Androst-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) are important drug intermediates that can be biosynthesized from phytosterols. However, the C9 hydroxylation of steroids via 3-ketosteroid 9α-hydroxylase (KSH) limits AD and 4-HBC accumulation. Five active KshAs, the oxidation component of KSH, were identified in Mycobacterium fortuitum ATCC 35855 for the first time. The deletion of kshAs indicated that the five KshA genes were jointly responsible for C9 hydroxylation during phytosterol biotransformation. MFKDΔkshA, the five KshAs deficient strain, blocked C9 hydroxylation and produced 5.37 g/L AD and 0.55 g/L 4-HBC. The dual function reductase Opccr knockout and 17β-hydroxysteroid dehydrogenase Hsd4A enhancement reduced 4-HBC content from 8.75 to 1.72% and increased AD content from 84.13 to 91.34%, with 8.24 g/L AD being accumulated from 15 g/L phytosterol. In contrast, hsd4A and thioesterase fadA5 knockout resulted in the accumulation of 5.36 g/L 4-HBC from 10 g/L phytosterol. We constructed efficient AD (MFKDΔkshAΔopccr_hsd4A) and 4-HBC (MFKDΔkshAΔhsd4AΔfadA5) producers and provided insights for further metabolic engineering of the M. fortuitum ATCC 35855 strain for steroid productions. KEY POINTS: • Five active KshAs were first identified in M. fortuitum ATCC 35855. • Deactivation of all five KshAs blocks the steroid C9 hydroxylation reaction. • AD or 4-HBC production was improved by Hsd4A, FadA5, and Opccr modification.

Androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) are valuable steroid pharmaceutical intermediates obtained by soybean phytosterol biotransformation by Mycobacterium Cyclodextrins (CDs) are generally believed to be carriers for phytosterol delivery and can improve the production of AD and ADD due to their effects on steroid solubilization and alteration in cell wall permeability for steroids. To better understand the mechanisms of CD promotion, we performed proteomic quantification of the effects of hydroxypropyl-β-CD (HP-β-CD) on phytosterol metabolism in Mycobacterium neoaurum TCCC 11978 C2. Perturbations are observed in steroid catabolism and glucose metabolism by adding HP-β-CD in a phytosterol bioconversion system. AD and ADD, as metabolic products of phytosterol, are toxic to cells, with inhibited cell growth and biocatalytic activity. Treatment of mycobacteria with HP-β-CD relieves the inhibitory effect of AD(D) on the electron transfer chain and cell growth. These results demonstrate the positive relationship between HP-β-CD and phytosterol metabolism and give insight into the complex functions of CDs as mediators of the regulation of sterol metabolism.IMPORTANCE Phytosterols from soybean are low-cost by-products of soybean oil production and, owing to their good bioavailability in mycobacteria, are preferred as the substrates for steroid drug production via biotransformation by Mycobacterium However, the low level of production of steroid hormone drugs due to the low aqueous solubility (below 0.1 mmol/liter) of phytosterols limits the commercial use of sterol-transformed strains. To improve the bioconversion of steroids, cyclodextrins (CDs) are generally used as an effective carrier for the delivery of hydrophobic steroids to the bacterium. CDs improve the biotransformation of steroids due to their effects on steroid solubilization and alterations in cell wall permeability for steroids. However, studies have rarely reported the effects of CDs on cell metabolic pathways related to sterols. In this study, the effects of hydroxypropyl-β-CD (HP-β-CD) on the expression of enzymes related to steroid catabolic pathways in Mycobacterium neoaurum were systematically investigated. These findings will improve our understanding of the complex functions of CDs in the regulation of sterol metabolism and guide the application of CDs to sterol production.

The ability of five fungal species belonging to two genera of Aspergillus and Fusarium has been examined in the microbial transformation of androst-4-ene-3, 17-dione (AD). Furthermore, the biotransformation of nandrolone decanoate (2) by F. fujikuroi has been studied. AD (1) was converted by cultures of Aspergillus sp. PTCC 5266 to form 11α-hydroxy-AD (3) as the only product, with a yield of 86% in 3 days. Moreover, two hydroxylated metabolites 11α-hydroxy-AD (3, 65%) and 7β-hydroxy-AD (4; 18%) were isolated in biotransformation of AD by A. nidulans. On the other hand, it was metabolized by F. oxysporum to produce 14α-hydroxy-AD (5; 38%) and testosterone (6; 12%). Microbial transformation of AD by F. solani led to the production of 11α-hydroxy-AD (3; 54%) and testosterone (6; 14%). AD was reduced at the 17-position by F. fujikuroi to produce testosterone in the yield of 42%. Finally, nandrolone decanoate was transformed by F. fujikuroi via hydrolysis and oxidation at the 17-position to produce two metabolites namely 17β-hydroxyestr-4-en-3-one (7, 25.4%) and estr-4-en-3,17-dione (8, 33%), respectively. The all metabolites were purified and subsequently identified based on their spectra data analysis and comparing them to the literature data.

Filamentous fungi is a huge phylum of lower eukaryotes with diverse activities towards various substrates, however, their biocatalytic potential towards steroids remains greatly underestimated. In this study, more than forty Ascomycota and Zygomycota fungal strains of 23 different genera were screened for the ability to catalyze structural modifications of 3-oxo-androstane steroids, - androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). Previously unexplored for these purposes strains of Absidia, Acremonium, Beauveria, Cunninghamella, Doratomyces, Drechslera, Fusarium, Gibberella genera were revealed capable of producing in a good yield valuable 7α-, 7β-, 11α- and 14α-hydroxylated derivatives, as well as 17β-reduced and 1(2)-dehydrogenated androstanes. The bioconversion routes of AD and ADD were proposed based on the key intermediates identification and time courses of the bioprocesses. Six ascomycete strains were discovered to provide effective 7β-hydroxylation of ADD which has not been so far reported. The structures of major products and intermediates were confirmed by HPLC, mass-spectrometry (MS), 1H and 13C NMR analyses. The results contribute to the knowledge on the functional diversity of steroid-transforming filamentous fungi. Previously unexplored fungal biocatalysts capable of effective performing structural modification of AD and ADD can be applied for industrial bioprocesses of new generation.

The microbial transformation of androst-4-ene-3,17-dione (4-AD; I) by three fungal species, involved Fusarium solani BH1031, Aspergillus awamori MH18 and Mucor circinelloides W12, has been studied. The latter two fungi were studied for the first time on biotransformation of 4-AD. The main product obtained by Fusarium solani BH1031 was 17α-oxa-D-homo-androst-1,4-diene-3,17-dione (testolactone; IV), which can be used as an anticancer agent. The main derivative yielded by Aspergillus awamori MH18 was 11α-hydroxyandrost-4-ene-3,17-dione (11α-OH-4-AD; VI), which was an important intermediate to produce Eplerenone. Meanwhile, the microbial transformation of 4-AD by Mucor circinelloides W12 produced three derivatives. Possible metabolic pathway of 4-AD via Fusarium solani BH1031 was proposed. Furthermore, the optimization for the production of 11α-OH-4-AD was carried out and the conversion rate reached to 84.0%. In this process, the dextrin and corn flour showed significant effects by response surface analysis.

3-Ketosteroid-Δ1-dehydrogenases (KsdD) from Mycobacterium neoaurum could transform androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione. This reaction has a significant effect on the product of pharmaceutical steroid. The crystal structure and active site residues information of KsdD from Mycobacterium is not yet available, which result in the engineering of KsdD is tedious. In this study, by the way of protein modeling and site-directed mutagenesis, we find that, Y122, Y125, S138, E140 and Y541 from the FAD-binding domain and Y365 from the catalytic domain play a key role in this transformation. Compared with the wild type, the decline in AD conversion for mutants illustrated that Y125, Y365, and Y541 were essential to the function of KsdD. Y122, S138 and E140 contributed to the catalysis of KsdD. The following analysis revealed the catalysis mechanism of these mutations in KsdD of Mycobacterium. These information presented here facilitate the manipulation of the catalytic properties of the enzyme to improve its application in the pharmaceutical steroid industry.

In steroid biotransformation, soybean oil can improve the productivity of steroids by increasing substrate solubility and strengthen the cell membrane permeability. However, little is known of its role as oxygen carrier and its mechanism of promoting the steroid biotransformation. In this work, soybean oil used as oxygen vector for the enhancement of androst-4-ene-3,17-dione (AD) production by Mycobacterium neoaurum TCCC 11979 (MNR) was investigated. Upon the addition of 16% (v/v) soybean oil, the volumetric oxygen transfer coefficient (K L a) value increased by 44%, and the peak molar yield of AD (55.76%) was achieved. Analysis of intracellular cofactor levels showed high NAD+, ATP level, and a low NADH/NAD+ ratio. Meanwhile, the two key enzymes of the tricarboxylic acid (TCA) cycle, namely, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, were upregulated after incubation with soybean oil. These enhancements induced by the increasing of oxygen supply showed positive effects on phytosterol (PS) bioconversion. Results could contribute to the understanding of effects of soybean oil as oxygen vector on steroid biotransformation and provided a convenient method for enhancing the efficiency of aerobic steroid biocatalysis.

The reduction of 17-oxosteroids to 17β-hydroxysteroids is one of the important transformations for the preparation of many steroidal drugs and intermediates. The strain Zygowilliopsis sp. WY7905 was found to catalyze the reduction of C-17 carbonyl group of androst-4-ene-3,17-dione (AD) to give testosterone (TS) as the sole product by the constitutive 17β-hydroxysteroid dehydrogenase (17β-HSD). The optimal conditions for the reduction were pH 8.0 and 30°C with supplementing 10g/l glucose and 1% Tween 80 (w/v). Under the optimized transformation conditions, 0.75g/l AD was reduced to a single product TS with >90% yield and >99% diastereomeric excess (de) within 24h. This strain also reduced other 17-oxosteroids such as estrone, 3β-hydroxyandrost-5-en-17-one and norandrostenedione, to give the corresponding 17β-hydroxysteroids, while the C-3 and C-20 carbonyl groups were intact. The absence of by-products in this microbial 17β-reduction would facilitate the product purification. As such, the strain might serve as a useful biocatalyst for this important transformation.

Rhodococcus rhodochrous DSM43269 is well known for its 3-ketosteroid-9α-hydroxylases. However, the function of its 3-ketosteroid-Δ(1)-dehydrogenases (KSDD) remains unknown. This study compared the involvement of ksdds in the strain\'s androst-4-ene-3,17-dione (AD) transformation via gene deletion. The conversion was performed using AD as substrate or directly with 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD). The single deletion of ksdd1 or ksdd3 did not appear to result in the accumulation of 9α-OH-AD, whereas the single mutant △ksdd2 could preserve this compound to some extent. To further compare the role of ksdds in this strain, double mutants were constructed. All ksdd2 mutants combined with ksdd1 and/or ksdd3 resulted in the accumulation of 9α-OH-AD, among which the double mutant △ksdd2,3 behaved similarly to the single mutant △ksdd2 in this process. The mutant that lacked both ksdd1 and ksdd3 was still displayed, with no effect on the degradation of 9α-OH-AD. The triple mutant △ksdd1,2,3 was then constructed and exhibited the same capability as △ksdd1,2, accumulating more 9α-OH-AD than △ksdd2,3 and △ksdd2. The transcription of KSDD1 and KSDD2 increased, whereas that of KSDD3 seemed to exhibit no change, despite the use of the inducer AD or 9α-OH-AD. Thus, only ksdd1 and ksdd2 were involved in the transformation of AD to 9α-OH-AD. ksdd2 had the main role, ksdd1 had a minor effect on 9α-OH-AD degradation, and ksdd3 did not exhibit any action in this course.

Musk is widely used as a traditional drug in Asia for the treatment of stroke, tumour, and cardiopathy with an oral dosage of 0.03-0.1 g per day. Because of the potential anabolic effect, musk preparations have been included in the list of medical products containing prohibited substances employed for doping. The application of musk pod formulation was regarded as the reason of some adverse analytical findings in the 2011 FIFA Women\'s World Cup. In order to investigate the influence of musk administration on the doping test, we executed a chemical analysis and excretion study. The gas chromatography/mass spectrometry (GC-MS) analysis demonstrated the diversity of steroid concentrations in musk samples. Furthermore, the δ(13)C-values of steroids from wild deer musk showed more depleted than those of domestic deer musk by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis. Because the steroids from some musk had δ(13)C-values in the range of naturally produced steroids in human body, the possible abuse of this kind of musk is very hard to be detected by isotope ratio mass spectrometry (IRMS) in doping control. Musk grains from wild and domestic deer were administrated for the excretion study respectively. Spot urine samples were collected from two male volunteers before and after 100 mg musk grains administration. The profiles and carbon isotope ratios of urinary steroids were determined by GC-MS and GC/C/IRMS. The ingestion of either wild or domestic deer musk did not lead to the adverse analytical finding of doping control in the single dosage of 100mg.