Functional reconstruction of bovine P450scc steroidogenic system in <i>Escherichia coli</i>

Abstract

Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis-cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components-the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin reductase, AdR) and an iron-sulfur protein (adrenodoxin, Adx). In this work, the three-component electron transport chain (AdR/Adx/CYP11A1) from bovine adrenal cortex has been implemented in Escherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins identical to the bovine CYP11A1, AdR and Adx on molecular weights and immuno-specificity. The experiments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the presence of cholesterol solubilizing agent-methyl-β-cyclodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for studies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.

Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line

“…Samples of cells carrying pcDNA3.1/CT‐GFP plasmid were used as a negative control. Along with the homogenates under consideration, we loaded the lysate of Escherichia coli cells transformed with pBar_Triple, which directed the synthesis of individual proteins P450scc, Adx and AdR that were used as reference proteins (Figure A–C, Lane 1).…”

“…1,3,4 The investigation and reconstruction of cytochrome P450 systems, as well as other multienzyme systems in model organisms and cell lines, require the application of methods that provide an opportunity to coexpress several proteins in the cell. There is a variety of approaches for the simultaneous expression of several heterologous genes: for example, the construction of multicistronic plasmid by integration of ribosomal binding sites (for prokaryotic organisms) 5 or IRES sequences (for eukaryotic organisms) 6 ; the application of vectors comprising the hybrid DNA constructs, encoding a fusion of several individual proteins 7 or single plasmids comprising expression cassettes for target genes in tandem under the control of different promoters and terminators 8 ; the application of vectors for multiple integration into the genome 9 or for the realization of CRISPR-Cas technology. 10 All of these approaches involve their benefits and limitations.…”

Functionalized PHB granules provide the basis for the efficient side-chain cleavage of cholesterol and analogs in recombinant Bacillus megaterium

“…Hence, to the best our knowledge, so far no recombinant whole-cell system has been described, which can efficiently convert these compounds to pregnenolone. Attempts to utilize recombinant E. coli for pregnenolone production yielded only product concentrations in the µg/L range after 24 h [ 5 ]. In recent years, efforts have been made to produce steroid hormones in Saccharomyces cerevisiae by engineering the sterol biosynthesis pathway of the organism itself and thus endogenously providing the substrates for the recombinant enzymes [ 6 – 8 ].…”

Pregnenolone Overproduction in Yarrowia lipolytica by Integrative Components Pairing of the Cytochrome P450scc System

“…13−15 However, the poor pregnenolone conversion from its precursors remains a big challenge. Makeeva et al 14 just obtained 420 μg/L pregnenolone from 500 μM (193 mg/L) cholesterol by using an E. coli biocatalyst with bovine P450scc system. Compared with E. coli, Saccharomyces cerevisiae has been served as a more successful platform to achieve a titer of 60 mg/L pregnenolone 13 and 11.5 mg/L hydrocortisone 16 from glucose.…”

“…Pregnenolone is the key precursor to synthesize all classes of steroidal drugs including glucocorticoids, mineralocorticoids, and sex hormones . As so far, pregnenolone production has been realized in several microbial platforms through expressing the mature form of P450scc proteins (mCYP11A1, mAdx, and mAdR) without N-terminal mitochondrial targeting sequence (MTS). − However, the poor pregnenolone conversion from its precursors remains a big challenge. Makeeva et al just obtained 420 μg/L pregnenolone from 500 μM (193 mg/L) cholesterol by using an E. coli biocatalyst with bovine P450scc system.…”