vestibИзвестия Национальной академии наук Беларуси. Серия биологических наукProceedings of the National Academy of Sciences of Belarus, Biological Series1029-89402524-230XThe Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1029-8940-2024-69-3-207-216vestib-930Research ArticleСтатьиПодавление побочных реакций ацетилирования прегненолона в трансгенных дрожжах с использованием конкурентного ингибирования и оптимальной системы «вектор – хозяин»Suppressing the side reactions of pregnenolone acetylation in transgenic yeast using competitive inhibition and optimal vector‒host systemФалетровЯ. В.FaletrovYa. V.

Фалетров Ярослав Вячеславович – канд. хим. наук, доцент, вед. науч. cотрудник.

ул. Ленинградская, 14, 220006, Минск

Yaroslav V. Faletrov – Ph. D. (Chem.), Associate Professor, Leading Researcher, Research Institute for Physicochemical Problems of Belarusian State University.

14, Leningradskaya Str., 220006, Minsk

yaroslav82@tut.byФроловаН. С.FrolovaN. S.

Фролова Нина Степановна – науч. сотрудник.

ул. Ленинградская, 14, 220006, Минск

Nina S. Frolova – Researcher, Research Institute for Physicochemical Problems of Belarusian State University.

14, Leningradskaya Str., 220006, Minsk

frolova_n_2006@bk.ruМауерсбергерШ.MauersbergerS.

Мауерсбергер Штефан – д-р наук, ст. науч. сотрудник.

Хедда Фогель, 01062, Дрезден

Stephan Mauersberger ‒ D. Sc., Senior Researcher, Institute of Microbiology, Dresden University of Technology.

Hedda Vogel, 01062, Dresden

ШкуматовВ. М.ShkumatovV. M.

Шкуматов Владимир Макарович – член-корреспондент, д-р биол. наук.

Vladimir M. Shkumatov – Corresponding Member, D. Sc. (Biol.).

Научно-исследовательский институт физико-химических проблем Белорусского государственного университетаResearch Institute for Physicochemical Problems of Belarusian State UniversityИнститут микробиологии Дрезденского технологического университетаInstitute of Microbiology, Dresden University of Technology202401082024693207216Copyright © Фалетров Я.В., Фролова Н.С., Мауерсбергер Ш., Шкуматов В.М., 20242024Фалетров Я.В., Фролова Н.С., Мауерсбергер Ш., Шкуматов В.М.Faletrov Y.V., Frolova N.S., Mauersberger S., Shkumatov V.M.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestibio.belnauka.by/jour/article/view/930

Известно, что прегненолон и продукты его превращения цитохромом Р450 17α-гидроксилаза/17,20-лиаза (P450c17) ‒ 17-гидроксипрегненолон, дегидроэпиандростерон ‒ подвергаются 3-O-ацетилированию ферментом Atf2p дрожжей Saccharomyces cerevisiae. Нами установлено, что добавление в среду для культивирования изоамилового или амилового спирта уменьшает образование 3-O-ацетилированных продуктов как неиндуцированными, так и индуцированными клетками трансгенных дрожжей S. cerevisiae GRF18/YEp5117α, экспрессирующими P450c17 под контролем промотора GAL10. Более эффективным оказалось использование микроорганизма-хозяина Yarrowia lipolytica и промотора экспрессии изоцитратлиаза ICL1. Анализ генома дрожжей Y. lipolytica с помощью программы BLAST показал отсутствие в них потенциальных аналогов Atf2p, что подтверждено отсутствием продуктов ацетилирования прегненолона. Выбор оптимального организма-хозяина является альтернативой методу получения штамма с удаленными генами.

The rational design of the vector‒host system and the conditions for its use is the key to its most effective use as a biocatalyst for obtaining valuable products or for testing potential bioregulators. In this work, we will consider examples of such solutions for some yeasts from the literature over the past 5 years and our experience in optimizing the reactions of 17α-hydroxylation of pregnanes with recombinant strains of the yeast S. cerevisiae and Y. lipolytica. Thus, it was known that pregnenolone and its product pregnenolone and the products of its transformation by cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17) ‒ 17 hydroxypregnenolone, dehydroepiandrosterone ‒ undergo 3-O-acetylation by the Atf2p enzyme of the yeast Saccharomyces cerevisiae. We have found that adding isoamyl or amyl alcohol to the cultivation medium significantly reduced the formation of 3-O-acetylated products by both uninduced and induced cells of transgenic yeast S. cerevisiae GRF18 YEp5117α expressing P450c17 under the control of the GAL10 promoter. In this case, the Atf2p substrate competition model was applied. A more effective solution was to use a different host microorganism and an expression promoter. The analysis of the genome of the yeast Y. lipolytica using the BLAST program showed the absence of potential analogues of Atf2p, which was confirmed by the absence of pregnenolone acetylation products. Selecting the optimal host organism is an alternative to using a strain with a deleted gene.

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