Ludmila A. Novikova

In this study, for the first time, the genes encoding the mammalian steroidogenesis system—cytochrome P450scc (CYP11A1), and its native redox partners adrenodoxin and adrenodoxin reductase—were successfully expressed in the methylobacterium Methylorubrum extorquens. The advantage of using methylobacteria as an expression chassis is that they grow on inexpensive mineral media, use methanol as a carbon and energy source, and do not possess their own sterol catabolism systems. Using recombinant methylobacteria, the valuable steroid pregnenolone was obtained as a sole metabolite from cholesterol. The effect of media composition, bioconversion conditions such as methanol and N-sources content, modes of substrate addition, detergents, methyl-β-cyclodextrin, biomass, and aeration on pregnenolone accumulation was investigated. Under optimized conditions, its yield exceeded 100 mg/L. The results demonstrate a proof of concept relating to the use of bacteria lacking their own steroid degradation systems as microbial chassis for heterologous steroidogenesis systems, including mammalian cytochrome CYP11A1.

This study investigates the optimization of human STARD3 cholesterol transporter expression to enhance bovine P450scc-mediated steroid biotransformation in Escherichia coli, co-expressing STARD3 and P450scc system proteins (cytochrome P450scc (CYP11A1), adrenodoxin and adrenodoxin reductase). We compared different expression strategies for STARD3, including the use of pelB and TorA signal peptides for potential periplasmic localization, and evaluated different E. coli strains to maximize cholesterol biotransformation. Although both signal peptides failed to efficiently transport STARD3 to the periplasm, with most of the protein remaining in the cytoplasm and membrane fractions, the STARD3 protein retained functionality in cholesterol transport. Auto-induction with lactose proved superior to IPTG-based induction, resulting in significantly higher expression levels of both STARD3 and P450scc. Among the E. coli strains tested (BL21(DE3), BL21(DE3)pLysS, HMS174(DE3), C41(DE3), and Rosetta(DE3)pLysS), Rosetta(DE3)pLysS demonstrated the highest STARD3 expression and the most significant impact of STARD3 on cholesterol biotransformation. It increased P450scc activity by approximately three times in Rosetta(DE3)pLysS compared to strains expressing only the P450scc system proteins. This strain showed a cholesterol biotransformation efficiency that was 6.97 ± 3.69 times higher compared to the previously used BL21(DE3). These results provide valuable insights for the optimization of recombinant steroidogenic systems in bacterial hosts for potential biotechnological applications.

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.

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.

The cholesterol hydroxylase/lyase (CHL) system, located in the mitochondria of the mammalian adrenal cortex cells, consists of cytochrome P450scc (CYP11A1), adrenodoxin (Adx), and adrenodoxin reductase (AdR) and performs the first stage of the steroidogenesis: AdR and Adx enable the electron transfer between NADPH and cytochrome P450scc, and P450scc catalyzes the conversion of cholesterol into pregnenolone. CHL system was reconstructed in Escherichia coli using the polycistronic plasmid pTrc99A/CHL. In E. coli cells, the recombinant proteins form the catalytically active system. CHL activity towards 22R-hydroxycholesterol was 4.0 ± 1.3 nmol pregnenolone/h per 1 mg homogenate protein. The alteration of the order of heterologous cDNAs in the expression cassette from AdR–Adx–P450scc to P450scc–Adx–AdR results in alteration of stoichiometric ratio P450scc/Adx/AdR from 1:1.45:4.2 to 1:1.67:0.98; the former ratio is more optimal for the functioning of the cytochrome P450scc. The application of modified cDNA of Adx (AdxS112W) does not increase the CHL activity; however, the introduction of the second copy of AdxS112W gene into the expression cassette increases both the expression level of АdxS112W and the CHL activity in comparison with P450scc/АdxS112W/AdR system. In vivo activity of the CHL system in bacteria is limited by the substrate uptake by bacterial cells: it varied in the range of 0.05–0.62 mg pregnenolone/l resting cell suspension per 1-day cultivation, depending on the type and concentration of permeabilizing agents in the medium. The obtained results contribute to the knowledge of CHL system functioning in living bacteria.

The cholesterol hydroxylase/lyase (CHL) system, consisting of cytochrome P450scc, adrenodoxin (Adx) and adrenodoxin reductase (AdR), initiates mammalian steroidogenesis, converting cholesterol to pregnenolone. The foot‐and‐mouth disease virus 2A‐based method allows to express multiple proteins from a single transcript. We developed a 2A‐based multicistronic system for the coexpression of three bovine CHL system proteins as the self‐processing polyprotein pCoxIV‐P450scc‐2A‐Adx‐2A‐AdR‐GFP (pCoxIV‐CHL‐GFP), with a cleavable N‐terminal mitochondrial targeting presequence. HEK293T cells transfected with plasmid, containing complementary DNA (cDNA) for pCoxIV‐CHL‐GFP, efficiently performed the expression of P450scc‐2A, targeted to mitochondria, and Adx‐2A, AdR‐GFP and the fusion protein Adx‐2A‐AdR‐GFP, which were predominantly localized in the cytosol. Despite the spatial separation of expressed P450scc and redox partners, the transfected HEK293T cells were able to convert the steroid substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T cells were not catalytically active. The presence of 2А peptide residue on the C‐terminus of P450scc did not preclude its enzymatic activity. HEK293T cells transfected with a vector directing the synthesis of only P450scc‐2A demonstrated cytochrome P450scc activity comparable to that of cells expressing all three CHL system components, and to that of nature steroidogenic cells. Thus, the P450scc activity detected in cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3β‐hydroxysteroid dehydrogenase. Therefore, HEK293T cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.

This review summarizes data about structural and functional organization of steroidogenic P450-dependent enzymatic systems. Problems of catalysis of steroid substrate transformation, special features...

Hybrid proteins consisting of the mature form of cytochrome P450scc (mP) and adrenodoxin (Ad), attached to either the NH2-or COOH-terminus (Ad-mP and mP-Ad, respectively), were expressed in E. coli....