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Expression genes encoding cholesterol carrier proteins to increase the efficiency of P450scc system in E. coli cellsстатья Тезисы
- Авторы: Zamalutdinova S., Isaeva L., Rubtsov M., Novikova L.
- Журнал: FEBS open bio
- Том: 12
- Номер: S1
- Год издания: 2022
- Издательство: John Wiley & Sons Inc.
- Местоположение издательства: United States
- Первая страница: 315
- Последняя страница: 315
- Номер статьи: P-05.3-001
- DOI: 10.1002/2211-5463.13440
- Аннотация: Cytochrome P450scc (CYP11A1) with its redox partners adren- odoxin reductase (AdR) and adrenodoxin (Adx) forms a com- plex of the P450scc system. This system provides the initial reactions of mammalian steroidogenesis, resulting in the forma- tion 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 (Pre- viously 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 trans- porter 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 endo- somes. Obtained DNA constructions provide synthesis in E. coli of StARD1 or StARD3. Both of them are predominantly local- ized in the membrane fraction. For the first time, human cholesterol-carrying proteins were shown to manifest their activ- ity in bacterial cells. According to the results of HPLC-MS and fluorescence spectroscopy, the carrier proteins expressed in bac- teria increase the intracellular level of both cholesterol and 22- NBD-hydroxycholesterol by ~2-fold 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 usefultool for the creation of artificial whole-cell biocatalysts. The research was supported by RFBR (20-08-00467A)
- Добавил в систему: Замалутдинова Софья Васильевна