Current Trends and Perspectives in Microbial Bioconversions of Steroids

Donova, Marina V.

doi:10.1007/978-1-0716-3385-4_1

Methods in Molecular Biology

2023

DOI: 10.1007/978-1-0716-3385-4_1

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Current Trends and Perspectives in Microbial Bioconversions of Steroids

Marina V. Donova

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2024

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Cited by 3 publications

References 66 publications

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A Novel A105Y Mutant of CYP17A1 Exhibits Almost Perfect Regioselectivity in the Production of 17α-Hydroxyprogesterone

Du,

Tian,

et al. 2024

J. Agric. Food Chem.

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17α-Hydroxyprogesterone (17α-OHP) is a steroid hormone with significant biological activity that can be obtained by catalyzing progesterone (PROG), the main product of sitosterol, through CYP17A1. However, increasing the catalytic specificity of HCYP17A1 for C17 hydroxylation of progesterone (PROG) poses a formidable challenge due to the close proximity of the C16 and C17 positions. In this study, a rational design was utilized to alter the spatial configuration of the substrate channel, leading to the complete abolition of its 16-hydroxylation activity. Subsequent molecular dynamics simulations revealed that the A105Y mutation heightened the rigidity of the G95−I112 region of CYP17A1, consequently regulating the direction of the entry of PROG into the catalytic pocket. Moreover, the establishment of hydrogen bonding between Y105 and R239, coupled with Pi-stacking of A105Y with F114, effectively immobilizes the substrate PROG in a fixed position, explaining the practically perfect regioselectivity observed in A105Y. Finally, a multifaceted enzymatic cascade system, incorporating A105Y, cytochrome P450 reductase (CPR), and glucose-6-phosphate dehydrogenase (ZWF) for NADPH cofactor regeneration, was constructed in Pichia pastoris GS115. The resulting biocatalyst produced 106 ± 3.2 mg L −1 17α-OHP, a 4.6-fold increase compared with A105Y alone. Thus, this study provides valuable insights for improving the regioselectivity and activity of P450 enzymes.

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A Novel A105Y Mutant of CYP17A1 Exhibits Almost Perfect Regioselectivity in the Production of 17α-Hydroxyprogesterone

Du,

Tian,

et al. 2024

J. Agric. Food Chem.

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

Tekucheva,

Karpov,

Fokina

et al. 2024

Microbiology

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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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Identification of Electron Transfer in the System of Ferredoxins and Ferredoxin Reductases from Mycolicibacterium smegmatis

Epiktetov,

Karpov,

Donova

2024

Microbiology

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Steroid-26-monooxygenases belong to the cytochrome P450 superfamily and function as part of three-component systems together with ferredoxins and ferredoxin reductases providing electron transport. The P450-dependent redox partners of the actinobacterial strain Mycolicibacterium smegmatis mc2155 were investigated. The genes encoding mycolibacterial ferredoxins (FdxD and FdxE) and ferredoxin reductases (FdrA and FprA) were overexpressed in E. coli cells. A scheme for isolation and purification of synthesized recombinant proteins using affinity chromatography was developed, resulting in electrophoretically homogeneous preparations. Spectral analysis of ferredoxin reductase showed absorption peaks characteristic of FAD-containing proteins. The reaction of cytochrome c reduction using recombinant proteins was carried out, demonstrating that FdxD, FdxE, FdrA, and FprA can act as components of electron transport from the reducing equivalents of NAD(P)H.

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Current Trends and Perspectives in Microbial Bioconversions of Steroids

Cited by 3 publications

References 66 publications

A Novel A105Y Mutant of CYP17A1 Exhibits Almost Perfect Regioselectivity in the Production of 17α-Hydroxyprogesterone

A Novel A105Y Mutant of CYP17A1 Exhibits Almost Perfect Regioselectivity in the Production of 17α-Hydroxyprogesterone

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

Identification of Electron Transfer in the System of Ferredoxins and Ferredoxin Reductases from Mycolicibacterium smegmatis

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