advances in microbial

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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, carbon- carbon 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 Com- pany 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 in- corporated 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 antican- cer agents as well as in other applications. Although intro- ductions 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 re-

25 v i e w s - covered literature of the period 1979-mid-1992.

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

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

Hydroxylation

The lloL-, 1113-, and 16c~-hydroxylations are now exclu- sively 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 mycell ium immobilized in Ca-alginate gel was used as a biocatalyst. Stabilization of steroid 1 l- hydroxylation activity of Cunninghamella elegans l~roto- plasts in organic osmotic stabilizers has been reported.' Pro- toplasts 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

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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 ben- zene-divinylbenzene resin coated with the polyethylene- polypropylene oxide adduct of N, N"-tetra (oxypropylenox- ide) 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.

Microb ia l t r ans fo rma t ion of 3[3-hydroxy-5c~-H- pregnanes 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-ene- 3,17-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 axial- flow turbine as a stirrer was used. The preparative yield of the product was 51%, and the adsorbent could be used repeatedly.

Carbon-Carbon bond cleavage

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

Biotransformation of steroids: Mahato and Garai

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 Mv- cobacterium vaccae ZIMET 11094. Strains and culture con- dition 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. Testoster- one 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 Myco- bacterium vaccae has been reported by Spassov et al. ~9 These mutants were resistant to rifampicin and streptomy- cin. 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 in- ducible cholesterol oxidase-producing bacterium, Arthro- bacter 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. NRRLB- 3683 in the presence of an androstenone adsorbent, Amber- lite 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

Steroids, 1997, vol. 62, April 333

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 ef- ficient than batch-wise reused adsorbed cultures.

Enzymatic catalysis in nonaqueous solvents

The application of nonaqueous environment for enzymatic catalysis has received much attention in recentyears. Ex-

24 2 J cellent reviews are available on this aspect. ' 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 bond- forming 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 de- velopments 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-

vents was investigated. 31 The highest degradation activities were obtained with cells immobilized in celite with bis (2- ethylhexyl) phthalate as the conversion medium.

A review has been published 32 that deals with biocatalysis with enzymes and microorganisms immobilized by en- trapment in lyotropic liquid crystals and substrates dis- solved 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.' '

Cyclodextrin medium

Cyclodextrins (CD) form inclusion compounds that are bio- compatible 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 Rhodococ- cus 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-CD- induced 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

334 Steroids, 1997, vol. 62, April

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, 1713- estradiol 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, hydroxypropyl- 13-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. fla- vam cells growing in the presence of podophyllotoxin, com- plexed with dimethyl-[3-CD.

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. Al- though 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 Ap- parently 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 metaboli tes: 3[3-hydroxy-5ot-pregnan-20-one; 1513- hydroxypregna- 1,4-diene-3,20-dione; 713,1513-dihydroxypregn-4-ene-3, 20-dione; and 11 et,1513-dihydroxypregn-4- ene-3,20-dione. However, fermentation of the substrate with acetone-dried cells furnished pregna-l,4-diene-3, 20- dione 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 consider- able interest.

New cell-immobilization techniques

A novel cell-immobilization technique was developed by Houng e t a l . 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


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 Rho- dococcus 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 19- hydroxysterols. Two novel microbial conversion products of progesterone derivatives were obtained by Krisch- enowski 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 1113,1213- dihydroxy-17e~-acetoxyprogesterone (8) in addition to the known l113-hydroxy compound. Incubation of 3[3- acetoxycholest-5-ene-19-ol with Moraxella sp. gave three neutral metabolites (9-11) of which compound 9 was un- known. 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 Iida 44 isolated a mutant H-45 of Rho- dococcus 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-oxo- campest-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 men- tioned 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 Acre- monium strictum NN 106 gave three known compounds, 14 ot-hydroxy-AD, 11 ~-hydroxy-AD, and 713,1 lc~-dihydroxy- AD, and two new steroids, 6[3,14~-dihydroxy-AD (16), and 613,1 lc~-dihydroxy-AD (2). 14~-Hydroxyandrost-4-ene- 3,6,17-trione (17) was prepared from 16 by oxidation. Com- pound 17 showed the highest inhibition against estrogen


Review

Table 1 Hydroxylation

Substrate Microorganism Product Reference

21-Acetoxy-17cx-hydroxypregn-4- Arthrobacter simplex (i) 11t3, 21-Dihydroxypregna-1, 4,17(20)-trien-3-one 55 ene-3,11,20-trione (ii) 11 [3-Hydroxypregna-1,4,17

(20)-trien-3-one-21-hemisuccinate 11 [3,17e,21-Trihydroxypregn-4-ene-3, 20-dione 21-Acetoxy-17~-hydroxypregn-4-

ene-3,20-dione 17~-acetoxy-21-hydroxypregna-1,

4-diene-3,20-dione 17e-Acetoxypregn-4-ene-3,20-dione

Absidia orchidis 56

57

42

17c~-Acetoxy- 11 [3,21 -dihyd roxyp reg n a- 1, 4-diene,3,20-dione

(i) 17e-Acetoxy-1113-hyd roxypreg n-4-ene-3,20-dione (ii) 17e-Acetoxy-1113,1213-dihydroxypregn-4-ene-3,

20-dione &x-Androstan-3-one Cephalosporium 17e-Hydroxy-5~-androstan-3-one 58

aphidicola 5e-And rostane-3,6-dione Cephalosporium (i) &x,1713-Dihydroxy-513-androstan-6-one 58

aphidicola (ii) 1713-Hydroxyandrost-4-ene-3, 6-dione (iii) 313,5~-Dihydroxyandrost-6,17-dione (iv) 313,5~,1713-Trihydroxya ndrostan-6-one

Androsta-1,4-diene-3,17-dione 1 lcx-Hyd roxyandrosta-1,4-diene-3,17-dione

Androst-4-ene-3,17-dione Androst-4-ene-3,17-dione Androst-4-ene-3,17-dione

Androst-4-ene-3,17-dione

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-5e- androst-l-en-3-one

17e,21-Dihydroxypregn-4-ene-3,20- dione




17cx,21-Dihyd roxypreg n-4-ene-3,20- dione

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

Cochiobolus lunatus

Pycnosporium sp ATCC 12231

Aspergillus ochraceus

Curvularia lunata Acremonium stricture Acremonium

strictum NN106

59

Myrothecium striatisporum

Eurotium sp. Myrothecium Aspergillus fumigatus

14c~-Hydroxya nd 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

Mucorpiriformis (i) 1713-Hyd roxya ndrost-4-en-3-one 61 (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-Trihyd roxyandrost-4-en-3-one 14e-Hydroxyandrost-4-ene-3,17-dione

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

14~x-Hyd roxya 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-&x-

androst-l-en-3-one (i) 11c~,17~,21 Trihydroxypregn-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

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-Hydroxya ndrost-4-ene-3,17-dione

(i) 3[3,7e-Dihydroxyandrost-5-en-17-one (ii) 313,7e,171~-Trihydroxyandrost-5-ene

613,1713-Dihyd roxya 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~-Dihyd roxypreg n-5-en-20-one (ii) 313,7e,1 le-Trihydroxypregn-5-en-20-one

17e-Hyd roxypreg n-4-ene-3,20-dione

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

Corynespora Melonis CBS 16260

Pycnosporium sp. ATCC 12231

60 11 45

8

9 10 38

62

7

21

6, 63-65

22

66

67

67

68-72 73 73 61

74

61

42

42

336 S te ro ids , 1997, vo l . 62, Ap r i l

Bio t rans format ion o f steroids: Mahato and Garai

Table 1 (continued)


11c(-Hydroxypregn-4-ene-3,20-dione Cephalosporium (i) 6#,11(x-Dihyd roxypreg n-4-ene-3,20-dione 75 aphidicola (ii) 66,11c(,2017,-Trihydroxypreg n-4-en-3-one

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

(iii) 1 l(~,2013-Dihydroxypregn-4-en-3-one (i) 1213,17(x-Dihyd roxypregn-4-ene-3,20-dione 75

(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 48 (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 48 (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 61 (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

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

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



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




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

Cephalosporium aphidicola

Mucor piriformis

Mucor piriformis

Mucor piriformis

Mucor piriformis Mucor piriformis

Rhizopus nigricans

Aspergillus ochraceus

Mortierella isobellina Rhizopus nigricans Aspergillus

ochraceus Acremonium stricture

Cochliobolus lunata

Aspergillus fumigatus

Aspergillus ochraceus Mucor piriformis Cochliobolus specifer


Cyanidiophyceac ernersonii

Muriella aurantiaca

Galdierla sulphuraria Scemedesmus

capricortum

(iv) 3c(,7(x,14~-Trihydroxy-5(x-preg n-16-en-20-one 14(x-Hydroxypregna-4,16-diene-3,20-dione 73 (i) 513,14c(-Dihydroxypreg nane-3,2O-dione 61

(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 76

(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 77

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

29 78 40

(i) 713,156-Dihydroxypregn-4-ene-3,20-dione 45 (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~,1 ll3-Dihydroxypregn-4-ene-3,20-dione 79

(ii) 14(x-Hydroxypregn-4-ene-3,11,20-trione (i) 1 lc~-Hydroxypregn-4-ene-3,20-dione 80

(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) 1 lc¢,156-Dihydroxypregn-4-ene-3,20-dione 1 lc~-Hydroxypregn-4-ene-3,20-dione 81 14(x-Hydroxypreg n-4-ene-3,20-dione 73

(i) 21-Hyd roxypregn-4-ene-3,2O-dione 82 (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) 613,1 l~x-Dihydroxypregn-4-ene-3,20-dione 75 (ii) 1 l(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 83

(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 83

(ii) 15(x-Hydroxypregn-4-ene-3,20-dione 6{3-Hydroxypregn-4-ene-3,20-dione 83

(i) 26-Hydroxypregn-4-ene-3,20-dione 83 (ii) 66-Hydroxypreg n-4-ene-3,20-dione

S te ro ids , 1997, vo l . 62, Ap r i l 337

Review

Table 2 Side-chain cleavage


313-Acetoxy-19-hydroxy- Moraxella sp. (i) 19-Hydroxy-5(~-androst-1-ene-3,17-dione 43 cholest-5-ene (ii) 19-Hydroxyandrost-4-ene-3,17-dione

313-Acetoxy-19-hydroxycholest-5-ene

(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 84 (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 85 Androsta-1,4-diene-3,17-dione 86 Androsta-1,4-diene-3,17-dione 20

Moraxella sp.

Cholesterol Mycobacterium sp. NRRLB-3805 Cholesterol Rhodococcus corallina Cholesterol Arthrobacter simplex and

Mycobacterium sp. NRRLB-3683 Cholesterol Rhodococcus equi (i) Androstane-3,17-dione 87

(ii) Androsta-1,4-diene-3,17-dione Cholesterol Mycobacterium sp. (i) Androst-4-ene-3,17-dione 88

(ii) Androsta-1,4-diene-3,17-dione (iii) 17#-Hyd roxyandrosta-l,4-dien-3-one (iv) 17#-Hydroxyandrosta-1,4-dien-3-one

313-Hyd roxya ndrost-5-en-17-one Cholesterol

Cholesterol Cholesterol

2e,3e-Dihydroxy- 5(~-cholestan-6-one

19-Hyd roxy-cholesterol

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)-estratriene- 17-yl)- heptanoic acid

19-Hydroxy-campesterol

Mycobacterium fortuitum NRRLB-8153

Mycobacterium sp. Rhodococcus equi

Mycobacterium vaccae

Rhodococcus mutant k-3

Rhodococcus mutant k-3

89

17 90

91

41

41

Lithocholic acid Mycobacter ium sp. 20(x-Hydroxymethylpregn-4-en-3-one 92 313-Methoxymetho- M y c o b a c t e r i u m 315-Methoxymethoxy-21 -hydroxy- 93

xyergosta-5,7,22-triene 20-methylpregna-5,7-diene Pregn-4-ene-3,20-dione Pseudomonas sp. (i) Androsta-1,4-diene-3,17-dione 94

(ii) 17L3-Hydroxyandrosta-1,4-dien-3-one Pregn-4-en-3,20-dione Cephalosporium aphidicola 1713-Acetoxyandrost-4-en-3-one 75 Sitosterol Mycobacterium fortuitum 9(x-Hydroxyandrost-4-ene-3,17-dione 13 Sitosterol Arthrobacter oxydans (i) 3-Oxochol-4-en-24-oic acid 95

(ii) 27-Norcholest-4-ene-3,24-dione Sitosterol Rhodococcus equi k-3 (i) 3-Oxo ergosta-1,4-dien-26-oic acid 44

(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 Sitosterol Mycobacterium sp. 9c~-Hydroxya ndrost-4-ene-3,17-dione 15 Sitosterol Mycobacterium NRRLB-3683 Androsta-1,4-diene-3,17-dione 96 Sitosterol Arthrobacter simplex (i) Androst-4-ene-3,17-dione 97

(ii) Androsta-1,4-diene-3,17-dione Sitosterol M y c o b a c t e r i u m Androst-4-ene-3,17-dione 98 Sitosterol Mycobacterium v a c c a e Androst-4-ene-3,17-dione 19 Solasodiene Mycobacterium sp. NRRLB-3805 Androst-4-ene-3,17-dione 99 Sterol Mycobacterium v a c c a e Androsta-1,4-diene-3,17-dione 16 Sterol Mycobacterium fortuitum 9(x-Hydroxyandrost-4-ene-3,17-dione 12, 100 Sterol Mycobacterium NRRLB-3805 Androst-4-ene-3,17-dione 101 Sterol Mycobacterium fortuitum 9(x-Hydroxyandrost-4-ene-3,17-dione 102 Sterol Mycobacterium v a c c a e Androsta-1,4-diene-3,17-dione 103 Sterol Arthrobacter simplex Androsta-1,4-diene-3,17-dione 104 Sterol Mycobacterium sp. (i) Androst-4-ene-3,17-dione 105

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



Table 3 Dehydrogenation


31~,-Acetoxy-513-preg n- Mycobacterium Pregna-4,16-diene-3,20-dione 106 16-en-20 -one N R R L B - 3 8 0 5

21-Acetoxy-1113,17(x- Arthrobactersimp/ex 21-Acetoxy-111%17~-dihydroxy-6~-methylpreg na- 107 dihydroxy-6c~-methyl 1,4-diene-3,20-dione preg n-4-ene-3,20-dione

513-Androstane-3,17-dione Mycobacterium sp. (i) Androst-4-ene-3,17-dione 108 (ii) 313-Hydroxy-5c~-androstan-17-one

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

And rost-4-ene-3,17-dione And rost-4-ene-3,17-dione

Aspergillus fumigatus Acetone dried cell of

A. fumigatus Chenodeoxycholic acid B a c i l l u s 3a-Hydroxy-7-oxo-513-cholan-24-oic acid 109 Chenodeoxycholic acid Bacillus sp. 3ct-Hydroxy-7-oxo-513-cholan-24-oic acid 110 Cholesterol Mycobacterium sp. Cholest-4-en-3-one 88 Cholesterol Rhodococcus e q u i Cholest-4-en-3-one 90 Cholesterol Escherichia (i) Cholest-4-en-3-one 111

(ii) Cholest-5-en-3-one Cholesterol Nocardia r u b r a Cholest-4-en-3-one 112 Cholesterol Arthrobacter simplex Cholest-4-en-3-one 113 Cholesterol Rhodococcus Cholest-4-en-3-one 34

erythropolis Cholesterol Rhodococcus e q u i Cholest-4-en-3-one 114 Cholic acid Bacillus (i) 3(x-Hydroxy-7,12-dioxo-513-cholan-24-oic acid 109

(ii) 3c(,12ct-Dihydroxy-7-oxo-513-cholan-24-oic acid Cholic acid Arthrobacter simplex (i) 3c~,7(x-Dihydroxy-12-oxo-513-chotan-24-oic acid 52

(ii) 7c(,12ct-Dihydroxy-3-oxo-4-cholenoic acid Cholic acid Bacillus sp. TTUR-2 3(x,7a-Dihydroxy-12-oxo-5~-cholan-24-oic acid 115 Cholic acid Bacillus sp. (i) 3(x,7(x-Dihydroxy-12-oxo-513-cholan-24-oic acid 110

313,1713-Dihydroxy-5ct-androstane 3ct-Hydroxy-5(x-androstan-17-one

3,3-(2,2-Dimethyltrimethyl- enedioxy)-5-10ct-epoxy-5(x- estr-9(11 )-en-1713-ol

3(x-Hydroxy-513-androstan-17-one 313-Hydroxypregn-5-en-20-one Lithocholic acid 19-Nor-1713-hydroxy-androst-

4-en-3-one Sitosterol

(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)-5,10tx- epoxy-5a-estr-9(11 )-en-17-one

1113,17(x,21-Trihydroxypregn- 4-ene-3,20-dione

1113,17(x-21-Trihydroxypregn- 4-ene-3,20-dione

1113,17(x,21-Trihydroxy-6c~- methyl-pregn-4-ene-3,20-dione

1113,17ct,21 -Trihydroxypreg n- 4-ene-3,20-dione

1113,17ct,21-Trihydroxypreg n- 4-ene-3,20-dione

Ursodeoxycholic acid

38 38

Nocardia rubra 112 Mycobacterium sp. 108

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


(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 Bacillus sp.

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 t r ihydroxyprogesterones, among which 713,1513,17a- trihydroxyprogesterone (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 mar- row 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 4e t - t r ihydroxy-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-

Ste ro ids , 1997, vo l . 62, Ap r i l 339

Review

Table 4 Miscellaneous reaction


313-Acetoxy-19-hydroxy- Moraxella sp. (i) 19-Hydroxycholestan-3-one 84 cholest-5-ene ~, c~'-bipyridyl (ii) 19-Hydroxycholest-4-en-3-one

5c~-Androstane-3,17-dione

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

5~-Androstan-17-one

(iii) Cholest-5-ene-313,19-diol (i) 1713-Hyd roxy-5a-androstan-3-one 58

(ii) 17~-Acetoxyandrosta n-3-one (i) 1713-Hydroxyandrosta-1,4-dien-3-one 124

(ii) Androst-4-ene-3,17-dione (iii) 1713-Hydroxya ndrost-4-en-3-one (i) 5e-Androstan-1713-ol 58

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

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-ene- 3,20-dione

17~-Hydroxypregn-4-ene- 3,20-dione

Lithocholic acid

Lithocholic acid 3-Methoxy-8,14-secoestra-

1,3,5(10),9(11 )-tetraene- 14,17-dione

I~-Methyldigitoxin Pregn-4-ene-3,20-dione Preg n-4-ene-3,20-dio ne

Sitosterol


Marchantia polymorpha


Mycobacterium sp. NRRLB-3683 Marchantia polymorpha Aspergillus fumigatus Acetone dried cells

of A. fumigatus Alcaligen recti

Arthrobacter-82 A/ca/igen recti


Corynebacterium


Digitalis lunata Pergularia tomentosa

Mycobacterium sp, Mucorpiriformis sp.

Nocardia DSM-43298

Mucor piriformis


Cunninghamela blackesleena Kloeekera magma

Digitalis Cyanidiophyceae caldium Scenedesmus

quadricauda Rhodococcus equi k-3

(ii) 5c~-And rosta ne- 1713-acetate 1713-Hydroxyandrost-4-en-3-one 18 17c~-Hydroxyandrost-4-ene-3,11 -dione 124 1717,-Hydroxyandrost-4-en-3-one 38

(i) 1713-Hydroxyandrost-4-en-3-one 38 (ii) 1713-Hydroxyand rosta-l,4-dien-3-one (i) 3~-Methoxy-7~,12~-dihydroxy-5~-cholan- 125

24-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

3-Oxo-23,24-dinorchola-1,4-diene-22-oic acid 126 (i) 3~-Methoxy-7~,12~-dihydroxy-513-cholan- 125

24-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 (i) 3,12-Dioxo-23,24-dinorchola-4,6-dienoic acid 52

(i i) 7c~-Hydroxy-3,12-dioxo-23,24-dinorcholenoic acid (iii) 3~,7e-Dihydroxy-12-oxo-513-23,24-

dinorcholan-22-oic acid (iv) 7tx,12~-Dihydroxy-3-oxo-513-cholan-24-oic acid (v) 7~,12~-Dihydroxy-3-oxo-23,24-dinorchol-

4-enoic acid (vi) Methyl-3~,7~,12~-trihydroxy-513-cholan-24-oate (vii) 21~-Hydroxy-3,12-dioxo-23,24-dinorcholan-

4,6-dienoic acid 3e,12~-Dihydroxy-7-oxo-23,24-dinor-513-cholan- 127

22-oic acid (i) Methyl-3~,12~-dihydroxy-513-cholan-24-oate 128

(ii) 3,12-Dioxo-513-cholan-24-oic acid (iii) 3cx-Hydroxy-12-oxo-51~-cholan-24-oic acid Digoxin 129

(i) Periplogenin 130 (ii) l~-Hydroxy digitoxigenin

(iii) Uzarigenin 3~-hydroxy-5¢x-androstan-17-one 108

(i) 3~,1713-Dihydroxyandrost-5-ene 61 (ii) 313-Hydroxyandrost-5-ene-7,17-dione

(iii) 313,1713-Dihyd roxyandrost-5-ene-7-one (i) 9,10-Seco-3,17~-dihydroxypregna-1,3,5 131

(10)-triene-9,20-dione (ii} 9,10-Seco-3,17c~,20-trihydroxy-pregna-1,

3,5(10)-trien-9-one 17e,20{x-Dihyd roxypreg n-4-en-3-one

(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~-Hyd roxypreg 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

48, 61

128

50 132

133 83 83

44

3 4 0 Ste ro ids , 1997, vo l . 62, A p r i l


I OH 2 3

R

~ J ~ C O O H ~llr~C00H i730

5 R • CH 3 6 7

ICH 3

8 H 9 I0

o~'"~v ~" g 0-~ ~'..~ V 0 "~ ~ V

II 12 13

0

0 17 ~H3 C-O

H 0 " ~ H 20

~ ~ ~ : 0 M COOH COOH 0

14 15 OH 16

--OH

OH OH 0 HO

18 19

COOH

CH20H ,,, . , , '" , . , , ,~ ~ . o ~ O , L I J

OH 21

"~ . ~ .H . ICOOH

2 5 26 2 2 R :COOH 2 3 R : COOMe 2 4 R : CHO

Figure 1 Structures of novel metabolites.


Review

formis. The structure of 20 was determined by X-ray crys- tallography. 49 A new bile acid glycoside lithocholic acid 3a-O-13-D-glucopyranoside (21) was produced by Cunning- hamella blakesleena ST-22 from lithocholic acid. 5° In a study on the sterol-transforming ability of fast growing My- cobacterium strains obtained by in vivo genetic recombina- tion 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 crys- tallography, and its absolute configuration was deduced to be 24(R) and 25(R). A novel metabolite, 213-hydroxy-3,12- dioxo-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.

Future possibilities

Since 1950, steroids have been the traditional field for in- dustrially 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. Con- tinuous emphasis on the application of genetic engineering of microorganisms for their improvement as steroid- transforming 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 commer- cial 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.

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

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