Zamalutdinova Sofia V, Isaeva Ludmila V, Golyshev Sergei A, Rubtsov Mikhail A, Novikova Ludmila A
Faculty of Biology, M.V. Lomonosov Moscow State University, Leninskie Gory, 1/12, Moscow, Russia, 119991.
Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1/40, Moscow, Russia, 119991.
Mol Biotechnol. 2025 Aug 22. doi: 10.1007/s12033-025-01490-w.
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.
本研究探讨人类STARD3胆固醇转运蛋白表达的优化，以增强牛P450scc介导的大肠杆菌中的类固醇生物转化，共表达STARD3和P450scc系统蛋白（细胞色素P450scc（CYP11A1）、肾上腺皮质铁氧化还原蛋白和肾上腺皮质铁氧化还原蛋白还原酶）。我们比较了STARD3的不同表达策略，包括使用pelB和TorA信号肽进行潜在的周质定位，并评估了不同的大肠杆菌菌株以最大化胆固醇生物转化。尽管两种信号肽都未能有效地将STARD3转运到周质中，大部分蛋白质保留在细胞质和膜组分中，但STARD3蛋白在胆固醇转运中保留了功能。乳糖自动诱导被证明优于基于IPTG的诱导，导致STARD3和P450scc的表达水平显著更高。在所测试的大肠杆菌菌株（BL21(DE3)、BL21(DE3)pLysS、HMS174(DE3)、C41(DE3)和Rosetta(DE3)pLysS）中，Rosetta(DE3)pLysS表现出最高的STARD3表达以及STARD3对胆固醇生物转化的最显著影响。与仅表达P450scc系统蛋白的菌株相比，它在Rosetta(DE3)pLysS中使P450scc活性提高了约三倍。该菌株的胆固醇生物转化效率比先前使用的BL21(DE3)高6.97±3.69倍。这些结果为优化细菌宿主中的重组类固醇生成系统以用于潜在的生物技术应用提供了有价值的见解。
