Regio- and stereospecificity of microbial hydroxylation was studied at the transformation of 3-keto-4-ene steroids of androstane and pregnane series by the filamentous fungus of Curvularia lunata VKM F-644. The products of the transformations were isolated by column chromatography and identified using HPLC, massspectrometry (MS) and proton nuclear magnetic resonance (¹H NMR) analyses. Androst-4-ene-3,17-dione (AD) and its 1(2)-dehydro- and 9α-hydroxylated (9-OH-AD) derivatives were hydroxylated by the fungus mainly in position 14α, while 6α-, 6β- and 7α-hydroxylated products were revealed in minor amounts. At the transformation of C₂₁-steroids (cortexolone and its acetylated derivatives) the presence of 17-acetyl group was shown to facilitate further selectivity of 11β-hydroxylation. Original procedures for protoplasts obtaining, mutagenesis and mutant strain selection have been developed. A stable mutant (M4) of C. lunata with high 11β-hydroxylase activity towards 21-acetate and 17α,21-diacetate of cortexolone was obtained. Yield of 11β-hydroxylated products reached about 90% at the transformation of 17α, 21-diacetate of cortexolone (1 g/l) using mutant strain M4.

A microbial catalysis is an effective tool for steroid structural modifications allowing generating the compounds which may be difficult to obtain by conventional synthetic methods. Selected fungal strains of Ascomycota and Zygomycota divisions (totally, 75) representing different phyla were screened for their activity toward 3β-hydroxypregn-5-ene-20-one (pregnenolone) focusing on the production of 11α-hydroxyprogesterone. Along with the ability to catalyze 7α-, 7β- and 11α-hydroxylation which was revealed for some representatives of Pleosporaceae, Trichocomaceae, Hypocreaceae, Clavicipitaceae, Microascaceae, Cunninghamellaceae, Mucoraceae and Phycomycetaceae families, micromycete strains belonging to Acremonium, Aspergillus, Curvularia, Rhizopus, and Scopulariopsis genera were selected capable of modifying 3β-ol-5-ene- to 3-keto-4-ene moiety, - this reaction being rather uncommon for fungi. The ability to catalyze double hydroxylation of pregnenolone at positions 7β and 11α by some tested micromycetes was revealed for the first time. Sustainable and high-efficient single-stage production of 11α-hydroxyprogesterone (up to 70%) from pregnenolone which was hitherto unreported was reached with the strain of Aspergillus niger VKM F-212 under the optimized conditions. The results demonstrate a highly diverse biocatalytic potential of fungal strains towards pregnenolone, reveal the existence of novel active biocatalysts that can be exploited for the synthesis of high-value hydroxysteroids and expand knowledge on structural modifications of steroid molecules in lower eukaryotes.

The hydroxylation of steroids in the C7β 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 (P450_cur) 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 P450_cur gene was increased more than 300-fold. A two-gene construct with a candidate P450_cur 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 P450_cur and CPR genes were obtained. The functional activity of the recombinant P450_cur was studied in vivo during the bioconversion of androstane steroids. The fungal 7-monooxygenase predominantly catalyzed the 7β-hydroxylation of androstadienedione (ADD), DHEA, and androstenediol, whereas 1-dehydrotestosterone was hydroxylated by P450_cur mainly at the C7-Hα position. To our knowledge, this is the first report of a recombinant yeast capable of catalyzing the 7α/β-hydroxylation of ADD and DHEA.