deletion mutants of parent strain R. erythropolis SQ1, designated strains RG2 (kshA mutant) and RG4 (kshB mutant), were unable to grow on steroid substrates AD(D), whereas growth on 9 alpha-hydroxy-4-androstene-3,17-dione (9OHAD) was not affected. Incubation of these mutant strains with AD resulted in the accumulation of ADD (30-50% conversion), confirming the involvement of KshA and KshB in AD(D) 9 alpha-hydroxylation. Strain RG4 was also impaired in sterol degradation, suggesting a dual role for KshB in both sterol and steroid degradation.     

Molecular characterization of three 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes of Rhodococcus ruber strain Chol-4

Rhodococcus ruber strain Chol-4 isolated from a sewage sludge sample is able to grow on minimal medium supplemented with steroids, showing a broad catabolic capacity. This paper reports the characterization of three different 3-ketosteroid-Δ(1)-dehydrogenases (KstDs) in the genome of R. ruber strain Chol-4. The genome of this strain does not contain any homologues of a 3-keto-5α-steroid-Δ(4)-dehydrogenase (Kst4d or TesI) that appears in the genomes of Rhodococcus erythropolis SQ1 or Comamonas testosteroni. Growth experiments with kstD2 mutants, either a kstD2 single mutant, kstD2 double mutants in combination with kstD1 or kstD3, or the triple kstD1,2,3 mutant, proved that KstD2 is involved in the transformation of 4-androstene-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) and in the conversion of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) to 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD). kstD2,3 and kstD1,2,3 R. ruber mutants (both lacking KstD2 and KstD3) did not grow in minimal medium with cholesterol as the only carbon source, thus demonstrating the involvement of KstD2 and KstD3 in cholesterol degradation. In contrast, mutation of kstD1 does not alter the bacterial growth on the steroids tested in this study and therefore, the role of this protein still remains unclear. The absence of a functional KstD2 in R. ruber mutants provoked in all cases an accumulation of 9OHAD, as a branch product probably formed by the action of a 3-ketosteroid-9α-hydroxylase (KshAB) on the AD molecule. Therefore, KstD2 is a key enzyme in the AD catabolism pathway of R. ruber strain Chol-4 while KstD3 is involved in cholesterol catabolism.     

Microbial transformation of sterols to C19-steroids by Rhodococcus equi

A wild-type strain of Rhodococcus equi, isolated from soil, degraded cholesterol, -sitosterol, stigmasterol and mixed sterois to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). A definite preference for a relatively simply structured cholesterol side chain was observed. Highest specific cholesterol side-chain cleavage was associated with active growth of the culture. Maximum yield of ADD was obtained when sodium acetate and cholesterol were incorporated together in the medium. Specific side-chain cleavage required the presence of 2,2-dipyridyl, an inhibitor of ring cleavage.S. Ahmad and B.N. Johri are with the Department of Microbiology, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantriagar 263 145, Nainital, UP, India. P.K. Roy, A.W. Khan and S.K. Basu are at Fermentation Technology Division, Central Drug Research Institute, Lucknow, India.     

Resistance to androstanes as an approach for androstandienedione yield enhancement in industrial mycobacteria

The resistance to androstandienedione (ADD) of industrial mycobacteria was demonstrated as a valuable approach to increasing ADD yield in sterol fermentations. Colonies growing at 1 mg/ml ADD in culture medium after nitrosoguanidine mutagenesis showed a differential behavior in respect to parentals in cholesterol biotransformation. In the presence of exogenous ADD, a substantial depletion of ADD production was observed in parental strains B3683 and Ex4, whereas it was unaffected, and even increased, in resistant colonies. An apparent reduction from ADD to androstandione and testosterone was also noticed. Furthermore, the ADD resistance phenotype may be related to the increase in steroid 1,2 dehydrogenase activity.     

Microbial transformations of testosterone to 5alpha-dihydrotestosterone by two species of Penicillium: P. chrysogenum and P. crustosum.

Two species of Penicillium--P. chrysogenum and P. crustosum--were cultured in presence of [3H]testosterone as a substrate. Both species were shown to reduce the 4,5-double bond in testosterone to give dihydrotestosterone (DHT). The steroids produced were 5alpha-dihydrotestosterone, DHT, 3alpha-hydroxy-5beta-androstan-17-one, 3alpha-hydroy-5alpha-androstan-17-one, 4-androstene-3,17-dione, and 5alpha-androstane-3,17-dione. These products implicate the presence of the 5alpha-reductase, with maximal activity at pH 6 and 8, in both species of Penicillium. The presence of DHT in the growth medium and not in the mycelium suggests that DHT is excreted into the medium.     

Efficient biotransformation of cholesterol to androsta-1,4-diene-3,17-dione by a newly isolated actinomycete <Emphasis Type="Italic">Gordonia neofelifaecis</Emphasis>

A newly isolated actinomycete, Gordonia neofelifaecis (NRRL B-59395) from the faeces of Neofelis nebulosa, was used to selectively degrade the side-chain of cholesterol. The intermediates were purified and characterized. Quantitative analysis of the accumulated metabolites from cholesterol side-chain cleavage was conducted during the biotransformation. The results showed that the profile of accumulated intermediates was different from those of other reported microorganisms. Among the five metabolites, androsta-1,4-diene-3,17-dione (ADD) was the main product of the side-chain degradation, with a high conversion rate (87.2%), indicating its potential for industrial production of ADD. At the end of transformation, the substrate cholesterol was completely consumed. The effect of some factors on the bioconversion was also investigated. To our best knowledge, this is the first report regarding cholesterol side-chain cleavage using bacteria belonging to Gordonia.     

Effect of inoculation strategies,substrate to biomass ratio and nitrogen sources on the bioconversion of wood sterols by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp.

4-Androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) are the main precursors in the production of steroidal drugs from phytosterols. To carry out the bioconversion, different inoculation strategies have been proposed. We compared the use of whole fermented broth and of free resting cells of two mutant strains of Mycobacterium sp. (DSMZ2966 and DSMZ2967) in shake flasks. Also the effect of the nitrogen source (ammonium sulfate, ammonium chloride and ammonium nitrate) and the sterol to biomass ratio at high substrate concentrations (19.2 g/l and 48.1 g/l) was evaluated. We found that the bioconversion with free resting cells (cell pellets) is more efficient than that with whole fermented broth, increasing both AD and ADD production. The use of ammonium nitrate in the culture medium and low substrate to biomass ratios (close to 1.0) increased the production yield. We also found that the bioconversion can be run at high substrate concentration under non-sterile conditions.     

Microbial conversion of solasodine to 1-androstene-3,17-dione (AD), a key intermediate for androgen synthesis

The spirosolane side chain of solasodine has been cleaved by cholesterol preinduced Mycobacterium sp. NRRL B-3805 to yield 1-androstene-3,17-dione (AD), a key intermediate for the synthesis of androgenic drugs. Conversion up to 34% has been recorded in shake flask culture after 192 h incubation period using dimethyl-formamide as carrier for solasodine addition.     

Microbial Degradation of Sterols

A process is described for the microbial degradation of cholesterol and plant sterols, to produce androsta-1, 4-diene-3, 17-dione and androst-4-ene-3, 17-dione, by two newly isolated bacteria designated Mycobacterium sp. NRRL B-3683 and Mycobacterium sp. NRRL B-3805. These myocbacteria produce substantial amounts of 17-ketonic compounds without appreciable degradation of the steroid nucleus. No ring degradation inhibitory agents are necessary. The first microbiological production of 20alpha-hydroxymethylpregna-1, 4-dien-3-one is also reported.     

Mycobacterium sp．高效转化植物甾醇为雄甾-1，4-二烯-3，17-二酮的诱变选育

采用紫外线、亚硝基胍复合诱变雄甾-4-烯-3,17-二酮（AD）和雄甾-1,4-二烯-3,17-二酮（ADD）的转化产生菌Mycobacterium sp．,结合平板筛选,获得一株遗传性状稳定单产ADD的突变菌株Mycobacterium sp．-11,其ADD质量浓度达到1246ms／L,比原始菌株（484mg／L）提高了150％,经初步优化后发酵液中ADD最高达到1430mg／L,发酵液中ADD质量占ADD、AD两产物质量总和的比例由70％提高到99．1％。     

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%.