17β-Hydroxysteroid Dehydrogenase from the Fungus Cochliobolus lunatus: Biosynthesis in Mycolicibacterium neoaurum Actinobacterial Cells and Functional Characterization

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Tatiana A. Timakova¹, Mikhail V. Karpov¹, Vera M. Nikolaeva¹, Daria N. Tekucheva¹, Andrei A. Shutov¹, Victoria V. Fokina^1,a*, and Marina V. Donova¹

¹G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”, 142290 Pushchino, Moscow Region, Russia

^* To whom correspondence should be addressed.

Received: July 7, 2025; Revised: September 1, 2025; Accepted: September 16, 2025

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17β-Hydroxysteroid dehydrogenase (17β-HSD) is an enzyme used in biotechnology for producing testosterone from phytosterol. Heterologous 17β-HSD from the fungus Cochliobolus lunatus catalyzes NADPH-dependent reduction of the 17-oxo group of androstenedione/androstadienedione formed in mycolicibacterial cells as a result of the inherent polyenzymatic process of side chain oxidation of phytosterols, yielding testosterone/Δ¹-dehydrotestosterone, respectively. The object of this study was heterologous 17β-HSD from the fungus C. lunatus (17β-HSD_Cl) with a 6×His tag (6×His-17β-HSD_Cl), synthesized in the cells of actinobacteria Mycolicibacterium neoaurum. Isolation and purification of the recombinant enzyme were performed using affinity chromatography. The 6×His-17β-HSD_Cl enzyme preparation exhibited the highest activity toward androstenedione. Activity of the 6×His-17β-HSD_Cl depended on NADPH and was observed in the pH range from 6.0 to 9.0 with an optimum at pH 7.0. Analysis of kinetic characteristics showed that the properties of the heterologous enzyme 6×His-17β-HSD_Cl synthesized in M. neoaurum cells are comparable with those reported for the 17β-HSD enzyme isolated from the fungus C. lunatus, as well as for the recombinant 17β-HSD_Cl enzymes synthesized in Escherichia coli and Mycolicibacterium smegmatis cells. The results expand our knowledge on microbial 17β-HSDs and suggest potential for the use of the recombinant M. neoaurum strains expressing a codon-optimized cDNA sequence encoding 17β-HSD_Cl from the fungus C. lunatus for producing testosterone from phytosterol.

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KEY WORDS: phytosterol, testosterone, 17β-hydroxysteroid dehydrogenase, actinobacteria, recombinant enzyme, biotechnology

DOI: 10.1134/S0006297925601959

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