Single-Stage Bioconversion of Phytosterol into Testosterone by Recombinant Strains of Mycolicibacterium neoaurum

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Abstract

A plasmid containing the genes of a fungal 17β-hydroxysteroid dehydrogenase, which catalyzes the reduction of the steroid core at the C17 position, and mycobacterial glucose-6-phosphate dehydrogenase, which promotes the recycling of the essential coenzyme NAD(P)H, was constructed. Its constitutive expression in well-studied Mycolicibacterium neoaurum strains made it possible to increase significantly the yield of C-17 hydroxysteroids. In particular, recombinant strains created on the basis of M. neoaurum VKM Ac-1815D and M. neoaurum NRRL B-3805 ΔkstD exhibited predominant accumulation of testosterone, while the strain based on M. neoaurum VKM Ac-1816D accumulated dehydrotestosterone and testosterone simultaneously.

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About the authors

D. N. Tekucheva

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences

Author for correspondence.
Email: tekuchevadn@gmail.com
Russian Federation, Pushchino, 142290

M. V. Karpov

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences

Email: tekuchevadn@gmail.com
Russian Federation, Pushchino, 142290

V. V. Fokina

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences

Email: tekuchevadn@gmail.com
Russian Federation, Pushchino, 142290

T. I. Timakova

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences

Email: tekuchevadn@gmail.com
Russian Federation, Pushchino, 142290

A. A. Shutov

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences

Email: tekuchevadn@gmail.com
Russian Federation, Pushchino, 142290

M. V. Donova

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences

Email: tekuchevadn@gmail.com
Russian Federation, Pushchino, 142290

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2. Fig. 1. Transformation of 5 g/l of phytosterol by the parent strains of M. neoaurum VKM Ac-1816D (1b), VKM Ac-1815D (2b), NRRL B-3805 ΔkstD (3b) and recombinant strains carrying a bicistronic genetic construct under the regulation of a constitutive promoter created on their basis (1a, 2a, 3a, respectively). The vertical dotted line indicates the moment of transition from the oxidative to the reducing mode. 1 — T; 2 — AD; 3 — ADD; 4 — dT.

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3. Figure S1. Structure of the recombinant plasmid pMVN 2٥. Synthetic genes encoding ١٧β-HSD from Cochliobolus lunatus (17beta-HSDCl) and bacterial type 2 G6PDH from Mycobacterium tuberculosis H37Rv (G6PDMt2) were cloned in the pMV261N vector under the control of the constitutive promoter Phsp60. The plasmid contains a kanamycin resistance cassette (KanR) and replication initiation points in E.coli (oriE) and mycobacteria (oriM) cells.

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4. Fig. S 2. Growth dynamics of the parent strains of M. neoaurum HCM Ac-١٨١٥D (١), Ac-١٨١٦D (2), NRRL B-٣٨٠٥ ΔkstD (3) and recombinant strains created on their basis by transformation by plasmid pMVN25 (N25)

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