One of the main obstacles to the successful use of Escherichia coli cells for steroid transformation in biotechnological processes is inefficient transport of steroid substrates into the cells. Here, we tested the possibility of using human cholesterol transfer protein STARD1 (steroidogenic acute regulatory protein) to increase the efficiency of steroid uptake by bacterial cells. Genetic constructs were obtained for the synthesis in E. coli BL21 (DE3) cells of a truncated version of STARD1 containing protein functional domain (residues 66-285) and STARD1 (66-285)-GFP fusion protein, both carrying bacterial periplasmic targeting sequence pelB at the N-terminus. Analysis of preparations of E. coli/pET22b/STARD1-GFP cells by fluorimetry and Western blotting confirmed that the used expression system ensured the synthesis of the heterologous protein. Using fluorescence spectroscopy, it was demonstrated that the presence of STARD1 in the cells increased the efficiency of assimilation of NBD-labeled cholesterol analogues by E. coli/pET22b/STARD1 cells 1.3-1.6 times (p < 0.05) compared to the wild-type cells, thus demonstrating that human STARD1 exhibits its functional activity in bacterial cells. This opens prospects for optimizing and using a fundamentally new approach to increase the efficiency of steroid uptake by cells – the inclusion of a specific carrier protein in the cell membrane, which can expand the arsenal of methods used to obtain strains of microorganisms for synthesis.

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.

Электроформование позволяет создавать полимерные мембраны, состоящие из нановолокон. Эти мембраны находят применение в различных задачах фильтрации, изготовления раневых покрытий и тканеинженерных конструкций, а кроме того, их рассматривают как перспективные подложки для иммуноанализа. Несмотря на активный интерес научного сообщества к применению электроформованных мембран для иммуноанализа, на настоящий момент не проведено их прямого сопоставления с мембранами, сформированными с помощью других технологий. В данной работе мы провели такое сопоставление и показали, что детекция мелатонина методом иммуноферментного анализа происходит практически одинаково на мембранах, изготовленных методом электроформования, и обычных коммерчески доступных мембранах.