Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system

Abstract

Background: Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones.

Results: Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and ¹H- and ¹³C-NMR analyses.

Conclusion: The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones-progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Keywords: Mycolicibacterium smegmatis mc2 155; 3-methoxymethylated steroids; Cytochrome P450scc; Pregnenolone; Progesterone; Sterols.

Fig. 1

Schematic representation of the general organization of the mammalian cholesterol hydroxylase/C20-C22 lyase system in adrenocortical mitochondria. Cytochrome P450scc catalyzes the reaction of the cleavage of cholesterol side chain to form pregnenolone

Fig. 2

Structures of the plasmids for gene expression in mycolicibacteria. a The plasmid pNS10 contains cDNA encoding bovine cytochrome P450scc. b The plasmid pNS11 contains cDNAs for bovine AdR, Adx (1–108 aa) and cytochrome P450scc in a single expression cassette driven by acetamidase promoter system (amiC, amiA, amiD and amiS). The plasmids include respective origins of replication (oriE and oriM) for propagation in E. coli and M. smegmatis, transcription terminator T1 from the E. coli rrnB gene (T), and hygromycin B resistance marker (hygR). NcoI, HindIII and BglII restriction sites used at cloning procedure are designated

Fig. 3

The conversion of sterols (2.33 mM) by the recombinant M. smegmatis mc² 155 pNS11 strain. a Curves of progesterone accumulation from cholesterol and phytosterol, b cholesterol and phytosterol utilization

Fig. 4

The conversion of MOM-sterols (2.33 mM) by the recombinant M. smegmatis mc² 155 pNS11 strain. a Curves of MOM-pregnenolone accumulation from MOM-cholesterol and MOM-phytosterol, b MOM-cholesterol and MOM-phytosterol utilization

Fig. 5

MOM-Cholesterol bioconversion by M. smegmatis mc² 155 pNS11 to form MOM-DHEA and MOM-pregnenolone

Fig. 6

The influence of adding acetamide on the bioconversion of MOM-cholesterol by M. smegmatis mc² 155 pNS11. a The yield of MOM-pregnenolone, b the yield of MOM-DHEA. Control—without adding acetamide; the single application of acetamide: at 0, 12 or 24 h; the double application of acetamide: at 0 and 12 h; 12 and 24 h or 0 and 24 h; the triple application of acetamide at 0, 12 and 24 h; the quadruple addition—at 0, 12, 24 and 36 h after the strain inoculation. The biotransformation time—48 h; final acetamide concentration in the medium—2 g/L

Fig. 7

HPLC and mass analysis of crystalline pregnenolone obtained after acid hydrolysis of MOM-pregnenolone. a HPLC profile; b mass spectrum