ELSEVIER

Review
Advances in microbial steroid biotransformation
Shashi B. Mahato and Subhadra Garai
Indian Institute of Chemical Biology, Calcutta, India Microbial biotransformations of various steroids are reviewed. Developmental studies on hydroxylation, carboncarbon bond cleavage, enzymatic catalysis in nonaqueous solvents, use of cyclodextrin medium, cell immobilization, and new microbial reactions are highlighted. Various steroid substrates, their metabolites and the microorganisms used for the transformations are compiled covering the literature for the period 1992-1995. (Steroids 62:332-345, 1997) 1997 by Elsevier Science Inc.

Keywords: microbial

biotransformation; review; substrate; metabolite; compilation

Introduction
The importance of microbial biotechnology in the production of steroid drugs and hormones was realized for the first time in 1952 when Murray and Peterson of Upjohn Company patented the process of 1 la-hydroxylation of progesterone by a Rhizopus speciesJ Since then, microbial reactions for the transformation of steroids have proliferated, and specific microbial transformation steps have been incorporated into numerous partial syntheses of new steroids for evaluation as drugs and hormones. A variety of steroids are widely used as anti-inflammatory, diuretic, anabolic, contraceptive, antiandrogenic, progestational, and anticancer agents as well as in other applications. Although introductions of new steroids into commerce is now limited, our interest lies in improvement in the yields of desired metabolites as well as preparation of novel steroids that are difficult to synthesize by chemical means. The areas of microbial biotechnology that are now receiving attention are: application of the newer concepts of genetic engineering of microorganisms for their improvement as steroid-transforming agents; solubility improvement for carrying out biotransformation of substrates that are sparingly soluble in water; immobilization of enzymes or whole cells in a suitable matrix for repetitive economic utilization of enzymes; development of a continuous process for economic product recovery; and manipulation of culture media for improvement in product yields by use of cyclodextrin. Our previous rev i e w s25 - covered literature of the period 1979-mid-1992.

This review attempts to present the situation during the period from late 1992 to 1995.
Hydroxylation

Address reprint requests to Dr. Shashi B. Mahato, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta-700032, India. Received August 19, 1996; accepted November 14, 1996. Steroids 62:332-345, 1997 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

The lloL-, 1113-, and 16c~-hydroxylations are now exclusively achieved in the steroid industry by microbial transformations operating at high yield and controlled costs. However, developmental activities for further cost reduction in these transformations are sometimes reported. The other hydroxylations that seem to have potential for industrial exploitation are 9o~- and 14ot-hydroxylations. Russian workers 6 studied the 1113-hydroxylation of cortexolone by the mycellium of Curvularia lunata VKMF-644 in the presence of 13-cyclodextrin (13-CD). The ratios of cortexolone to [3-CD varying from 1.0:0.1 to 1.0:1.0 were used. The yield of hydrocortisone by cortexolone biotransformation at a concentration of 4 g/L (the ratio of cortexolone to [3-CD was 1.0:0.6) was 70-75%. When 13-CD was not used, the yield was 40-45%. No stimulation of the process was observed when the mycellium immobilized in Ca-alginate gel was used as a biocatalyst. Stabilization of steroid 1 lhydroxylation activity of Cunninghamella elegans l~rotoplasts in organic osmotic stabilizers has been reported.' Protoplasts of C. elegans showing 1 lo~-, and 1113-hydroxylating ability of substance S (cortexolone) preserved high transformation activity when dispersed in glucose-enriched organic osmotic stabilizers. The observation is interesting and requires further investigation. 14o~-hydroxyandrost-4-ene-3,17-dione is a useful intermediate for the preparation of hormones. A patent has been taken out 8 for the manufacture of this product by fermentation of androst-4-ene-3,17-dione (AD) with a strain of Myrothecium striatisporum. Another patent was taken 9 for
0039-128X]97/$17.00 PII S0039-128X(96)00251-6

Biotransformation of steroids: Mahato and Garai

manufacture of the same product by fermentation of the same substrate but using a number of different microbial strains. 613,14ot-Dihydroxyandrost-4-ene-3,17-dione and 14ct-hydroxyandrost-4-ene-3,6,17-trione possess androgen activity and are useful inhibitors for breast cancer cells. The process for production of the diketone as well as the triketone has been patented. 1 The process involves culturing a microorganism belonging to the genus Myrothecium in a medium supplemented with AD and isolating the diketone from the culture medium. The diketone is then converted to the triketone in a simple manner. Another process for the preparation of the triketone has also been patented. 1~ The process comprises fermentation of AD with Acremonium strictum NN 106 to yield 14o~-hydroxy-AD. Alkoxylation of ketone group at the 3 position of the latter led to the formation of 3-alkoxy-14a-hydroxyandrosta-3,5-dien-17-one, which was subsequently oxidized by oxidizing agents to the desired product. 9oL-Hydroxyandrost-4-ene-3,17-dione is a useful intermediate that is easily dehydrated into the 9,11-dehydro system. Such 9-halogenated corticoids as well as 11-keto structures can be produced without the traditional l l hydroxylations. Microbial processes are now available for production of this intermediate. A process for manufacture of the compound by fermentation of a sterol mixture containing [3-sitosterol, campesterol, and stigmasterol with Mycobacterium fortuitum has been patented. ~2 Thus, the microorganism was cultured aerobically in 2.5 L medium containing 22.5 g sterol mixture and polyethylene polypropylene glycol for 72 h. The product (yield 31.1%) was isolated from the culture medium using adsorbent at the end of the fermentation. Another patent of similar nature has been taken for the production of the compound. ~3 According to this process, Mycobacterium fortuitum was cultured for 72 h in a medium containing sitosterol and an ethylvinyl benzene-divinylbenzene resin coated with the polyethylenepolypropylene oxide adduct of N, N"-tetra (oxypropylenoxide) N'-(2-oxo-propyl) diethylene-triamine. The isolation and purification of the compound from the fermentation broth was facilitated by including a surfactant-coated adsorbent in the medium. M i c r o b i a l t r a n s f o r m a t i o n of 3[3-hydroxy-5c~-Hpregnanes to their 9et-hydroxy-4-ene-3-keto derivatives has also been reported. 14 Biotransformation of 5et-H-steroids at 0.5-3.0 g/L by a wild Rhodococcus species strain isolated from the soil resulted in the formation of the corresponding 4-ene-3-keto and 9et-hydroxy-4-ene-3-keto analogs. The conversion of 5(x-H-steroids into 9et-hydroxy derivatives as final products depended on the functionality of the D-ring. Transformation of sitosterol to 9ot-hydroxyandrost-4-ene3,17-dione by Mycobacterium sp. 207 cells in the presence of an adsorption resin has been published. ~5 A bioreactor containing an adsorption resin and with a two-stage axialflow turbine as a stirrer was used. The preparative yield of the product was 51%, and the adsorbent could be used repeatedly. the 22-double bond to suitable starting structures, sitosterol, campesterol, and cholesterol were considered waste products because of the lack of economic routes for the cleavage of their saturated side chain. Development of the selective microbial degradation of the sterol side chain helped the utilization of these cheap and readily available natural products. However, continued attempts are being made for further cost reduction in the process. A process for conversion of sterols to androsta-1,4-diene-3,17-dione (ADD) has been patented.16 The process involves two-phase culture of Mvcobacterium vaccae ZIMET 11094. Strains and culture condition were carefully standardized to ensure the prevention of inhibition of the sterol side chain degradation by ADD. An adsorbent resin was used in the final culture medium, and an aqueous suspension of sterol (10 g/L) was added. The product ADD was recovered from the adsorbent by elution with MeOH. Liu reported the transformation of cholesterol to testosterone by a Mycobacterium sp. 17 A supplement of 4% glucose and 1% peptone into a synthetic medium, pH control of 6.0, and continuous feeding of cholesterol were the most important parameters. Under optimal conditions, the maximum molar conversion rate of testosterone from cholesterol was upto 42.68% in a 2.5 L surface-aerated fermentor after 100 h cultivation. Testosterone was isolated from the fermentation broth by the addition of XAD-7 resin. Testosterone production by Mycobacterium sp. NRRLB-3683 has been discussed by Hung et al. TM It has been shown that testosterone is not formed by single reduction of the 17-keto group of AD but by a double reduction of both the 17-keto group and the 1-2 double bond of ADD. Bioconversion of sitosterol to AD by mutants of Mycobacterium vaccae has been reported by Spassov et al. ~9 These mutants were resistant to rifampicin and streptomycin. Production of ADD from cholesterol using two-step microbial transformation has been reported. 2 Cholesterol (20 g/L) was initially converted to cholestenone by an inducible cholesterol oxidase-producing bacterium, Arthrobacter simplex U-S-A-18. The maximum productivity of cholestenone was 8 g/L-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 to ADD by Mycobacterium sp. NRRLB3683 in the presence of an androstenone adsorbent, Amberlite XAD-7. The maximum productivity of ADD was 0.91 g/L-day, and the molar conversion rate was 35%.

Mixed culture
Developmental studies are continuing for conducting two or more microbial steps in one fermentation using mixed cultures. In mixed culture fermentation, it is essential that the two microorganisms are capable of inducing the desired enzymes in one another's presence. Transformation of cortexolone into prednisolone by mixed culture has been reported. 2~ Curvularia lunata was the most active organism for 11 [3-hydroxylation of cortexolone to cortisol; whereas, Mycobacterium smegmatis was the most potent bacterium converting the latter to prednisolone. A mixed culture of C. lunata grown for 48 h and allowed to transform cortexolone for another 24 h and M. smegmatis was a combination that

Carbon-Carbon bond cleavage

Although stigmasterol has long been used as a starting material for its chemical modification by oxidative cleavage of

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Review
gave 51% yield of prednisolone. The yield of the product was markedly affected by the composition of the fermentation medium. Some nutritional requirements for the mixed culture have been described. 22 Transformation of cortexolone into prednisolone in a single-step fermentation using immobilized mixed culture, entrapped in different gels or adsorbed on clay particles has also been reportedY Calcium alginate at a gel concentration of 2% gave the highest transformation activities and prednisolone yields. The entrapped mixed cultures could be repeatedly used in batch-wise transformation for at least six times when suspended in diluted nutrient medium and for three times when suspended in distilled water. In the latter case, the entrapped cultures had to be reactivated in nutrient medium for future use. Mixed cultures adsorbed on clay particles were successfully reused 18 times with reactivation after the ninth and fifteenth uses. Continuous transformation of cortexolone into prednisolone by mixed cultures adsorbed on clay particles was more efficient than batch-wise reused adsorbed cultures. vents was investigated. 31 The highest degradation activities were obtained with cells immobilized in celite with bis (2ethylhexyl) phthalate as the conversion medium. A review has been published 32 that deals with biocatalysis with enzymes and microorganisms immobilized by entrapment in lyotropic liquid crystals and substrates dissolved in a co-existing solvent phase from which products may be isolated. Examples are given for the continuous enzymic production of (R)-cyanohydrins and for the microbial degradation of steroids. A new method for the microbial transformation on interface between hydrophilic carriers and hydrophobic organic solvents has been proposed. 33 Microorganisms were placed on the interface between such hydrophilic carriers as agar and hydrophobic organic solvents. These microorganisms could catalyze various transformations of such synthetic substrates as hydrolysis, esterification, oxidation, and reduction more efficiently than emulsion systems. This new type of bioreactor was tentatively named an "interface bioreactor.' '

Enzymatic catalysis in nonaqueous solvents

The application of nonaqueous environment for enzymatic catalysis has received much attention in recentyears. Excellent reviews are available on this aspect. 24 '2 J Enzymes acquire remarkable novel properties such as enhanced stability, altered substrate specificity, and ability to catalyze new reactions. 26 However, progress on the application of these promising features is restricted so far, because our fundamental understanding of this area is very limited. Zaks and Klibanov 27 studied the dependence of catalytic activity of several unrelated enzymes in a number of organic solvents as a function of the water content. From their experiment on replacement of water with other hydrogen bondforming additives, and titration of enzyme amino acids in an organic medium, as well as the literature on dehydrated enzymes, they concluded that the water required by enzymes in nonaqueous solvents provides them with sufficient conformational flexibility needed for catalysis. The results of a study on the control of enzyme enantioselectivity by the reaction medium by Sakurai et al. 28 opined that predictable and rational control of enantioselectivity of enzymes by changing the reaction medium rather than the enzyme itself is possible. The effect of cell immobilization and organic solvents on sulfoxidation and steroid hydroxylation by Mortierella isabellina ATCC 42613 was investigated by Holland et al. 29 They observed that the 14a-hydroxylation of progesterone and the sulfoxidation of thioanisole proceeded in high yield using resting-cell bioconversions but were not carried out by alginate bead preparations in the absence of an organic solvent. The best results were obtained with 5 or 10% aqueous methanol, and the stereoselectivity of sulfoxidation of thioanisole was found to be dependent upon the nature and concentration of organic co-solvent. A review on the recent development of microbial sterol biotransformation systems 3 particularly emphasizes the new enzymic approach of investigating the cleavage of sterol side chains, new developments in the area of immobilized cell systems, and use of organic media. Sterol side-chain cleavage with immobilized Mycobacterium cells in water-immiscible organic sol-

Cyclodextrin medium
Cyclodextrins (CD) form inclusion compounds that are biocompatible with microbes. This property prompted their use in microbial transformation of water-insoluble organic compounds. An intensive and systematic investigation of the oxidation of cholesterol to cholest-4-en-3-one by Rhodococcus erythropolis was undertaken in the presence of natural and chemically modified CDs in a stirred bioreactor. 34 The biotransformation was strongly affected by the mode of addition of the natural CDs. Although simultaneous addition of cholesterol with either 13- or -/-CD led to a limited enhancement effect, the microbial oxidation of [3- or ~-CD complexes of cholesterol was totally inhibited. In contrast, the alkylated CDs, dimethyl-, trimethyl-, and hydroxy-, propyl-13-CD exhibited a remarkable enhancement of the microbial oxidation, irrespective of their mode of addition. A comparative study was conducted by the same authors 35 on two biocatalysts, resting R. erythropolis cells and soluble cholesterol oxidase, both catalyzing cholesterol oxidation in cyclodextrin medium. The enzyme-mediated sterol oxidation was clearly enhanced by the dimethylated 13-CD, as in the microbial oxidation. However, the microbial transformation was subject to a larger enhancement effect than the enzymic one with respect to corresponding transformation without CD. The larger dimethylated 13-CDinduced effect exerted on the microbial system was attributed to the stronger affinity of dimethylated [3-CD to the microbial cell. This CD--cell interaction was thought to be manifested through a slightly inhibited microbial growth and a limited leakage of cellular proteins and cholesterol oxidase, Alekhina et al. 36 studied microbiological transformation of steroid-13-CD inclusion compounds. They observed that [3-CD at 7-8 g/L promoted 1,2-dehydrogenation of hydrocortisone to prednisolone and of 6a-methyl hydrocortisone to 6c~-methylprednisolone by Corynbacterium

simplex.
A review on the use of cyclodextrins in bioconversions in plant cell biotechnology has appeared. 37 The application of cyclodextrins as precursor solubilizers in biotechnology

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processes, in which plant cells are used, is new. The paper describes the use of CD in the bioconversions by freely suspended and/or immobilized plant cells or plant enzymes. After complexation with [3-CD, the phenolic steroid, 1713estradiol could be ortho-hydroxylated into a catechol, mainly 4-hydroxyestradiol by a phenoloxidase from grown cells of Mucuna pruriens. The glucosylation of podophyllotoxin by cell cultures from Linumflavum was also investigated. Four cyclodextrins, [3-CD, ~,-CD, hydroxypropyl13-CD, and dimethyl-[3-CD were used to improve solubility of podophyllotoxin. Dimethyl-13-CD met the needs the best, and the solubility of podophyllotoxin could be enhanced. Podophyllotoxin-13-D-glucoside was formed by the L. flavam cells growing in the presence of podophyllotoxin, complexed with dimethyl-[3-CD. markedly enhance the bioconversion rate with the existence of a hydrophobic layer, most likely resulting from the increasing portion of substrate to gel matrix. Bioconversion with higher substrate concentration was possible using ethyl acetate water medium. The conversion rate increased almost linearly with increasing substrate concentration from 10 to 80 g/L. The rate with 80 g/L progesterone increased up to six times greater than the rate with the immobilized cells without coating. The method could be performed under mild conditions. It has potential for industrial application.

New microbial reactions and novel metabolites

Although introduction of new steroids into commerce is now limited, there are continuing reports on new microbial reactions as well as the production of new steroids by microbial transformation that are otherwise difficult to synthesize. Murohisa and lida 41 reported production of four new key intermediates in the microbial degradation of cholesterol and campesterol sitosterol side chains. Exposure of 19-hydroxycholesterol and 19-hydroxycampesterol to Rhodococcus mutant K-3 afforded four new steroid carboxylic acids (3-6) in addition to estrone. The formation of these products helped to propose a degradation pathway for 19hydroxysterols. Two novel microbial conversion products of progesterone derivatives were obtained by Krischenowski and Kieslich 42 during screening of 131 microorganisms under aerobic conditions. While fermentation of 17~-hydroxyprogesterone with Co~nespora melonis CBS 16260 yielded 8[3,17~x-dihydroxyprogesterone (7), fermentation of 17o~-acetoxyprogesterone furnished 1113,1213dihydroxy-17e~-acetoxyprogesterone (8) in addition to the known l113-hydroxy compound. Incubation of 3[3acetoxycholest-5-ene-19-ol with Moraxella sp. gave three neutral metabolites (9-11) of which compound 9 was unknown. 43 Acidic metabolites were not formed. Thus, the authors suggested that complete removal of C17-side chain takes place prior to aromatization of the ring A in 11. Murohisa and Iida44 isolated a mutant H-45 of Rhodococcus equi K-3, which produced the new compound 3-oxostigmasta-1, 4-dien-26-oic acid (12) in high yield from sitosterol (containing 40% campesterol) and three other new compounds 3-oxocampesta- 1, 4-dien-26-oic acid (13), 3-oxostigmast-4-en-26-oic acid (14), and 3-oxocampest-4-en-26-oic acid (15) along with a number of known compounds. It should be noted that in the original paper, 44 compounds (13) and (15) have wrongly been mentioned as ergostane derivatives. The formation of the intermediates helped the authors to propose a degradation pathway of sitosterol side chain by Rhodococcus equi. Yoshihama obtained new steroids in his investigation on the preparation of steroids by microbial transformation of AD and progesterone and evaluation of their pharmaceutical properties. 45 Microbial hydroxylation of AD by Acremonium strictum NN 106 gave three known compounds, 14 ot-hydroxy-AD, 11 ~-hydroxy-AD, and 713,1lc~-dihydroxyAD, and two new steroids, 6[3,14~-dihydroxy-AD (16), and 613,1 lc~-dihydroxy-AD (2). 14~-Hydroxyandrost-4-ene3,6,17-trione (17) was prepared from 16 by oxidation. Compound 17 showed the highest inhibition against estrogen

Acetone-dried vegetative cells

Cells from microorganisms dried with acetone are useful for special preparations, although this process does not compete with vegetative cell fermentations for manufacture. Although fermentation of AD with acetone-dried cells of a fungal strain, Aspergillus fumigatus yielded ADD as the major product and testosterone and its A ~ anolog as minor ones, usual fermentation of AD with the strain furnished a new dihydroxy derivative, 7[3, l la-dihydroxy AD (1) and another steroid 6[3, 1 l~x-dihydroxy AD (2) along with minor metabolites, ADD, A ~ testosterone, and testosterone. 38 Apparently in acetone-dried cells, the hydroxylase enzymes are inactivated, but the dehydrogenase and reductase enzymes remains active. Selective 1-dehydrogenation of progesterone by a strain of Aspergillus fumigatus was achieved by fermentation of progesterone with acetone-dried cells of the fungus in the presence of [3-cyclodextrin.39 Usual fermentation of progesterone with the strain yielded four isolable metabolites: 3[3-hydroxy-5ot-pregnan-20-one; 1513hydroxypregna- 1,4-diene-3,20-dione; 713,1513-dihydroxypregn-4-ene-3, 20-dione; and 11 et,1513-dihydroxypregn-4ene-3,20-dione. However, fermentation of the substrate with acetone-dried cells furnished pregna-l,4-diene-3, 20dione and AD. On the other hand, fermentation with acetone-dried cells in presence of [3-cyclodextrin afforded pregna-1,4-diene-3,20-dione as the only isolable metabolite. The results demonstrated that in acetone-dried cells, the hydroxylase and A4-reductase enzymes are inactivated. The activity of the enzyme system responsible for the degradation of the side chain of progesterone is inhibited in the presence of 13-cyclodextrin. Inhibition of certain enzymes in presence of a cyclodextrin is a phenomenon of considerable interest.

New cell-immobilization techniques

A novel cell-immobilization technique was developed by Houng e t al. 4 that involves increase of substrate partition to the gel matrix by coating a polyurea thin layer on the surface of the calcium alginate beads. The bioconversion of progesterone to 1 le~-hydroxyprogesterone with these polyurea-coating alignate-entrapped Aspergillus ochraceous cells was investigated using different organic solvents in biphasic media. The reaction medium of ethyl acetate could

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Table 1 Hydroxylation Substrate 21-Acetoxy-17cx-hydroxypregn-4ene-3,11,20-trione 21-Acetoxy-17~-hydroxypregn-4ene-3,20-dione 17~-acetoxy-21-hydroxypregna-1, 4-diene-3,20-dione 17e-Acetoxypregn-4-ene-3,20-dione &x-Androstan-3-one 5e-And rostane-3,6-dione Microorganism Arthrobacter simplex Absidia orchidis Cochiobolus lunatus Pycnosporium sp ATCC 12231 Cephalosporium aphidicola Cephalosporium aphidicola Aspergillus ochraceus Curvularia lunata Acremonium stricture Acremonium strictum NN106 Mucorpiriformis Product (i) 11t3, 21-Dihydroxypregna-1, 4,17(20)-trien-3-one (ii) 11 [3-Hydroxypregna-1,4,17 (20)-trien-3-one-21-hemisuccinate 11 [3,17e,21-Trihydroxypregn-4-ene-3, 20-dione 17c~-Acetoxy-11 [3,21-dihyd roxyp reg na- 1, 4-diene,3,20-dione (i) 17e-Acetoxy-1113-hydroxypreg n-4-ene-3,20-dione (ii) 17e-Acetoxy-1113,1213-dihydroxypregn-4-ene-3, 20-dione 17e-Hydroxy-5~-androstan-3-one (i) &x,1713-Dihydroxy-513-androstan-6-one (ii) 1713-Hydroxyandrost-4-ene-3, 6-dione (iii) 313,5~-Dihydroxyandrost-6,17-dione (iv) 313,5~,1713-Trihydroxyandrostan-6-one 1lcx-Hyd roxyandrosta-1,4-diene-3,17-dione 14c~-Hydroxyand rost-4-ene-3,17-dione 14~-Hydroxyand rost-4-ene-3,6,17-trione (i) 14{x-Hydroxyandrost-4-ene-3,17-dione (ii) 11cx-Hydroxyandrost-4-ene-3,17-dione (iii) 713,11cx-Dihydroxyandrost-4-ene-3,17-dione (iv) 613,14e-Dihydroxyandrost-4-ene-3,17-dione (v) 613,11c~-Dihydroxyandrost-4-ene-3,17-dione (i) 1713-Hydroxya ndrost-4-en-3-one (ii) 1413-Hydroxyandrost-4-ene-3,17-dione (iii) 7~-Hyd roxyandrost-4-ene-3,17-dione (iv) 1413,1713-Dihydroxyandrost-4-en-3-one (v) 7e,1413,1713-Trihydroxyandrost-4-en-3-one 14e-Hydroxyandrost-4-ene-3,17-dione 14~x-Hydroxya nd rost-4-ene-3,17-dione 613,14cx-Dihydroxyandrost-4-ene-3,17-dione (i) 713,11~-Dihydroxyandrost-4-ene-3,17-dione (ii) 613,11e-Dihydroxyandrost-4-ene-3,17-dione 1713-(N-butylcarbamoyl)-1 lcx-hydroxy-4-aza-&xandrost-l-en-3-one (i) 11c~,17~,21Trihydroxypregn-4-ene-3,20-dione (ii) 1113,17~,21-Trihydroxypregn-4-ene-3,20-dione 11[3,17c~,21-Trihyd roxypreg na-1,4-diene-3, 20-dione 1113,17e,21-Trihydroxypregn-4-ene-3,20-dione 11[3,17e,21-Trihydroxypregna- 1,4-diene-3, 20-dione Reference 55 56 57 42 58 58

Androsta-1,4-diene-3,17-dione Androst-4-ene-3,17-dione Androst-4-ene-3,17-dione Androst-4-ene-3,17-dione

59 60 11 45

Androst-4-ene-3,17-dione

61

Androst-4-ene-3,17-dione Androst-4-ene-3,17-dione Androst-4-ene-3,17-dione Androst-4-ene-3,17-dione 1713-(N-Butylcarbamoyl)-4-aza-5eandrost-l-en-3-one 17e,21-Dihydroxypregn-4-ene-3,20dione 17e,21-Dihydroxypregn-4-ene-3,20dione 17e,21-Dihydroxypregn-4-ene-3,20dione 17e,21-Dihydroxypregn-4-ene-3,20dione 17cx,21-Dihydroxypreg n-4-ene-3,20dione 24-Epibrassinolide 24-Epicastasterone 13-Ethylgon-4-ene-3,17-dione 1713-Hydroxyandrosta-1,4-dien-3-one 17[3-Hyd roxya nd rost-4-en-3-one 313-Hydroxyandrost-5-en-17-one 1713-Hydroxyandrost-4-en-3-one 17c~-Hydroxyp reg n-4-ene-3,20-dio ne
17e-Hyd roxypreg n-4-ene-3,20-dione

Myrothecium striatisporum Eurotium sp. Myrothecium Aspergillus fumigatus Selenastrum capricornutum Cunninghamella elegans Curvularia lunata and Mycobacterium smegmatis Curvularia lunata Curvularia lunata and Mycobacterium smegmatis Beauveria bassiana Cunninghamella echinulata Cunninghamella echinulata Penicillium raistrickfi Mucor piriformis Mucor piriformis Mucor piriformis Marchantia polymorpha Mucor piriformis Corynespora Melonis CBS 16260 Pycnosporium sp. ATCC 12231

9 10 38 62 7 21
6, 63-65

22

11cx,17~,21-Trihydroxypregn-4-ene-3,20-dione 121~-Hydroxy-24-epibrassinolide 1213-Hydroxy-24-epicastasterone 15e-Hydroxy-13-ethylgon-4-ene-3,17-dione 14~,1713-Dihydroxyandrosta-1,4-dien-3-one 14~x-Hydroxyandrost-4-ene-3,17-dione (i) 3[3,7e-Dihydroxyandrost-5-en-17-one (ii) 313,7e,171~-Trihydroxyandrost-5-ene 613,1713-Dihydroxya nd rost-4-en-3-one (i) 7e,17o~-Dihydroxypregn-4-ene-3,20-dione (ii) 613,17e,20e-Trihyd roxypreg n-4-en-3-o ne (iii) 11e,17c~,20-Trihydroxypregn-4-en-3-one 813,17e-Dihydroxypregn-4-ene-3,20-dione (i) 3[3,7c~-Dihydroxypreg n-5-en-20-one (ii) 313,7e,1le-Trihydroxypregn-5-en-20-one

66 67 67 68-72 73 73 61 74 61 42 42

3[3-Hydroxypregn-5-en-20-one

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B i o t r a n s f o r m a t i o n o f steroids: M a h a t o a n d Garai
Table 1

(continued) Microorganism Cephalosporium aphidicola Cephalosporium aphidicola Mucor piriformis Mucor piriformis Product (i) 6#,11(x-Dihyd roxypreg n-4-ene-3,20-dione (ii) 66,11c(,2017,-Trihydroxypreg n-4-en-3-one (iii) 1 l(~,2013-Dihydroxypregn-4-en-3-one (i) 1213,17(x-Dihydroxypregn-4-ene-3,20-dione (ii) 66,17c(-Dihydroxypregn-4-ene-3,20-dione (iii) 613,11(x,17(x-Trihydroxypregn-4-ene-3,20-dione (i) 7c~,17(x-Dihydroxypregn-4-ene-3,20-dione (ii) 6#,17(x,20(x-Trihydroxypreg n-4-en-3-one (iii) 11e~,17e,20(x-Trihydroxypregn-4-en-3-one (i) 14(x-Hydroxypregna-4,16-diene-3,20-dione (ii) 7(x,14(x-Dihydroxypregn-4,16-diene-3,20-dione (iii) 36,7c(,14c~-Trihydroxy-5(x-pregn-16-en-20-one (iv) 3(x,7(x,14(x-Trihyd roxy-5(x-preg n- 16-en-20-o ne (i) 7(x,14(x-Dihyd roxypregna-4,16-diene-3,20-dione (ii) 14(x-Hyd roxypregna-4,16-diene-3,20-dione (iii) 313,7(x,14c~-Trihydroxy-5(x-preg n-16-en-20-one (iv) 3c(,7(x,14~-Trihydroxy-5(x-preg n-16-en-20-one 14(x-Hydroxypregna-4,16-diene-3,20-dione (i) 513,14c(-Dihydroxypreg nane-3,2O-dione (ii) 14(x-Hydroxypreg n-4-ene-3,20-dione (iii) 66,14c~-Dihydroxypreg n-4-ene-3,20-dione (iv) 7(x,14(x-Dihydroxypreg n-4-ene-3,20-dione (v) 713,14(x-Dihydroxypregn-4-ene-3,20-dione (i) 1 l(x-Hydroxypregn-4-ene-3,20-dione (ii) 1 l(x-Hydroxy-5(x-pregnane-3,20-dione (iii) 66,1 l(x-Dihydroxypregn-4-ene-3,20-dione 1 l(x-Hydroxypreg n-4-ene-3,20-dione 14(x-Hydroxypreg n-4-ene-3,20-dione 1 l(x-Hydroxypreg n-4-ene-3,20-dione 1 ltx-Hyd roxypreg n-4-ene-3,20-dione (i) 713,156-Dihydroxypregn-4-ene-3,20-dione (ii) 66,1 l(x-Dihydroxypregn-4-ene-3,20-dione (iii) 11c~,1513-Dihydroxypregn-4-ene-3,20-dione (iv) 613,11(x,17c~-Trihydroxypregn-4-ene-3,20-dione (v) 11 e,156,17(x-Trihydroxypregn-4-ene-3,20-dione (vi) 7{3,1513,17(x-Trihydroxypreg n-4-ene-3,20-dione (i) 7c~,1ll3-Dihydroxypregn-4-ene-3,20-dione (ii) 14(x-Hydroxypregn-4-ene-3,11,20-trione (i) 1lc~-Hydroxypregn-4-ene-3,20-dione (ii) 156-Hydroxypregn-4-ene-3,20-dione (iii) 76-Hydroxypregn-4-ene-3,20-dione (iv) 76,15i~-Dihydroxypregn-4-ene-3,20-dione (v) 1lc,156-Dihydroxypregn-4-ene-3,20-dione 1 lc~-Hydroxypregn-4-ene-3,20-dione 14(x-Hydroxypreg n-4-ene-3,20-dione (i) 21-Hyd roxypregn-4-ene-3,2O-dione (ii) 17(x-Hydroxypregn-4-ene-3,20-dione (iii) 116-Hydroxypregn-4-ene-3,20-dione (iv) 11c(,17c~-Dihydroxypregn-4-ene-3,20-dione (v) 1113,17c(,21-Trihydroxypregn-4-ene-3,20-dione (vi) 11(x,17(x,21-Trihydroxypregn-4-ene-3,20-dione (i) 613,1l~x-Dihydroxypregn-4-ene-3,20-dione (ii) 1l(x-Hydroxypregn-4-ene-3,20-dione (iii) 126,17-Dihydroxypregn-4-ene-3,20-dione (iv) 206-Hydroxypreg n-4-en-3-one (v) 66,1 l(x,2013-Trihydroxypregn-4-en-3-one (i) 26-Hydroxypregn-4-ene-3,20-dione (ii) 66-Hydroxypregn-4-ene-3,20-dione (iii) 9(x-Hydroxypreg n-4-ene-3,20-dione (iv) 14(x-Hydroxypreg n-4-ene-3,20-dione (v) 16(x-Hydroxypregn-4-ene-3,20-dione (i) 613-Hydroxypregn-4-ene-3,20-dione (ii) 15(x-Hydroxypregn-4-ene-3,20-dione 6{3-Hydroxypregn-4-ene-3,20-dione (i) 26-Hydroxypregn-4-ene-3,20-dione (ii) 66-Hydroxypreg n-4-ene-3,20-dione Reference 75 75 48 48

Substrate 11c(-Hydroxypregn-4-ene-3,20-dione 17(x-Hydroxypreg n-4-ene-3,20-dione 17(x-Hyd roxypreg n-4-ene-3,20-dione Preg na-4,16-diene-3,20-dione

Preg na-4,16-diene-3,20-dione

Mucor piriformis

61

Pregna-4,16-diene-3,20-dione Preg n-4-ene-3,20-dione

Mucor piriformis Mucor piriformis

73 61

Pregn-4-ene-3,20-dione Preg n-4-ene-3,20-dione Preg n-4-ene-3,20-dione Preg n-4-ene-3,20-dione Preg n-4-ene-3,20-dione Preg n-4-ene-3,20-dione

Rhizopus nigricans Aspergillus ochraceus Mortierella isobellina Rhizopus nigricans Aspergillus ochraceus Acremonium stricture

76 77 29 78 40 45

Pregn-4-ene-3,20-dione Pregn-4-ene-3,20-dione

Cochliobolus lunata Aspergillus fumigatus

79 80

Pregn-4-ene-3,20-dione Pregn-4-ene-3,20-dione Preg n-4-ene-3,20-dione

Aspergillus ochraceus Mucor piriformis Cochliobolus specifer

81 73 82

Pregn-4-ene-3,20-dione

Cephalosporium aphidicola

75

Pregn-4-ene-3,20-dione

Cyanidiophyceac ernersonii

83

Pregn-4-ene-3,20-dione Pregn-4-ene-3,20-dione Pregn-4-ene-3,20-dione

Muriella aurantiaca Galdierla sulphuraria Scemedesmus capricortum

83 83 83

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Review
Table 2 Substrate 313-Acetoxy-19-hydroxycholest-5-ene 313-Acetoxy-19-hydroxycholest-5-ene Side-chain cleavage Microorganism Product (i) 19-Hydroxy-5(~-androst-1-ene-3,17-dione (ii) 19-Hydroxyandrost-4-ene-3,17-dione (iii) 9(x,19-Dihydroxyandrost-4-ene-3,17-dione (iv) 3-Hyd roxyestra-1,3,5(10)-trien-17-one (i) 3-Hydroxyestra-1,3,5(10)-trien-17-one (ii) 19-Hydroxy-5(x-androst-1-ene-3,17-dione (iii) 19-Hydroxyandrost-4-ene-3,17-dione (iv) 9(x,19-Dihydroxyand rost-4-ene-3,17-dione (v) 19-Hydroxya ndrosta ne-3,17-dione Androst-4-ene-3,17-dione Androsta-1,4-diene-3,17-dione Androsta-1,4-diene-3,17-dione (i) Androstane-3,17-dione Reference 43

Moraxella sp.

Moraxella sp.

84

Cholesterol Cholesterol Cholesterol Cholesterol Cholesterol

Mycobacterium sp. NRRLB-3805 Rhodococcus corallina Arthrobacter simplex and Mycobacterium sp. NRRLB-3683 Rhodococcus equi Mycobacterium sp.

85 86 20 87 88

(ii) Androsta-1,4-diene-3,17-dione
(i) Androst-4-ene-3,17-dione

(ii) Androsta-1,4-diene-3,17-dione
Cholesterol Cholesterol Cholesterol 2e,3e-Dihydroxy5(~-cholestan-6-one 19-Hyd roxy-cholesterol

Mycobacterium fortuitum NRRLB-8153 Mycobacterium sp. Rhodococcus equi Mycobacterium vaccae Rhodococcus mutant k-3

(iii) 17#-Hyd roxyandrosta-l,4-dien-3-one (iv) 17#-Hydroxyandrosta-1,4-dien-3-one 313-Hydroxya ndrost-5-en-17-one 1713-Hydroxyandrost-4-en-3-one (i) Androsta-1,4-diene-3,17-dione (ii) Androst-4-ene-3,17-dione (i) 2(x,3(x,6(x-Trihyd roxy-5(x-a nd rosta n- 17-o ne (ii) 2c(-Hydroxyandrost-4-ene-3,17-dione (i) Estra-l,3,5(10)-trien-3-ol (ii) 2(3-Hyd roxy-1,3,5(10)-estra-trien-17-yl)propionic acid (iii) 2-Methyl-6(3-hydroxy-1,3,5(10)-estratriene-17-yl)heptanoic acid (iv) 2(3-Hyd roxy-1,3,5(10),17-estra-tetraen-17-yl)propionic acid (i) 2(3-Hyd roxy-1,3,5(10),17-estra-tetraen-17-yl)propionic acid (ii) 2,3-Dimethyl-6-(3-hyd roxy-1,3,5(10)-estratriene17-yl)- heptanoic acid 20(x-Hydroxymethylpregn-4-en-3-one 315-Methoxymethoxy-21 -hydroxy20-methylpregna-5,7-diene (i) Androsta-1,4-diene-3,17-dione (ii) 17L3-Hydroxyandrosta-1,4-dien-3-one 1713-Acetoxyandrost-4-en-3-one 9(x-Hydroxyandrost-4-ene-3,17-dione (i) 3-Oxochol-4-en-24-oic acid (ii) 27-Norcholest-4-ene-3,24-dione (i) 3-Oxo ergosta-1,4-dien-26-oic acid (ii) 3-Oxo ergost-4-en-26-oic acid (iii) 20-Ca rboxypreg n-4-en-3-one (iv) 20-Ca rboxypreg na-l,4-dien-3-one (v) Androst-4-en-3,17-dione (vi) And rosta-1,4-diene-3,17-dione (vii) Propionic acid (viii) Acetic acid 9c~-Hydroxya ndrost-4-ene-3,17-dione Androsta-1,4-diene-3,17-dione (i) Androst-4-ene-3,17-dione (ii) Androsta-1,4-diene-3,17-dione Androst-4-ene-3,17-dione Androst-4-ene-3,17-dione Androst-4-ene-3,17-dione Androsta-1,4-diene-3,17-dione 9(x-Hydroxyandrost-4-ene-3,17-dione Androst-4-ene-3,17-dione 9(x-Hydroxyandrost-4-ene-3,17-dione Androsta-1,4-diene-3,17-dione Androsta-1,4-diene-3,17-dione (i) Androst-4-ene-3,17-dione (ii) Androsta-1,4-diene-3,17-dione

89 17 90 91 41

19-Hydroxy-campesterol

Rhodococcus mutant k-3

41

Lithocholic acid 313-Methoxymethoxyergosta-5,7,22-triene Pregn-4-ene-3,20-dione Pregn-4-en-3,20-dione Sitosterol Sitosterol Sitosterol

Mycobacterium sp. Mycobacterium Pseudomonassp. Cephalosporium aphidicola Mycobacterium fortuitum Arthrobacter oxydans Rhodococcus equi k-3

92 93 94 75 13 95 44

Sitosterol Sitosterol Sitosterol Sitosterol Sitosterol Solasodiene Sterol Sterol Sterol Sterol Sterol Sterol Sterol

Mycobacterium sp. Mycobacterium NRRLB-3683 Arthrobacter simplex Mycobacterium Mycobacterium v a c c a e Mycobacterium sp. NRRLB-3805 Mycobacterium v a c c a e Mycobacterium fortuitum Mycobacterium NRRLB-3805 Mycobacterium fortuitum Mycobacterium v a c c a e Arthrobacter simplex Mycobacterium sp.

15 96 97 98 19 99 16 12, 100 101 102 103 104 105

338

Steroids, 1997, vol. 62, April

Biotransformation of steroids: Mahato and Garai

Table 3 Substrate 31~,-Acetoxy-513-pregn16-en-20-one 21-Acetoxy-1113,17(xdihydroxy-6c~-methyl preg n-4-ene-3,20-dione 513-Androstane-3,17-dione And rost-4-ene-3,17-dione And rost-4-ene-3,17-dione Chenodeoxycholic acid Chenodeoxycholic acid Cholesterol Cholesterol Cholesterol Cholesterol Cholesterol Cholesterol Cholesterol Cholic acid Cholic acid Cholic acid Cholic acid 313,1713-Dihydroxy-5ct-androstane 3ct-Hydroxy-5(x-androstan-17-one 3,3-(2,2-Dimethyltrimethylenedioxy)-5-10ct-epoxy-5(xestr-9(11 )-en-1713-ol 3(x-Hydroxy-513-androstan-17-one 313-Hydroxypregn-5-en-20-one Lithocholic acid 19-Nor-1713-hydroxy-androst4-en-3-one Sitosterol 1113,17(x,21-Trihydroxypregn4-ene-3,20-dione 1113,17(x-21-Trihydroxypregn4-ene-3,20-dione 1113,17(x,21-Trihydroxy-6c~methyl-pregn-4-ene-3,20-dione 1113,17ct,21-Trihydroxypreg n4-ene-3,20-dione 1113,17ct,21-Trihydroxypreg n4-ene-3,20-dione Ursodeoxycholic acid Dehydrogenation Microorganism Mycobacterium NRRLB-3805 Arthrobactersimp/ex Mycobacterium sp. Aspergillus fumigatus Acetone dried cell of A. fumigatus Bacillus Bacillus sp. Mycobacterium sp. Rhodococcus e q u i Escherichia Nocardia r u b r a Arthrobacter simplex Rhodococcus erythropolis Rhodococcus e q u i Bacillus Arthrobacter simplex Bacillus sp. TTUR-2 Bacillus sp. Nocardia rubra Mycobacterium sp. Mycobacterium NRRLB-3683 Mycobacterium sp. Nocardia rubra Mycobacterium sp. Rhodococcus sp. DSM 92344 Rhodococcus equi k-3 Arthrobacter simplex Corynebacterium simplex Corynebacterium simplex Corynebacterium equi Arthrobacter simplex Bacillus sp. Product Pregna-4,16-diene-3,20-dione 21-Acetoxy-111%17~-dihydroxy-6~-methylpreg na1,4-diene-3,20-dione (i) Androst-4-ene-3,17-dione (ii) 313-Hydroxy-5c~-androstan-17-one Androsta-1,4-diene-3,17-dione Androsta-1,4-diene-3,17-dione 3a-Hydroxy-7-oxo-513-cholan-24-oic acid 3ct-Hydroxy-7-oxo-513-cholan-24-oic acid Cholest-4-en-3-one Cholest-4-en-3-one (i) Cholest-4-en-3-one (ii) Cholest-5-en-3-one Cholest-4-en-3-one Cholest-4-en-3-one Cholest-4-en-3-one Cholest-4-en-3-one (i) 3(x-Hydroxy-7,12-dioxo-513-cholan-24-oic acid (ii) 3c(,12ct-Dihydroxy-7-oxo-513-cholan-24-oic acid (i) 3c~,7(x-Dihydroxy-12-oxo-513-chotan-24-oic acid (ii) 7c(,12ct-Dihydroxy-3-oxo-4-cholenoic acid 3(x,7a-Dihydroxy-12-oxo-5~-cholan-24-oic acid (i) 3(x,7(x-Dihydroxy-12-oxo-513-cholan-24-oic acid (ii) 3a,12ct-Dihydroxy-7-oxo-513-cholan-24-oic acid (iii) 3ct-Hydroxy-7,12-dioxo-513-cholan-24-oic acid Androst-4-ene-3,17-dione (i) Androst-4-ene-3,17-dione (ii) 5a-Androsta ne-3,17-dione 3,3-(2,2-Dimethyltrimethylenedioxy)-5,10txepoxy-5a-estr-9(11 )-en-17-one (i) And rost-4-ene-3,17-dione Pregn-4-ene-3,20-dione 3-Oxo-4-cholenoic acid (i) 3-Hydroxyandrosta-1,3,5(10)-trien-17-one (ii) Androsta-1,3,5(10)-triene-3,1713-diol (i) Sitosta-l,4-dien-3-one (ii) Sitos-4-en-3-one 1113,17(~,21-Trihydroxypregna-1,4-diene-3, 20-dione 1113,17a,21-Trihydroxypregna-1,4-diene-3, 20-dione 1113,17(x,21-Trihydroxy-6(x-methyl-pregna-1, 4-diene-3,20-dione 1113,17(x,21-Trihydroxypregna-1,4-diene-3, 20-dione 1113,17c~,21-Trihydroxypregna-1,4-diene-3, 20-dione 713-Hydroxy-3-oxo-513-cholan-24-oic acid Reference 106 107 108 38 38 109 110 88 90 111 112 113 34 114 109 52 115 110 112 108 116 92 112 92 117 44 118 36 36 119, 120 121,122 123

synthetase (aromatase). It also exhibited aromatase inhibition in rat ovaries, depression in serum estrogen levels, and strong antitumor activity against human ovarian cancer on nude mice. Hydroxylation of progesterone by the same fungal strain yielded three dihydroxyprogesterones and three trihydroxyprogesterones, among which 713,1513,17atrihydroxyprogesterone (18) was claimed to be a new compound. The compound (18) has also been prepared by Mahato et al. 46 by microbial hydroxylation of 17c~hydroxyprogesterone by Aspergillus fumigatus. It is re-

ported to have the property of suppressing ovulation and showing high differentiation induction activity in bone marrow leukemia MI cells. 47 The compound also showed strong immunosuppressive activity on mouse lymphocytes and weak toxicity against lymphocytes compared to progesterone. 45 Madyastha and Joseph 48 obtained two new steroids, 3 13,7c~, l 4et-trihydroxy-5 ot-pregn- 16-en-20-one (19) 3o~,7o~,14et-trihydroxy-5o~-pregn- 16-en-20-one (20) by transformation of 16-dehydroprogesterone by Mucor piri-

S t e r o i d s , 1997, v o l . 62, A p r i l

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Review Table 4
Substrate 313-Acetoxy-19-hydroxycholest-5-ene 5c~-Androstane-3,17-dione Androsta-1,4-diene-3,17-dione 5~-Androstan-17-one Androsta-1,4-diene-3,17-dione Androst-4-ene-3,11,17-trione Androst-4-ene-3,17-dione Androst-4-ene-3,17-dione Chenodeoxycholic acid Miscellaneous reaction Microorganism Product Reference 84 58 124 58 18 124 38 38 125

Cholesterol Cholic acid

Cholic acid

Cholic acid Deoxycholic acid Digitoxin Digitoxigenin 3~-Hyd roxy-5~-androstan-17-one 313-Hydroxyandrost-5-en-17-one 17~-Hydroxypregn-4-ene3,20-dione 17~-Hydroxypregn-4-ene3,20-dione Lithocholic acid Lithocholic acid 3-Methoxy-8,14-secoestra1,3,5(10),9(11 )-tetraene14,17-dione I~-Methyldigitoxin Pregn-4-ene-3,20-dione Preg n-4-ene-3,20-dio ne Sitosterol

(i) 19-Hydroxycholestan-3-one (ii) 19-Hydroxycholest-4-en-3-one (iii) Cholest-5-ene-313,19-diol Cephalosporium (i) 1713-Hydroxy-5a-androstan-3-one aphidicola (ii) 17~-Acetoxyandrosta n-3-one Marchantia (i) 1713-Hydroxyandrosta-1,4-dien-3-one polymorpha (ii) Androst-4-ene-3,17-dione (iii) 1713-Hydroxyandrost-4-en-3-one Cephalosporium (i) 5e-Androstan-1713-ol aphidicola (ii) 5c~-Androsta ne- 1713-acetate Mycobacterium sp. NRRLB-3683 1713-Hydroxyandrost-4-en-3-one Marchantia polymorpha 17c~-Hydroxyandrost-4-ene-3,11 -dione Aspergillus fumigatus 1717,-Hydroxyandrost-4-en-3-one Acetone dried cells (i) 1713-Hydroxyandrost-4-en-3-one of A. fumigatus (ii) 1713-Hydroxyand rosta-l,4-dien-3-one Alcaligen recti (i) 3~-Methoxy-7~,12~-dihydroxy-5~-cholan24-oic acid (ii) 3e-Methoxy-7~-hydroxy-5~-cholan-24-oic acid (iii) 3cx-Methoxy-713-hydroxy-513-cholan-24-oic acid (iv) 3cx-Methoxy-12ct-hydroxy-513-cholan-24-oic acid (v) 7~-Hydroxy-3-oxo-4-cholenoic acid (vi) 7c~-Hydroxy-3-oxo-4-cholenoic acid Arthrobacter-82 3-Oxo-23,24-dinorchola-1,4-diene-22-oic acid A/ca/igen recti (i) 3~-Methoxy-7~,12~-dihydroxy-513-cholan24-oic acid (ii) 3tx-Methoxy-7c~-hydroxy-513-cholan-24-oic acid (iii) 3~-Methoxy-713-hydroxy-513-cholan-24-oic acid (iv) 3e-Methoxy-12~-hydroxy-513-cholan-24-oic acid (v) 7e,12~-Dihydroxy-3-oxo-4-cholenoic acid Arthrobacter simplex (i) 3,12-Dioxo-23,24-dinorchola-4,6-dienoic acid (i i) 7c~-Hydroxy-3,12-dioxo-23,24-dinorcholenoicacid (iii) 3~,7e-Dihydroxy-12-oxo-513-23,24dinorcholan-22-oic acid (iv) 7tx,12~-Dihydroxy-3-oxo-513-cholan-24-oic acid (v) 7~,12~-Dihydroxy-3-oxo-23,24-dinorchol4-enoic acid (vi) Methyl-3~,7~,12~-trihydroxy-513-cholan-24-oate (vii) 21~-Hydroxy-3,12-dioxo-23,24-dinorcholan4,6-dienoic acid Corynebacterium 3e,12~-Dihydroxy-7-oxo-23,24-dinor-513-cholan22-oic acid Arthrobacter simplex (i) Methyl-3~,12~-dihydroxy-513-cholan-24-oate (ii) 3,12-Dioxo-513-cholan-24-oic acid (iii) 3cx-Hydroxy-12-oxo-51~-cholan-24-oic acid Digitalis lunata Digoxin Pergularia tomentosa (i) Periplogenin (ii) l~-Hydroxy digitoxigenin (iii) Uzarigenin Mycobacterium sp, 3~-hydroxy-5x-androstan-17-one Mucorpiriformis sp. (i) 3~,1713-Dihydroxyandrost-5-ene (ii) 313-Hydroxyandrost-5-ene-7,17-dione (iii) 313,1713-Dihydroxyandrost-5-ene-7-one Nocardia DSM-43298 (i) 9,10-Seco-3,17~-dihydroxypregna-1,3,5 (10)-triene-9,20-dione (ii} 9,10-Seco-3,17c~,20-trihydroxy-pregna-1, 3,5(10)-trien-9-one Mucor piriformis 17e,20{x-Dihyd roxypreg n-4-en-3-one

Moraxella sp. ~, c~'-bipyridyl

126 125

52

127 128 129 130 108 61 131

48, 61 128 50 132 133 83 83 44

Arthrobacter simplex Cunninghamela blackesleena Kloeekera magma Digitalis Cyanidiophyceae caldium Scenedesmus quadricauda Rhodococcus equi k-3

(i) Methyl-3~-acetoxy-5~-cholan-24-oate (ii) n-Butyl 3~-acetoxy-513-cholan-24-oate Lithocholic acid 3~-O-13-D-glucopyranoside 17~-Hyd roxy-3-met hoxy-8,14-secoestra-1, 3,5(10),9(11 )-tetraen-14-0ne 13-Methyl digoxin 20c~-Hydroxypreg n-4-en-3-o ne 3-Hydroxy-9,10-secopregna-1,3,5(10)triene-9,20-dione (i) Stigmasta-1,4-dien-3-on-26-oic acid (ii) Stigmast-4-en-3-on-26-oic acid

340

S t e r o i d s , 1997, v o l . 62, A p r i l

Biotransformation of steroids: Mahato and Garai

I R

OH

~J~COOH

~llr~C00H

i730

5 R CH3

3 ICH

I0

o~'"~v ~" g II

0-~ ~'..~ V 12

0"~ ~

V 13

~ H O O C

COOH

M 0

Figure 1 Structures of novel metabolites.

14

15

OH

16

--OH
OH 0 HO
18 19

OH

17

~H3

C-O
COOH

H 0 " ~ H 20
"~

.o~O,

CH20H OH
.

L I J
~ .H

,,,.,,'",.,,,~ ~ 21
.

ICOOH

25 2 2 R :COOH 2 3 R : COOMe 2 4 R : CHO

26

Steroids, 1997, vol. 62, April

341

Review

formis. The structure of 20 was determined by X-ray crystallography. 49 A new bile acid glycoside lithocholic acid 3a-O-13-D-glucopyranoside (21) was produced by Cunninghamella blakesleena ST-22 from lithocholic acid. 5 In a study on the sterol-transforming ability of fast growing Mycobacterium strains obtained by in vivo genetic recombination experiment Ambrus et al. 5 ~isolated novel intermediates of microbial side-chain degradation of sitosterol. They reported the structure and stereochemistry of the new 26-oxygenated steroid derivatives 22-25, The molecular structure of compound 25 was determined by X-ray crystallography, and its absolute configuration was deduced to be 24(R) and 25(R). A novel metabolite, 213-hydroxy-3,12dioxo-23,24-dinorchola-4,6-dienoic acid 26 was produced along with a few other metabolites by transformation of cholic acid by Arthrobacter simplex. The pathway of formation of these metabolites of cholic acid has also been proposed. 52'53 Cholic acid-induced N-methylhydantoin synthesis by a strain ofAlcaligens recti in nutrient medium has been reported. 54 The formation of this product has been attributed to the amino acids contained in the nutrient medium under the catalysis of various enzymes at different stages in the presence of cholic acid. It is noteworthy that cholic acid and other bile acids are present in the mammalian liver where creatine is synthesized. It has been suggested that in mammalian liver, cholic acid acts as an inducer for the synthesis of creatinine, the immediate precursor of N-methylhydantoin.

Acknowledgments
Financial supports from the Council of Scientific and Industrial Research (CSIR), New Delhi, India in the form of Senior Research Fellowship (SG) and Emeritus Scientist (SBM) are gratefully acknowledged.

References
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Future possibilities
Since 1950, steroids have been the traditional field for industrially used microbial transformation, and remarkable progress already has been made. However, further developmental activities relating to controlled multiple transformation for the purpose of cost reduction are anticipated. Continuous emphasis on the application of genetic engineering of microorganisms for their improvement as steroidtransforming agents is expected. Development of new steroids possessing useful biological activity may receive greater attention. Continuing attempts are expected for utilization of sterol fractions obtained as industrial by-product in the production of steroid intermediates. There is a distinct possibility of further development on conducting two or more microbial steps in a single-step fermentation using mixed culture or immobilized mixed culture. The commercial success of microbial steroid transformation so far has been achieved by flourishing vegetative cell cultures. Among the alternative processes, the present interest centers on the development of process using immobilized cells for industrial exploitation. Further emphasis on this process is expected to achieve the objective. Further developmental studies on the use of cyclodextrins, nonaqueous, low water, or mixed solvent systems in microbial transformations of water insoluble organic compounds are anticipated. Microbial hydroxylations of various steroid substrates are shown in Table 1. Tables 2 and 3 show the side-chain cleavages and the dehydrogenations, respectively. Table 4 shows miscellaneous reactions. In each table, the substrates are arranged alphabetically.

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