Biotransformation of Steroids Using Different Microorganisms

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 stereo selective 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.


Introduction
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 [6], 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 [12], for prevention of heart disease [13], as antifungal agents [14], and as active ingredients used for the treatment of obesity [15] and AIDS [16]. Recently, the antiviral activity against the herpes simplex virus type I of some steroid glycosides was determined [17].
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 [18]. 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 [19].
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 [23].
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 [26], which can be difficult to obtain by conventional chemical methods and have been widely used in the bioconversion of steroids [25]. 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.
Obtaining hydroxylated derivatives in a specific position is one of the objectives of the steroid industry; for example, 14α-hydroxysteroids are shown to have antiinflammatory, 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-1,4dien-3,17-dione (111, 70%) was obtained (Figure 13) [74].
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   Compounds 197 and 198 showed a potent growth inhibition against drug-resistant strains of S. aureus (Figure 26) [94].

Conclusions
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 Δ 5(6) to Δ 4(5) , hydrogenation of double bonds Δ 1(2) and Δ 4 (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.
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Author details
Arturo Cano-Flores*, Javier Gómez and Rigoberto Ramos Laboratorio de Biotransformaciones y Química de Productos Naturales, L-314, Facultad de Estudios Superiores Zaragoza, UNAM, México, D.F., Mexico *Address all correspondence to: acano1750@gmail.com 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.