Expression of the Curvularia sp. P450 Monooxygenase Gene in Escherichia coli and Confirmation of Its 7-Hydroxylation Function

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Resumo

The diversity and uniqueness of fungal cytochromes P450 (CYP), capable of catalyzing the regio- and stereospecific hydroxylation of steroids, makes them important for microbiological synthesis of valuable hydroxysteroids. In the present work, the function of recombinant fungal P450 monooxygenase (CYPI) of Curvularia sp. strain VKM F-3040, a promising biocatalyst of 7-hydroxylation of androstane steroids, was studied. RT-PCR amplification of cDNA of the candidate CYPI gene and of the gene of its natural redox partner (POR), their cloning and heterologous expression in the cells of E. coli BL 21 DE(3) was carried out. In vitro experiments showed the ability of the obtained recombinant monooxygenase to catalyze hydroxylation of dehydroepiandrosterone at positions 7α and 7β. Our results expand the knowledge about fungal steroid hydroxylases and open up the prospects for the synthesis of valuable 7-hydroxysteroids by using recombinant producers.

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Sobre autores

V. Kollerov

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

Autor responsável pela correspondência
Email: svkollerov@rambler.ru
Rússia, Pushchino, 142290

S. Tarlachkov

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

Email: svkollerov@rambler.ru
Rússia, Pushchino, 142290

A. Shutov

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

Email: svkollerov@rambler.ru
Rússia, Pushchino, 142290

M. Donova

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

Email: svkollerov@rambler.ru
Rússia, Pushchino, 142290

Bibliografia

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2. Fig. 1. Visualization of PCR products of the CYPI(a) and POR(b) genes amplified using the first chain of cDNA and recombinant plasmid pET-22b(+)-CYPI(c) and pET-22b(+)-POR(d) as a matrix after treatment with NdeI restriction endonucleases/NotI and EcoRI/NotI, respectively; gel electrophoresis on 0.8% agarose (120 V; 40 min) with ethidium bromide staining; M — DNA marker (~0.5 mcg).

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3. Fig. 2. TLC profile of samples of the transformation liquid of the DHEA bioconversion (0.1 g/l) with sediment R20 000 (variants 1, 3) and supernatant S20 000 (variants 2, 4) obtained from the homogenate of destroyed recombinant E. coli BL 21 DE(3)-pET-22b(+) cells bearing an empty vector (variants 1, 2) and a homogenate of destroyed recombinant E. coli BL 21 DE(3)-pET-22b(+)-CYPI and E. coli BL 21 DE(3)-pET-22b(+)-POR cells carrying the target genes CYPI and POR, respectively (variants 3 4) (72 hours of transformation); C is a mixture of standard steroids at the point (from top to bottom): DHEA (2 mcg), androstenediol (3 mcg), 7β-OH-DHEA (2 mcg), 7α-OH-DHEA (2 mcg) (a); Comparative HPLC analysis of compounds I and II with control samples 7β-OH-DHEA (b) and 7α-OH-DHEA (c).

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