Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system.

BACKGROUND: Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. RESULTS: Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. CONCLUSION: The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones-progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Identification of a Gene Encoding a New Fungal Steroid 7-Hydroxylase and Its Functional Characterization in Pichia pastoris Yeast.

The hydroxylation of steroids in the C7ß position is one of the rare reactions that allow the production of value-added precursors in the synthesis of ursodeoxycholic acid and other pharmaceuticals. Recently, we discovered this activity in the ascomycete Curvularia sp. VKM F-3040. In this study, the novel gene of 7-hydroxylase (P450cur) was identified as being heterologously expressed and functionally characterized in Pichia pastoris. Transcriptome data mining and differential expression analysis revealed that 12 putative genes in Curvularia sp. mycelia significantly increased their expression in response to dehydroepiandrosterone (DHEA). The transcriptional level of the most up-regulated cytochrome P450cur gene was increased more than 300-fold. A two-gene construct with a candidate P450cur gene and the gene of its natural redox partner, NADPH-cytochrome P450 reductase (CPR), which is interconnected by a T2A element, was created. Using this construct, recombinant P. pastoris strains co-expressing fungal P450cur and CPR genes were obtained. The functional activity of the recombinant P450cur was studied in vivo during the bioconversion of androstane steroids. The fungal 7-monooxygenase predominantly catalyzed the 7ß-hydroxylation of androstadienedione (ADD), DHEA, and androstenediol, whereas 1-dehydrotestosterone was hydroxylated by P450cur mainly at the C7-Hα position. To our knowledge, this is the first report of a recombinant yeast capable of catalyzing the 7α/ß-hydroxylation of ADD and DHEA.

Draft genome sequence of soil isolate Mycolicibacterium fortuitum DVD-1301.

Some strains of Mycolicibacterium possess high sterol-oxidizing activity and are used in the pharmaceutical industry for the production of steroid precursors. Herein, we report a draft genome sequence of the soil-dwelling Mycolicibacterium fortuitum DVD-1301 isolated in the floodplain of the river Oka. The genome contains a full set of steroid catabolic genes.

Reconstruction of the Steroid 1(2)-Dehydrogenation System from Nocardioides simplex VKM Ac-2033D in Mycolicibacterium Hosts.

Microbial 1(2)-dehydrogenation of 3-ketosteroids is an important basis for the production of many steroid pharmaceuticals and synthons. When using the wild-type strains for whole cell catalysis, the undesirable reduction of the 20-carbonyl group, or 1(2)-hydrogenation, was observed. In this work, the recombinant strains of Mycolicibacterium neoaurum and Mycolicibacterium smegmatis were constructed with blocked endogenous activity of 3-ketosteroid-9α-hydroxylase, 3-ketosteroid-1(2)-dehydrogenase (3-KSD), and expressing 3-KSD encoded by the gene KR76_27125 (kstD2NS) from Nocardioides simplex VKM Ac-2033D. The in vivo activity of the obtained recombinant strains against phytosterol, 6α-methyl-hydrocortisone, and hydrocortisone was studied. When using M. smegmatis as the host strain, the 1(2)-dehydrogenation activity of the constructed recombinant cells towards hydrocortisone was noticeably higher compared to those on the platform of M. neoaurum. A comparison of the strengths of inducible acetamidase and constitutive hsp60 promoters in M. smegmatis provided comparable results. Hydrocortisone biotransformation by M. smegmatis BD/pMhsp_k expressing kstD2NS resulted in 95.4% prednisolone yield, and the selectivity preferred that for N. simplex. Mycolicibacteria showed increased hydrocortisone degradation at 35 °C compared to 30 °C. The presence of endogenous steroid catabolism in Mycolicibacterium hosts does not seem to confer an advantage for the functioning of KstD2NS. The results allow for the evaluation of the prospects for the development of simple technological methods for the selective 1(2)-dehydrogenation of 3-ketosteroids by growing bacterial cells.

De novo transcriptome assembly of Curvularia sp. VKM F-3040, a promising steroid-modifying ascomycete.

This research presents de novo transcriptome shotgun assembly for Curvularia sp. VKM F-3040, which is a putative fungal strain able to modify androstane steroids with production of 7-hydroxy and 17-hydroxylated derivatives-key intermediates in the synthesis of pharmaceutical ingredients. The data are of importance for creating novel microbial biocatalysts.

Expression of Synthetic cyp102A1-LG23 Gene and Functional Analysis of Recombinant Cytochrome P450 BM3-LG23 in the Actinobacterium Mycolicibacterium smegmatis.

Cytochrome CYP102A1 (P450 BM3) of Priestia megaterium (bas. Bacillus megaterium) has several unique functional features and thus provides an ideal object for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme part was obtained that hydroxylates several androstanes at C7ß with the formation of products with the anti-inflammatory and neuroprotective activities. In this study, synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant was expressed as a component of either monocistronic operon or bicistronic operon containing the gdh (glucose dehydrogenase, GDH) or zwf2 (glucose 6-phosphate dehydrogenase, G6PD) gene in Mycolicibacterium smegmatis BD cells. The recombinant bacteria were able hydroxylate androst-4-ene-3,17-dione (AD) into 7ß-OH-AD. Their biocatalytic activity was increased twice by increasing the solubility of CYP102A1-LG23 protein in the cells and supplementing the cells with the additional cofactor regeneration system by introducing GDH and G6PD. The maximum 7ß-OH-AD yield (37.68 mol%) was achieved by co-expression of cyp102A1-LG23 and gdh genes in M. smegmatis. These results demonstrate the possibility of using synthetic genes to obtain recombinant enzymes and expand our understanding of the processes involved in steroid hydroxylation by bacterial cytochromes. The data obtained can be used to develop new approaches for microbiological production of 7ß-hydroxylated steroids in genetically modified Mycolicibacterium species.

Current Trends and Perspectives in Microbial Bioconversions of Steroids.

The microbiological transformation of sterols is currently the technological basis for the industrial production of valuable steroid precursors, the so-called synthons, from which a wide range of steroid and indane isoprenoids are obtained by combined chemical and enzymatic routes. These compounds include value-added corticoids, neurosteroids, sex hormones, bile acids, and other terpenoid lipids required by the medicine, pharmaceutical, food, veterinary, and agricultural industries.Progress in understanding the molecular mechanisms of microbial degradation of steroids, and the development and implementation of genetic technologies, opened a new era in steroid biotechnology. Metabolic engineering of microbial producers makes it possible not only to improve the biocatalytic properties of industrial strains by enhancing their target activity and/or suppressing undesirable activities in order to avoid the formation of by-products or degradation of the steroid core, but also to redirect metabolic fluxes in cells towards accumulation of new metabolites that may be useful for practical applications. Along with whole-cell catalysis, the interest of researchers is growing in enzymatic methods that make it possible to carry out selective structural modifications of steroids, such as the introduction of double bonds, the oxidation of steroidal alcohols, or the reduction of steroid carbonyl groups. A promising area of research is strain engineering based on the heterologous expression of foreign steroidogenesis systems (bacterial, fungal, or mammalian) that ensure selective formation of demanded hydroxylated steroids.Here, current trends and progress in microbial steroid biotechnology over the past few years are briefly reviewed, with a particular focus on the application of metabolic engineering and synthetic biology techniques to improve existing and create new whole-cell microbial biocatalysts.

Cholesterol Assay Based on Recombinant Cholesterol Oxidase, ABTS, and Horseradish Peroxidase.

Cholesterol determination by cholesterol oxidase reaction is a fast, convenient, and highly specific approach with widespread use in clinical diagnostics. Routinely, endpoint measurements with 4-aminophenazone or 4-aminoantipyrine as chromogens and sodium cholate, surfactants, or alcohols as solubilizing agents are used. Here we describe a novel kinetic method to determine cholesterol in 0.05-0.75 mM range in neutral or acidic buffers by use of recombinant cholesterol oxidase from Nocardioides simplex in a coupled reaction with horseradish peroxidase, ABTS as a chromogen, and methyl-ß-cyclodextrin as a solubilizing agent.

Selective Microbial Conversion of DHEA into 7α-OH-DHEA.

7α-Hydroxy dehydroepiandrosterone (7α-OH-prasterone, 7α-OH-DHEA) is a key steroid intermediate in the synthesis of valuable pharmaceuticals widely used in the treatment of autoimmune illness, rheumatoid arthritis, colitis, and other severe diseases. The steroid can be produced using a filamentous fungus, which is capable of regio- and stereospecific hydroxylation of the steroid 3ß-alcohol (DHEA) in the allylic position C7. Here, we describe a method for highly selective microbial production of 7α-OH-DHEA from DHEA using the zygomycete Backusella lamprospora VKM F-944. The method ensures high yield of 7α-OH-DHEA (up to 89%, mol/mol) even at high concentration of the substrate DHEA (15 g/L).

Obtaining of 24-Norchol-4-ene-3,22-dione from Phytosterol with Mutants of Mycolicibacterium neoaurum.

Engineered mutants of Mycolicibacterium spp. are known producers of valuable steroid synthons with C19 or C22 skeleton. Here we describe a method for site-directed mutagenesis of Mycolicibacterium neoaurum strains, bioconversion from phytosterol, and selective purification of C23 steroid 24-norchol-4-ene-3,22-dione (24-NCED) and C22 steroid 20-hydroxymethylpregn-4-ene-3-one (20-HMP). The yields of crystalline products with 95% purity by the method here described are 2.74 ± 0.085 g for 24-NCED and 1.42 ± 0.085 g for 20-HMP from 10 g/L phytosterol. 20-HMP is recognized as the key precursor in chemical syntheses of pharmaceutical corticosteroids and 24-NCED is a promising synthon for the synthesis of valuable steroids and own potent biological activity.

Insight into Different Stages of Steroid Degradation in Thermophilic Saccharopolyspora hirsuta VKM Ac-666T Strain.

Steroids are abundant molecules in nature, and various microorganisms evolved to utilize steroids. Thermophilic actinobacteria play an important role in such processes. However, very few thermophiles have so far been reported capable of degrading or modifying natural sterols. Recently, genes putatively involved in the sterol catabolic pathway have been revealed in the moderately thermophilic actinobacterium Saccharopolyspora hirsuta VKM Ac-666T, but peculiarities of strain activity toward sterols are still poorly understood. S. hirsuta catalyzed cholesterol bioconversion at a rate significantly inferior to that observed for mesophilic actinobacteria (mycobacteria and rhodococci). Several genes related to different stages of steroid catabolism increased their expression in response to cholesterol as was shown by transcriptomic studies and verified by RT-qPCR. Sequential activation of genes related to the initial step of cholesterol side chain oxidation (cyp125) and later steps of steroid core degradation (kstD3, kshA, ipdF, and fadE30) was demonstrated for the first time. The activation correlates with a low cholesterol conversion rate and intermediate accumulation by the strain. The transcriptomic analyses revealed that the genes involved in sterol catabolism are linked functionally, but not transcriptionally. The results contribute to the knowledge on steroid catabolism in thermophilic actinobacteria and could be used at the engineering of microbial catalysts.

Draft Genome Sequence of Mycolicibacterium smegmatis VKM Ac-1171 Contains Full Set of Sterol Catabolic Genes.

Mycolicibacterium smegmatis VKM Ac-1171 is a saprotrophic bacterium that was isolated several decades ago and is deposited in microbial collections around the world. We report here a draft genome sequence of the strain. Annotation of the genome revealed the presence of a complete set of genes related to the sterol catabolic pathway.

Recombinant Extracellular Cholesterol Oxidase from Nocardioides simplex.

Cholesterol oxidase is a highly demanded enzyme used in medicine, pharmacy, agriculture, chemistry, and biotechnology. It catalyzes oxidation of 3ß-hydroxy-5-ene- to 3-keto-4-ene- steroids with the formation of hydrogen peroxide. Here, we expressed 6xHis-tagged mature form of the extracellular cholesterol oxidase (ChO) from the actinobacterium Nocardioides simplex VKM Ac-2033D (55.6 kDa) in Escherichia coli cells. The recombinant enzyme (ChONs) was purified using affinity chromatography. ChONs proved to be functional towards cholesterol, cholestanol, phytosterol, pregnenolone, and dehydroepiandrosterone. Its activity depended on the structure and length of the aliphatic side chain at C17 atom of the steroid nucleus and was lower with pregnenolone and dehydroepiandrosterone. The enzyme was active in a pH range of 5.25÷6.5 with the pH optimum at 6.0. Kinetic assays and storage stability tests demonstrated that the characteristics of ChONs were generally comparable with or superior to those of commercial ChO from Streptomyces hygroscopicus (ChOSh). The results contribute to the knowledge on microbial ChOs and evidence that ChO from N. simplex VKM Ac-2033D is a promising agent for further applications.

Ursodeoxycholic acid production by Gibberella zeae mutants.

Ursodeoxycholic acid (UDCA) is a highly demanded pharmaceutical steroid widely used in medicine. An ascomycete Gibberella zeae VKM F-2600 is capable of producing UDCA by 7ß-hydroxylation of lithocholic acid (LCA). The present study is aimed at the improvement of the fungus productivity. The original procedures for the protoplast obtaining followed by UV mutagenesis and screening of ketoconazole-resistant mutant clones have been applied. The highest yield of G. zeae protoplasts was obtained when using the mycelium in the active growth phase, ammonium chloride as an osmotic stabilizer and treatment of the fungal cells by the lytic enzymes cocktail from Trichoderma hurzanium. The conditions for effective protoplast regeneration and the UV-mutagenesis were found to provide 6-12% survival rate of the protoplasts with superior number of possible mutations. Three of 27 ketoconazole-resistant mutant clones obtained have been selected due to their increased biocatalytic activity towards LCA. The mutant G. zeae M23 produced 26% more UDCA even at relatively high LCA concentration (4 g/L) as compared with parent fungal strain, and the conversion reached 88% (w/w). The yield of UDCA reached in this study prefers those ever reported. The results contribute to the knowledge on ascomycete mutagenesis, and are of importance for biotechnological production of value added cholic acids.

Steroid modification by filamentous fungus Drechslera sp.: Focus on 7-hydroxylase and 17ß-hydroxysteroid dehydrogenase activities.

Fungal strain Drechslera sp. Ph F-34 was shown to modify 3-oxo- and 3-hydroxy steroids of androstane series to form the corresponding allylic 7-alcohols and 17ß-reduced derivatives thus evidencing the presence of 7α-, 7ß-hydroxylase and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) activities. The growing mycelium predominantly hydroxylated androsta-1,4-diene-3,17-dione (ADD) at the 7ß-position, while much lower 7α-hydroxylation was observed. Along with 7ß-hydroxy-ADD and its corresponding 7α-isomer, their respective 17ß-alcohols were produced. In this study, transformation of ADD, androst-4-en-17ß-ol-3-one (testosterone, TS) and 3ß-hydroxyandrost-5-en-17-one (dehydroepiandrosterone, DHEA) by resting mycelium of Drechslera sp. have been estimated in different conditions with regard to the inducibility and functionality of the 17ß-HSD and 7-hydroxylase enzyme systems. Steroids of androstane, pregnane and cholane series were evaluated as inducers. The inhibitory analysis was provided using cycloheximide (CHX). Steroids were assayed using TLC and HPLC methods, and the structures were confirmed by mass-spectrometry, 1H and 13C NMR spectroscopy data. 17ß-HSD of the mycelium constitutively reduced 17-carbonyl group of ADD and DHEA to form the corresponding 17ß-alcohols, namely, androsta-1,4-diene-17ß-ol-3-one (1-dehydro-TS), and androst-5-ene-3ß,17ß-diol. Production of the 7α- and 7ß-hydroxylated derivatives depended on the induction conditions. The inducer effect relied on the steroid structure and decreased in the order: DHEA > pregnenolone > lithocholic acid. ß-Sitosterol did not induce hydroxylase activity in Drechslera sp. CHX fully inhibited the synthesis of 7-hydroxylase in Drechslera mycelium thus providing selective 17-keto reduction. Results contribute to the diversity of steroid modifying enzymes in fungi and can be used at the development of novel biocatalysts for production of valuable steroid 7(α/ß)- and 17ß-alcohols.

Hydroxylation of pregnenolone and dehydroepiandrosterone by zygomycete Backusella lamprospora VKM F-944: selective production of 7α-OH-DHEA.

In this paper, we studied the transformation of two 3ß-hydroxy-5-ene-steroids-pregnenolone and dehydroepiandrosterone (DHEA) by Backusella lamprospora VKM F- 944. The soil-dwelling zygomycete wild-type strain has been earlier selected during the screening and previously unexplored for this purpose. The fungus fully converted pregnenolone to form a mixture of axial 7α-hydroxy-pregnenolone and 7α,11α-dihydroxy-pregnenolone, while no metabolites with ß-orientation of the hydroxyl group were detected. The pathway to 7α,11α-diOH-pregnenolone seems to include 7α-hydroxylation of 11α-hydroxylated derivative. The only product from DHEA was identified as 7α-hydroxy-DHEA. The structures of steroid metabolites were confirmed by HPLC, mass-spectrometry (MS), and 1H and 13C NMR analyses. Under the optimized conditions, the yield of 7α-OH-DHEA reached 94% (w/w) or over 14 g/L in absolute terms, even at high concentration of the substrate (DHEA) (15 g/L). To our knowledge, it is the highest yield of the value-added 7α-OH-DHEA reported so far. The results contribute to the knowledge of the diversity of the wild-type fungal strains capable of effective steroid hydroxylation. They could be applied for the production of allylic steroid 7α-alcohols that are widely used in medicine. KEY POINTS: • Zygomycete Backusella lamprospora actively hydroxylates 3ß-hydroxy-5-en-steroids. • Axial 7α-hydroxylation is the preferable reaction by the strain towards pregnenolone and DHEA. • The strain selectively produces 7α-OH-DHEA even at high substrate concentrations (up to 15 g/L).

Bioproduction of testosterone from phytosterol by Mycolicibacterium neoaurum strains: "one-pot", two modes.

The main male hormone, testosterone is obtained from cheap and readily available phytosterol using the strains of Mycolicibacterium neoaurum VKM Ac-1815D, or Ac-1816D. During the first "oxidative" stage, phytosterol (5-10 g/L) was aerobically converted by Ac-1815D, or Ac-1816D to form 17-ketoandrostanes: androstenedione, or androstadienedione, respectively. At the same bioreactor, the 17-ketoandrostanes were further transformed to testosterone due to the presence of 17ß-hydroxysteroid dehydrogenase activity in the strains ("reductive" mode). The conditions favorable for "oxidative" and "reductive" stages have been revealed to increase the final testosterone yield. Glucose supplement and microaerophilic conditions during the "reductive" mode ensured increased testosterone production by mycolicibacteria cells. Both strains effectively produced testosterone from phytosterol, but highest ever reported testosterone yield was achieved using M. neoaurum VKM Ac-1815D: 4.59 g/l testosterone was reached from 10 g/l phytosterol thus corresponding to the molar yield of over 66%. The results contribute to the knowledge on phytosterol bioconversion by mycolicibacteria, and are of significance for one-pot testosterone bioproduction from phytosterol bypassing the intermediate isolation of the 17-ketoandrostanes.

Steroid Metabolism in Thermophilic Actinobacterium Saccharopolyspora hirsuta VKM Ac-666T.

The application of thermophilic microorganisms opens new prospects in steroid biotechnology, but little is known to date on steroid catabolism by thermophilic strains. The thermophilic strain Saccharopolyspora hirsuta VKM Ac-666T has been shown to convert various steroids and to fully degrade cholesterol. Cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3ß-hydroxy-cholest-5-en-26-oic acid were identified as intermediates in cholesterol oxidation. The structures were confirmed by 1H and 13C-NMR analyses. Aliphatic side chain hydroxylation at C26 and the A-ring modification at C3, which are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively, occur simultaneously in the strain and are followed by cascade reactions of aliphatic sidechain degradation and steroid core destruction via the known 9(10)-seco-pathway. The genes putatively related to the sterol and bile acid degradation pathways form three major clusters in the S. hirsuta genome. The sets of the genes include the orthologs of those involved in steroid catabolism in Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 and related actinobacteria. Bioinformatics analysis of 52 publicly available genomes of thermophilic bacteria revealed only seven candidate strains that possess the key genes related to the 9(10)-seco pathway of steroid degradation, thus demonstrating that the ability to degrade steroids is not widespread among thermophilic bacteria.

Different genome-wide transcriptome responses of Nocardioides simplex VKM Ac-2033D to phytosterol and cortisone 21-acetate.

BACKGROUND: Bacterial degradation/transformation of steroids is widely investigated to create biotechnologically relevant strains for industrial application. The strain of Nocardioides simplex VKM Ac-2033D is well known mainly for its superior 3-ketosteroid Δ1-dehydrogenase activity towards various 3-oxosteroids and other important reactions of sterol degradation. However, its biocatalytic capacities and the molecular fundamentals of its activity towards natural sterols and synthetic steroids were not fully understood. In this study, a comparative investigation of the genome-wide transcriptome profiling of the N. simplex VKM Ac-2033D grown on phytosterol, or in the presence of cortisone 21-acetate was performed with RNA-seq. RESULTS: Although the gene patterns induced by phytosterol generally resemble the gene sets involved in phytosterol degradation pathways in mycolic acid rich actinobacteria such as Mycolicibacterium, Mycobacterium and Rhodococcus species, the differences in gene organization and previously unreported genes with high expression level were revealed. Transcription of the genes related to KstR- and KstR2-regulons was mainly enhanced in response to phytosterol, and the role in steroid catabolism is predicted for some dozens of the genes in N. simplex. New transcription factors binding motifs and new candidate transcription regulators of steroid catabolism were predicted in N. simplex. Unlike phytosterol, cortisone 21-acetate does not provide induction of the genes with predicted KstR and KstR2 sites. Superior 3-ketosteroid-Δ1-dehydrogenase activity of N. simplex VKM Ac-2033D is due to the kstDs redundancy in the genome, with the highest expression level of the gene KR76_27125 orthologous to kstD2, in response to cortisone 21-acetate. The substrate spectrum of N. simplex 3-ketosteroid-Δ1-dehydrogenase was expanded in this study with progesterone and its 17α-hydroxylated and 11α,17α-dihydroxylated derivatives, that effectively were 1(2)-dehydrogenated in vivo by the whole cells of the N. simplex VKM Ac-2033D. CONCLUSION: The results contribute to the knowledge of biocatalytic features and diversity of steroid modification capabilities of actinobacteria, defining targets for further bioengineering manipulations with the purpose of expansion of their biotechnological applications.

Microbial Steroid Production Technologies: Current Trends and Prospects.

This Special Issue aims to collect articles and reviews on new methodologies, research, and achievements in the field of steroid microbial biotechnologies [...].