Zamalutdinova Sofia V, Isaeva Ludmila V, Faletrov Yaroslav V, Eroshchenko Nikolay N, Kirushin Alexey N, Tashlitsky Vadim N, Rubtsov Mikhail A, Novikova Ludmila A
Faculty of Biology, M.V. Lomonosov Moscow State University, Leninskie Gory, 1/12, Moscow, 119991, Russia.
Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1/40, Moscow, 119991, Russia.
Bioresour Bioprocess. 2025 Jul 21;12(1):77. doi: 10.1186/s40643-025-00909-1.
Synthesis of steroid drugs is possible through biotechnological methods, however, the low efficiency of transporting steroid substrates across microbial membranes is the challenge in using microorganisms for their production. STARD1 and STARD3 proteins (members of the START domain family) work in some mammalian cells in conjunction with the steroidogenic P450scc enzyme system (cytochrome P450scc, adrenodoxin reductase, and adrenodoxin), transporting cholesterol to the mitochondria, where cytochrome P450scc converts it into pregnenolone. In this study, we investigated the effect of STARD1 and STARD3 on P450scc efficiency in E. coli expression models. The combination of the STARD3(216-444) or STARD1(53-285) protein bearing an N-terminal periplasm-targeting sequence, with the P450scc system reconstituted in E. coli, resulted in an increase of pregnenolone synthesis by 3.2- and 1.9-fold, respectively, on a laboratory scale. STARD3(216-444) showed higher levels of expression and a greater effect on the incorporation of sterols into cells and whole-cell biotransformation of cholesterol, compared to STARD1(53-285). This study proposes a fundamentally new approach to optimizing the functioning of the P450scc system in microbial cells, which uses the cholesterol transfer protein to increase the uptake efficiency of a poorly soluble steroid substrate by bacteria. The demand for steroid medications is increasing, and the use of specific carrier proteins could be a useful tool to enhance the efficiency of whole-cell biosynthesis of various steroid compounds.
通过生物技术方法合成甾体药物是可行的，然而，甾体底物跨微生物膜运输效率低是利用微生物生产甾体药物面临的挑战。STARD1和STARD3蛋白（START结构域家族成员）在一些哺乳动物细胞中与甾体生成P450scc酶系统（细胞色素P450scc、肾上腺皮质铁氧化还原蛋白还原酶和肾上腺皮质铁氧化还原蛋白）协同作用，将胆固醇转运至线粒体，在那里细胞色素P450scc将其转化为孕烯醇酮。在本研究中，我们在大肠杆菌表达模型中研究了STARD1和STARD3对P450scc效率的影响。带有N端周质靶向序列的STARD3(216 - 444)或STARD1(53 - 285)蛋白与在大肠杆菌中重构的P450scc系统相结合，在实验室规模上分别使孕烯醇酮合成增加了3.2倍和1.9倍。与STARD1(53 - 285)相比，STARD3(216 - 444)表现出更高的表达水平，并且对甾醇掺入细胞以及胆固醇的全细胞生物转化具有更大的影响。本研究提出了一种全新的方法来优化微生物细胞中P450scc系统的功能，该方法利用胆固醇转运蛋白来提高细菌对难溶性甾体底物 的摄取效率。对甾体药物的需求正在增加，使用特定的载体蛋白可能是提高各种甾体化合物全细胞生物合成效率的有用工具。
