Optimization of Human STARD3 Cholesterol Transporter Expression for Enhancing Bovine P450scc-Mediated Steroid Biotransformation in E. coli

Zamalutdinova, S.V.; Isaeva, L.V.; Golyshev, S.A.; Rubtsov, M.A.; Novikova, L.A.

Авторы: Zamalutdinova Sofia V., Isaeva Ludmila V., Golyshev Sergei A., Rubtsov Mikhail A., Novikova Ludmila A.
Журнал: Molecular Biotechnology
Год издания: 2025
Издательство: Springer International Publishing AG
Местоположение издательства: Cham, Switzerland
DOI: 10.1007/s12033-025-01490-w
Аннотация: This study investigates the optimization of human STARD3 cholesterol transporter expression to enhance bovine P450scc-mediated steroid biotransformation in Escherichia coli, co-expressing STARD3 and P450scc system proteins (cytochrome P450scc (CYP11A1), adrenodoxin and adrenodoxin reductase). We compared different expression strategies for STARD3, including the use of pelB and TorA signal peptides for potential periplasmic localization, and evaluated different E. coli strains to maximize cholesterol biotransformation. Although both signal peptides failed to efficiently transport STARD3 to the periplasm, with most of the protein remaining in the cytoplasm and membrane fractions, the STARD3 protein retained functionality in cholesterol transport. Auto-induction with lactose proved superior to IPTG-based induction, resulting in significantly higher expression levels of both STARD3 and P450scc. Among the E. coli strains tested (BL21(DE3), BL21(DE3)pLysS, HMS174(DE3), C41(DE3), and Rosetta(DE3)pLysS), Rosetta(DE3)pLysS demonstrated the highest STARD3 expression and the most significant impact of STARD3 on cholesterol biotransformation. It increased P450scc activity by approximately three times in Rosetta(DE3)pLysS compared to strains expressing only the P450scc system proteins. This strain showed a cholesterol biotransformation efficiency that was 6.97 ± 3.69 times higher compared to the previously used BL21(DE3). These results provide valuable insights for the optimization of recombinant steroidogenic systems in bacterial hosts for potential biotechnological applications.
Добавил в систему: Рубцов Михаил Александрович

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Optimization of Human STARD3 Cholesterol Transporter Expression for Enhancing Bovine P450scc-Mediated Steroid Biotransformation in E. coliстатья Исследовательская статья

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