17β-HYDROXYSTEROID DEHYDROGENASE FROM THE FUNGUS Cochliobolus lunatus: BIOSYNTHESIS IN Mycolicibacterium neoaurum ACTINOBACTERIAL CELLS AND FUNCTIONAL CHARACTERISTICS

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Abstract

17β-Hydroxysteroid dehydrogenase (17β-HSD) is an enzyme used in biotechnology for obtaining testosterone from phytosterol. Heterologous 17β-HSD from the fungus Cochliobolus lunatus catalyzes the NADPH-dependent reduction of the 17-oxo group of androstenedione/androstadienedione, formed in the cells of mycolicibacteria as a result of their inherent polyenzyme process of oxidation of the aliphatic side chain of phytosterol, with the formation of testosterone/Δ1-dehydrotestosterone, respectively. The object of the study was heterologous 17β-HSD from the fungus C. lunatus (17β-HSDCl) with a 6xHis tag (6×His-17β-HSDCl), synthesized in the cells of actinobacteria Mycolicibacterium neoaurum. Isolation and purification of the recombinant enzyme was carried out using affinity chromatography. The enzyme preparation 6×His-17β-HSDCl exhibited the highest activity towards androstenedione. The activity of 6×His-17β-HSDCl depended on NADPH and was manifested 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 6×His-17β-HSDCl enzyme synthesized in M. neoaurum cells are comparable to those obtained for the 17β-HSD enzyme isolated from the fungus C. lunatus, as well as for the recombinant 17β-HSDCl enzymes synthesized in Escherichia coli and Mycolicibacterium smegmatis cells. The results expand our knowledge of microbial 17β-HSDs and indicate the potential for using recombinant M. neoaurum strains expressing a codon-optimized cDNA sequence encoding 17β-HSDCl from the fungus C. lunatus for producing testosterone from phytosterol.

About the authors

T. A Timakova

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center "Pushchino Center for Biological Research of the Russian Academy of Sciences"

Pushchino, Russia

M. V Karpov

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center "Pushchino Center for Biological Research of the Russian Academy of Sciences"

Pushchino, Russia

V. M Nikolaeva

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center "Pushchino Center for Biological Research of the Russian Academy of Sciences"

Pushchino, Russia

D. N Tekucheva

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center "Pushchino Center for Biological Research of the Russian Academy of Sciences"

Pushchino, Russia

A. A Shutov

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center "Pushchino Center for Biological Research of the Russian Academy of Sciences"

Pushchino, Russia

V. V Fokina

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center "Pushchino Center for Biological Research of the Russian Academy of Sciences"

Email: 2vvfokina@gmail.com
Pushchino, Russia

M. V Donova

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences; Federal Research Center "Pushchino Center for Biological Research of the Russian Academy of Sciences"

Pushchino, Russia

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