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.     

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.     

Microbial transformation of phytosterols mixture from rice bran oil unsaponifiable matter by selected bacteria

Rice bran sample (12 Kg) was extracted and rice bran oil (RBO ≅ 76.8 g) was saponified. The resulted unsaponifiable matter of RBO (RBO unsap) was qualitatively and quantitatively estimated using different chromatographic analyses. RBO, produced 9.65% unsaponifiable matter with the following contents, cholesterol, 6.75%; stigmasterol, 3.4%; β. sitosterol, 10.23% and campesterol, 4.2%, in addition to unknown phytosterols, hydrocarbons and waxes. Microbial transformation process started by screening of 35 bacterial strains, locally isolated from rice bran, air and soil, using RBO unsap as a carbon and an energy source to produce some pharmaceutically useful C₁₈ and C₁₉ steroids. Moraxella ovis was the most potent isolate for its highest capability to utilize RBO unsap and selectively degrade the phytosterols side-chain producing androst-4-ene-3,17-dione (AD), androsta-1,4-diene-3,17-dione (ADD), testosterone (T) and estrone (E). The RBO unsap was the best carbon and energy source. Maximum production of the desired products was observed in 36 h, pH 7 and at 30°C by M. ovis.     

Steroidal Metabolites Transformed by <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> Cultures Block Breast Cancer Estrogen Biosynthesis

Suspension of cultured cells of Marchantia polymorpha have the potential to hydrogenate the olefinic bonds present in androst-1,4-dien-3,17-dione (boldione, 1) to afford dihydroandrost-3,17-dione derivatives including: androst-4-ene-3,17-dione (androstenedione, 4-AD, 2), 5α-androstane-3,17-dione (androstenedione, AD, 4), and the less abundant metabolite 5α-androst-1-ene-3,17-dione (1-androstenedione, 1-AD, 3). After isolation and purification, these metabolites were characterized on the basis of spectroscopic analyses using 1D and 2D NMR as well as mass spectrometry. Cytotoxicity of the biotransformation products against breast adenocarcinoma cells (MCF-7) was assessed by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and cell death (apoptosis or necrosis) was assayed by acridine orange/ethidium bromide staining. Aromatase (cytochrome P450 19 enzyme, CYP19) inhibitory activity was measured by a tritiated water release assay and by direct measurement of bio-transformed steroids using the tritium labeled substrate ³H-androst-4-ene-3,17-dione. CYP19 mRNA expression in MCF-7 cells was analyzed by real-time PCR. Steroidal products 3 and 4 revealed a highly significant inhibition of MCF-7 cell growth that was predominantly due to apoptosis not necrosis. Steroidal products 3 and 4 are both potent inhibitors of aromatase activity and CYP19 mRNA expression, while 2 is a known substrate for aromatase. These data establish that metabolites 3 and 4 are potent chemical agents against breast cancer via aromatase inhibitory mechanism. Results were interpreted via virtual docking of the biotransformation products to the human placental aromatase active site.     

Mycobacterium sp. mutant strain producing 9α-hydroxyandrostenedione from sitosterol

Mycobacterium sp. VKM Ac-1815D and its derivatives with altered resistance to antibacterial agents were able to produce androst-4-ene-3,17-dione (AD) as a major product from sitosterol. In this study, those strains were subjected to subsequent mutagenization by chemical agents and UV irradiation in combination with sitosterol selection pressure. The mutant Mycobacterium sp. 2-4 M was selected, being capable of producing 9-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol, with a 50% molar yield. Along with 9-OH-AD, both AD and 9-hydroxylated metabolites with a partially degraded side-chain were formed from sitosterol by the mutant strain. The strain was unable to degrade 9-OH-AD, but degraded androsta-1,4-diene-3,17-dione (ADD), thus indicating a deficiency in steroid 1(2)-dehydrogenase and the presence of 9-hydroxylase activity.     

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.     

Microbiological hydroxylation of androst-1,4-dien-3,17-dione by Neurospora crassa

Nine hydroxy-derived androstadiene compounds were isolated from the fermentation broth of Neurospora crassa when incubated in the presence of androst-1,4-dien-3,17-dione (ADD; I) for 7 days. Hydroxylations at 6β, 7β, 11α, 14α- positions and 17-carbonyl reduction of the substrate were the characteristics observed in this biotransformation. Their structures were determined by spectroscopic methods as 17β-hydroxyandrost-1,4-dien-3-one (II), 14α-hydroxyandrost-1,4-dien-3,17-dione (III), 6β-hydroxyandrost-1,4-dien-3,17-dione (IV), 11α-hydroxyandrost-1,4-dien-3,17-dione (V), 6β,17β-dihydroxyandrost-1,4-dien-3-one (VI), 7β-hydroxyandrost-1,4-dien-3,17-dione (VII), 14α,17β-dihydroxyandrost-1,4-dien-3-one (VIII), 6β,14α-dihydroxyandrost-1,4-dien-3,17-dione (IX), and 11α,17β-dihydroxyandrost-1,4-dien-3-one (X). A new steroid substance, 6β,14α-dihydroxyandrost-1,4-dien-3,17-dione (IX), was also characterized during this study. The best fermentation condition was found to be 7-day incubation at 25°C and pH values of 5.0–6.0 in the presence of 0.05 g 100 mL^?1 of the substrate. At a concentration above 0.075 g 100 mL^?1, the biotransformation was completely inhibited.     

Bioconversion of lipophilic compounds by immobilized microbial cells in organic solvents

Microbial cells were gel-entrapped with photo-crosslinkable resin prepolymers or urethane prepolymers, respectively. The resulting gels have different tailor-made hydrophobic or hydrophilic character. They were used for successful bioconversion of hydrophobic steroids and terpenoids in watersaturated mixtures of organic solvents. The experiments show the influence of the hydrophobicity of the gels and the polarity of the solvent mixtures, respectively. Use of hydrophobic gels and less polar solvents is preferable for bioconversion of hydrophobic compounds. The selective formation of a desired product among diverse products from a single substrate by appropriate use of hydrophobic or hydrophilic gels is possible. In each case, tests should be made to select the appropriate gel and solvent mixture. Bioconversions tested are: dehydroepiandrosterone to 4-androstene-3,17-dione; cholesterol to cholestenone; β-sitosterol to β-sitostenone; stigmasterol to stigmastenone; pregnenolone to progesterone; testosterone to Δ¹-dehydrotestosterone or 4-androstene-3,17-dione, respectively; all with immobilized cells of Nocardia rhodocrous; and stereoselective hydrolysis of dl-menthyl-succinate to yield l-menthol with immobilized cells of Rhodotorula minuta var. texensis.     

Entrapment of microbial cells and organelles with hydrophilic urethane prepolymers

Summary Microbial cells and cellular organelles were immobilized by mixing aqueous suspensions of the biocatalysts with water-miscible urethane prepolymers. Thus immobilized preparations of acetone-dried cells of Arthrobacter simplex and thawed cells of Nocardia rhodocrous showed appreciable {ie351-1} activities in the transformation of hydrocortisone into prednisolone and 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione, respectively. The activities of catalase and alcohol oxidase were observed in the immobilized peroxisomes (microbodies) of a methanol-grown yeast Kloeckera sp. No. 2201. Yeast mitochondria entrapped with the prepolymer showed adenylate kinase activity. These results indicate the usefulness of the urethane prepolymers as convenient materials for entrapment of not only enzymes, but also organelles and microbial cells.     

Production of 11α-hydroxysteroids from sterols in a single fermentation step by Mycolicibacterium smegmatis

11α-hydroxylated steroid synthons are one of the most important commercially pharmaceutical intermediates used for the production of contraceptive drugs and glucocorticoids. These compounds are currently produced by biotransformation using fungal strains in two sequential fermentation steps. In this work, we have developed by a rational design new recombinant bacteria able to produce 11α-hydroxylated synthons in a single fermentation step using cholesterol (CHO) or phytosterols (PHYTO) as feedstock. We have designed a synthetic operon expressing the 11α-hydroxylating enzymes from the fungus Rhizopus oryzae that was cloned into engineered mutant strains of Mycolicibacterium smegmatis that were previously created to produce 4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD) from sterols. The introduction of the fungal synthetic operon in these modified bacterial chassis has allowed producing for the first time 11αOH-AD and 11αOH-ADD with high yields directly from sterols in a single fermentation step. Remarkably, the enzymes of sterol catabolic pathway from M. smegmatis recognized the 11α-hydroxylated intermediates as alternative substrates and were able to efficiently funnel sterols to the desired hydroxylated end-products.     

Studies on the Microbiological Transformation of Steroids

An attempt was made to clarify how Pellicularia filamentosa f. sp. microsclerotia IFO 6298 capable of hydroxylating C₂₁-steroids at the C-19 position converts C₁₉-steroids, especially monohydroxyderivatives of androst-4-ene-3, 17-dione. Such substrates as 11β-hydroxyandrost-4-ene-3,17-dione (I), androst-4-ene-3, 11, 17-trione (II), androsta-1,4-diene-3, 17-dione (III), 11β-hydroxyandrosta-1,4-diene-3,17-dione (IV), 14α-hydroxyandrost-4-ene-3, 17-dione (V), 15α-hydroxyandrost-4-ene-3, 17-dione (VI) and 9α-hydroxyandrost-4-ene-3, 17-dione (VII) were converted by the organism. All the main and several minor products were then isolated and identified. As a result it is concluded that this organism converts I and II into 14α-hydroxyandrost-4-ene-3,11,17-trione, III and IV into 14α-hydroxyandrosta-1,4-diene-3,1l,17-trione, V into 11α 14α dihydroxyandrost-4-ene-3, 17-dione (main) and 11β, 14α-dihydroxyandrost-4-ene-3, 17-dione (minor, a tentative structure), VI into 11β, 15α-dihydroxyandrost-4-ene-3,17-dione (main) and 15α-hydroxyandrost-4-ene-3,11,17-trione (minor, a tentative structure) and VII into 9α, 14α-dihydroxyandrost-4-ene-3, 17-dione (main) and 6β, 9α-dihydroxyandrost-4-ene-3,17-dione (minor).

In addition, the structural requirement of substrate for the 19-hydroxylation catalyzed by the organism and the influence of a hydroxyl group on steroid nucleus upon the 11β- and 14α-hydroxylations and the 11β-OH-dehydrogenation was discussed.     

Identification and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum

Mycobacteria have been modified to transform sterols to produce valuable steroids. Here, we demonstrated that the oxidation of sterols to sterones is a rate-limiting step in the catabolic pathway of sterols in Mycobacterium neoaurum. Two cholesterol oxidases ChoM1 and ChoM2 involved in the step were identified in M. neoaurum and the ChoM2 shared up to 45% identity with other cholesterol oxidases. We demonstrated that the combination of ChoM1 and ChoM2 plays a significant role in this step. Accordingly, we developed a strategy to overcome this rate-limiting step by augmenting the activity of cholesterol oxidases in M. neoaurum strains to enhance their transformation productivity of sterols to valuable steroids. Our results indicated that the augmentation of ChoM2 achieved 5.57 g/l androst-1,4-diene-3,17-dione in M. neoaurum NwIB-01MS and 6.85 g/l androst-4-ene-3,17-dione in M. neoaurum NwIB-R10, greatly higher than the original yield, 3.87 g/l androst-1,4-diene-3,17-dione and 4.53 g/l androst-4-ene-3,17-dione, respectively.     

Microbial 16β-Hydroxylation of Steroids with Aspergillus niger

Microbial 16β-hydroxylation of some steroids with Wojnowicia graminis, Corticium centrifugum and Bacillus megaterium has been reported, but not 16β-hydroxylation of normal 17-oxo steroids with Aspergillus niger. This time, we tried microbial transformation of dehydroepiandrosterone with this fungus, and obtained 4-androstene-3,17-dione, 17β-hydroxy-4-androstene-3,16-dione, 16β,17β-dihydroxy-4-androsten-3-one and a new compound, 16β-hydroxy-4-androstene-3,17-dione. This new compound was also obtained by the fermentation of 4-androstene-3,17-dione and testosterone.     

植物甾醇微生物转化制备甾体药物中间体的研究进展

微生物选择性降解植物甾醇侧链获取甾体药物合成的重要中间体雄甾-4-烯-3,17-二酮（4-AD）和雄甾-1,4-二烯-3,17-二酮（ADD）对于我国制药行业具有重要意义。现存文献资料对该领域缺乏全面系统的分析总结,从甾醇侧链微生物转化的机理、途径及其收率的影响因素等几个方面综述了近几年的研究进展,并对此领域的发展趋势进行了展望。     

Role of Neutral Metabolites in Microbial Conversion of 3β-Acetoxy-19-Hydroxycholest-5-Ene into Estrone

Biotransformation of 3β-acetoxy-19-hydroxycholest-5-ene (19-HCA, 6 g) by Moraxella sp. was studied. Estrone (712 mg) was the major metabolite formed. Minor metabolites identified were 5α-androst-1-en-19-ol-3,17-dione (33 mg), androst-4-en-19-ol-3,17-dione (58 mg), androst-4-en-9α,19-diol-3,17-dione (12 mg), and androstan-19-ol-3,17-dione (1 mg). Acidic metabolites were not formed. Time course experiments on the fermentation of 19-HCA indicated that androst-4-en-19-ol-3,17-dione was the major metabolite formed during the early stages of incubation. However, with continuing fermentation its level dropped, with a concomitant increase in estrone. Fermentation of 19-HCA in the presence of specific inhibitors or performing the fermentation for a shorter period (48 h) did not result in the formation of acidic metabolites. Resting-cell experiments carried out with 19-HCA (200 mg) in the presence of α,α′-bipyridyl led to the isolation of three additional metabolites, viz., cholestan-19-ol-3-one (2 mg), cholest-4-en-19-ol-3-one (10 mg), and cholest-5-en-3β,19-diol (12 mg). Similar results were also obtained when n-propanol was used instead of α,α′-bipyridyl. Resting cells grown on 19-HCA readily converted both 5α-androst-1-en-19-ol-3,17-dione and androst-4-en-19-ol-3,17-dione into estrone. Partially purified 1,2-dehydrogenase from steroid-induced Moraxella cells transformed androst-4-en-19-ol-3,17-dione into estrone and formaldehyde in the presence of phenazine methosulfate, an artificial electron acceptor. These results suggest that the degradation of the hydrocarbon side chain of 19-HCA does not proceed via C₂₂ phenolic acid intermediates and complete removal of the C₁₇ side chain takes place prior to the aromatization of the A ring in estrone. The mode of degradation of the sterol side chain appears to be through the fission of the C₁₇-C₂₀ bond. On the basis of these observations, a new pathway for the formation of estrone from 19-HCA in Moraxella sp. has been proposed.     

Cholesterol biotransformation to androsta-1,4-diene-3,17-dione by growing cells of <Emphasis Type="Italic">Chryseobacterium gleum</Emphasis>

Cholesterol oxidase activity was studied during biotransformation of cholesterol to androsta-1,4-diene-3,17-dione (ADD) by Chryseobacterium gleum. Spent LB media, containing cholesterol (3 mM≈1 g l⁻¹) where the bacterium was grown for 24 h, at 30°C with constant shaking at 120 rpm, had the highest enzyme activity (167 U mg⁻¹). The growing cells produced 0.076 g ADD from 1 g cholesterol l⁻¹.     

Preparation of androsta-1,4-diene-3,17-dione from sterols using <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis> VKPM Ac-1656 strain

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM Ac-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI₃ in situ.     

Hormonal steroids in the tissue of induced rat mammary tumours

The possible presence of steroids in the tissue of induced hormone-dependent rat mammary tumours was investigated. The method used involves a preliminary extraction of tumours followed by chemical separation and thin-layer chromatography. The identified compounds were cholesterol, androst-4-ene-3,17-dione, 5β-androst-1-ene-3,17-dione, androsta-1,4-diene-3,17-dione and oestrone. This is the first report of the presence of these steroids in the tissue of an experimental tumour of a non-endocrine organ. In particular 5β-androst-1-ene-3,17-dione has not previously been identified from natural sources.     

Biosynthesis of androgens and pheromonal steroids in neonatal porcine testicular preparations

The biosynthesis of testosterone and 4-androstene-3,17-dione and some 16-androstenes has been studied in homogenates or subcellular fractions of testes from 3-week-old Landrace piglets. Pregnenolone was converted into 5,16-androstadien-3 beta-ol, 4,16-androstadien-3-one, 5 alpha-androst-16-en-3-one and 5 alpha-androst-16-en-3 alpha- and 3 beta-ols, but the quantities were some 50 times less than those formed in the mature boar testis. Androgens were also formed in the microsomal fractions but the quantities of 4-androstene-3,17-dione (from side-chain cleavage of 17-hydroxyprogesterone) and of testosterone (from reduction of 4-androstene-3,17-dione) were 50-70 times lower than in the adult animal. The kinetic parameters and cofactor preference of the 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were determined in the cytosolic, microsomal and mitochondrial fractions of neonatal porcine testes.     

Fermentation kinetics of free and immobilized Penicillium raistrickii able perform the 15α-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione

Growing Penicillium raistrickii i 477 cells immobilized by microencapsulation, entrapment in calcium alginate beads and photopolymerization were used for the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione (I) to 15α-hydroxy-13-ethyl-gon-4-en-3,17-dione (II). The immobilized cells had lower maximum specific growth rates and yield coefficients when cultivated on the carbon source glucose than the non-immobilized cells, which leads to lower volumetric productivities than the use of nonimmobilized cells. However, the cells immobilized by microencapsulation and calcium alginate entrapment showed a specific productivity equal to that of the respective non-immobilized cells based on product formation per dry biomass and time. Photommobilized cells were not able to grow in the presence of the steroid because the substrate concentrations within the polymer reached inhibiting amounts for growth and product formation. In the absence of the steroid, the growing photoimmobilized cells showed a prolonged lag-phase in comparison with the free cells.