ИСТИНА

The construction of microorganisms capable of carrying out processes going in the steroidogenic organs of mammals is an important area of scientific research. Such microorganisms are increasingly being used in biotechnological processes for the synthesis of steroid drugs. Cytochrome P450scc (CYP11A1), as part of the mammalian enzymatic system (including P450scc, AdR and Adx), catalyzes the first reactions of the steroidogenesis cascade, which ensures the transformation of cholesterol into pregnenolone. This research wascarried out in order to optimize the functioning of the bovine P450scc system, reconstructed in E. coli cells. It has been shown that the synthesis of human cholesterol tranfer proteins STARD1 or STARD3 in BL21(DE3) cells leads to an increase in the efficiency of assimilation of sterols by bacteria. Using model systems of heterologous expression based on E. coli BL21(DE3), it was demonstrated that cells carrying out simultaneous expression of the gene encoding STARD1 or STARD3 with the pelB sequence and genes encoding the P450scc system proteins more effectively carry out the bioconversion of cholesterol into pregnenolone in comparison with cells carrying only genes of the P450scc system proteins. As a result of the expression of the STARD1 or STARD3 gene, the level of cholesterol bioconversion by the P450scc system is increased in 1,8 and 4,8 times, respectively (determined using ELISA and HPLC). A search was carried out for the E. coli host strain that provides the most optimal conditions for the functioning of STARD3 in conjunction with cytochrome P450scc. Using polycistronic plasmids, a panel of E. coli strains was obtained - BL21(DE3)pLys, C41, Rosetta(DE3)pLys, HMS174 and BL21(DE3), synthesizing proteins of the P450 system and the STARD3 protein. It was found that E. coliRosetta(DE3)pLysSstrain is an optimal for the functioning of the reconstructed P450 system. The use of this strain allowed to increase the yield of pregnenolone by 6,5 times in comparison with the strain BL21(DE3).