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Abstract <p>17β-Hydroxysteroid dehydrogenase (17β-HSD) is an enzyme used in biotechnology for producing testosterone from phytosterol. Heterologous 17β-HSD from the fungus <i>Cochliobolus lunatus</i> 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/Δ<sup>1</sup>-dehydrotestosterone, respectively. The object of this study was heterologous 17β-HSD from the fungus <i>C.&#xa0;lunatus</i> (17β-HSD<sub>Cl</sub>) with a 6×His tag (6×His-17β-HSD<sub>Cl</sub>), synthesized in the cells of actinobacteria <i>Mycolicibacterium neoaurum</i>. Isolation and purification of the recombinant enzyme were performed using affinity chromatography. The 6×His-17β-HSD<sub>Cl</sub> enzyme preparation exhibited the highest activity toward androstenedione. Activity of the 6×His-17β-HSD<sub>Cl</sub> depended on NADPH and was observed in the pH range from 6.0 to 9.0 with an optimum at pH&#xa0;7.0. Analysis of kinetic characteristics showed that the properties of the heterologous enzyme 6×His-17β-HSD<sub>Cl</sub> synthesized in <i>M.&#xa0;neoaurum</i> cells are comparable with those reported for the 17β-HSD enzyme isolated from the fungus <i>C.&#xa0;lunatus</i>, as well as for the recombinant 17β-HSD<sub>Cl</sub> enzymes synthesized in <i>Escherichia coli</i> and <i>Mycolicibacterium smegmatis</i> cells. The results expand our knowledge on microbial 17β-HSDs and suggest potential for the use of the recombinant <i>M.&#xa0;neoaurum</i> strains expressing a codon-optimized cDNA sequence encoding 17β-HSD<sub>Cl</sub> from the fungus <i>C.&#xa0;lunatus</i> for producing testosterone from phytosterol.</p>

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

  • 期刊论文

  • 发表日期：

    2025年9月1日

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  • Tatiana A. Timakova,
  • Mikhail V. Karpov,
  • Vera M. Nikolaeva,
  • Daria N. Tekucheva,
  • Andrei A. Shutov,
  • Victoria V. Fokina,
  • Marina V. Donova

摘要

Abstract

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-HSDCl) with a 6×His tag (6×His-17β-HSDCl), synthesized in the cells of actinobacteria Mycolicibacterium neoaurum. Isolation and purification of the recombinant enzyme were performed using affinity chromatography. The 6×His-17β-HSDCl enzyme preparation exhibited the highest activity toward androstenedione. Activity of the 6×His-17β-HSDCl 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-HSDCl 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-HSDCl 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-HSDCl from the fungus C. lunatus for producing testosterone from phytosterol.