1.1. Microbiological transformation of steroids.
Steroids are small organic molecules that are synthesized in steroidogenic tissues and act on target sites to regulate a cascade of physiological functions . Examples of natural occurring steroids include: sterols, steroidal saponins, cardioactive glycosides, bile acids, corticosteroids and mammalian sex hormones . They are based on the steran skeleton which is composed of three six-carbon ring units and one five-carbon ring unit. The rings are labelled A, B, C, and D beginning from the far left (see fig. 1). In naturally occurring steroids, all four rings are in the chair conformation  with rings B, C, and D in trans- configuration with respect to each other. For rings A and B the position of the C-19 methyl group attached to C-10 and the hydrogen attached to C-5 determines the structure and their cis-/trans- configuration. Overall, neighbouring substituent are trans- if they are diaxial or diequatorial like in fig. 1a, and are cis- if they are axial-equatorial (fig. 1b).
However, the two methyl groups attached to C-10 and C-13 are always axial in relative to rings B and D, with C-10 substituent being the conformational reference point . Hence, the 5α- steroid skeleton (see fig. 1a) is in the ‘trans-trans-tans-‘ configuration, and thus is broadly planar. The knowledge of the stereochemistry of steroid molecules is highly significant in understanding its biotransformation reactions which is the basis of this study.
Steroids represent a class of natural products with diverse therapeutic properties. It has been observed that minor changes in the molecular structure of steroids can affect their biological activity [4,5]. Hence numerous research have been conducted to improve the activity of existing steroid compounds and to synthesize novel steroidal compounds with pharmacological activity, and thus the most significant area of these research is the transformation of steroids using biocatalysts.
Biotransformation could be defined as the modification of an organic compound into a recoverable product by chemical reactions catalysed by enzymes originating from a biological system . It should be noted that the organic compound which is the substrate is not involved in the primary or secondary metabolism of the biological system concerned, and thus distinguishes this process from biosynthesis. The biotransformation of steroids is one of the most important microbial processes that are highly regio- and stereospecific, involving chemical modifications (e.g. oxidation, reduction, hydrolysis, isomerisation, epoxidation, etc.) to the parent steroid which are catalysed by the microbial enzymes. In addition, the features which govern their regiospecificity differ from those controlling chemical specificity, and so it is possible to obtain biotransformation at centres that are chemically unreactive . For example, in the study conducted by Peterson and Murray using Rhizopus arrhizus, it was observed that progesterone was hydroxylated at C-11 which is an ureactive site in this steroid molecule . Therefore, these characteristics alongside the rapid growth and high metabolic rates of microorganisms give biotransformation reactions an advantage over conventional chemical processes as a tool in the production of therapeutic agents (e.g. anti-inflammatory, diuretics, anabolic, contraceptive, anti-cancer, anti-androgenic, postgestational etc.) in the pharmaceutical industry. The ever growing research into the study of microbial transformation of steroids have led to newer technology in this area of science such as: genetically modification of microorganisms to improve their steroid transforming capabilities, the immobilization of whole cells or isolated enzymes in a suitable matrix for repetitive economic utilization of the enzymes, manipulation of culture media to improve product yields by the use of enhancers e.g. cyclodextrin, and the improvement of the solubility of substrates are insoluble (or sparingly soluble) in water . Furthermore, the advances in microbial steroid biotransformation have led to the discovery of new microbial reactions and novel metabolites which may be of interest within academia and clinical medicine.
1.2. The mechanism of Hydroxylation
The hydroxylation of a compound is a very important metabolic process, in humans; this process is catalysed by cytochrome P450 enzymes and results in products with a higher polarity than the parent compound, and thus aiding its excretion from the body [1,3]. The process of hydroxylation, involves the conversion of a carbon-hydrogen to a carbon-hydroxyl bond, and when catalysed by the enzyme hydroxylase, the reaction is more regio- and stereospecific in contrast to the conventional chemical process [8-12]. As a result, microbial hydroxylation is rather used for the synthesis of hydroxysteroid.
Fungal hydroxylation of steroids continues to be the focus of attention at different levels of research and product development. In spite of its popularity this process is not fully understood because few studies have been conducted on the hydroxylase enzyme due to the difficulty in isolating this enzyme [10,11]. However, most studies have shown that the cytochrome P450 enzyme is also responsible for steroid hydroxylation in filamentous fungi [9-11,13,22].
Cytochrome P450 (CYP 450) enzyme is an iron-haem system which carries out a wide range of biocatalytical transformation. These enzymes are also known as monooxygenases because they transfer one atom of molecular oxygen to an organic substrate. The catalytic mechanism for this reaction involves the binding of the substrate to the active site of the enzyme and then the displacement of a water molecule (see fig.2). This is followed by a reduction of the iron in the CYP 450-haem complex to its ferrous state (Iron II) by an electron transfer. The ferrous state then binds to molecular oxygen to form a ferrous-dioxy (Iron (III)-OOH) species. This species then loses a hydroxyl anion to form an iron (IV)-oxygen radical. This radical may withdraw a hydrogen atom from the substrate to generate a carbon radical and an iron (IV)-hydroxyl species. The carbon radical then accepts a hydroxyl radical from the iron (IV)-hydroxyl species to form a hydroxylated product and iron (III). A simple general reaction equation for this process is summarised below: (where R represents the substrate and NADPH is the electron transferring species).
RH + NADPH + H+ + O2 → ROH + NADP+ + H2O
In other to fully understand the mechanism of fungal hydroxylation of steroids, the relationship between the structure of the CYP 450 hydroxylase enzyme and its regio- and stereoselective characteristic has to be defined. However, as mentioned earlier not much studies have been conducted on the structural features of this enzyme, and so ‘active site models’ was developed to grasp the concept of the regio- and stereoselective outcome of microbial hydroxylation reactions.
The first model, postulated by Brannon et al suggested the possibility for a steroidal substrate to be bound by a single steroid hydroxylase in more than one orientation due to two- sites binding, which could result in hydroxylation taking place at more than one position given the appropriate geometrical relationship between the active site of the enzyme and the carbon atom of the substrate undergoing the reaction [9,14]. These four orientations are represented as normal, reverse, inverted and reverse inverted (see fig. 3) and has been observed in the metabolic handling of 3β-hydroxy-17a-oxa-D-homo-5α-androstan-17-one by a filamentous fungus; Aspergillus tamarii .
The other model, Jones’ model takes into account only the normal and reverse binding orientations . It requires the existence of three active centres on the steroid hydroxylase enzyme. These active centres have dual roles and could act both as a binding site or a hydroxylating site . However, these roles are mutually exclusive, and so hydroxylation would occur at the closest nuclear centre to the steroid. Hence the enzyme-substrate interaction proposed by Jones would suggest a triangular location with an approximate spatial correspondence to C-3, C-11 and C-16 atoms of the steroid nucleus  (fig. 4).
This model could not explain the hydroxylation reactions by some microorganisms. Therefore another theory was developed by McCrindle et al using both models above and taking into account the 3- D nature of the steroid compound and hydroxylase enzyme . In this model, the steroid ring acts as a planar reference point (fig. 5). Binding site A favours oxygen atoms below the plane of the ring and hydroxylation is alpha. Binding site B is similar to A but can also hyroxylate alpha (axial or equatorial) or beta (equatorial) atoms. Whereas, binding site C binds preferentially to oxygen atoms above the plane of the steroid ring and hydroxylate with -beta orientation. Overall, this model tends to fit the hydroxylation pattern of most microorganisms.
The hydroxylation outcome of some steroids can be predicted based on the oxygen functions or ‘directing groups’ on the steroid skeleton. As a rule of thumb mono- oxygenated substrates are dihydroxylated and their transformation products are often in low yields . This is as result of the presence of one oxygen function on the steroid compound making it less polar and thus decreasing its solubility which hinders its permeation into the microbial cell. In addition to this, the presence of only one oxygen function allows the steroid to bind to the enzyme at only one centre, thereby increasing its rotation and oscillation about the active site which makes it more likely to be dihydroxylated. Whereas, di- oxygenated substrates are monohydroxylated because the presence of two oxygen functions reduces the chance of multiple hydroxylations due to the reduction in the possible number of binding orientations . Furthermore, the presence of two binding oxygen groups increases the rate of reactivity of microbiological transformation as the increased substrate polarity improves solubility and thus permeation into the cell membrane of the microorganism is very likely. A wide variety of organisms have shown this pattern of hydroxylation with a wide range of substrates [15,16].
Hydroxylated steroids possess useful pharmacological activities, for example, C-11 hydroxylation is regarded as essential for anti- inflammatory action, and 16α- hydroxylated steroids have increased glucocorticoid activity [8,12]. Hence the steroid industry exploits the use of 11α-, 11β-, 15α- and 16α- hydroxylation mainly for the production of adrenal cortex hormones and their analogues . A range of microorganisms have been observed to affect this type of hydroxylations. For example, 11α- hydroxylation is performed using Rhizopus sp. Or Aspergillus sp., Cuvularia sp. or Cunninghamella sp. and Streptomyces sp. generates 11β- and 16α- hydroxylations respectively [8,18]. Further research has shown other hydroxylations (e.g. 7α-, 9α- and 14α- hydroxylations) of having the potential for industrial exploitation .
1.3. The mechanism of Baeyer- Villiger Oxidation
Baeyer- Villiger oxidation is the oxidative cleavage of a carbon-carbon bond adjacent to a carbonyl, which converts ketones to esters and cyclic ketones to lactones [19,20]. The mechanism of this chemical process was originally proposed by Criegee . It involves a two step process: a nucleophillic attack of a carbonyl by a peroxo species resulting in the formation of a ‘Criegee’ intermediate, which then undergoes rearrangement to the corresponding ester. Commonly used peracids or oxidants include: m-chloroperoxybenzoic acid, hydrogen peroxide, peroxyacetic acid and trifluoroperoxy acetic acid. This chemical process is highly significant, because the products generated are compounds which are intermediates in the synthesis of natural products or bioactive compounds. However, the oxidants used in chemical Baeyer- Villiger oxidation (BVO) are expensive and hazardous and also the reaction generates a large amount of waste products . Hence biological (or enzymatic) BVO offers a ‘greener’ approach for the production of chiral lactones.
Biological Baeyer- Villiger oxidations are mediated by flavin- dependent monooxygenase enzymes i.e. Baeyer- Villiger monooxygenases (BVMOs) [19,21,22]. As a result of the versatile nature of flavoproteins , BVMOs have been shown to perform a variety of catalytic reactions including BVO of steroidal systems.
The mechanism of microbial Baeyer- Villiger’s oxidation (fig. 6) is based on results obtained with cyclohexanone monooxygenase (CHMO) isolated from Acinetobacter calcoaceticus [19,22]. This enzyme was shown to possess flavin adenine dinucleotide (FAD) as a prosthetic group and was also found to be dependent on NADPH and oxygen. The enzymatic process is initiated by the reduction of the tightly bound FAD by NADPH followed by rapid oxidation by molecular oxygen to produce flavin 4a- peroxide anion, which acts as the oxygenating species. Nucleophillic attack of the substrate carbonyl group by the flavin 4a- peroxide anion results in the ‘Criegee’ intermediate. This intermediate then undergoes rearrangement to form the product lactone and 4a- hydroxy- flavin. The catalytic cycle is terminated by elimination of water to form FAD and the release of the product and co-factor. It should be noted that the mechanism for microbial BVO based on CHMO serves as a model for other BVMOs. However, there are some differences such as the co-factor NADPH can be replaced by NADH and the prosthetic group FAD can be replaced by FMN . Overall, there are no significant changes to the mechanism.
Microbial Baeyer- Villiger’s oxidation is highly regio- and stereoselective [4,19-22] and as result it is commonly utilized for the biotransformation of steroidal compounds. It has also been shown in various studies, the ability of microbial BVMOs to attack the different ring systems of the steroid skeleton. Glomerella fusarioides was observed to biotransform eburicoic acid through an attack on the ring- A system by way of BVO to form a lactone, followed by a ring- cleavage to produce carboxylic acid . In addition, 3-ketosteroids were observed to undergo Baeyer- Villiger’s oxidation with an isolated Baeyer- Villiger monooxygenase enzyme from Pseudomonas sp. attacking the C-3 ketone group on ring- A . Ring- B lactone formation has also been observed in the steroid system using tomato cell suspension cultures to produce 24- epibassinolide . Ring- D lactonization is very common and has been demonstrated by quite a few fungal species such as Pencillium sp., Cylindrocarim sp., Mucor sp. and Aspergillus sp. These fungi were able to biotransform progesterone to testololactone by way of Baeyer- Villiger’s oxidation via the intermediate steroid androst-4-ene-3,17-dione . So far, ring- C lactonization has not been observed, although studies have been conducted to view this ring attack but none have proven its possibility . Overall, several research have been undertaken and are still been conducted to explore the catalytic repertoire of Baeyer- Villiger monooxygenase enzymes, and these studies have shown the ability of this enzyme to catalyse the oxidation of 3- keto and 17- keto steroids with full control of the regiochemistry of the produced lactone thus allowing its application as an alternative to the conventional chemical process.
1.4. The mechanism of alcohol oxidation
Alcohol oxidation is an important reaction in organic chemistry. It leads to the production of aldehydes or carboxylic acids from primary alcohol and ketones from secondary alcohol. Tertiary alcohols are resistant to oxidation because it is impossible to remove a hydrogen ion or add an oxygen atom to the compound without breaking the C-C bond. The commonly used reagents for the oxidation of alcohol are Jones’ reagent, potassium permanganate and chromium- based reagents. However, the oxidation of primary alcohols to aldehydes creates a problem for the organic chemist because aldehydes are not stable when produced in the conventional chemical oxidation process thus the use of microbial cells is preferred to overcome this problem . The enzymes used in the oxidation of alcohol by microorganisms are alcohol dehydrogenases (ADH) which are dependent on the co-factors NAD+ or NADP+. The mechanism of this reaction consists of a series of equilibrium where the hydride from the alcohol substrate is transferred to NAD(P)+ in the ternary complex ‘enzyme- NAD+- alcohol’ complex . In humans, this process is carried in the same fashion and is extremely important for several endogenous as well as drug metabolism. Therefore, microorganisms could serve as models for human metabolism using this process. An unprecedented level of regioselctivity of microbial oxidation of the alcoholic group in bile acids has been observed . Some fungal species are known to have the ability to oxidise the C-3 and C-17 hydroxyl groups of steroidal compounds. Aspergillus tamarii has been shown to possess the enzyme 3β- hydroxy- steroid- dehydrogenases which catalyses the 3β- hydroxyl group to a C-3 ketone . Oxidation of the 17β- hydroxyl group has also been observed in a number of fungal species e.g. Penicillium sp., Aspergillus sp. and Mucor sp [24,25]. In general, a number of microorganisms have shown the ability to oxidise the alcohol groups on a steroid compound to generate the ketone analogue, which could serve as an intermediate in the synthesis of lactones.
1.5. The mechanism of carbonyl reduction
The reverse reaction of oxidation is reduction. It involves the transfer of one hydride ion to the carbonyl group. In conventional chemical reaction, the catalysts commonly used are sodium borohydride (NaBH4) and Lithium aluminium hydride (LiAlH4), aldehydes are easily reduced to primary alcohols using these catalysts. However, the high stereoselective reduction of ketones to chiral secondary alcohols is better performed with microbial enzymes [20,22]. This process is catalyzed by alcohol dehydrogenases (ADHs), requiring the co-enzymes NADH or NADPH which transfers the hydride ion to the Si- or Re- face of the carbonyl group resulting in the formation of the corresponding (S)- or (R)- alcohol [22,25]. Microbial reduction of ketones to secondary alcohols normally proceeds in accordance with Prelog’s rule to give secondary alcohols in the main (S)- enantiomer [25,26]. However, only a very limited number of microbial enzyme (ADHs) is available to allow anti- Prelog activity and have been demonstrated in the fungus Myceliophthora thermophila .
The ability of microorganisms to reduce the carbonyl groups on steroid compounds was reported in 1937 by Mamoli and Vercelloni who described the reduction of the 17- keto group in androst-4-ene-3,17-dione to testosterone by Saccharomyces cerevisiae . Since then this process has been demonstrated for a wide variety of substrates and microorganisms of different species. Carbonyl reduction often accompanies other reactions in steroid biotransformation, and thus acts as one of the processes in the production of hydroxysteroids.
1.6. The microorganism: Myceliophthora thermophila
Thermophilic fungi are among the few fungal species of eukaryotic organism that are able to survive at temperatures as high as 60 – 62oC . However, Cooney and Emerson’s definition of thermophilic fungi is: fungi that have a growth temperature minimum at or above 20oC and a growth temperature maximum at or above 50oC . These fungi have a widespread distribution in both tropical and temperate regions, inhabiting various types of soil and places where decomposition of plant material and organic matter occur thus providing the warm, humid and aerobic environment which are the basic conditions for their development [28,29]. The enzymes of thermophilic fungi have been studied to explore their contribution in biotechnology, and these studies have identified a remarkable range of extracellular enzymes (e.g. proteases, lipases, α-amylases, glucoamylases, cellulases, cellobiose dehydrogenases, xylanases, α- D-glucuronidase, polygalacturonase, laccases, phytase and D-glucosyltransferase) and intracellular enzymes (e.g. trehalases, invertases, β-glycosidases, lipoamide dehydrogenases, ATP sulfurylases and protein disulfide isomerases) . The majority of these enzymes are appreciably thermostable which have resulted in its application in sugar and paper industries .
So far only two studies to date have been conducted to investigate the steroid biotransformation abilities of thermophilic fungi. The first study used the thermophilic filamentous fungus, Rhizomucor tauricus and it was observed that all transformations were oxidative producing mono- and dihydroxylated products with allylic hydroxylation been the predominant route of attack on the steroid compounds . The second study was conducted using Myceliophthora thermophila  on which this present study is based.
Myceliophthora thermophla is a thermophilic filamentous fungus classed as an ascomycete within the phyla of fungi . It has another name which is sometimes used, Sporotrichum (Chrysosporium) thermophile [28,29]. However, M. thermophila is the sexual (telomorph) stage of the fungi, while Sporotrichum (Chrysosporium) thermophile is the asexual (anamorph) stage . Its main habitat is in the soil and it is found in the following countries: USA, Canada, India, UK, Japan and Australia . But this fungus can grow on simple media containing carbon, nitrogen and essential mineral salts such as Czapek- dox agar (CDA). The optimum growth temperature for M. thermophila is within the range 45 – 50oC . It grows rapidly on CDA at 45oC, producing colonies that vary in surface texture from cottony to granular and its colour changes from white to cinnamon brown . This fungus has also been observed to generate extracellular enzymes such as laccases, xylanases, cellulases and phytase which have been exploited for use in the food industry and as biocatalyst in biotechnological processes .
This present study is a continuation of the research into steroid biotransformation by M. thermophila. Previously, a series of steroids (progesterone, testosterone acetate, 17β-acetoxy-5α-androstan-3-one, testosterone and androst-4-ene-3,17-dione) were incubated with this fungus, and a wide range of biocatalytical activity was observed with enzymatic attack at all four rings of the steroid nucleus and the C-17β side- chain. This fungus demonstrated an unusual ring- A opening following incubation of the steroid 17β-acetoxy-5α-androstan-3-one, and thus generating 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid. It was also identified to be the first thermophilic fungus to cleave the side- chain of progesterone. M. thermophila also demonstrated reversible acetylation and oxidation of the 17β- alcohol of testosterone  (fig. 8).
Further investigation into the diverse biocatalytical activity of this organism has led to the incubation of six saturated steroids namely: 17β-hydroxy-5α-androstan-3-one, 5α-prgnane-3,20-dione, 3β-hydroxy-5α-androstan-17-one, 3α-hydroxy-5α-androstan-17-one, 5α-androstan-3,6,17-trione and 5α-androstan-3,17-dione with M. thermophila
The proposed hypothesis from previous study is outlined as follows:
* Presumed lactonohydrolase activity evident from the isolation of an open lactone ring.
* Enzymes responsible for the reduction of C3 ketone to a 3α- alcohol and hydrogenation of the C-4-C-5 alkene are induced by progesterone.
* Organism’s ability for reverse metabolism, which is evident from the acetylation of testosterone to generate testosterone acetate and the reduction of the C-17 ketone of androst-4-ene-3,17-dione to produce testosterone which further undergoes acetylation.
* Preference for stereochemistry of hydroxylation with attack at axial protons (6β, 7α, 11β, 14α).
Therefore, the main aim of this study is to observe the effect of saturated steroids on the biocatalytical activity of Myceliophthora thermophila CBS 117.65 and to prove the hypothesis from the previous study.
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