ИСТИНА

Cytochrome P450scc (CYP11A1) with its redox partners adrenodoxin reductase (AdR) and adrenodoxin (Adx) forms a complex of the P450scc system. This system provides the initial reactions of mammalian steroidogenesis, resulting in the formation of pregnenolone. Earlier it has been shown that the P450scc system can function in E. coli cells. However, transport of P450scc substrate – cholesterol, into cells is inefficient (Previously published in Efimova et al. (2018) Mol Biotechnol, 61(4), 261­ 273), thereby placing a limit on the yield of the target product. To solve this problem, we used a fundamentally new approach – the incorporation of the human cholesterol transporter into the bacterial membrane. We were examining the potential of the mitochondrial protein StARD1 (steroidogenic acute regulatory protein 1), a native partner of cytochrome P450scc, as well as StARD3, delivering cholesterol to endosomes. Obtained DNA constructions provide synthesis in E. coli of StARD1 or StARD3. Both of them are predominantly localized in the membrane fraction. For the first time, human cholesterol­carrying proteins were shown to manifest their activity in bacterial cells. According to the results of HPLC­MS and fluorescence spectroscopy, the carrier proteins expressed in bacteria increase the intracellular level of both cholesterol and 22­NBD­hydroxycholesterol by ~2­fold compared to the control strain. Most notably, this is the first study demonstrating the functional coupling of recombinant StARD1 and StARD3 with the P450scc system. Also, it was shown that StARD1 and StARD3 increase the whole cell cholesterol biotransformation activity of recombinant strain of E. coli with reconstructed P450scc system by ~2.5 and ~6 times. Pregnenolon is excreted in the culture medium and is almost absent in cells. The data obtained demonstrates that the approach used, may be a useful tool for the creation of artificial whole­cell biocatalysts. The research was supported by RFBR (20­08­00467A)