The introduction of a hydroxyl group “biohydroxylation” in the steroid skeleton is an important step in the synthesis of new steroids used physiologically as hormones and active drugs. There are currently about 300 known steroid drugs whose production constitutes the second category within the pharmaceutical market after antibiotics. Several biotransformations at industrial scale have been applied in the production of steroid hormones and drugs, which have functionalized different types of raw materials by means of chemo-, regio-, and stereoselective reactions (hydroxylation, Baeyer-Villiger oxidation, oxidation reactions, reduction of group carbonyl, isomerization, and Michael additions, condensation reactions, among others). In Green Chemistry, biotransformations are an important chemical methodology toward more sustainable industrial processes.
- steroid compounds
- biological transformation
Steroids (stereos = solids) are organic compounds derived from alcohols, which are widely distributed in the animal and plant kingdoms. Their base skeleton has 17 carbon atoms in a tetracyclic ring system known as cyclopentanoperhydrophenanthrenes (gonane and estrane). In this group of substances, life-vital compounds are categorized, such as cholesterol, bile acids, sex hormones, vitamin D, corticosteroids, cardiac aglycones, and antibiotics, among others.
Some of the most potent toxins are steroid alkaloids. Steroids are responsible for important biological functions in the cell; for example, the steroids derived from androstane, pregnane, and estrane have hormonal activity [1, 2, 3, 4, 5]; bile acids are important for the digestion and absorption of fats; and cardiotonic aglycones are used for the treatment of heart disease. Sterols are constituents of the cell membrane, essential for cell stability and development; also, they are precursors of bile acids and steroid hormones.
A large number of steroids are used as anti-inflammatory agents , immunosuppressants, progestational agents, diuretics, anabolics, and contraceptives [7, 8, 9]. Some are used for the treatment of prostate and breast cancer [10, 11], for adrenal insufficiency , for prevention of heart disease , as antifungal agents , and as active ingredients used for the treatment of obesity  and AIDS . Recently, the antiviral activity against the herpes simplex virus type I of some steroid glycosides was determined .
The therapeutic action of some steroid hormones has been associated with their interaction with intracellular receptors, which act as transcription factors in the regulation of gene expression . It has been reported that some steroids, such as dehydroepiandrosterone (DHEA), progesterone, pregnenolone and its sulfated derivatives [19, 20], as well as, 17β-estradiol, allopregnanolone and its synthetic derivatives (afoxolaner and ganaxolone) are considered neurosteroids, due to their action at the level of the CNS .
The physiological activity of steroids depends on their structure, the type, number, spatial orientation, and reactivity of the different functional groups present in the tetracyclic core as well as the oxidation state of the rings. For example, the presence of an oxygenated function in C-11β is crucial for the anti-inflammatory activity; the hydroxyl function in C-17β determines androgenic properties; the aromatization of ring A confers estrogenic effect; and corticosteroids have the 3-keto-4-ene group and the pregnane side chain at C-17 [21, 22].
Currently, about 300 steroid drugs are known, and this number tends to grow. Their production represents the second category in the pharmaceutical market after antibiotics [24, 25]. Nowadays, steroids represent one of the largest sectors in pharmaceutical industry with world markets in the region of US$ 10 billion and the production exceeding 1,000,000 tons per year .
The production of steroid drugs and hormones is one of the best examples of the applications that biotransformations have on an industrial scale [3, 21]. Microbiological transformations are an effective tool for the preparation of various compounds , which can be difficult to obtain by conventional chemical methods and have been widely used in the bioconversion of steroids . In 1950, the pharmacological effects of cortisol and progesterone were reported, in addition to the hydroxylation of the latter in C-11α using Rhizopus species. This began a very important stage in the development of the synthesis of steroids with biological activity [4, 5].
Currently, a great versatility of microbial systems in the pharmaceutical industry for the commercial production of steroids and other drugs is recognized [27, 28]. Several hundreds of microbiological transformations of steroids have been reported in the literature; also, many bioconversions have been incorporated into numerous partial syntheses of new compounds for their evaluation such as hormones or drugs [21, 29, 30, 31, 32]. Chemical derivatives of some steroids are reported to have better therapeutic advantages than the starting materials.
However, the main objectives in the research and development of the steroid drug industry currently consist of the detection and isolation of microbial strains with novel activity or more efficient transformation capacity, where genetic engineering and metabolic engineering can play a prominent role in the metabolism of bacteria, fungi, and plants [33, 34, 35, 36].
The aim of the present review is to emphasize the importance of biotransformation using microorganisms to obtain steroid compounds with pharmaceutical interest, as a chemical-biological strategy that alternates with the chemical synthesis, and to highlight the chemical reaction made by different types of microorganisms in the functionalization of the steroid skeleton.
2. Microbiological transformations of steroids
In Green Chemistry, biotransformations constitute an important methodology in organic chemistry . The microbiological transformations of steroids have been an essential chemical tool used for the preparation of many intermediaries and in the generation of new drugs, where chemical functionalization-hydroxylation, Baeyer-Villiger oxidation, reduction, isomerization, Michael additions, and condensation reactions can be carried out in different positions of the steroid skeleton in chemo-, regio-, and stereoselective ways, being very complicated or even impossible by the classic chemical methods. Currently, any stereogenic center of the steroid skeleton can be specifically hydroxylated stereoselectively. Nowadays, biohydroxylations in C-11α, 11β, 15α, and 16α are industrially carried out via a microbial hydroxylation with good yields and enantiomeric excess (ee). Below are some of the microbiological transformations performed on different natural and synthetic steroids .
In the literature, it is the well-documented regio- and stereoselective hydroxylation in C-14 with α orientation in progesterone (1) and other steroids by well-functioning fungi, such as Thamnostylum piriforme (ATCC 8992), Mucor griseocyanus (ATCC 1207a), Actinomucor elegans (MMP 3132), and Zygodesmus sp. (ATCC 14716).
From the incubation of 1 with T. piriforme, 14α-hydroxyprogesterone (2, 32%) and 9α-hydroxyprogesterone (3, 1.4%) were obtained; whereas in the incubation of 1 with M. griseocyanus,2 (13.4%), 7α,14α-dihydroxyprogesterone (4, 6.5%) and 6β,14α-dihydroxyprogesterone (5, 2.8%) were obtained. In the biotransformation of 1 using A. fumigatus after 24 h of incubation, different mono-and dihydroxylated products were obtained: 11α-hydroxyprogesterone (6, 33%), 11α,15β-dihydroxyprogesterone (7, 17%), 7β,15β-dihydroxyprogesterone (8, 14%), 15β-hydroxyprogesterone (9), 7β-hydroxyprogesterone (10), where 9 and 10 were detected in minimal quantity. Finally, at 72 h, the main products were 7 (48%) and 8 (25%), with the positions 11α and 15β being hydroxylated more easily than the position 7β in 1 [38, 39].
In the incubation of 1 with Saprolegnia hypogyna, 4-androstene-3,17-dione (11), testosterone (12), and testolactone (13) were obtained . The compounds 13 (98%) were also obtained from the bioconversion of 1 using A. sojae (PTCC 5196). The biotransformation pathway indicating the presence of Baeyer-Villiger monooxygenase (BVMO) can carry out both oxygenative esterification of 20-ketosteroids and oxygenative lactonization of 17-ketosteroids . The compounds 15α-hydroxyprogesterone (14, 47%) and 12β,15α-dihydroxyprogesterone (15, 25%) were isolated in the biotransformation of 1 using Fusarium culmorum . In the biotransformation of 1 using the bacterium, thermophilic Bacillus stearothermophilus, four products of monohydroxylation, 20α-hydroxyprogesterone (16, 61%), 6β-hydroxyprogesterone (17, 21%) and 6α-hydroxyprogesterone (18, 14%), and 9,10-seco-pregnen-3,9,20-trione (19, 4%), were isolated .
An efficient regio- and stereoselectivity was observed in the biotransformation of 1 on a large scale by the system Mucor 881 (M881) to give the hydroxylated derivatives 6, 6β,11α-dihydroxyprogesterone (20), and 6β-hydroxypregn-4-ene-3,11,20-trione (21). In the literature, it is described that species of the genus Mucor and Rhizopus can hydroxylate said positions but with lower yields. The fungal system M881 showed the ability to carry out hydroxylation at 6β and 11α positions of 4-ene-3-one steroids (1, 11, 12 and 211) .
Recently, it was reported that in the biotransformation of 1 using Penicillium aurantiogriseum for 10 days, 11 and androsta-1.4-dien-3,17-dione (22) were obtained. These products were observed in the biotransformation of 1 using Bacillus sphaericus; the hydroxylation in C-17 was mainly observed [45, 46]. Biotransformation of 1 using Geobacillus gargensis (DSM 15378) has resulted in the production of secoderivatives: 19 and 23 (9,10-seco-4-pregnene-20α-hydroxy-3,9-dione), which are produced by the rupture of the ring B of 1 (Figure 1) . Secosteroids are an important group, which exhibits a variety of different biological activities [48, 49].
In the biotransformation of 5β-dihydroprogesterone (24) using T. piriformis, 14α-hydroxy-5β-pregnan-3,20-dione (25, 11.8%), 3β,14α-dihydroxy-5β-pregnan-20-one (26, 0.5%), and 14α,15β-dihydroxy-5β-pregnan-3,20-dione (27, 0.4%) were characterized, while in the biotransformation of 3β-hydroxy-5β-pregnan-20-one (28), 26(0.6%) and 3β,9α,14α-trihydroxy-5β-pregnan-20-one (29, 16%) were isolated, after being incubated for 96 h. The microbiological transformation of 28 using Actinomucor elegans produced the compounds 25 and 28 in lower yield than T. piriforme and a minor product identified as 3β,9α-dihydroxy-5α-pregnan-20-one (30) (Figure 2) .
The biotransformation of 16-dehydroprogesterone (4,16-pregnadien-3,20-dione, 31) using Mucor piriformis has been reported to give different hydroxylation products: 14α-hydroxypregna-4,16-dien-3,20-dione (32, 1%), 7α,14α-dihydroxypregna-4,16-dien-3,20-dione (33, 78%), 3β,7α,14α-trihydroxy-5α-pregna-16-en-20-one (34, 3%), and 3α,7α,14α-trihydroxy-5α-pregna-16-en-20-one (35, 2%); while the microsomes prepared from 31 transformed the hydroxylate to 14α-hydroxy derivative (32). Incubation of 32 with M. piriformis resulted in the formation of 33–35 (Figure 3) .
In contrast, in the biotransformation of 17α-hydroxyprogesterone (36) using M. piriformis, after 48 h of incubation, four compounds were obtained: 17α,20α-dihydroxypregn-4-en-3-one (37, 19%), 7α,17α-dihydroxypregn-4-en-3,20-dione (38, 25%), 6β,17α,20α-trihydroxy-pregn-4-en-3-one (39, 18%), and 11α,17α,20α-trihydroxypregn-4-en-3-one (40, 25%); it was observed that M. piriformis was able to hydroxylate the C-6, C-7, C-11, and C-14 positions stereospecifically, in addition to reducing the 4-en-3-one system in ring A and the keto group of C-20 (Figure 4) . The biotransformation of 36 using Fusarium culmorum led to the formation of 14 (47%) and 15 (25%) .
Pregnenolone (3β-hydroxypregn-5-en-20-one, 41), the precursor of many steroid hormones, was biotransformed by Mucor piriformis to obtain two metabolites, 3β,7α-dihydroxypregn-5-en-20-one (42) and 3β,7α,11α-trihydroxypregn-5-en-20-one (43) , where 43 (46.4%) was also a bioconversion product of 41 using Mucor circinelloides var. lusitanicus . Two metabolites of pregnenolone (41) obtained from biotransformation of B. cinereae were characterized as 3β,11α,16β-trihydroxypregn-5-en-20-one (44, 39%) and 11α,16β-dihydroxypregn-4-en-3,20-dione (45, 6%). The formation of the hydroxylation products in C-11 and C-16 by B. cinereae can be determined by the presence of the acetyl group in C-20 . The biotransformation of 41 using different microorganisms (Cunninghamella elegans, R. stolonifer, and G. fujikuroi) was reported by Choudhary et al. . Incubation of 41 with C. elegans produced 3β,7β,11α-trihydroxypregn-5-en-20-one (46, 28%), 3β,6α,11α,12β,15β-pentahydroxypregn-4-en-20-one (47, 4%), and 3β,6β,11α-trihydroxypregn-4-en-20-one (48, 2%), while incubation with G. fujikuroi, two products 3β,7β-dihydroxypregn-5-en-20-one (49, 3%) and 6β,15β-dihydroxypregn-4-en-3,20-dione (50, 2%) were obtained. In the microbiological transformation of 41 using different Bacillus strains, 42, 49, and 7-oxo-pregnenolone (51) were the major products obtained , while by using Fusarium oxysporum var. cubense,42 was the only product obtained . The biotransformation of pregnenolone acetate (52) using C. elegans generated 41, 22, 6β,15β-dihydroxyandrosta-4-en-3,17-dione (53), and 11α,15β-dihydroxypregn-4-en-3,20-dione (54), while by using R. stolonifer, 11α-hydroxypregn-4-en-3,20-dione (55) and 53 were obtained (Figure5) .
The microbiological transformation of the racemic mixture of 13-ethyl-17β-hydroxy-18,19-dinor-17α-pregn-4-en-20-yn-3-one (56) was tested with different fungi Rhizopus nigricans, R. arrhizus, Aspergillus niger, A. ochraceus, and Curvularia lunata. The bioconversion of the racemic mixture of 53 by R. arrhizus produced only one major product, (±)-13-ethyl-10β,17β-dihydroxy-18,19-dinor-17α-pregn-4-en-20-yn-3-one (57, 28.4%), whereas R. nigricans, A. niger, and C. lunata biotransformed 56 to 57 more slowly and inefficiently .
The racemic mixture (±)-13-ethyl-7β,17β-dihydroxy-18,19-dinor-17α-pregn-4-en-20-yn-3-one (58, 4.3%) was obtained as product of incubating mixture 56 with A. ochraceus; none of the fungi tested were able to differentiate the two enantiomers of 56 in the course of the hydroxylation reaction; in addition, the absence of the hydroxylated derivative in C-11 is due to the presence of the ethyl group in C-13 or the ethynyl group in C-17 . The microbiological transformation of the racemic mixture and the dextro enantiomer of compound 56 has been described using different species of Cunninghamella . For example, the transformation of the racemic mixture of 56 by C. blakesleeana (AS 3.910) produced 57 (5.3%), 13-ethyl-6β,17β-dihydroxy-18,19-dinor-17α-pregn-4-en-20-yn-3-one (59, 3.6%), 13-ethyl-15α,17β-dihydroxy-18,19-dinor-17α-pregn-4-en-20-yn-3-one (60, 3.0%), and 13-ethyl-6β,10β,17β-trihydroxy-18,19-dinor-17α-pregn-4-en-20-yn-3-one (61, 3.6%), while by using C. echinulata (AS 3.1990), 61 (3.2%), 57 (1.2%), and enantiomer dextro of 58 (2.9%) were obtained. The transformation of the enantiomer dextro of 56 using C. blakesleeana produced 57 (1.2%), 58 (2.9%), and 61 (3.2%), by using C. echinulata, the same compounds were obtained but in lower yield. Therefore, the microbial transformation of the racemic mixture and the d-enantiomer of 56 using different Cunninghamella species gave poor yields and poor resolutions, which were obtained for the hydroxylation reaction (Figure 6) .
The biotransformation of danazol (17β-hydroxy-17α-pregna-2,4-dien-20-yno-[2,3-d]-isoxazole, 62), a heterocyclic steroid drug in which an isoxazole ring is fused with ring-A of a steroid nucleus, using Fusarium lini, A. niger and Cephalosporium aphidicola yielded 17β-hydroxy-2-(hydroxymethyl)-17α-pregn-4-en-20-yn-3-one (63) and 17β-hydroxy-2-(hydroxymethyl)-17α-pregn-1,4-dien-20-yn-3-one (64); while Bacillus cereus afforded 64, as the only product . Microbial transformation of danazol (62) using C. blakesleeana yielded four compounds: 14β,17β-dihydroxy-2-(hydroxymethyl)-17α-pregn-4-en-20-yn-3-one (65, 1.2%), 1α,17β-dihydroxy-17α-pregna-2,4-dien-20-yno-[2,3-d]-isoxazole (66, 1.2%), and 6β,7β-dihydroxy-17α-pregna-2,4-dien-20-yno-[2,3-d]-isoxazole (67, 0.8%) and 64 (1.2%). This involves hydroxilations al C-1, C-6 and C-15, whereas oxidation at C-3, and N-O bond cleavage has also occurred (Figure 7) .
Norethisterone (17α-ethynyl-19-nortesterone, 68) is a potent progestin used as a contraceptive agent; its biotransformation with Cephalosporium aphidicola (IMI 68689) produced the aromatization of ring A that yielded 17α-ethynylestradiol (69), whereas 69 was biotransformed by Cunninghamella elegans (NRRL 1392) producing the compounds 19-nor-17α-pregna-1,3,5(10)-trien-20-yn-3,4,17β-triol (70), 19-nor-17α-pregna-1,3,5(10)-trien-20-yn-3,7α,17β-triol (71), 19-nor-17α-pregna-1,3,5(10)-trien-20-yn-3,11α,17β-triol (72), 19-nor-17α-pregna-1,3,5(10)-trien-20-yn-3,6β,17β-triol (73), and 19-nor-17α-pregna-1,3,5(10)-trien-20-yn-3,17β-diol-6β-methoxy (74) (Figure 8) .
Mestranol (75) and 17β-methoxymestranol (76) are the mono- and dialkylated derivatives of 69, respectively. In incubating 75 with C. elegans, two hydroxylated compounds were obtained: 6β-hydroxymestranol (77, 2.8%) and 6β,12β-dihydroxymestranol (78, 3.6%), inferring that the presence of the methoxyl group in C-3 reduces the number of biotransformation products and introduces hydroxyl groups in C-6 and C-12 with β orientation, while 76 was not biotransformed due to the presence of the methoxyl group in C-17 (Figure 9) .
Microbial transformation of 6-dehydroprogesterone (79) using A. niger yielded five metabolites: 6β-chloro-7α,11α-dihydroxypregna-4-en-3,20-dione (80, 1.0%), 7α-chloro-6β,11α-dihydroxypregna-4-en-3,20-dione (81, 1.33%), 6α,7α,-epoxy-11α-hydroxypregna-4-en-3,20-dione (82, 1.33%), 6α,7α,-epoxy-pregna-4-en-3,20-dione (83, 2.0%), and 11α-hydroxypregna-4,6-dien-3,20-dione (84, 2.33%). Compound 11α-hydroxyandrosta-4,6-dien-3-one (85, 15.4%) was obtained through whole cell biotransformation of 79 by G. fujikuroi (ATCC 10704). The formation of 80 and 81 is an interesting finding. This route provides an efficient method for the obtention of chlorohydrins from alkene functionality . The compound 84 was obtained through the microbial transformation of 79 using R. nigricans , Nigrospora sphaerica, Mucor racemosus, and Botryosphaeria obtusa. 6-dehydroprogesterone (79) is a synthetic derivate of progesterone. Botryodiplodia theobromae was used for the synthesis of 6-DPH from progesterone (Figure 10) .
Incubation of melengestrol acetate (86) with C. blakesleeana, which provides an route for the monohydroxylation of the (86) at C-11, yielded a 17α-acetoxy-11β-hydroxy-6-methylenepregna-4,6-diene-3,20-dione (87) (Figure 11) .
Biotransformation of 3β-hydroxy-17β-carboxyethyl-5β-androstenol (88) using T. pyriformis resulted in the mixture of 3β,14α-dihydroxy-17β-carboxyethyl-5β-androstenol (89, 9%) with 9α,14α-dihydroxy derivative (90, 12%) and two minor products 14α,15α-dihydroxy (91) and 15β-hydroxy (92). Compound 92 was identified as a product of biotransformation using A. elegans, M. griseocyamus, and Zygodesmus sp. (Figure 12) .
Androst-4-en-3,17-dione (11), which plays an important role in the metabolism of drugs, among many other functions, was biotransformed using M. piriformis to give one main product, 6β-hydroxyandrost-4-en-3,17-dione (93, 13%), and four minor products, 14α-hydroxyandrost-4-en-3,17-dione (94, 2%), 7α-hydroxyandrost-4-en-3,17-dione (95, 2%), testosterone (12, 3%), and 6β-hydroxytestosterone (96, 1%). In the biotransformation of 11 using M. griseocyamus94 (9%), 95 (4%) and 14α-hydroxytestosterone (97, 9%) were the major products obtained; likewise, 11 and 93 were identified in the mixture of biotransformation products . From the incubation of 11 with M. piriformis, 94–97 and 7α,14α-dihydroxytestosterone (98) were obtained . Hydroxylated steroids in C-9 are important intermediaries in the synthesis of highly effective anti-inflammatory drugs. The microbiological transformation of 11 to 9α-hydroxyandrost-4-en-3,17-dione (99) was studied using Rhodococcus sp. in a low-nutrient culture medium at a fixed pH (Figure 13) . When 11 was incubated with Bacillus strain HA-V6–3, the metabolites 12, 93–97, 6β,14α-dihydroxyandrost-4-en-3,17-dione (100), 11α-hydroxyandrost-4-en-3,17-dione (101), androst-4-en-3,6,17-trione (102), and 5α-androst-3,6,17-trione (103) were produced as described by Schaaaf and Dettner .
In the bioconversion of 11 using C. aphidicola, 93 and 94 were obtained , while in the fermentation of 11 using Curvularia lunata, the products 101 (4%), 17β-hydroxyandrost-1,4-dien-3-one (104, 4.4%), androsta-1,4-dien-3,17-dione (105, 3%), 11α,17β-dihydroxyandrost-4-en-3-one (106, 4%), and 107 (15α-hydroxyandrost-1,4-dien-3,17-dione, 2.8%) were obtained (Figure 13) . Biotransformation of 11 using Beauveria bassiana was studied in times and with culture media at different pH (pH 6 and 7) . At pH 6, two products were obtained: 106 and 6β,11α-dihydroxyandrost-4-en-3,17-dione (108), where the stereoselective hydroxylation was observed at C-11α and C-6β; while at pH 7, the compounds 12, 106, 3α,11α,17β-trihydroxy-5α-androstane (109), and 6β,11α,17β-trihydroxy-androst-4-en-3-one (110) were obtained. Products 93 (14%) and 94 (75%) were isolated from the biotransformation of 11 using Chaetomium sp. (Figure 13) .
Obtaining hydroxylated derivatives in a specific position is one of the objectives of the steroid industry; for example, 14α-hydroxysteroids are shown to have anti-inflammatory, contraceptive, and antitumor activities. With the biotransformation of 11 and 105 using different strains of the fungus, C. lunata allowed in the case of 11, the production of a major product, 94; while with 105, 14α-hydroxyandrost-1,4-dien-3,17-dione (111, 70%) was obtained (Figure 13) .
Androsta-1,4-dien-3,17-dione (105) is a useful precursor in the chemical or microbiological preparation of other steroid hormones and pharmaceutical. Transformation of 105 by Colletotrichum lini (As3.486) produced the hydroxylated compounds at C-11α and C-15α: 15α-hydroxyandrost-1,4-dien-3,17-dione (107), 11α,15α-dihydroxyandrost-1,4-dien-3,17-dione (112), and 15α,17β-dihydroxyandrost-1,4-dien-3-one (113) (Figure 14) .
Testosterone (12) was metabolized by M. griseocyamus and T. piriforme. In the biotransformation of 12 using M. griseocyamus, 97 (35%) and other products were obtained, where 94 was identified as the major product. Conversely, the microbiological transformation of 12 using T. piriforme produced 97 (10%), as the main product at 24 h; after 72 h of biotransformation, four products were obtained: 93 (13%), 96 (7%), 97 (13%), and 111 (5%). It was discovered that T. piriforme produced smaller quantity of 14α-hydroxy derivatives (Figure 15) .
In the biotransformation of 12 using Nectria haematococca, four substances were isolated, whose performance was dependent on the incubation time; majority of the products were produced at 72 h. The hydroxylated derivatives in C-11 with α orientation and dehydrogenation in C1-C2 resulted in the following compounds: 11α-hydroxyandrost-1,4-dien-3,17-dione (114, 8.0%), 11α,17β-dihydroxyandrost-1,4-dien-3-one (115, 4.3%), 101 (1.9%), and 104 (2.3%) . Incubation of 12 with Fusarium culmorum produced 93 (10%) and 96 (32%) with hydroxylated derivatives at C-6β, including the products, 15α,17β-dihydroxyandrost-4-en-3-one (116, 22%) and 15α-hydroxyandrost-4-en-3,17-dione (117). Selective hydroxylation of 103 at C-6 with a β orientation and allylic position at the unsaturated 3-keto-system is favored by the system π and the presence of the hydroxyl group at C-17, while hydroxylation at C-15 is a very frequent process carried out by fungi of the genus Fusarium . Metabolites 11, 85, 105, and 115 were obtained as oxidation and hydroxylation products of 12 using the fungus F. oxysporum var. cubense . The fungus, Cephalosporium aphidicola, was hydroxylated with 12 to give the products 96 (47%) and 97 (3%), with hydroxylated derivatives in C-6β and C-14α, respectively . Incubation of 12 with C. lunata and Pleurotus ostreatus yielded compounds 11 (17%) and 115 (13%), respectively . The phytopathogenic fungus, Botrytis cinerea, produced 7β,17β-dihydroxyandrost-3-one (118, 73%), as the only biotransformation product of 12. It seems that the presence of the hydroxyl group in C-17 in the androstane skeleton directed the hydroxylation at C-7 with a β orientation (Figure 15) .
In the biotransformation of 12 using Bacillus stearothermophilus, thermophilic bacterium, the major product obtained was 11 (90.2%); it was generated by the oxidation of C-17, and the hydroxylated derivatives of 11 in C-6 (93, C-6β, 1.1%) and (119, C-6α, 0.9%) include two monohydroxy derivatives of 12, 96 (C-6β, 3.9%) and 120 (C-6α, 3.9%). This indicates that hydroxylation with α orientation in C-6 may be a common action of some thermophilic bacteria . Biotransformation of 11 using B. stearothermophilus in the presence of hydrolase inducers—salicylic acid, chloramphenicol, cyclodextrin, dexamethasone, riboflavin, and rifampicin—resulted in obtaining a higher concentration of the compounds: 9,10-seco-4-androst-3,9,17-trione (121), 5α-androst-3,6,17-trione (103), 17β-hydroxy-5α-androst-3,6-dione (122), 3β,17β-dihydroxyandrost-4-en-6-one (123), and 17β-hydroxyandrost-4,6-dien-3-one (124). For example, the presence of glucose and cycloheximide favored the obtaining of 123, while the production of 124 was achieved in the presence of rifampicin . The products isolated from the biotransformation of 12 using Chaetomium sp. were 93 (21%), 94 (39%), and 99 (19%); after 24 h of incubation, the presence of 11 was detected. Janeczko et al.  concluded that the steric factors associated with the substrate determine the location and orientation of the hydroxyl group. For example, the carbonyl group in C-17 at 11 directs the entry of the hydroxyl group at C-14 with α orientation, while the hydroxylation in C-6β is favored by the presence of the hydroxyl group in C-17, as in 12. In the case of progesterone (1), which has an acyl group, dihydroxylated derivatives were observed in C-6 and C-14 (Figure 15) .
Incubation of 11 and 12 with C. lini ST-1 displayed different catalytic characteristics. Biotransformation of 11 afforded two products: 15α-hydroxyandrost-4-en-3,17-dione (117, 5%) and 11α,15α-dihydroxyandrost-4-en-3,17-dione (125, 64%), while 12 yielded 15α-hydroxyandrost-4-en-3,17-dione (117, 60%). Incubation of 1 resulted in the isolation of 14. Wu et al.  concluded that the different hydroxylation sites between 11 and 12 suggested that the hydroxyl group or carbonyl group on the substrate at C-17 had influence on the location of introduced hydroxyl groups (Figure 15).
Dehydroepiandrosterone (3β-hydroxyandrost-5-en-17-one, 126) endogenous prohormone secreted by the adrenal glands is a precursor of androgens and estrogens. Incubation with M. piriformis allowed the isolation of five compounds: 3β,17β-dihydroxyandrost-5-ene (127), 3β,7α-dihydroxyandrost-5-en-17-one (128), 3β-hydroxyandrost-5-en-7,17-dione (129), 3β,17β-dihydroxyandrost-5-en-7-one (130), and 3β,7α,17β-trihydroxyandrost-5-ene (131). The action of the fungus was the stereospecific hydroxylated products at C-7α (128 and 131) and the reduction of the carbonyl group at C-17 . From the microbiological transformation of 126 using Rhizopus stolonifer, six poducts were isolated: 127 (20%), 128 (12%), 129 (20%), 3β,17β-dihydroxyandrost-4-ene (132, 12%), 17β-hydroxyandrost-4-en-3-one (133, 34%), and 3β,11β-dihydroxyandrost-4-en-17-one (134, 15%) . Fusarium oxysporum biotransformed to 126 in a mixture of four hydroxylated derivatives (127–129 and 130), which were characterized as their acetylated derivatives; the hydroxylation was favorably in C-7 stereospecifically (α orientation) in the 3β-hydroxy-Δ5-steroids, while Colletotrichum musae biotransformed to 126–127 by reducing the carbonyl group in C-17 (Figure 16) .
In the biotransformation of 126 using Penicillium griseopurpureum and P. glabrum, the following was produced; hydroxylated derivatives in C-7α (95), C-14α (94) and C-15α (117), with 11 being the main product. In addition, P. griseopurpureum generated products for the Baeyer Villiger oxidation to give the lactone D ring (testolactone, 13) and its hydroxylated derivative at C-15α (15α-hydroxy-17α-oxa-
The biotransformation of 17α-ethynyl-17β-hydroxyandrost-4-en-3-one (ethisterone, 138) and 17α-ethyl-17β-hydroxyandrost-4-en-3-one (139) was described using the fungi Cephalosporium aphidicola and Cunninghamella elegans. The bioconversion of 138 using C. aphidicola yielded 17α-ethynyl-17β-hydroxyandrost-1,4-dien-3-one (140, 5.5%), while by using C. elegans, 17α-ethynyl-11α,17β-dihydroxyandrost-4-en-3-one (141, 3.4%) was obtained. The biotransformation of 138 using C. aphidicola generated 17α-ethyl-17β-hydroxyandrost-1,4-dien-3-one (142, 2.2%). In contrast, when incubating 139 with C. elegans, two new products were obtained: 17α-ethyl-11α,17β-dihydroxyandrost-4-en-3-one (143, 2.8%) and 17α-ethyl-6α,17β-dihydroxy-5α-androstan-3-one (144, 1.6%) (Figure 17) .
Adrenosterone (145) is an inhibitor of the enzyme estrogen synthetase responsible for the formation of estrogen, and it has a great clinical application. Biotransformation of 145 using C. aphidicola produced androst-1,4-dien-3,11,17-trione (146, 3%), 17β-hydroxyandrost-4-en-3,11-dione (147, 2%), and 17β-hydroxyandrost-1,4-dien-3,11-dione (148, 17%). 145 (11.2%) and 12 (8.1%) were obtained from the biotransformation of 145 using Fusarium lini, while 147 (36.8%) was obtained from the biotransformation of 145 using Trichothecium roseum (Figure 18) .
The biotransformation of mesterolone (1α-methyl-17β-hydroxy-5α-androst-3-one, 149), a synthetic androgenic steroid, was performed using different fungi as described by Choudhary et al. . From the biotransformation of 149 using C. aphidicola, the compounds 1α-methyl-5α-androst-3,17-dione (150), 1α-methyl-5α-androst-3,17-diol (151), and 1α-methyl-15α-hydroxy-5α-androst-3,17-dione (152) were obtained. Incubation of 149 with Fusarium lini produced the compounds 152, 1-methyl-5α-androst-1-en-3,17-dione (153), 1α-methyl-6α,17β-dihydroxy-5α-androst-3-one (154), 1α-methyl-15α,17β-dihydroxy-5α-androst-3-one (155), and 1-methyl-15α,17β-dihydroxy-5α-androst-1-en-3-one (156). The products obtained from the biotransformation of 149 using R. stolonifer were 150, 154, 156, 1α-methyl-7α,17β-dihydroxy-5α-androst-3-one (157), and 1α-methyl-11α,17β-dihydroxy-5α-androst-3-one (158) . Bioconversion of 149 using C. blakesleeana produced seven biotransformation products, such as 154, 157, 158, in addition to 1α-methyl-1β,11β,17β-trihydroxy-5α-androst-3-one (159), 1α-methyl-7α,11β,17β-trihydroxy-5α-androst-3-one (160), 1α-methyl-1β,6α,17β-trihydroxy-5α-androst-3-one (161), and 1α-methyl-1β,11α,17β-trihydroxy-5α-androst-3-ona (162). Macrophomina phaseolina biotransformed 149 to obtain 1α-methyl-17β-hydroxy-5α-androst-3,6-dione (155) . Additionally, the biotransformation of 141 using C. blakesleeana (ATCC 8688A) yielded three metabolites: 1α-methyl-11β,14α,17β-trihydroxy-5α-androstan-3-one (163, 0.4%), 1α-methyl-7β,17β-dihydroxy-5α-androstan-3-one (164, 0.47%), and 1α-methyl-17β-hydroxy-5α-androstan-3,7-dione (165, 0.67%). C. blakesleeana catalyzed the β-hydroxylation in C-11, and dihydroxylation and oxidations at various positions of steroid skeleton (Figure 19) .
In the microbiological transformation of 3-hydroxyestra-1,3,5-(10)-trien-17-one (166) using Fusarium oxysporum var. cubense, the compounds, reduced in C-17 (3,17-dihydroxyestra-1,3,5-(10)-triene, 167) and hydroxylated in C-15 (3,15α-dihydroxiestra-1,3,5-(10)-triene, 168), were isolated (Figure 20) .
Prednisone (169) is a synthetic corticosteroid (prodrug) used for the treatment of autoimmune, inflammatory and kidney diseases, among others. Biotransformation of 169 using C. elegans occurred by hydrogenation of the Δ4(5) and reduction of C-20, to produce the compounds 17α,21-dihydroxy-5α-pregn-1-en-3,11,20-trione (170, 15.6%) and 17α,(20S),21-trihydroxy-5α-pregn-1-en-3,11-dione (171, 6.5%); whereas as the only biotransformation product, 169 using F. lini (5.2%), R. stolonifer (5.5%) and C. lunata (6.2%), was 1,4-pregnadien-17α,(20S),21-trihydroxy-3,11-dione (172) (Figure 21) .
The main chemical transformation carried out by different Acremonium species in various steroid compounds have been oxidations, reductions, hydroxylations in different positions, isomerizations, and hydrolysis of the chain in C-17. Hydrocortisone (173) is an important anabolic, used clinically as anti-inflammatory and antiallergic drug, besides being a raw material for the synthesis of many steroid hormones. Biotransformation of 173 using Acremonium strictum generated the products 11β,17β-dihydroxyandrost-4-en-3-one (174, 8%), 11β,17α,20β,21-tetrahydropregn-4-en-3-one (175, 11.2%), and 21-acetoxy-17β,17α,20-trihydroxypregn-4-en-3-one (176, 7.6%); it was observed that the actions of the said species were as the reduction, acetylation and degradation of the chain in C-17, without modification of the unsaturated ketone-α,β . Biotransformation of 173 using Gibberella fujikuroi yielded 11β-hydroxyandrost-4-en-3,17-dione (177, 41%), while B. subtilis and R. stolonifer yielded 175 (15%). The products 173 (45%) and 3β,11β,17α,21-tetrahydroxy-5α-pregnan-20-one (178, 31%) were obtained from the bioconversion of 173 using Bacillus cereus (Figure 22) .
Incubation of 17β-methoxy-5α-androst-3-one (179) with Cephalosporium aphidicola produced 17β-methoxy-5α-androst-3β-ol (180) and 6β,11α-dihydroxy-17β-methoxy-5α-androst-3-one (181); while the biotransformation of 17β-methoxyestra-4-en-3-one (182) using C. aphidicola produced a major metabolite 6β-hydroxy-17β-methoxyestra-4-en-3-one (183). Similarly, the microbiological transformation of 3β-methoxyandrost-5-en-17-one (184) gave a mixture of products: 7α-hydroxy-3β-methoxyandrost-5-en-17-one (185) and 7β-hydroxy-3β-methoxyandrost-5-en-17-one (186) (Figure 23) .
In the literature, several species of fungi belonging to the genera Aspergillus, Fusarium, Mortierella, and Penicillium and capable of hydroxylating various steroids in C-15 have been described. For example, Jekkel et al.  described that more than 3000 fungi hydroxylate 13β-ethyl-4-gonene-3,17-dione (187) in C-15 position, the genus being Fusarium, particularly F. nivale; the fungus preferentially hydroxylated 187 with an α orientation in C-15 (15α-hydroxy-13β-ethyl-4-gonene-3,17-dione, 188, 77%) and C-7β (7β,15α-dihydroxy-13β-ethyl-4-gonene-3,17-diona, 189). On the other hand, the biotransformation of 187 using Mortierella pusilla produced 188, 190 (10β-hydroxy-13β-ethyl-4-gonene-3,17-dione) and 191 (6β-hydroxy-13β-ethyl-4-gonene-3,17-dione) (Figure 24).
The ethynodiol diacetate (192) is a synthetic derivative 1, used as an oral contraceptive because it inhibits the ovulation process. The microbiological transformation of 192 using Cunninghamella elegans produced four hydroxylated compounds characterized as: 17α-ethynylestr-4-en-3β,17β-diacetoxy-6α-ol (193, 0.5%), 17α-etynylestr-4-en-3β,17β-diacetoxy-6β-ol (194, 1.0%), 17α-etynylestr-4-en-3β,17β-diacetoxy-10β-ol (195, 0.5%), and 17α-ethynyl-17β-acetoxiestr-4-en-3-one (196, 1.4%) (Figure 25) .
Desogestrel (13-ethyl-17-methylene-18,19-dinor-17α-pregn-4-en-20-yn-17-ol, 197) is an orally active third-generation contraceptive steroid drug. Conversion of 197 by C. blackesleeana (ATCC 8688 A) yielded four metabolites: 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-6β,15β,17β-triol (198), 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-3β,6β,17β-triol (199), 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-3α,5α,6β,17β-tetraol (200), and 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-6β,17β-dihydroxy-3-one (201). Compounds 197 and 198 showed a potent growth inhibition against drug-resistant strains of S. aureus (Figure 26) .
The drugs mexrenone (202) and canrenone (203) are steroids with a spironolactone in C-17 and are potent antagonists of mineralocorticoids . The biotransformation of 202 and 203 using a wide variety of microorganisms resulted in the production of monohydroxylated products in different positions, where Beauveria bassiana generated 11α-hydroxymexrenone (204, 67%) as the major product, while 12β-hydroxymexrenone (205, 50%) and 6β-hydroxymexrenone (206, 33%) were obtained using Mortierella isabellina. The dehydrogenation product (Δ1(2)-mexrenone, 207, 15%) was favored with Bacterium cyclooxidants as well as Rhodococcus equi, Nocardia aurentia, and Comamonas testosteroni. From the biotransformation of 203 using Corynespora cassiicola, 9α-hydroxycanrenone (208, 30%) was obtained, . Conversion of canrenone (203) by Colletotrichum lini ST-1 gave two hydroxyl compounds, 15α-hydroxy-canrenone (209, 22%) and 11α,15α-dihydroxy-canrenone (210, 47%) (Figure 27) .
One of the steroids used in the treatment of breast cancer is exemestane (211), an inhibitor of steroidal aromatase. From the transformation of 211 using Macrophomina phaseolina, 16β,17β-dihydroxy-6-methylene-androsta-1,4-diene-3-one (212), 17β-hydroxy-6-methylene-androsta-1,4-diene-3,16-dione (213), and 17β-hydroxy-6-methylene-androsta-1,4-diene-3-one (214) were obtained, while by using Fusarium lini, the only product obtained was 11α-hydroxy-6-methylene-androsta-1,4-diene-3,17-dione (215) (Figure 28) .
4-Hydroxyandrost-4-ene-3,17-dione (formestane, 216) is an irreversible aromatase inhibitor and therapeutically used in breast cancer treatment in postmenopausal women. Bioconversion of 216 using Rhizopus oryzae (ATCC 1145) resulted in the production of 4β,5α-dihydroxyandrost-3,17-dione (217, 8.6%) and 3,5α-dihydroxyandrost-2-ene-4,17-dione (218) , while the biotransformation of 217 using Beauveria bassiana produced 4,17β-dihydroxyandrost-4-en-3-one (219, 5.3%), 3α,17β-dihydroxy-5β-androstan-4-one (220, 0.9%), and 4,11α,17β-trihydroxyandrost-4-en-3-one (221, 2.4%) (Figure 29) .
Methyltestosterone (222), an anabolic steroid, was transformed by Mucor racemosus in 5 days to produce two monohydroxylated products in the C-7 (7α-hydroxymethyltestosterone, 223, 35%) and C-15 (15α-hydroxymethyltestosterone, 224, 21%) positions, plus a dihydroxylated product (12,15α-dihydroxymethyltestosterone, 225, 22%) . Recently, three additional products were identified: 11α-hydroxy-17α-methyltestosterone (226), 6β-hydroxy-17α-methyltestosterone (227), and 6β,11α-dihydroxy-17α-methyltestosterone (228). Isolation of hydroxylation products have been reported in different carbons from 222 with different orientations, C-6β, C-7β, C-9α, C-11α, C-12β, and C-15α (Figure 30).
Dianabol (methandrostenolone, 17α-methyl-17β-hydroxyl-androst-1,4-dien-3-on, 229) is an oral anabolic steroid that promotes the synthesis of proteins (increasing the muscle tissue). From the biotransformation of 229 using Cunninghamella elegans, five bioconversion products were obtained: 6β-hydroxydianabol (230), 15α-hydroxydianabol (231), 11α-hydroxydianabol (232), 6β,12β-dihydroxydianabol (233), and 6β,15α-dihydroxydianabol (234). The products 17β-hydroxy-17α-methyl-5α-androst-1,4-dien-3,6-dione (235), 7β-hydroxydianabol (236), 15β-hydroxydianabol (237), 17β-hydroxy-17α-methyl-5α-androst-1,4-dien-3,11-dione (238), and 11β-hydroxydianabol (239) were obtained from the biotransformation of 229 using Macrophomina phaseolina . Biotransformation of 229 using several microorganisms has been reported, for example, Penicillium notatum  transformed 229 into 230 and 231, while Trichoderma hamatum produced 232 . Similarly, B. bassiana, A. ochraceus, Colletotrichum lagenarium, and Sporotrichum sulfurreducens gave a biotransformed product 232 . Absidia glauca metabolized 229 in compounds 230, and 236–237 . In contrast, the biotransformation of 229 using A. coerula yielded 239 along with 7α-hydroxydianabol (240) , while by using B cinerea, 237 was obtained as the only product (Figure 31) .
Methasterone (241) is a synthetic anabolic steroid, known to gain muscle mass. Microbial transformation of 241 using M phaseolina yielded 17β-hydroxy-17α(hydroxymethyl)-2α-methyl-5α-androstane-3,6-dione (242), while by using C. blakesleeana, 7α-hydroxymethasterone (243, 2.0%), 7α,16β-dihydroxymethasterone (244, 0.7%), 5α,12β-dihydroxymethasterone (245, 1.0%), 7α,12β-dihydroxymethasterone (246, 1.5%), and 7α,9α-dihydroxy-methasterone (247, 0.5%) were obtained. Incubation of 241 with Fusarium lini yielded different metabolites with dehydrogenation in ring A and D: 6β,17β-dihydroxy-2,17α-dimethyl-5α-androst-1,4-diene-3-one (248, 1.0%), 15α,17β-dihydroxy-2α,17α-dimethyl-5α-androst-1,4-diene-3-one (249, 0.6%), 6β,17β-dihydroxy-2,17α-dimethylandrost-1,4-diene-3-one (250, 0.4%), 14α,15α-dihydroxy-2,17-dimethyl-5α-androst-1,4,16-trien-3-one (251, 0.3%), 17β-hydroxy-2,17α-dimethyl-5α-androst-5α-1,4-dien-3,6-dione (252, 0.3%), and 17β-hydroxy-2,17α-dimethyl-5α-androst-1,4-dien-3-one (253, 1.0%) (Figure 32) .
The biotransformation processes of different steroid compounds described in this review, although not exhaustive, aim to highlight the importance of biotransformation through different microorganisms, as a useful chemical-biological tool for obtaining novel derivatives for research purpose and as industrial applications. An example includes obtaining steroid compounds for the pharmaceutical industry.
Biotransformation of steroids has been implemented in an important way in the partial synthesis of new steroids, for their evaluation as hormones and drugs. Currently, there is a wide variety of steroids used as diuretics, anabolic, anti-inflammatory, antiandrogenic, anticontraceptive, antitumor, among other applications. Chemical functionalization in different carbon atoms of the sternum skeleton is related to the biological activity of the molecule. This is why microbiological transformations play an important role in obtaining these compounds through chemical transformations, such as the oxidation of hydroxyl group at C-3 and C-17, isomerization of the double bond Δ5(6) to Δ4(5), hydrogenation of double bonds Δ1(2) and Δ4(5), and reduction of the carbonyl group at C-17 and C-20 with β orientation. Biohydroxylations performed in different positions of the steroid skeleton—C-11α, C-11β, C-15β, and C-16α—using different species of fungi of the genera Rhizopus, Aspergillus, Curvularia, Cunninghamella, and Streptomyces with high yields are an important chemical transformation in many synthesis schemes of new steroids with a determined biological activity.
Hydroxylation of steroids—progesterone, testosterone, 17α-methyltestosterone, and 4-androsten-3,17-dione—presenting the 4-en-3-one system, proceeds with a high stereo- and regioselectivity in the C-6 and C-11 positions, with a β orientation in C-6 and α orientation in C-11. The presence of the methyl group in C-10 is necessary for the hydroxylation in C-11, as can be seen in the derivatives of 19-nortesterone.
The interest in the biotransformation of steroid compounds has been increasing in recent years, due to the obtaining of new and useful pharmacologically active compounds. In addition to the development of new genetically modified strains, there is an increase in the availability of immobilized enzymes and the manipulation of culture media.
Biotransformation of steroids proceeds with low to moderate yields in general. One of the main causes is their low solubility in water. Currently, methodologies are developed that allow the incorporation of chemicals—surfactants, ionic liquids, cyclodextrins, liposomes, among others—that contribute to improve the yields of each biotransformation process and the processes friendly to the environment.
A la Carrera de Biología, FES-Zaragoza, UNAM. Ms. Fabiola Cano thank for her suggestions and comments toward improving the manuscript.