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

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.

Obtaining of 24-Norchol-4-ene-3,22-dione from Phytosterol with Mutants of Mycolicibacterium neoaurum.

Engineered mutants of Mycolicibacterium spp. are known producers of valuable steroid synthons with C19 or C22 skeleton. Here we describe a method for site-directed mutagenesis of Mycolicibacterium neoaurum strains, bioconversion from phytosterol, and selective purification of C23 steroid 24-norchol-4-ene-3,22-dione (24-NCED) and C22 steroid 20-hydroxymethylpregn-4-ene-3-one (20-HMP). The yields of crystalline products with 95% purity by the method here described are 2.74 ± 0.085 g for 24-NCED and 1.42 ± 0.085 g for 20-HMP from 10 g/L phytosterol. 20-HMP is recognized as the key precursor in chemical syntheses of pharmaceutical corticosteroids and 24-NCED is a promising synthon for the synthesis of valuable steroids and own potent biological activity.

Obtaining of 11α-Hydroxyandrost-4-ene-3,17-dione from Natural Sterols.

Two-step one-pot microbial transformation enables obtaining of valuable steroids that are difficult to produce chemically. Here we describe a method for obtaining 11α-hydroxyandrost-4-ene-3,17-dione (11α-HAD) from cheap and available natural sterols (phytosterols or cholesterol).11α-HAD is a primary adrenal steroid in mammals and also a key precursor in the syntheses of halogenated corticoids. Conventional routes for its obtaining are based on chemical synthesis, or microbial hydroxylation of androst-4-ene-3,17-dione (AD). AD in turn is produced primarily with microbial biotransformation of natural sterols by some actinobacteria.Consequent bioconversions of sterols using two microbial strains in one bioreactor vessel without separation and purification of AD provides high yield of 11α-HAD. At the first fermentation step, phytosterol is converted to AD with Mycobacterium neoaurum NRRL 3805B, or relative strains, to yield about 70% (mol/mol). At the second step, AD is almost fully (98%) hydroxylated at the position 11α with Aspergillus ochraceus VKM F-830, or other suitable organisms, in the same bioreactor. At the average, 30% (w/w) of the high-purity crystalline 11α-HAD can be obtained.The method can be exploited for production of 11α-HAD for practical use.

Synthesis of 3beta-hydroxy-androsta-5,7-dien-17-one from 3beta-hydroxyandrost-5-en-17-one via microbial 7alpha-hydroxylation.

The synthesis of 3beta-hydroxy-androsta-5,7-dien-17-one from 3beta-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA) via microbial 7alpha-hydroxylation has been accomplished. At the first stage, 3beta,7alpha-dihydroxy-androst-5-en-17-one was obtained in high yield (71.2%) using a strain of Gibberella zeae VKM F-2600, which was first applied for DHEA conversion. The further route included the substitution of 7alpha-hydroxyl group with chlorine followed by a dehydrochlorination stage, and required minimal purifications of the intermediate products. The steroids obtained at every step were characterized by TLC,1H NMR, MS, UV- and IR-spectrometry. The combination of microbial and chemical steps ensured 54.6% yield of the target 3beta-hydroxy-androsta-5,7-dien-17-one from DHEA and can be applied for obtaining novel vitamin D derivatives.