Microbiological synthesis of stereoisomeric 7(α/ß)-hydroxytestololactones and 7(α/ß)-hydroxytestolactones.

Microbiological synthesis of 7α- and 7ß-hydroxy derivatives of testololactone and testolactone was developed based on bioconversion of dehydroepiandrosterone (DHEA) by fungus of Isaria fumosorosea VKM F-881 with subsequent modification of the obtained stereoisomers by actinobacteria. The first stage included obtaining of the stereoisomers of 3ß,7(α/ß)-dihydroxy-17a-oxa-D-homo-androst-5-en-17-ones in the preparative amounts. Then the conversion of 7-hydroxylated D-lactones obtained by selected actinobacteria of Nocardioides simplex VKM Ac-2033D, Saccharopolyspora hirsuta VKM Ac-666, and Streptomyces parvulus MTOC Ac-21v was studied. Under the transformation of 3ß,7α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one and its corresponding 7ß-stereoisomer by N. simplex VKM Ac-2033D and S. hirsuta VKM Ac-666 the 7α- and 7ß-hydroxy-17a-oxa-D-homo-androst-4-ene-3,17-dione (7α- and 7ß-hydroxytestololactone), 7α- and 7ß-hydroxy-17a-oxa-D-homo-androsta-1,4-diene-3,17-dione (7α- and 7ß-hydroxytestolactone) were obtained with molar yields in a range of 60.3-90.9 mol%. The crystalline products of 7α-hydroxytestololactone, 7α-hydroxytestolactone, and their corresponding 7ß-hydroxy stereoisomers were isolated, and their structures were confirmed by mass spectrometry and 1H-NMR spectroscopy analyses. The strain of Str. parvulus MTOC Ac-21v transformed 3ß,7(α/ß)-dihydroxy-17a-oxa-D-homo-androst-5-en-17-ones into the corresponding 3-keto-4-ene analogs and did not show 3-ketosteroid 1(2)-dehydrogenase activity. The activity of actinobacteria towards steroid D-lactones was hitherto unreported.The results contribute to the knowledge of metabolic versatility of actinobacteria capable of transforming steroid substrates and may be applied in the synthesis of potential aromatase inhibitors.

[Formation of hydroxylated steroid lactones from Dehydroepiandrosterone by Spicaria fumoso-rosea F-881].

The transformation of dehydroepiandrosterone by Spicaria fumoso-rosea VKM F-881 produced 7alpha- and 7beta-hydroxy-dehydroepiandrosterone, 3beta,7alpha-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one, and 3beta,7beta-dihydroxy- 17a-oxa-D-homo-androst-5-en-17-one. The yield of the main product-3beta,7beta-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one-was 49.5-72 mol % at substrate loadings of 5-20 g/L. Lactone formation proceeded through 7alpha- and 7beta-hydroxy derivatives of dehydroepiandrosterone. The structure of the products was determined by mass spectrometry, 1H-NMR spectroscopy, and 13C-NMR spectroscopy. The proposed microbiological method for producing steroid lactones opens prospects for the syn- thesis of novel steroid compounds.

[Dihydroxylation of dehydroepiandrosterone in positions 7alpha and 15alpha by mycelial fungi].

The ability of 485 fungal strains is studied for catalysis of the process of 7alpha, 15alpha-dihydroxylation of dehydroepiandrosterone (DHEA, 3alpha-hydroxy-5-androstene-17-one), a key intermediate of the synthesis of physiologically active compounds. The ability for the formation of 3alpha, 7alpha, 15alpha-trihydoxy-5-androstene-17-one (7alpha, 15alpha-di-OH-DHEA) was found for the first time for representatives of 12 genera, eight families, and six orders of ascomycetes, eight genera, four families, and one order of zygomycetes, one genus, one family, and one order of basidiomycetes, and four genera of mitosporous fungi. The most active strains are found among genera Acremonium, Gibberella, Fusarium, and Nigrospora. In the process of transformation of DHEA (2 g/l) by strains of Fusarium oxysporum RKM F-1600 and FGibberella zeae BKM F-2600, the molar yield was 63 and 68%, respectively. Application of the revealed active strains of microorganisms opens prospects for the efficient production of key intermediates of synthesis of modern medical preparations.

Deoxycholic acid transformations catalyzed by selected filamentous fungi.

More than 100 filamentous fungi strains, mostly ascomycetes and zygomycetes from different phyla, were screened for the ability to convert deoxycholic acid (DCA) to valuable bile acid derivatives. Along with 11 molds which fully degraded DCA, several strains were revealed capable of producing cholic acid, ursocholic acid, 12-keto-lithocholic acid (12-keto-LCA), 3-keto-DCA, 15ß-hydroxy-DCA and 15ß-hydroxy-12-oxo-LCA as major products from DCA. The last metabolite was found to be a new compound. The ability to catalyze the introduction of a hydroxyl group at the 7(α/ß)-positions of the DCA molecule was shown for 32 strains with the highest 7ß-hydroxylase activity level for Fusarium merismoides VKM F-2310. Curvularia lunata VKM F-644 exhibited 12α-hydroxysteroid dehydrogenase activity and formed 12-keto-LCA from DCA. Acremonium rutilum VKM F-2853 and Neurospora crassa VKM F-875 produced 15ß-hydroxy-DCA and 15ß-hydroxy-12-oxo-LCA, respectively, as major products from DCA, as confirmed by MS and NMR analyses. For most of the positive strains, the described DCA-transforming activity was unreported to date. The presented results expand the knowledge on bile acid metabolism by filamentous fungi, and might be suitable for preparative-scale exploitation aimed at the production of marketed bile acids.

Hydroxylation of lithocholic acid by selected actinobacteria and filamentous fungi.

Selected actinobacteria and filamentous fungi of different taxonomy were screened for the ability to carry out regio- and stereospecific hydroxylation of lithocholic acid (LCA) at position 7ß. The production of ursodeoxycholic acid (UDCA) was for the first time shown for the fungal strains of Bipolaris, Gibberella, Cunninghamella and Curvularia, as well as for isolated actinobacterial strains of Pseudonocardia, Saccharothrix, Amycolatopsis, Lentzea, Saccharopolyspora and Nocardia genera. Along with UDCA, chenodeoxycholic (CDCA), deoxycholic (DCA), cholic (CA), 7-ketodeoxycholic and 3-ketodeoxycholic acids were detected amongst the metabolites by some strains. A strain of Gibberella zeae VKM F-2600 expressed high level of 7ß-hydroxylating activity towards LCA. Under optimized conditions, the yield of UDCA reached 90% at 1g/L of LCA and up to 60% at a 8-fold increased substrate loading. The accumulation of the major by-product, 3-keto UDCA, was limited by using selected biotransformation media.