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.

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.

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).

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).

Biotransformation of androstenedione and androstadienedione by selected Ascomycota and Zygomycota fungal strains.

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-, 7ß-, 11α- and 14α-hydroxylated derivatives, as well as 17ß-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 7ß-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.

Obtaining of 11α-Hydroxyandrost-4-ene-3,17-dione from Natural Sterols.

Two-step one-pot microbial transformation enables obtaining of valuable steroids that are difficult to produce chemically. Here we describe a method for obtaining 11α-hydroxyandrost-4-ene-3,17-dione (11α-HAD) from cheap and available natural sterols (phytosterols or cholesterol).11α-HAD is a primary adrenal steroid in mammals and also a key precursor in the syntheses of halogenated corticoids. Conventional routes for its obtaining are based on chemical synthesis, or microbial hydroxylation of androst-4-ene-3,17-dione (AD). AD in turn is produced primarily with microbial biotransformation of natural sterols by some actinobacteria.Consequent bioconversions of sterols using two microbial strains in one bioreactor vessel without separation and purification of AD provides high yield of 11α-HAD. At the first fermentation step, phytosterol is converted to AD with Mycobacterium neoaurum NRRL 3805B, or relative strains, to yield about 70% (mol/mol). At the second step, AD is almost fully (98%) hydroxylated at the position 11α with Aspergillus ochraceus VKM F-830, or other suitable organisms, in the same bioreactor. At the average, 30% (w/w) of the high-purity crystalline 11α-HAD can be obtained.The method can be exploited for production of 11α-HAD for practical use.