Steroid Intermediates Research Articles

Improvement of 9α-hydroxyandrost-4-ene-3,17-dione production in Mycolicibacterium neoaurum by regulation of cell wall formation and transcriptional regulator PadR

The biotransformation of phytosterol into high value steroid intermediates such as 9α-hydroxyandrost-4-ene-3,17-dione (9-OHAD) in Mycolicibacterium is the cornerstone of the steroid pharmaceuticals. However, the limited permeability of the dense mycobacterial cell wall severely hinders the efficient transportation of phytosterol and their bioconversion to 9-OHAD. In this study, we disrupted the genetic pathways involved in trehalose biosynthesis, trehalose recycle and by-product formation, leading to alterations in cell wall formation, cell permeability and 9-OHAD productivity. This manipulation led to an increase of 63.7% in the yield of 9-OHAD, reaching 10.8 g/L at a phytosterol concentration of 20 g/L in shake flask. The enhancement of cell permeability and 9-OHAD production were achieved through the deletion of genes TreS, TreY, OtsA, LpqY, and SugC, as well as the inactivation of regulator PadR. Notably, it was found that the increase in TMM content of cell wall components via TLC analysis directly affected the distribution of 9-OHAD within and outside the cell, ultimately leading to an increase in extracellular production of 9-OHAD from 12% to 32.1%. Therefore, this study provides with an effective strategy for enhancing 9-OHAD production by increasing cell permeability while minimizing by-product 4-AD formation.

Functional characterization of two reductase KshBs in Mycobacterium fortuitum and their applications to C9 non-hydroxylated steroid production

3-ketosteroid-9α-hydroxylase (KSH), a two-component monooxygenase consisting of a Rieske oxygenase KshA and a ferredoxin reductase KshB, is a crucial enzyme involved in C-9 hydroxylation and is indispensable in the microbial catabolism of sterols. However, the in vivo function of KshB remains unclear. In this study, two reductase subunits, KshB1 and KshB2 from Mycobacterium fortuitum ATCC 35855, were first characterized and then engineered in the strain MFΔkstD, which produces 9-OHAD (9α-hydroxy-4-androstene-3,17-dione), to construct the strains producing AD (4-androstene-3,17-dione). The transcriptional level and specific enzyme activity of KshB1 under phytosterols induction was significantly higher compared to KshB2. After knocking out kshB1, AD peaked at 1.77 g/L at 48 h, subsequently being fully converted to 9-OHAD. Furthermore, we developed the stable AD-producer MFΔkstDΔkshB by null mutation both kshB1 and kshB2, although no AD was detected by single knocking out kshB2. Through gene complementation and the bioconversion of phytosterols, KshB1 emerged as the primary reductase in the KSH system, with KshB2 serving a complementary role. The stable AD-producer MFΔkstDΔkshB could produce 5.18 g/L, 6.91 g/L, and 9.03 g/L AD from the transformation of 10 g/L, 15 g/L, and 20 g/L phytosterols, respectively. In conclusion, these findings highlight a new strategy for the metabolic engineering of Mycobacterium fortuitum, involving the inactivation of the reductase KshB to facilitate the production of C9 non-hydroxylated steroidal intermediates.

A practical approach to supported liquid extraction and measurement of 18 steroids in plasma and serum by targeted liquid chromatography tandem mass spectrometry

Chromatography combined with mass spectrometry is a gold standard technique for steroid measurement, however the type of sample preparation, the dynamic range and reliability of the calibration curve, the chromatographic separation and mass spectrometry settings ultimately determine the success of the method. The steroid biosynthetic pathway is conserved in higher mammals and literature demonstrates that the concentration ranges of different steroid groups are relatively comparable across species. We sought to develop a robust and reliable multi steroid targeted analysis method for blood that would have wide application across higher mammals. The method was developed following bioanalytical method validation guidelines to standards typically applied to human clinical studies, including isotopically labelled internal standards where at all possible. Here we describe the practical approach to a 96-well supported liquid extraction (SLE) method of extraction from plasma (200 µL) using an Extrahera liquid handling robot (Biotage, Sweden), including quality control samples, followed by a comprehensive separation and targeted LC-MS/MS analysis of 18 steroids in plasma (pregnenolone, progesterone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, aldosterone, 11-deoxycortisol, 21-deoxycortisol, cortisol, cortisone, androstenedione, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, estrone, 17β-estradiol and estriol).•SLE in a 96-well format of up to 74 biological plasma samples, enriched with multiple isotopically labelled internal standards, a 12-point aqueous calibration curve, and 6 serum quality controls, designed to monitor long-term performance of the method•Chromatographic separation of multiple steroids along the gradient, with ammonium fluoride mobile phase additive to improve sensitivity, followed by electrospray ionisation and constant polarity switching•Aqueous calibration standards that cover physiologically relevant ranges - high nanomolar glucocorticoids, low nanomolar androgens and picomolar ranges for estrogens and steroid intermediates.

Aldosterone and aldosterone synthase inhibitors in cardiorenal disease.

Modulation of the renin-angiotensin-aldosterone system is a foundation of therapy for cardiovascular and kidney diseases. Excess aldosterone plays an important role in cardiovascular disease, contributing to inflammation, fibrosis, and dysfunction in the heart, kidneys, and vasculature through both genomic and mineralocorticoid receptor (MR)-mediated as well as nongenomic mechanisms. MR antagonists have been a key therapy for attenuating the pathologic effects of aldosterone but are associated with some side effects and may not always adequately attenuate the nongenomic effects of aldosterone. Aldosterone is primarily synthesized by the CYP11B2 aldosterone synthase enzyme, which is very similar in structure to other enzymes involved in steroid biosynthesis including CYP11B1, a key enzyme involved in glucocorticoid production. Lack of specificity for CYP11B2, off-target effects on the hypothalamic-pituitary-adrenal axis, and counterproductive increased levels of bioactive steroid intermediates such as 11-deoxycorticosterone have posed challenges in the development of early aldosterone synthase inhibitors such as osilodrostat. In early-phase clinical trials, newer aldosterone synthase inhibitors demonstrated promise in lowering blood pressure in patients with treatment-resistant and uncontrolled hypertension. It is therefore plausible that these agents offer protection in other disease states including heart failure or chronic kidney disease. Further clinical evaluation will be needed to clarify the role of aldosterone synthase inhibitors, a promising class of agents that represent a potentially major therapeutic advance.

Identification of a new C-23 metabolite in sterol degradation of Mycobacterium neoaurum HGMS2 and analysis of its metabolic pathways

Mycobacterium neoaurum has the ability to produce steroidal intermediates known as 22-hydroxy-23, 24-bisnorchol-4-en-3-one (BA) upon the knockout of the genes for either the hydroxyacyl-CoA dehydrogenase (Hsd4A) or acyl-CoA thiolase (FadA5). In a previous study, we discovered a novel metabolite in the fermentation products when the fadA5 gene was deleted. This research aims to elucidate the metabolic pathway of this metabolite through structural identification, homologous sequence analysis of the fadA5 gene, phylogenetic tree analysis of M. neoaurum HGMS2, and gene knockout. Our findings revealed that the metabolite is a C23 metabolic intermediate, named 24-norchol-4-ene-3, 22-dione (designated as 3-OPD). It is formed when a thioesterase (TE) catalyzes the formation of a β-ketonic acid by removing CoA from the side chain of 3, 22-dioxo-25, 26-bisnorchol-4-ene-24-oyl CoA (22-O-BNC-CoA), followed by spontaneously undergoing decarboxylation. These results have the potential to contribute to the development of novel steroid intermediates.

A fungal CYP from Beauveria bassiana with promiscuous steroid hydroxylation capabilities

Hydroxylation of steroid core is critical to the synthesis of steroid drugs. Direct sp3 C–H hydroxylation is challenging through chemical catalysis, alternatively, fungal biotransformation offers a possible solution to this problem. However, mining and metabolic engineering of cytochrome P450 monooxygenases (CYPs) is usually regarded as a more eco-friendly and efficient strategy. Herein, we report the mining and identification of a new steroid CYP (CYP68BE1) from Beauveria bassiana by transcriptomics, heterologous expression, in vivo and in vitro functional characterization. The catalytic promiscuity of CYP68BE1 was explored, and CYP68BE1 showed promiscuously and catalytically versatile, which is qualified for monohydroxylation on C11α, C1α, C6β and dihydroxylation on C1β,11α and C6β,11α of six steroids, leading to the production of key steroid intermediates required in the industrial synthesis of some indispensable steroid drugs. Molecular dynamics simulations were performed, revealing the molecular basis of different binding orientations of CYP68BE1 with different substrates. The discovery of CYP68BE1 offers a promising biocatalyst for enriching the steroid structural and functional diversity, which also can be applied to biosynthesize valuable steroid drug intermediates.

Process development of transforming phytosterols to steroidal pharmaceutical intermediates by Mycolicibacterium neoaurum with L-carnitine

The process of phytosterol microbial transformation in aqueous phase is the most important pathway to prepare steroidal pharmaceutical intermediates. However, the low solubility of phytosterols and difficult contact between substrate and cells become the main obstacles in practical application. In this study, a comparison of commonly used vegetable oils, surfactants, cosolvents and ionic liquids identified L-carnitine as a novel zwitterionic additive to enhance phytosterol bioconversion by growing and resting cells of Mycolicibacterium neoaurum LLZ2. The addition of L-carnitine had significance in obtaining more biomass and increasing glucose metabolic velocity. Moreover, the surface of cells cultivated with L-carnitine was smoother, which probably enhanced the contact of phytosterols and cells. In other aspects, the dispersion of substrate and several key enzymes involving in the synthesis of steroidal intermediates were not influenced by L-carnitine. Through the optimization of reaction parameters, the total yield of androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) was improved by 39%. This study provides a green approach for reaction engineering of Mycolicibacterium-mediated phytosterol bioconversion, and new insights into possible mechanism.

Biosynthesis of steroidal intermediates using Mycobacteria: a review

Steroids are a class of medicines with important physiological and pharmacological effects. In pharmaceutical industry, steroidal intermediates are mainly prepared through Mycobacteria transformation, and then modified chemically or enzymatically into advanced steroidal compounds. Compared with the "diosgenin-dienolone" route, Mycobacteria transformation has the advantages of abundant raw materials, cost-effective, short reaction route, high yield and environmental friendliness. Based on genomics and metabolomics, the key enzymes in the phytosterol degradation pathway of Mycobacteria and their catalytic mechanisms are further revealed, which makes it possible for Mycobacteria to be used as chassis cells. This review summarizes the progress in the discovery of steroid-converting enzymes from different species, the modification of Mycobacteria genes and the overexpression of heterologous genes, and the optimization and modification of Mycobacteria as chassis cells.

Whole-Genome Analysis of Mycobacterium neoaurum DSM 1381 and the Validation of Two Key Enzymes Affecting C22 Steroid Intermediates in Sterol Metabolism.

Mycobacterium neoaurum DSM 1381 originated from Mycobacterium neoaurum ATCC 25790 by mutagenesis screening is a strain of degrading phytosterols and accumulating important C22 steroid intermediates, including 22-hydroxy-23, 24-bisnorchola-4-en-3-one (4-HP) and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (HPD). However, the metabolic mechanism of these C22 products in M. neoaurum DSM 1381 remains unknown. Therefore, the whole-genome sequencing and comparative genomics analysis of M. neoaurum DSM 1381 and its parent strain M. neoaurum ATCC 25790 were performed to figure out the mechanism. As a result, 28 nonsynonymous single nucleotide variants (SNVs), 17 coding region Indels, and eight non-coding region Indels were found between the genomes of the two strains. When the wild-type 3-ketosteroid-9α-hydroxylase subunit A1 (KshA1) and β-hydroxyacyl-CoA dehydrogenase (Hsd4A) were overexpressed in M. neoaurum DSM 1381, the steroids were transformed into the 4-androstene-3, 17- dione (AD) and 1,4-androstadiene-3,17-dione (ADD) instead of C22 intermediates. This result indicated that 173N of KshA1 and 171K of Hsd4A are indispensable to maintaining their activity, respectively. Amino acid sequence alignment analysis show that both N173D in KshA1 and K171E in Hsd4A are conservative sites. The 3D models of these two enzymes were predicted by SWISS-MODEL and AlphaFold2 to understand the inactivation of the two key enzymes. These results indicate that K171E in Hsd4A may destroy the inaction between the NAD+ with the NH3+ and N173D in KshA1 and may disrupt the binding of the catalytic domain to the substrate. A C22 steroid intermediates-accumulating mechanism in M. neoaurum DSM 1381 is proposed, in which the K171E in Hsd4A leads to the enzyme's inactivation, which intercepts the C19 sub-pathways and accelerates the C22 sub-pathways, and the N173D in KshA1 leads to the enzyme's inactivation, which blocks the degradation of C22 intermediates. In conclusion, this study explained the reasons for the accumulation of C22 intermediates in M. neoaurum DSM 1381 by exploring the inactivation mechanism of the two key enzymes.

A Keto Reductase Involved in Steroid Degradation in Mycolicibacterium neoaurum.

Phytosterols can be used by microorganisms as carbon and energy sources and completely degraded into CO2 and H2 O. The catabolic pathway of phytosterols was well characterized in many microorganisms. Blocking the steroid core ring degradation by deletions of fadE30 and fadD3 genes, two important steroid intermediates, 3aα-H-4α-(3'-Propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone-δ-lactone (sitolactone, or HIL) and 3aα-H-4α-(3'-propionic acid)-7aβ-methylhexahydro-1,5-indanedione (HIP) can be accumulated. They are currently used to synthesize nor-steroid drugs with an α-methyl group or without the methyl group at the C10 -position, such as estrone and norethindrone. In this study, a key gene involved in the bioconversion of HIP to HIL was identified in Mycolicibacterium neoaurum. Through heterologous expression, gene hipR was found to be involved in the reduction of the C5 keto group of HIP to a hydroxy group, leading to spontaneously lactonization into HIL in vitro. Through gene complementation and knockout, HipR functions were verified and two HIP degradation pathways in vivo were elucidated. The finding of this research facilitated the understanding of the metabolic pathway of sterols, and was directly applied to engineering robust production strains by overexpression or knockout of related genes.

Scale-Up of Phytosterols Bioconversion into Androstenedione.

Phytosterols, coming as a by-product of vegetable oils or wood pulp, contain the cyclopentanoperhydrophenanthrene nucleus and can be bioconverted into steroid intermediates by removing the C17 side chain. This chapter shows the scale-up, from flask to bioreactor, of phytosterols bioconversion into 4-androstene-3,17-dione (androstenedione; AD) using Mycolicibacterium neoaurum B-3805. Due to the fact that phytosterols and AD are nearly insoluble in water, two-phase systems and the use of chemically modified cyclodextrins have been described as methods to solve it. Here, we use a water-oil two-phase system that allows the bioconversion of up to 20g/L of phytosterols into AD in 5L and 20L bioreactors.

Bioconversion of Phytosterols into Androstenedione by Mycolicibacterium.

The chapter describes the bioconversion of phytosterols into androstenedione (AD) by Mycolicibacterium spp. in shake flasks and fermenters, as well as LC-MS-based methods for analysis of phytosterols and steroid products. Phytosterols are derived as by-products of vegetable oil refining and manufacture of wood pulp. They contain the same four-ring nucleus as steroids and may be converted to high-value steroids by removing the sidechain at C17 and minor changes at other sites in the ring structure. Many bacteria, including Mycolicibacterium spp., can degrade phytosterols. Mutants of Mycolicibacterium spp. unable of ring cleavage can, when growing on phytosterols, accumulate the steroid intermediates androstenedione (AD) and androstadienedione (ADD). The practical challenge with microbial conversion of phytosterols to steroids is that both the substrate and the product are virtually insoluble in water. In addition, some steroids, notably ADD, may be toxic for the cells. Two main strategies have been employed to overcome this challenge: the use of two-phase systems and the addition of chemically modified cyclodextrins. The latter method is used here. Defined cultivation and bioconversion media for both shake flask and fermenter are given, as well as hints how to minimize the practical problems due to the water-insoluble phytosterol. Sampling, sample extraction, and quantification of substrates and products using LC-MS analysis are described.

Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells

Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans.

Whole-genome analyses and metabolic modification of Mycobacterium sp. LY-1 to enhance yield of 9α-OH-AD

With the increasing application of steroid drugs as therapeutics, the demand for steroid drugs is increasing. In recent years, biological synthesis has become the standard approach to produce steroid intermediates, while this method still faces some problems such as unclear metabolic pathway and low yield. Mycobacterium sp. LY-1 can convert phytosterols into 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD) which is a key intermediate for the synthesis of steroid drugs with long effective time and significant pharmacological activity. In this work, the whole-genome sequence of the Mycobacterium sp. LY-1 was analyzed, and the side-chain degradation pathway of phytosterols in Mycobacterium sp. LY-1 was proposed. Meanwhile, the related key enzymes of phytosterol metabolism were identified through qRT-PCR. Through overexpressing the key enzymes including KshA2, KshB, and HsdB, the yield of 9α-OH-AD increased by 12.7% compared to that of the control. Furthermore, by optimizing the medium and culture conditions, the yield of 9α-OH-AD reached 50.4%. The maximum yield was 30.7% higher than that of the original strain. The results are of significance for the industrial production of 9α-OH-AD using metabolic engineering methods.

Biotransformation Enables Innovations Toward Green Synthesis of Steroidal Pharmaceuticals.

Steroids have been widely used in birth-control, prevention, and treatment of various diseases, representing the largest sector after antibiotics in the global pharmaceutical market. The steroidal active pharmaceutical ingredients (APIs) have been produced via partial synthetic processes first mainly from sapogenins, which was converted into 16-dehydropregnenolone by the famous "Marker Degradation". Traditional mutation and screening, and process engineering have resulted in the industrial production of 4-androstene-3,17-dione (AD), androst-1,4-diene-3,17-dione (ADD), 9α-hydroxy-androsta-4-ene-3,17-dione (9α-OH-AD), and so on, which serve as the key intermediates for the synthesis of steroidal APIs. Recently, genetic and metabolic engineering have generated highly efficient microbial strains for the production of these precursors, leading to the replacement of sapogenins with phytosterols as the starting materials. Further advances in synthetic biology hold promise in the design and construction of microbial cell factories for the industrial production of steroidal intermediates and/or APIs from simple carbon sources such as glucose. Integration of biotransformation into the synthesis of steroidal APIs can greatly reduce the number of reaction steps, achieve lower waste discharge and higher production efficiency, thus enabling a greener steroidal pharmaceutical industry.

Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment

BackgroundSteroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain.ResultsThe Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP.ConclusionKstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Enhancement of Ultraviolet B Irradiation with a Photoluminescent Composite Film and Its Application in Photochemical Microfluidic Synthesis

Ultraviolet B (UVB) irradiation is of great importance for photochemical synthesis of steroidal intermediates, which could be efficiently provided by neither traditional mercury lamps nor novel lig...

Enhancing the bioconversion of phytosterols to steroidal intermediates by the deficiency of kasB in the cell wall synthesis of Mycobacterium neoaurum

BackgroundThe bioconversion of phytosterols into high value-added steroidal intermediates, including the 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), is the cornerstone in steroid pharmaceutical industry. However, the low transportation efficiency of hydrophobic substrates into mycobacterial cells severely limits the transformation. In this study, a robust and stable modification of the cell wall in M. neoaurum strain strikingly enhanced the cell permeability for the high production of steroids.ResultsThe deletion of the nonessential kasB, encoding a β-ketoacyl-acyl carrier protein synthase, led to a disturbed proportion of mycolic acids (MAs), which is one of the most important components in the cell wall of Mycobacterium neoaurum ATCC 25795. The determination of cell permeability displayed about two times improvement in the kasB-deficient strain than that of the wild type M. neoaurum. Thus, the deficiency of kasB in the 9-OHAD-producing strain resulted in a significant increase of 137.7% in the yield of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). Ultimately, the 9-OHAD productivity in an industrial used resting cell system was reached 0.1135 g/L/h (10.9 g/L 9-OHAD from 20 g/L phytosterol) and the conversion time was shortened by 33%. In addition, a similar self-enhancement effect (34.5%) was realized in the 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain.ConclusionsThe modification of kasB resulted in a meaningful change in the cell wall mycolic acids. Deletion of the kasB gene remarkably improved the cell permeability, leading to a self-enhancement of the steroidal intermediate conversion. The results showed a high efficiency and feasibility of this construction strategy.

Steroidogenesis and its regulation in teleost-a review.

Steroid hormones modulate several important biological processes like metabolism, stress response, and reproduction. Steroidogenesis drives reproductive function wherein development and differentiation of undifferentiated gonads into testis or ovary, and their growth and maturation, are regulated. Steroidogenesis occurs in gonadal and non-gonadal tissues like head kidney, liver, intestine, and adipose tissue in teleosts. This process is regulated differently through multi-level modulation of promoter motif transcription factor regulation of steroidogenic enzyme genes to ultimately control enzyme activity and turnover. In view of this, understanding teleostean steroidogenesis provides major inputs for technological innovation of pisciculture. Unlike higher vertebrates, steroidal intermediates and shift in steroidogenesis is critical for gamete maturation in teleosts, more essentially oogenesis. Considering these characteristics, this review highlights the promoter regulation of steroidogenic enzyme genes by several transcription factors that are involved in teleostean steroidogenesis. It also addresses different methodologies involved in promoter regulation studies together with glucocorticoids and androgen relationship with reference to teleosts.

Soluble expression, purification and biochemical characterization of a C-7 cholesterol dehydrogenase from Drosophila melanogaster

The C-7 cholesterol dehydrogenase (NVD), which converts cholesterol to 7-dehydrocholesterol (7-DHC) by 7,8-dehydrogenation, plays a pivotal role in the metabolism of cholesterol and steroid intermediates. The NVD protein was successfully expressed in insect Sf9 cells. To reduce the production cost for industrial application, the NVD gene was cloned into E. coli BL21(DE3). However, the His-tagged NVD protein showed poor binding to Ni-NTA resin, mainly due to the formation of inclusion bodies. Consequently, the expression and solubility of NVD were optimized by respectively fusing it with maltose-binding protein (MBP), glutathione S-transferase (GST), and the nonspecific DNA binding protein from Sulfolobus solfataricus (Sso7d) as solubility tags. The NVD fusion with MBP at the N-terminus and His-tag at the C-terminus achieved a high yield of the soluble enzyme. It was further purified by ion-exchange chromatography with 95.4% purity and with a 10.4% yield. The product 7-DHC, which is produced in a reaction catalyzed by NVD and ferredoxin reductase KshB, was initially identified by GC–MS. An analysis of the amino acid sequence alignment revealed a distinct Rieske-type iron-sulfur cluster and non-heme Fe2+-binding domain, which are evolutionarily conserved among NVD family enzymes.