Steroids comprise a wide range of naturally common compoundsdistributed in all the animal and plant kingdoms, with huge physiologically active derivatives that play crucial roles in biological systems[1-4].Steroids are key components of cell membranes, for stability and growth in cellularand development. Steroidsare precursors to bile acids and steroid hormones[5-8].Steroids have base structure consisting of 17 carbon atoms in a tetracyclic ring system well known as cyclopentanoperhydrophenanthrene, now as gonane and estrane[3-6].Steroid products are found indiversityof living species, ecdysteroids in insects, phytosterols and diosgenin in plants, cholesterol and corticosteroids: glucocorticoids, mineralocorticoids as well as sex hormones, bile acids, and vitamin D; neurosteroids, in vertebrates, and in yeasts and fungi are ergosterol and ergosteroids as part of its membrane cells [7,8,10].Steroids and its diversity areessential in medical practice, functioning as scaffolds for synthesizing new pharmacologically potent compounds [5,11,13,14].Steroids control a cascade of physiological activities at target sites and play key roles in cancer research [5,8,11,12].The physiological activity of steroids is closely associated to their molecular structure, as well as, the number, spatial orientation, and reactivity of functional groups in the steroid nucleus, as well as the oxidation state of the rings [1,4,13,15-17].For example, the presence of an oxygenated group at C-11β is essential for anti-inflammatory activity, besides a hydroxyl group at C-17β determines androgenic properties [3,5,8,19,20]. Aromatization of steroids at the A-ring affects estrogenic activity, and corticosteroids feature a 3-keto-5-ene group or a pregnane side chain at C-17 [2,11,14,21,22].In steroids functional modifications involve simple, chemically defined reactions catalyzed by microbial enzymes [1,4,13,15].Genetic MIT ability provides these enzymes to facilitate the transformation reactions, enhancing the efficiency and specificity of steroidsby MIT[6,7,9,23,24].The chemical modification of steroids, which requires high temperatures, pH, expensive reagents, and protective groups for reactive centers, has been a chemical method to obtain valuable new or improved drugs [3,8,16,17,25]. However, MIT offers an alternative approach that enables the production of biologically active steroid derivatives with high regio- and stereoselectivity under mild, environmentally friendly conditions [17-19,27-31].The aim of this short review is to analyze the potential of microbial biotransformation of steroidal compounds of value in the pharmaceutical industry and its connection with other related industries.

Microbial transformation of steroids

There are currently around 300 known steroidal drugs, used for several aims: immunosuppression, anti-inflammation, and contraception. Steroid applications have expanded to treating cancers, osteoporosis, human immunodeficiency virus (HIV)Infections or Acquired Immune Deficiency Syndromeor AIDS[3-5,7,8,32]. The therapeutic effects of certain steroid hormones are related to its interaction to intracellular receptors that regulate gene expression as transcription proprieties[13,20-22].Some steroids, as well as dehydroepiandrosterone, progesterone, pregnenolone, and itsproducts, like 17β-estradiol and allopregnanolone, are classified as neurosteroids due to steroidsactivity on the central nervous system[1,2,14].The MIT of exogenous steroid compounds is commonly by wide groups of bacteria and fungi, to enhancepharmacological activityand efficiency[27,30,33,34]. Several types of MIT reactions, as well as hydroxylation, dehydrogenation, side-chain degradation, ring A aromatization, reduction and esterification are used to achieve specific modifications [16-19, 22,23].MIT techniques diverse processes in culture media with microorganisms, free enzymes, biphasic systems, liposomes, microemulsions, methods altering cell wall permeability and the use of immobilized cells and enzymes [1,6,15,17,24].The spectrum of steroids that can be transformed by microbial cells is wide [4,7,9,18,25].Most advances in steroid happenedin 1950 at that time researchers had not clear idea about the pharmacological properties of cortisol and progesterone [8,14,16,30].

Researchers also discovered that genus fungi as well-knownspecies, could biotransform11α-hydroxylation, a critical reaction essential for synthesizing biologically active steroids [11,25,34,36,]; includingfungal transformation of Azorellane and Aqulinanetypes diterpenoids have unique tricyclic fused 5-, 6-, and 7-membered systems and a wide spectrum of biological properties: antimicrobial, antiprotozoal, spermicidal, gastroprotective [3,8,9,17,26].These discoveries marked the onset of a basic of development of steroids as a pharmaceutical, and the main point potential of microbial systems in the synthesis of valuable steroid compounds[10,11,13,32].Currently, the main objectives in steroid pharmaceutical research and development in target on identifying, and isolating microbial strains with unique activities or improving transformation capabilities [33-35, 37-39]. Genetic engineering and metabolic engineering of bacteria, fungi, and plants play a keyrole in these tasks[15,22,24,28,40]. Industrially, microbial hydroxylation activities, like are:  C-11α, C-11β, C-15α, and C-16α, are performed with high yields and enantioselectivity [2,9,13,14, 22,27]. Since steroids have hydrophobicity, which caused steroids to be tolerant to biodegradation, the mechanisms of steroid metabolism by both aerobic and anaerobic microorganisms have been investigated [18,23,26,28-30].For effective MIT, precursor steroids are required, that are then converted into valuable intermediates and final products [7,11,17,25,31].MIT are:  regiospecific and stereospecific, allowing the modification of compounds into suitable isomers through simple, chemically defined reactions catalyzed by microbial enzymes [1,3,15,32-33]. These enzymes act on compounds to design highly selective reactions, with easy techniques of isolation and purification of the new target compounds [3,6,17,19,22,27]. Besides, MITare is easy to use with necessary sterility conditions and allows for repeated working withthese enzymes [15,31,34].  SteroidMITare possible under several conditions of pressure and temperature, which is a viable alternative to chemical and ecological synthesis [2,23,24,40,41]. Although challenges such as productivity and chemical purity of steroids released, have non risk of contamination, microbial cells are systems can optimize and reduce costs by eliminating the need for isolating, purifying, and stabilizing pure enzymes [1,7,9,25]. Microbes naturally secrete all necessary cofactors and provide a stable environment for the enzymes, preventing protein structural changes and maintaining enzyme reactivity for many repeated processingto optimize steroid transformation[26,30,34,].Oxidation of steroid[6,12,35,].Common steroid precursors including cholesterol, steroidal alkaloids, steroidal sapogenins, and phytosterols, are readily available for MIT processes [16,18,19].

Types of steroids

Cholesterols and corticosteroid

Figure 1 illustrates the classification of steroids according to their biological functions or activities, including: bile acids, steroid hormones, cardioactive glycosides, aglycones, and steroid saponins[2,6,25].

Steroid hormones

Estrogens and androgens play a crucial role in maintaining homeostasis and regulating development [34,36,40]. The gut microbiota significantly influences systemic sex hormone levels by metabolizing these hormones into various derivatives [24,27,32,37]. Under normal physiological conditions, estrogens undergo rapid deactivation in the liver through processes such as glycosylation, sulfation, or methylation, followed by their elimination via urine and feces [30,31,37,38]. Gut microbes can alter this process by enzymatically reactivating estrogens, thereby modulating their bioavailability [8,32,35].

Microbial β-glucuronidase enzymes, primarily found in genera such as Clostridium and Bacteroides, facilitate the removal of glucuronides from deactivated estrogens, thereby restoring their activity [1,3,42]. Similarly, sulfatase-producing bacteria like Bacteroides fragilis and Peptococcus niger have been shown to convert estrone sulfate back into estrone, further influencing systemic estrogen levels [9,11,39]. Additionally, Bacteroides thetaiotaomicron and other gut microbes can act on sulfonated estrogen precursors, such as dehydroepiandrosterone (DHEA), altering hormone metabolism and potentially affecting inflammatory pathways [12-14,24]. Estrogen metabolism, gut microbes also impact androgen regulation [16,40-43]. For example, Mycobacterium neoaurum-derived 3β-HSDH (3β-hydroxysteroid dehydrogenase) has been implicated in the conversion of testosterone to androstenedione, a process linked to behavioral changes, including depression-like phenotypes in animal studies [16,40-43]. Collectively, these microbial interactions with sex steroid hormones highlight the critical role of the gut microbiome in endocrine regulation, with potential implications for metabolic, reproductive, and neuropsychiatric health.

Steroid saponins

For the steroid industry, natural steroid sapogenin, diosgenin is one more important raw material and it can be used for the microbial production of some new steroids of useful therapeutic action [21,44–45].The developing alternative methods for synthesizing therapeutically effective steroids, such as prednisone and prednisolone, has gained significant attention [42,46,47]. The glucocorticoids cortisone and hydrocortisone exhibit potent anti-inflammatory activity; however, their clinical use is often limited due to numerous side effects. Consequently, the development of improved derivatives, such as prednisone and prednisolone, allows for lower therapeutic doses, thereby reducing adverse effects [18,19,21].

Recent studies have highlighted the potential of microbial biotransformation in steroid synthesis. Among 13 screened Rhodococcus strains, Rhodococcus coprophilus DSM 43347 demonstrated the highest catalytic activity, efficiently performing Δ1-dehydrogenation of cortisone and hydrocortisone. This process resulted in the formation of prednisone with a 94% yield and prednisolone with a 97% yield, offering a promising biotechnological approach to steroid drug development [47-49].

Phytosterols

Phytosterols are plant-derived sterols, primarily sourced from Glycine max (soybean) or produced from tall oil or pitch [9,20,34,55].These plant steroids are extracted from specific parts of domestic plants for this purpose, as well as from press mud, generated during the extraction of edible oils [5,37,45,50].MIT of phytosterols has high economic value for the pharmaceutical industry, especially those derived from the activity of Mycobacteriumspp [12,27,51-53]. Besides sterol-containing waste from food, agricultural, and cellulose manufacturing processes can be utilized to produce valuable steroid compounds without requiring extensive purification of the phytosterols [28,33,35,47,54].

Ecdysterols

Ecdysteroids are defined according to their chemical structure and/or biological action (metamorphosis hormones), in insects and crustaceans, that is a cause of controversy[22,30,33,34,57].Ecdysteroids are organic compounds structurally similar to ecdysone, they are polyhydroxysteroids and are widely distributed in nature[4,5,55,56,58]. More than 250 ecdysteroids of different origins have been extracted and purified, especially from plants [7,8,10]. Ecdysteroids possess one or more vicinal diols, notably at the C-2 and C-3 positions of the A ring in the steroid nucleus, as well as at C-20 and C-22 in the side chain. Recent findings indicate that the molecular chaperone 4-phenylbutyrate (PBA) enables the selective extraction of ecdysteroids containing a C-20,22 diol group, while those with only a C-2,3 diol structure remain unextracted. This selective affinity offers a promising approach for the targeted isolation of specific ecdysteroid derivatives[14,17,30, 32,42]. In plants, it is attributed to chemical defense activity against predatory insects [34,37,43,55-58].

Ergoestrols

Ergosterol (ergosta-5,7,22-trien-3β-ol) is a sterol that serves as a biological precursor to vitamin D₂, by the action of ultraviolet light into ergocalciferol, or vitamin D2, through a photochemical reaction involving the cleavage of the B ring [7,9,10,12]. Ergosterol is the sterol that composes the cell membranes of fungi and certain protists such as trypanosomatids [5,6,14,17,32]. For these organisms, the synthesis of ergosterol is essential, it is a source of sterols[22,35,36,40,50]. Ergosterol is synthesized and exists in the cell membranes of fungi and plants as beta-sitosterol [53,55,56,59-61].

Types of steroids and its microbial transformation

Currently, there is a growing trend to isolate, analyze, and heterologously express enzymes that catalyze the microbial transformation of steroids [44,45,47,51,62,63].This approach not only expands the wide specter of target steroidsbut also holds the potential for scaling up production in the future [33,52,53].Several reactions are involved in the biotransformation of steroids, with key sites of these reactions on this specific molecule illustrated in Figure 2 [44,5254,61,64].

Figure 2.Show main sites of MITreactions,it has been described and discovered as a result of extensive and detailed research with a wide diversity of microorganisms that indicate that these changes could have occurred in the evolutionary past of these biological processes [9,40,49,50,65,66–70]. With the use of genetically improved microorganisms or those obtained by genetic engineering to produce molecules that are naturally difficult to obtain, as results indicate that it is even feasible to synthesize these molecules into bioreactors for safe pharmaceutical use,7α-Dihydroxylation[11,19,31,71,72].This pathway facilitates the removal of the 7α-hydroxy or 7β-hydroxy group from primary bile acids, leading to the formation of secondary bile acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA)[8,16,18,20,22,73].While the production of secondary bile acids via 7α-dehydroxylation is specific, deconjugation, oxidation, and epimerization are commonly carried out by several genera and species of intestinal bacteria, such as Bacteroides, Clostridium, Bifidobacterium and others bacterial genus [64,66,70,73-75].

The secondary bile acids, DCA and LCA, undergo oxidation and epimerization mediated by 3α/β-HSDH, resulting in the formation of various bile acid derivatives, including isoDCA, isoLCA, 3-oxoLCA, and isoalloLCA, which have recently been identified for their immunomodulatory functions in the gut[54,73,75-77,81]. Besides this other source of these organic compounds as derivates of cholesterol and hormones such as sulfonated cholesterol and estrogen, can be readily isolated from waste blood collected at slaughterhouses [24,69,71,78–81].In vertebrates, microbial lipids not only modulate host metabolism but also act as immune signaling molecules in the gut mucosa. Microorganisms regulate the development and functionality of wide intestinal immune cells and contribute to mucosal homeostasis and disease susceptibility [17, 20,37, 77,79,80]. Recent research has shown that working withRhodococcuspre-grown on n-alkanes can increase the yield of 9α-hydroxy androstenedione, achieving double the efficiency compared to cells grown on glucose [30,33,35,8,87].

Oxidation

A fundamental aspect for the MIT of steroids is to select microorganisms, that not only have the capacity in the molecular structure of these compounds but, also have or develop natural or induced tolerance of their enzymes that, facilitate MIT [1,5,8,9,29,33], under different chemical conditions, biochemical actions that are necessary for microorganisms involved in MIT of steroids [12,44, 46,82]. As well as oxidation of alcohols to ketone: 3β-OH to 3-keto by Aspergillussppor Rhizopus sppgenus fungi [7,35,44,47,52,61].

Hydroxylation

The substitution of a hydroxyl group directly for a hydrogen atom at a specific position in a steroid molecule, whether in the α or β configuration, can be achieved while retaining the molecule’s overall structure. Hydroxylation processes at positions 11α, 11β, 15α, and 16α are well-developed and are primarily used in the production of adrenal cortex hormones and their analogs [10,14,53,54,58]. In that sense, fungi are among the most active microorganisms for catalyzing hydroxylation reactions [25,32,43,46,48,83].Certain bacteria genera, as well as Pseudomonas aeruginosa[3,7,11,84]the spore-forming Bacillus or genus of actinomycetes and its specieslike:Streptomyces and Nocardia, exhibiting notable hydroxylation activity [32,34,41. Specifically, 11α- and 11β-hydroxylations are generally performed by some fungi genus as well known: as Rhizopus spp or Aspergillus spp [6,9,13,32,85]. While 16α-hydroxylation is carried out by other fungi genus as:Curvulariaspp., and Cunninghamellaspp [36,44,51]also is included the actinomycete genus Streptomycessp [2,3,16, 40,47].

Dehydrogenation

An example of bacterial dehydrogenation is the Δ1-dehydrogenation of 6α-methyl-cortisol, to 6α-methyl-prednisolone by resting cells of Arthrobacter globiformisand another bacterial genus [7,59,61,86].Based on a gene encoding 17β-hydroxysteroid dehydrogenase (17β-HSD), that was identified in the genome of Rhodococcussp P14. Recombinant of E. coli BL21 cells, expressing this enzyme successfully transformed estradiol, into estrone with up to 94% efficiency. TheRhodococcussp P14, could utilize other steroids, such as estriol and testosterone, as sole carbon sources[50,52,53,87].The genome screening also led to the identification of a gene for a short-chain dehydrogenase, that catalyzes the conversion of estradiol to estrone, estriol to 16-hydroxyestrone and testosterone to androst-4-en-3,17-dione [62,63,88-89]. Microbial dehydrogenation using Escherichia coligenetically modified has yielded promising results.[90]also reported in other bacteria[54,57,59,64,65,91], can also be applied to produce derivatives of cortisone and hydrocortisone having improved anti-inflammatory properties and reduced side effects [5,8,10]. This is achieved by introducing double bonds at specific positions in the steroid’s ring A structure [74,85,86,88,92].In that sense genera of both fungi and bacteria are able to dehydrogenate the secondary alcohol groups of steroids, generating corresponding carbonyl derivatives is well-known that microbial whole cells are particularly effective at performing Δ1-dehydrogenation [22,43,92,100,102].

Side chain biodegradation

Side-chain cleavage of steroidsis performed generally by fungi genus:Curvularia spp. or Cunninghamellaspp, however [3,7,9,16]: such reactions are described also differentanother genus of bacteria for example:actinobacteria, that is able to performthe basic step of the side-chain oxidation of sterols and other C-27 steroids is hydroxylation at C 26 or C-27 [27-30,34]. The aliphatic side-chain is degraded by an array of β-oxidation reactions [36,41,48,102].The terminal hydroxylation of C-27sterol is catalyzed by an enzyme known as cytochrome P450125 [97]That enzyme, also known as steroid 26-monooxygenase [51-53,59,64], is produced by various actinomycetes, including Rhodococcus jostii. It has been successfully purified and characterized [71,72,84,93].

Oxidation to ketone through hydroxylation

The chemical functionalization of different carbon atoms in the sterane skeleton is closely related to the MIT activity of the molecule [88,89,93,99,102]. Microbial transformations play a crucial role in obtaining these compounds through chemical processes, including the oxidation of hydroxyl groups at C-3 and C-17, isomerization of the double bond from Δ5-6 to Δ4-5, hydrogenation of double bonds at Δ1-2 and Δ4-5, and reduction of the carbonyl group at C-17 and C-20 with a β orientation[82-6,94,];with relative success, reason why new strategies with natural microorganisms and genetically modified microorganisms of plant origin, are still being investigated at MIT [95,96,100-103].Specifically, to achieve MIT in specific steroid sites that ensure chemical stability and sufficient product performance, through processes based on enzymes and microorganisms. that involve those naturally selected by genetic engineering to achieve efficiency in an effective hydroxylation of the steroid [14,16,97,98, 104-107]. 

RingA aromatization

Ring aromatization of suitable substrates by microorganisms forms aromatic compounds. For example, steroids like estrogens and estrones can be produced by ring Aromatization of steroid precursors or intermediates [21-25]. MIT of steroids has been successful even in aromatic molecules on functional groups, that determine specific pharmaceutical properties, which increases the spectrum of use in medicine [48,50,99–100].This is the case of MIT from diosgeninsuccessfully in other steroid compounds[13,51-53,101].

Reduction

This type of MITas well as the reduction of ketones and aldehydes to alcohols [62-65,69-71]. This process could be done by some genera and species of: algae, bacteria andfungi, which commonly undergo the reduction of exogenous compounds, like some important androgens can be produced by making use of these processes [7,84-86,93, 100,102],especially under the consideration of the natural microbial potential, to make direct modifications on steroids directly or indirectly with the immobilized enzymes, thus, the genetic design of microorganisms that make precise changes in the molecules to expand, or improve their pharmaceutical spectrum in medicine [12,13,16,17,30,103].

Hydrolysis of esters

The systemic side effects of steroids applied as drugs is to design molecules, of relatively easy metabolic deactivation after performing the action, in the pharmaceutical target site [104-105].For this purpose, during the MIT of steroids, it is feasible to apply: esterase enzymes for control and regulation of the activity of the steroid, with pharmacological value, since the esters are located in specific sites [106].It is possible to use microorganisms that act, on ester bonds sensitive to enzymatic activity, it has been a subject of research, thus microbial actions have been evaluated, with enzymes of such selectivity that its facilitate the hydrolysis activity of esters, with esterasesdue bacterial genera and species as well as other microbial species in the generation of polyester, today with better results with the use of genetic engineering[21,64,107].

Esterification

It is possible to obtain, through MIT, molecules similar to those reported in steroids, to make esterification with esterase enzymes by protein engineering in enteric bacteria, that synthesize them by optimizing  genetic potential for manipulation and complementing it with the use of organic solvents[108-110].In this sense, microbial synthesis of esters is relevant, from organic acids and alcohols, that accumulate in high concentration by bacteria such as Escherichia coli, capable of direct esterification by means of esterase enzymes efficient by expressing genes of the ethanol and lactic acid metabolic pathway for MIT steroids[1,64,121].

Microbial transformation of Steroid Compounds 

For sterol MIT, some bacterial genera and specieshave been identified, as effective biocatalysts as well as: Arthrobacter oxydans 317,A.rubellus,Brevibacteriumsp,Mycobacterium smegmatis [27,48,67,100,111-113];Pseudomonassppand Rhodococcus spp. However, to prevent the degradation of the steroid nucleus during these transformations, inhibitors are often required [16-18,22-25]. For example,estrogen, a major component in orally administered contraceptives, also plays a crucial role in hormone replacement therapy for menopause [3,5,8,40]. Estrone can be produced through the biotransformation of 19-nor-testosterone by cell-free extracts of Pseudomonas testosterone, with a small amount of estradiol-17β also produced in this reaction [40-43,92,94]. Recently, the 4,5-seco pathway of 17β-estradiol biodegradation by Rhodococcusequi DSSKP-R-001 was discovered, identifying the enzymes and genes involved in the initial stages of catabolism, this process begins with the dehydrogenation of 17β-estradiol by a short-chain dehydrogenase encoded by the hsd17b14 gene, to form estrone [5,10]. Also, Estrone could be transformed by flavin-binding monooxygenase (At1g12200), into 4-hydroxyestrone [28,29,31,33]. Subsequent cleavage of the steroid ring A, is catalyzed by 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione monooxygenase,encoded by the hsaC and catechol-1,2-dioxygenase encoded by the catA [35,43,49,112]. Özçınaret al., 2018 identified 14 biotransformed products based on neoruscogenin, a major spirostanol steroid from Ruscus aculeatus(butcher’s broom), as a substrate [36]. As well as withRhodococcusequi[87].R. aculeatus this plant, used in pharmaceutical preparations to treat conditions such as: chronic venous insufficiency, varicose veins, hemorrhoids, and orthostatic hypotension, was processed by the endophytic fungus Alternaria eureka,thatprimarily facilitated oxygenation, oxidation, and epoxidation reactions.Recent metabolomics and bioinformatics studies, have revealed that certain uncultured members of Cluster IV of bacterial anaerobic genus, belong toClostridium within the human gut microbiota carry genes for cholesterol dehydrogenases, specifically intestinal sterol metabolism A (ismA), that can convert cholesterol to coprostanol[11,16,17,114-115]. The regulation and transcription mechanisms of various isoforms of the KshAB gene are of particular interest to researchers [62-64]. According to data obtained by Baldanta et al. (2021), the KshA2 and KshA3 isoforms serve as the primary enzymes responsible for the degradation of androst-4-ene-3,17-dione and cholesterol, respectively, while KshA1plays a supportive role in these processes [85,86,9,116]. Beyond C-19 steroids, valuable 23,24-dinorcholane derivatives—important precursors for corticosteroid synthesis—have been microbially derived from sterols [4,66,86,93,97,103,117-118,123]. These transformations have also been reported in other bacterial genera [91,100-103,107-108,122], as well as through the catabolism of diosgenin by Mycolicibacterium sp. mHust-ΔkstD1,2,3 [68-69,101,124-130].

Conclusion

Steroids are naturally occurring compounds found throughout all living kingdoms. The chemical modifications of various carbon atoms, in the steroid framework are closely linked to the biological activity of these molecules. This is why MIT is avery important for producing these compounds, since the improvements facilitate specific chemical alterations that enhance its biological functions.Steroids can be synthesized through both chemical and microbial processes. Industrially, while well-known chemical methods are commonly used, microbial processes haveinteresting and valuable alternatives by enabling biotransformation or bioconversion. MIThas better and certain advantages over the chemical transformation, becauseits ability to produce steroids with precise modifications and high selectivity, making it essential for developing steroid-based pharmaceuticals, with better and widely targeted therapeutic effects.Microbial diversity such as microalgae, bacteria, and fungi are commonly employed for steroidbiotransformation. These processes are fundamental for releasing steroids used in a wide rangeof therapeutic applications, more than drug manufacturing, partial synthesis of new steroids, and evaluation for diverse applications such as hormones, diuretics, anabolic agents, anti-inflammatory agents, anti-androgens, contraceptives, and anti-tumor treatments.Interest in steroid MIT has increased in recent years due to the development of new, pharmacologically active compounds. This growth is supported by advancements in MITstrategies, as well as techniques of genetically modified microbial, newly isolated strains, immobilized enzymes, and optimized micriobiologicalculture conditions. These improvements enhance the efficiency and specificity of the MIT processes, making them quite essential for producing high-value new steroid products.

Acknowledgments

To Project 2.7 (2025) supported by the Scientific Research Coordination-UMSNH: “Aislamientoyseleccióndemicrorganismosendófitospromotoresdecrecimientovegetal para la agricultura y biorecuperacion de suelos. ToPhytonutrimentos de México and BIONUTRA, S.A de CV, Maravatío, Michoacán, México.

Conflicts of Interest: The authors declare no conflicts of interest

Cited literature

1. Sonawane, P.D., Heinig, U., Panda, S., Gilboa, N.S., Yona, M., Kumar, S.P et al., 2018. Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus Solanum. Proceedings of theNational Academy of Sciences of the United States of America. 115: E5419–E5428.

• Peng, H., Wang, Y., Jiang, K., Chen, X., Zhang, W., Zhang, Y et al., 2021. A dual role reductase from phytosterols catabolismenables the efficient production of valuable steroid precursors. AngewandteChemie International Edition 60: 5414–5420

• Bano S, et al., 2016. New Anti-Inflammatory Metabolites by Microbial Transformation of Medrysone. PLoS ONE 11(4): e0153951. https://doi.org/10.1371/journal.pone.0153951.

• Donova MV. 2017. Steroid bioconversions. Methods Mol Biol. 1645:1–13. https://doi.org/10.1007/978-1-4939-7183-1_1 PMID: 28710617

• Herráiz, I. 2017. Chemical pathways of corticosteroids, industrial synthesis from sapogenins. Methods in Molecular Biology. 1645: 15–27.

• Maltseva, P.Y.,    Plotnitskaya, N.A.&Ivshina, I.B. 2024. Transformation of Terpenoids and Steroids Using Actinomycetes of the GenusRhodococcus. Molecules, 29, 3378. doi:10.3390/ molecules2914337

• Jabbar, Z., Maryam, H., Maqsood, S & Ali, S. 2017. Microbial transformation of steroids: a focus on types and techniques. World Journal of Pharmaceutical 3(5): 193-203.

• Ericson‐Neilsen, W.& Kaye, A.D. 2014. Steroids: Pharmacology, Complications, and Practice Delivery Issues. Ochsner J., 14, 203–207

• Nassiri-Koopaei, N., & Faramarzi, M. A. 2015. Recent developments in the fungal transformation of steroids. Biocatalysis and Biotransformation, 33(1): 1-28.

1. Broekman, M. 2023. Applications of Steroid in Clinical Practice. Ann. Clin. Trials Vaccines Res. 13: 7–10. https://doi.org/10.37532/actvr.2023.13(1).07‐010

1. García, J. L., Ramos, R., Gómez, J., Vázquez, J. C., & Cano, A. 2015. Biotransformación de esteroides con diferentes microrganismos. Revista mexicana de ciencias farmacéuticas 46(1):17-32.

1. Liu, N., Feng, J., Zhang, R., Chen, X., Li, X., Yao, P. et al., 2019.Efficient microbial synthesis of key steroidal intermediates from biorenewable phytosterols by genetically modified Mycobacterium fortuitumstrains. Green Chemistry21: 4076–4085.

1. Cheng, L., Zhang, H., Cui, H., Davari, M.D., Wei, B., Wang, W et al., 2021.Efficient enzyme-catalyzed production of diosgenin: inspired by the biotransformation mechanisms of steroid saponins in TalaromycesstolliiCLY-6.Green Chemistry 23: 5896–5910.

1. Cano-Flores, A., Gómez, J., & Ramos, R. 2019. Biotransformation of steroids using different microorganisms. Chemistry and Biological Activity of Steroids. 5.Doi:10.5772/intechopen.85849

1. Willrodt C, Groning JAD, Nerke P, Koch R, Scholtissek A, Heine T et al., 2020. Highly efficient access to (S)-sulfoxides utilizing a promiscous flavoprotein monooxygenase in a whole-cell biocatalyst format. Chem Cat Chem 12:4664–4671. https://doi.org/10.1002/cctc.201901894

1. Aminudin, N.I., Ridzuan, M., Susanti, D.& Zainal, Z.A. 2022. Biotransformation of Sesquiterpenoids: A Recent Insight. J. Asian Nat. Prod. Res., 24, 103–145.https://doi.org/10.1080/10286020.2021.1906657.

1. Feng, J., Wu, Q., Zhu, D.& Ma, Y. 2022. Biotransformation Enables Innovations toward Green Synthesis of Steroidal Pharmaceuticals. ChemSusChem, 15, e202102399. https://doi.org/10.1002/cssc.202102399.

1. Luchnikova, N.A., Grishko, V.V., Ivshina, I.B. 2020. Biotransformation of Oleanane and UrsaneTriterpenic Acids. Molecules 25: 5526. https://doi.org/10.3390/molecules2523552

1. Xu, L., Wang, D., Chen, J., Li, B., Li, Q., Liu, P et al. 2022. Metabolic engineering of Saccharomyces cerevisiae for gram-scale diosgenin production. MetabolicEngineering, 70: 115–128.

• Cheng, J., Chen, J., Liu, X., Li, X., Zhang, W., Dai, Z et al., 2021.The origin and evolution of the diosgenin biosynthetic pathway in yam. PlantCommunications 2:100079

• Nasiri, A., Rashidi-Monfared, S., Ebrahimi, A., FalahiCharkhabi, N. &Moieni, A. 2022. Metabolic engineering of the diosgenin biosynthesis pathway in Trigonella foenum-graceum hairy root cultures. PlantScience 323: 111410.

• Liu, Y.J., Ji, W.T., Song, L., Tao, X.Y., Zhao, M., Gao, B et al., 2022.Transformation of phytosterols into pregnatetraenedione by acombined microbial and chemical process. Green Chemistry. 24: 3759–3771.

• Xiao, X.; He, J.-K.; Guan, Y.-X.; Yao, S.J. 2020. Effect of Cholinium Amino Acids Ionic Liquids As Cosolvents on the Bioconversion of Phytosterols by Mycobacterium sp. RestingCells. ACS Sustain. Chem. Eng 8: 17124–17132. [CrossRef]

• Yao, L., D’Agostino, G.D., Park, J., Hang, S., Adhikari, A.A., Zhang, Y., Li, W., Avila-Pacheco, J., Bae, S., Clish, C.B et al., 2022. A biosynthetic pathway for the selective sulfonation of steroidal metabolites by human gut bacteria. Nat Microbiol., 7(9):1404–1418. doi:10.1038/s41564- 022-01176-y

• Gao Q, Qiao Y, Shen Y, Wang M, Wang X, Liu Y. 2017. Screening for strainswith 11α-hydroxylase activity for 17α-hydroxy progesterone biotransformation.Steroids. 124:67–71. http://doi.org/10.1016/j.steroids.2017.05.009.

• Herrera-Canché S.G et al., 2023. Microbial Transformation of Azorellane and Mulinane. Diterpenoids. RevistaBrasileria de Farmacognosiahttps://Doi.org/10.1007./s43460-023-00364-z

• Galán, B.; Uhía, I.; García-Fernández, E.; Martínez, I.; Bahíllo, E.; de la Fuente, J.L.; Barredo, J.L.; Fernández-Cabezón, L.; García, J.L. 2017. Mycobacterium smegmatis Is a Suitable Cell Factory for the Production of Steroidic Synthons.Microb. Biotechnol10:138-150

• Shao, M.; Zhang, X.; Rao, Z.; Xu, M.; Yang, T.; Li, H.; Xu, Z.; Yang, S. A. 2016.  Mutant Form of 3-Ketosteroid-D1-Dehydrogenase Gives Altered Androst-1,4-Diene-3, 17-Dione/Androst-4-Ene-3,17-Dione Molar Ratios in Steroid Biotransformations by Mycobacterium neoaurum ST-095. J. Ind. Microbiol. Biotechnol. 43: 691–701. [CrossRef]

• Wang, H.; Yang, F.; Cheng, X.; Huang, Y.; Su, Z.2018. Crystal Structure and Characterization of 3-Ketosteroid-D1-Dehydrogenase from Mycobacterium Strain HGMS2GL. Biophys. J.  114: 583a. [CrossRef]

• Olivera, E.R.& Luengo, J.M. 2019. Steroids as Environmental Compounds Recalcitrant to Degradation: Genetic Mechanisms of Bacterial Biodegradation Pathways. Genes, 10, 512. doi:10.3390/genes10070512

• Ge J, Wang X, Bai Y, Wang Y, Wang Y, Tu T et al., 2023. Engineering Escherichia coli for efficient assembly of heme proteins. Microb Cell Fact. 22:59. https://doi.org/10.1186/s12934-023-02067-5 PMID:36978060

• Donova MV.2018. Microbiotechnologies for steroid production. Microbiology Australia. 10.1071/MA18040

• Donova, M. 2023. Current Trends and Perspectives in Microbial Bioconversions of Steroids. In Microbial Steroids: Methods and Protocols; Barreiro, C., Barredo, J.L., Eds.; Springer US: New York, NY, USA; pp. 3–21. ISBN 978‐1‐0716‐3384‐7.

• De Carvalho, C.C.C.R.; Da Fonseca, M.M.R. Biotransformations. In Comprehensive Biotechnology, 2nd ed.; Elsevier: Amsterdam. The Netherlands, 2011; Vol 2, pp: 451–460. https://doi.org/10.1016/B978‐0‐08‐088504‐9.00109‐4.

• Swizdor Aet al., 2014.Microbial Baeyer-Villiger oxidation of 5a steroid using Beauveria bassiana a Stereochemical requirement for the 11 a lactonization pathway. Steroid 82: 44-52

• Özçınar, O., Tağ, O., Yusufoglu, H., Kivçak, B. &Bedir, E. 2018. Biotransformation of neoruscogenin by the endophytic fungus Alternaria eureka. Journal of natural products 81(6): 1357-1367

• Zhang, H., Xie, Y., Cao, F., & Song, X. 2024. Gut microbiota-derived fatty acid and sterol metabolites: biotransformation and immunomodulatory functions. Gut Microbes 16(1): 2382336

• Sui, Y., Wu, J. & Chen J. 2021. The role of gut microbial beta-glucuronidase in estrogen reactivation and breast cancer. Front Cell Dev Biol 9: 631552. doi:10. 3389/fcell.2021.631552

• Ervin, S.M., Simpson, J.B., Gibbs, M.E., Creekmore, B.C., Lim, L., Walton, W.G., Gharaibeh, R.Z.&Redinbo, M.R. 2020. Structural insights into endobiotic reactivation by human gut microbiome-encoded sulfatases. Biochemistry, 59 (40):3939–3950. doi: 10.1021/acs.biochem.0c00711

• Fernández-Cabezón, L.; Galán, B.; García, J.L.2017. Engineering Mycobacterium smegmatis for Testosterone Production. Microb. Biotechnol. 10: 151–161. [CrossRef] [PubMed]

• Rodríguez-García, A. Fernández-Alegre, E, Morales, A.; Sola-Landa, A.; Lorraine, J.; McDonald, S.; Dovbnya, D.; Smith, M.C.M.; Donova, M.; Barreiro, C.2016. Complete Genome Sequence of Mycobacterium neoaurumNRRL B-3805, an Androstenedione (AD)Producer for Industrial Biotransformation of Sterols. J. Biotechnol 224: 64–65. [CrossRef]

• Zhou, X.; Zhang, Y.; Shen, Y.; Zhang, X.; Xu, S.; Shang, Z.; Xia, M.; Wang, M. 2019. Efficient Production of Androstenedione by Repeated Batch Fermentation in Waste Cooking Oil Media through Regulating NAD+/NADH Ratio and Strengthening Cell Vitality of Mycobacterium neoaurum. Bioresour. Technol. 279: 209–217. [CrossRef]

• Li, D., Liu, R., Wang, M., Peng, R., Fu, S., Fu, A., Le, J., Yao, Q., Yuan, T., Chi, H et al.,2022. 3beta-hydroxysteroid dehydrogenase expressed by gut microbes degrades testosterone and is linked to depression in males. Cell Host Microbe30(3): 329-339.doi: 10.1016/j.chom.2022.01.001

• Kozłowska E, Natalia Hoc, Jordan Sycz, Monika Urbaniak, Monika Dymarska, Jakub Grzeszczuk, Edyta Kostrzewa‑Susłow, ŁukaszStępień, ElżbietaPląskowska and Tomasz Janeczko. 2018. Biotransformation of Steroids by entomopathogenic strains of Isariafarinosa. Microb Cell Fact 17:71 https://doi.org/10.1186/s12934-018-0920-0

• Xiong, L.B., Liu, H.H., Zhao, M., Liu, Y.J., Song, L., Xie, Z.Y. et al., 2020. Enhancing the bioconversion of phytosterols to steroidal intermediates by the deficiency of kasB in the cell wall synthesis of Mycobacterium neoaurum. Microbial Cell Factories, 19: 80.

• Costa, S.; Zappaterra, F.; Summa, D.; Semeraro, B.; Fantin, G. 2020.  Δ1‐Dehydrogenation and C20 Reduction of Cortisone and Hydrocortisone Catalyzed by Rhodococcus Strains. Molecules, 25: 2192. https://doi.org/10.3390/molecules25092192.

• Tang, R.; Shen, Y.; Xia, M.; Tu, L.; Luo, J.; Geng, Y.; Gao, T.; Zhou, H.; Zhao, Y.; Wang, M.2019. A Highly Efficient Step-Wise Biotransformation Strategy for Direct Conversion of Phytosterol to Boldenone. Bioresour. Technol.  283: 242–250. [CrossRef] [PubMed]

• Sun, H.; Yang, J.; He, K.; Wang, Y.-P.; Song, H. 2021. Enhancing Production of 9_Hydroxy-Androst-4-Ene-3,17-Dione (9-OHAD) from Phytosterols by Metabolic Pathway Engineering of Mycobacteria. Chem. Eng. Sci.  230: 116195. [CrossRef]

• Tian, K., Meng, Q., Li, S., Chang, M., Meng, F.; Yu, Y., Li, H., Qiu, Q., Shao, J. &Huo, H. 2022. Mechanism of 17β-estradiol degradation by Rhodococcusequi via the 4, 5-seco pathway and its key genes. Environ. Pollut 312: 120021.  https://doi.org/10.1016/j.envpol.2022.120021.

• Mondaca, M. A., Vidal, M., Chamorro, S., and Vidal, G. 2017. Selection of biodegrading phytosterol strains.Methods Mol. Biol. 1645, 143–150. doi: 10.1007/978-1-4939-7183-1_9

• Shtratnikova, V.Y.; Schelkunov, M.I.; Dovbnya, D.V.; Bragin, E.Y.; Donova, M.V. 2017. Effect of Methyl-_-Cyclodextrin on Gene Expression in Microbial Conversion of Phytosterol. Appl. Microbiol. Biotechnol10: 4659–4667.

• Wang, X.; Hua, C.; Xu, X.; Wei, D.2019. Two-Step Bioprocess for Reducing Nucleus Degradation in Phytosterol Bioconversion by Mycobacterium neoaurumNwIB-R10hsd4A. Appl. Biochem. Biotechnol.  188:138–146.

• Oliveria, N.Vet al., 2022. Biotransformation of Phytosterols into Androstenedione—A Technological Prospecting Study. Molecules 27:3164https://doi.org/10.3390/molecules27103164.

• Li, X.; Chen, T.; Peng, F.; Song, S.; Yu, J.; Sidoine, D.N.; Cheng, X.; Huang, Y.; He, Y.; Su, Z. 2021. Efficient Conversion of Phytosterols into 4-Androstene-3,17-Dione and Its C1,2-Dehydrogenized and 9_-Hydroxylated Derivatives by Engineered Mycobacteria. Microb. Cell Fact.  20: 158.

• Christenhusz, M.J.M.; Byng, J.W. 2016. The number of known plant species in the world and its annual increase. Phytotaxa 261: 201–217.

• Antonelli, A.; Fry, C.; Smith, R.J.; Simmonds, M.S.J.; Kersey, P.J.; Pritchard, H.W.; Abbo, M.S.; Acedo, C.; Adams, J.; Ainsworth, A.M., et al., 2020. State of the World’s Plants and Fungi. Royal Botanic Gardens: Kew, UK [CrossRef]

• Tarkowska D, Strnad M. 2016. Plant ecdysteroids: plant sterols with intriguing distributions, biological effects and relations to plant hormones. Planta 244:545–555. doi: 10.1007/s00425-016-2561-z. 

• Dinan, L., Lafont, F., Lafont, R. 2023.The Distribution of Phytoecdysteroids among Terrestrial Vascular Plants: A Comparison of Two Databases and Discussion of the Implications for Plant/Insect Interactions and Plant Protection Plants 12 (4): 776 doi10.3310/plants1204776

• Dupont S, Lemetais G, Ferreira T, Cayot P, Gervais P, Beney L.2012 Ergosterol biosynthesis: a fungal pathway for life on land? Evolution.66: 2961–2968. doi: 10.1111/j.1558-5646.2012.01667.x.

• Krumpe K, Frumkin I, Herzig Y, Rimon N, Özbalci C, Brügger B, Rapaport D, Schuldiner M. 2012. Ergosterol content specifies targeting of tail-anchored proteins to mitochondrial outer membranes. Mol Biol Cell 23:3927–3935. doi: 10.1091/mbc. E11-12-0994.

• Chang, H.; Zhang, H.; Zhu, L.; Zhang, W.; You, S.; Qi, W.; Qian, J.; Su, R.; He, Z. 2020. A Combined Strategy of Metabolic Pathway Regulation and Two-Step Bioprocess for Improved 4-Androstene-3,17-Dione Production with an Engineered Mycobacterium neoaurum.Biochem. Eng. J 164: 107789. [CrossRef]

• Shao, M.; Zhang, X.; Rao, Z.; Xu, M.; Yang, T.; Li, H.; Xu, Z.2015. Enhanced Production of Androst-1,4-Diene-3,17-Dione by Mycobacterium neoaurum JC-12 Using Three-Stage Fermentation Strategy. PLoS ONE  10: e0137658 [CrossRef]

• Gupta, R.S.; Lo, B.; Son, J. 2018. Phylogenomics and Comparative Genomic Studies Robustly Support Division of the Genus Mycobacterium into an Emended Genus Mycobacterium and Four Novel Genera. Front. Microbiol.  9:67. [CrossRef]

• Sarnaik AP. Somnath Shinde Apurv Mhatre, Abigail Jansen, Amit Kumar Jha, Haley McKeownRyan Davis & Arul M. Varman. 2023. Unravelling the hidden powerofesterases for biomanufacturing of short‑chain esters. SciRep 13: 10766. https://doi.org/10.1038/s41598-023-37542-x

• Xu, L.; Yang, H.; Kuang, M.; Tu, Z.; Wang, X. 2017. Comparative Genomic Analysis of Mycobacterium neoaurum MN2 and MN4 Substrate and Product Tolerance. 3 Biotech 7: 181.

• Yan, M.Y., Yan, H.Q., Ren, G.X., Zhao, J.P., Guo, X.P. &Sun, Y.C. 2017. CRISPR-Cas12a-assisted recombineering in bacteria. Applied and Environmental Microbiology 83: e00947-17.

• Yan, M.Y., Li, S.S., Ding, X.Y., Guo, X.P., Jin, Q. & Sun, Y.C. 2020. A CRISPR-assisted nonhomologous end-joining strategy for efficient genome editing in Mycobacterium tuberculosis. MBio11: e02364-19.

• Zhang, Y., Zhou, X., Yao, Y., Xu, Q., Shi, H., Wang, K. et al., 2021. Coexpression of VHb and MceG genes in Mycobacterium sp strain LZ2 enhances androstenone production via immobilized repeated batch fermentation. BioresourTechnol 342: 125965.

• Zhao, A., Zhang, X., Li, Y., Wang, Z., Lv, Y., Liu, J. et al.,2021.Mycolicibacterium cell factory for the production of steroid-based drug intermediates. BiotechnologyAdvances 53:107860.

• Xie Can, Gangtian Zhu, Yuguang Hou, Zhiliang He. 2024. Fossil steroid acids can arise from microbial alteration of steranes. Organic Geochemistry 194: 104816

• Aggett, R., Mallette, E., Gilbert, S.E., Vachon, M.A., Schroeter, K.L., Kimber, M.S et al., 2019. The steroid side-chain-cleaving aldolase Ltp2-ChsH2DUF35 is a thiolase superfamily member with a radically repurposed active site. TheJournalofBiologicalChemistry 294: 11934–11943

• Xia, J., Ni, G., Wang, Y., Zheng, M. &Hu, S. 2022. Mycolicibacteracidiphilussp. nov., an extremely acid-tolerantmember of thegenusMycolicibacter. International JournalofSystematic and EvolutionaryMicrobiology, 72: 005419.

• Ridlon, J.M., Kang, D.J. &Hylemon, P.B. 2006. Bile salt biotransformations by human intestinal bacteria. J. Lipid Res47(2):241–259. doi:10.1194/jlr. R500013-JLR200

• Kozłowska E, Dymarska M, Kostrzewa-Susłow E, Janeczko T.2017. IsariafumosoroseaKCh J2 Entomopathogenic strain as an effective biocatalystfor steroid compound transformations. Molecules22:1511. http://www.mdpi.com/1420-3049/22/9/1511

• Dong, Z.& Lee, B.H. 2018.Bile salt hydrolases: structure and function, substrate preference, and inhibitor development. Protein Sci27(10):1742–1754. doi:10.1002/pro.3484

• Campbell, C., McKenney, P.T., Konstantinovsky, D., Isaeva, O.I., Schizas, M., Verter, J., Mai, C., Jin, W.B., Guo, C.J., Violante, Set al., 2020. Bacterial metabolism of bile acids promotes generation of peripheral regulatory T cells. Nature.581(7809):475–479. doi:10.1038/ s41586-020-2193-0

• Paik, D., Yao, L., Zhang, Y., Bae, S., D’Agostino, G.D., Zhang, M., Kim, E., Franzosa, E.A., Avila-Pacheco, J., Bisanz, J.Eet al., 2022. Human gut bacteria produce Tau (Eta) 17-modulating bile acid metabolites. Nature, 603(7903):907–912. doi:10.1038/s41586-022-04480-z

• Jan, H.M., Chen, Y.C., Shih, Y.Y., Huang, Y.C., Tu, Z., Ingle, A.B., Liu, S.W., Wu, M.S., Gervay-Hague, J., Mong, K.Tet al., 2016. Metabolic labelling of cholesteryl glucosides in Helicobacter pylori reveals how the uptake of human lipids enhances bacterial virulence. Chem Sci, 7(9):6208–6216. doi:10.1039/C6SC00889E

• Pellock, S.J. &Redinbo, M.R. 2017. Glucuronides in the gut: sugar-driven symbioses between microbe and host. J Biol Chem, 292(21):8569–8576. doi:10.1074/jbc. R116.767434

• Le, H.H., Lee, M.T., Besler, K.R., Comrie, J.M.C. & Johnson, E.L. 2022. Characterization of interactions of dietary cholesterol with the murine and human gut microbiome. Nat Microbiol7(9):1390–1403. doi:10.1038/s41564- 022-01195-9.

• Mutafova, B., Momchilova, S., Pomakova, D., Avramova, T. &Mutafov, S. 2018.Enhanced Cell Surface Hydrophobicity Favors the 9α‐Hydroxylation of Androstenedione by Resting Rhodococcus sp. Cells. Eng. Life Sci., 18: 949–954.  doi:10.1002/elsc.201800089

• Volmer J, Lindmeyer M, Seipp J, Schmid A, Buhler B. 2019. Constitutively solvent-tolerant Pseudomonas taiwanensisVLB120 ΔC ΔttgVsupports particularly high-styrene epoxidation activities when grownunder glucose excess conditions.BiotechnolBioeng 116 (5):1089–1101. https://doi.org/10.1002/bit.26924 PMID: 30636283

• González R, Nicolau F, Peeples TL. 2017. Optimization of the 11α-hydroxylation of steroid DHEA by solvent-adapted Beauveria bassiana. BiocatalBiotransformation. 35:103–9. https ://doi.org/10.1080/10242 422.2017.1289183.

• Eisa, M.; El-Refai, H.; Amin, M.2016. Single Step Biotransformation of Corn Oil Phytosterols to Boldenone by a Newly Isolated Pseudomonas aeruginosa.Biotechnol. Rep.  11: 36–43.

• Ortega-de los Ríos L, Luengo, J. L and Fernández-Cañón J. M. 2017. Steroid 11-Alpha-Hydroxylation by the Fungi Aspergillus nidulans and Aspergillus ochraceusMicrobial Steroids: Methods and Protocols, Methods in Molecular Biology vol. 1645. DOI 10.1007/978-1-4939-7283-1_19 Springer Science + Business LLC

• Kiss FM, Lundemo MT, Zapp J, Woodley JM, Bernhardt R. 2015. Process development for the production of 15β-hydroxycyproterone acetate using Bacillus megaterium expressing CYP106A2 as whole-cell biocatalyst. Microb Cell Fact 14:28. https://doi.org/10.1186/s12934-015-0210-z PMID:25890176

• Kim, Y.U., Han, J., Lee, S.S., Shimizu, K., Tsutsumi, Y. & Kondo, R. 2017. Steroid 9 alpha-hydroxylation during testosterone degradation by resting Rhodococcusequicells. ArchivderPharmazie -ChemistryinLifeSciences 340: 209–214.

• Shao, M., Sha, Z., Zhang, X, Rao, Z., Xu, M., Yang, T et al., 2017. Efficient androst-1,4-diene-3,17-dione production by co-expressing 3-ketosteroid-11-dehydrogenase and catalase in Bacillus subtilis. J. Appl. Microbiol. 122: 119–128.doi: 10.1111/jam.13336

• Szaleniec M, Wojtkiewicz AM, Bernhardt R, Borowski T, Donova M.2018. Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms.ApplMicrobiolBiotechnol. 102(19):8153–8171. https://doi.org/10.1007/s00253-018-9239-3 PMID: 30032434

• Lundemo MT, Notonier S, Striedner G, Hauer B, Woodley JM.2016. Process limitations of a whole-cell P450 reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli. ApplMicrobiolBiotechnol. 100 (3):1197–1208. https://doi.org/10.1007/s00253-015-6999-x PMID: 26432459

• Fernández-Cabezón, L., García-Fernández, E., Galán, B. & García, J.L. 2017. Molecular characterization of a new gene cluster for steroid degradation in Mycobacterium smegmatis. EnvironmentalMicrobiology 19: 2546–2563.

• Wu, C.; Xu, J.; Xie, J.; Wang, Z. 2018. A Streamlined High Throughput Screening Method for the Mycobacterium neoaurum Mutants with Expected Yield of Biotransformation Derivatives from Sterols.Chin. Chem. Lett. 29: 1251–1253. [CrossRef]

• Yang, Y.; Yang, S. Wu, Z. 2015. Development of 9_-Hydroxy-Androst-4-Ene-3,17-Dione (9_-OH-AD) through Cleaving Sterol Sidechain by Fermentation of Mycobacterium fortuitum. Chin. J. Appl. Environ. Biol.  21: 256–262. [CrossRef]

• Yuan, J.; Chen, G.; Cheng, S.; Ge, F.; Qiong, W.; Li, W.; Li, J.  2015. Accumulation of 9_-Hydroxy-4-Androstene-3,17-Dione by Co-Expressing KshA and KshB Encoding Component of 3-Ketosteroid-9-Hydroxylase in Mycobacterium sp. NRRL B-3805. Chin. J. Biotechnol 31:  523–533

• Bretschneider L, Heuschkel I, Wegner M, Lindmeyer M, Buhler K, Karande Ret al., 2021. Conversion of cyclohexane to 6-hydroxyhexanoic acid using recombinant Pseudomonas taiwanensisin a stirred tank bioreactor. Front Catal. 1:683248. https://doi.org/10.3389/fctls.2021.683248

• Li, W., Hang, S., Fang, Y., Bae, S., Zhang, Y., Zhang, M., Wang, G., McCurry, M.D., Bae, M., Paik, D et al., 2021. A bacterial bile acid metabolite modulates T(reg) activity through the nuclear hormone receptor NR4A1. Cell Host Microbe, 29(9):1366–1377 e1369. doi:10. 1016/j.chom.2021.07.013

• Bertelmann C, Mock M, Koch R, Schmid A, Buhler B. 2022. Hydrophobic outer membrane pores boost testosterone hydroxylation by cytochrome P450 monooxygenase BM3 containing cells. Front Catal. 2:887458.https://doi.org/10.3389/fctls.2022.887458

• Bertelmann C &Buhler B. 2024. Strategies found not to be suitable for stabilizing high steroid hydroxylation activities of CYP450 BM3-based whole-cellbiocatalysts. PLoS ONE 19(9): e0309965.https://doi.org/10.1371/journal.pone.0309965

• Ruff AJ, Arlt M, Van Ohlen M, Kardashliev T, Konarzycka-Bessler M, Bocola M, et al., 2016. An Engineered outer membrane pore enables an efficient oxygenation of aromatics and terpenes. J Mol Catal B Enzym.134:285–294. https://doi.org/10.1016/j.molcatb.2016.11.007

1. Fernández-Cabezón, L., Galán, B. & García, J.L. 2018. Unravelling a new catabolic pathway of C-19 steroids in Mycobacteriumsmegmatis. EnvironmentalMicrobiology, 20, 1815–1827.

1. Wang Zhikuan, HailiangQiu,YulongChen,Xuemin Chen, Chunhua Fu, Longjiang Yu. 2024. Microbial metabolism of diosgenin by a novel Isolated Mycolicibacteriumsp. HK-90: A promising biosynthetic platform to produce 19-carbon and 21-carbon steroids. Microbial Biotechnology 17: e14415 https://doi.org/10.1111/1751-7915.14415

1. Fernández-Cabezón, L., Galán, B. & García, J.L. 2018. New insights on steroid biotechnology. Frontiers in Microbiology 9: 958.

1. Hu B, Yu H, Zhou J, Li J, Chen J, Du G, et al., 2023. Whole-cell P450 biocatalysis using engineered Escherichia coli with fine-tuned heme biosynthesis. AdvSci.  10 (6): e2205580. https://doi.org/10.1002/advs.202205580 PMID: 36526588

1. Bednarek, M. 2016. Branched aliphatic polyesters by ring-opening (co)polymerization. Prog. Polym. Sci.58: 27– 58. DOI: 10.1016/j.progpolymsci.2016.02.002

1. Almeida, B. C., Figueiredo, P., and Carvalho, A. T. P. 2019. Polycaprolactone enzymatic hydrolysis: a mechanistic study. ACS Omega. 4: 6769–6774. doi: 10.1021/acsomega.9b00345

1. Kobayashi, S. 2015. Enzymatic ring-opening polymerization and polycondensation for the green synthesis of polyesters. Polym. Adv. Technol.26: 677– 686, DOI: 10.1002/pat.3564

1. Austin, H. P.; Allen, M. D.; Donohoe, B. S.; Rorrer, N. A.; Kearns, F. L.; Silveira, R. L.; Pollard, B. C.; Dominick, G.; Duman, R.; Omari, K. E.; Mykhaylyk, V.; Wagner, A.; Michener, W. E.; Amore, A.; Skaf, M. S.; Crowley, M. F.; Thorne, A. W.; Johnson, C. W.; Woodcock, H. L.; McGee han, J. E.; Beckham, G. T. 2018. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc. Natl. Acad. Sci. U.S.A.  115: E4350– E4357, DOI: 10.1073/pnas.1718804115
2. Maester, T. C. et al 2020.Exploring metagenomic enzymes: A novel esterase useful for short-chain ester synthesis. Catalysts 10: 1100

1. Sarnaik, A., Liu, A., Nielsen, D. & Varman, A. M. 2020. High-throughput screening for efficient microbial biotechnology. Curr. Opin. Biotechnol. 64: 141–150. https://doi.org/10.1016/j.copbio.2020.02.019

1. Walls, L. E. & Rios-Solis, L.2020. Sustainable production of microbial isoprenoid derived advanced biojet fuels using different generation feedstocks: A review. Front. Bioeng. Biotechnol 8:599560–599560.https://doi.org/10.3389/fbioe.2020.599560

1. Qin, N.; Shen, Y.; Yang, X.; Su, L.; Tang, R.; Li, W; Wang, M. 2017. Site-Directed Mutagenesis under the Direction of in Silico Protein Docking Modeling Reveals the Active Site Residues of 3-Ketosteroid-D1-Dehydrogenase from Mycobacterium neoaurum. World J. Microbiol. Biotechnol 33: 146. [CrossRef]

1. Shao, M., Zhang, X., Rao, Z., Xu, M., Yang, T., Xu, Z. et al. 2019. Identification of steroid C27 monooxygenase isoenzymes involved in sterol catabolism and stepwise pathway engineering of Mycobacterium neoaurumfor improved androst-1,4-diene-3,17-dioneproduction. Journalof Industrial Microbiology&Biotechnology 46: 635–647.

1. Zhou, X.; Zhang, Y.; Shen, Y.; Zhang, X.; Zan, Z.; Xia, M.; Luo, J.; Wang, M.2020. Efficient Repeated Batch Production of Androstenedione Using Untreated Cane Molasses by Mycobacterium neoaurum Driven by ATP Futile Cycle. Bioresour. Technol. 309: 123307. [CrossRef] [PubMed]

1. Kenny, D.J., Plichta, D.R., Shungin, D., Koppel, N., Hall, A.B., Fu, B., Vasan, R.S., Shaw, S.Y., Vlamakis, H., Balskus, E.P et al.,2020. Cholesterol metabolism by uncultured human gut bacteria influences host cholesterol level. Cell Host Microbe28(2):245–257 e246. doi:10.1016/j. chom.2020.05.013. 70.

1. Li, C., Strazar, M., Mohamed, A.M.T., Pacheco, J.A., Walker, R.L., Lebar, T., Zhao, S., Lockart, J., Dame, A., Thurimella, K et al.,2024. Gut microbiome and metabolome profiling in Framingham heart study reveals cholesterol-metabolizing bacteria. Cell187(8):1834–1852.e19. doi: 10.1016/j.cell.2024.03.014

1. Baldanta, S., Navarro Llorens, J.M.& Guevara, G. 2021. Further Studies on the 3‐Ketosteroid 9α‐Hydroxylase of Rhodococcusruber Chol‐4, a Rieske Oxygenase of the Steroid Degradation Pathway. Microorganisms9: 1171.https://doi.org/10.3390/microorganisms9061171.

1. Zhou, P.; Fang, Y.; Yao, H.; Li, H.; Wang, G.; Liu, Y. 2018. Efficient Biotransformation of Phytosterols to Dehydroepiandrosterone by Mycobacterium sp. Appl. Biochem Biotechnol186(2):496-506. doi: 10.1007/s12010-018-2739-x.

1. Hermann C., Lang S., Popp T., Hafner S., Steinritz D., Rump A., Port M., Eder S. 2021. Bardoxolone-Methyl (CDDO-Me) Impairs Tumor Growth and Induces Radiosensitization of Oral Squamous Cell Carcinoma Cells. Front. Pharmacol,11:607580. doi: 10.3389/fphar.2020.607580. 

1. Qing-Chang R., Hong-Jian, Y., H., Sheng-Li, L., S., & Jia-Qi, W. 2014. Diurnal variations of progesterone, testosterone, and androsta-1, 4-diene-3, 17-dione in the rumen and in vitro progesterone transformation by mixed rumen microorganisms of lactating dairy cows.Journal of Dairy Science97(5): 3061-3072

1. Putkaradze, N., Kiss, F. M., Schmitz, D., Zapp, J., Hutter, M. C., & Bernhardt, R. 2017. Biotransformation of prednisone and dexamethasone by cytochrome P450 based systems–Identification of new potential drug candidates. Journal of Biotechnology, 242: 101-110.

      121.Zuriat Jabbar, Hannana Maryam, Sana Maqsood and Sikander Al. 2017.Microbial transformation of steroids: a focus on types and

techniques.World Journal of Pharmaceutical and Life Sciences3(5): 193-203

1. KozłowskaEwa, Monika Urbaniak,  Anna Kancelista,  Monika Dymarska, EdytaKostrzewa-Susłow,  ŁukaszSteand Tomasz Janeczko.2017.Biotransformationofdehydroepiandrosterone (DHEA) byenvironmentalstrainsoffilamentousfungi. RSC Advances7: 31493

1. Su, L.; Xu, S.; Shen, Y.; Xia, M.; Ren, X.; Wang, L.; Shang, Z.; Wang, M.2020.TheSterol Carrier Hydroxypropyl-β-CyclodextrinEnhances

theMetabolismofPhytosterolsbyMycobacteriumneoaurum.Appl. Environ. Microbiol 86: e00441-20.

124.Awadhiya, P.; Banerjee, T. 2018. Tween 80 AlterstheProduction Ratio ofPharmaceuticallyImportantSteroidIntermediates, 4-ADand ADD duringBiotransformationof Soy SterolbyMycobacteriumsp. NRRL B-3805. Int. J. Pharm. Sci. Res 9:1935–1941.

125. Sripalakit, P.; Saraphanchotiwitthaya, A. 2016. UtilizationofPhytosterol-Containing Vegetable Oils as a SubstrateforProductionofAndrost-4-Ene-3,17-Dione and Androsta-1,4-Diene-3,17-Dione byUsingMycobacteriumsp.Biocatal. Agric. Biotechnol. 18–23.

126. Thygs, F.B.; Merz, J. 2017. DownstreamProcessSynthesisforMicrobialSteroids. Methods Mol. Biol.  1645: 321–345.

127.  Ahmed, E.M. 2014.Utilizationof Concrete as a Carrier forBacterialCellsduringBioconversionofSomeSterols.Int. J. Chem. Sci12: 413–428.

128.  Liu, X.; Hao, X.; Zhang, R.; Feng, W.; Zhu, C.; Zhang, B.; Shi, J. 2018.ConstructionofEngineering Bacteria forDegradingPhytosteroltoAndrost-1, 4-Diene-3, 17-Dione and theOptimizationofTransformation Medium. Sci. Technol. FoodInd. 39: 110–116.

129. Zhang, Y.; Zhou, X.; Wang, X.; Wang, L.; Xia, M.; Luo, J.; Shen, Y.; Wang, M. 2020. Improving Phytosterol Biotransformation at Low Nitrogen Levels by Enhancing the Methylcitrate Cycle with Transcriptional Regulators PrpR and GlnR of Mycobacterium neoaurum. Microb. Cell Fact 19: 13.

130. Xiong, L.-B.; Liu, H.-H.; Song, X.-W.; Meng, X.-G.; Liu, X.-Z.; Ji, Y.-Q.; Wang, F.-Q.; Wei, D.-Z. 2020. Improving the Biotransformationof Phytosterols to 9α-Hydroxy-4-Androstene-3,17-Dione by Deleting EmbC Associated with the Assembly of Cell Envelope inMycobacterium neoaurum. J. Biotechnol 323: 341–346.