Abstract
BackgroundAerobic side chain degradation of phytosterols by actinobacteria is the basis for the industrial production of androstane steroids which are the starting materials for the synthesis of steroid hormones. A native strain of Mycobacterium sp. VKM Ac-1817D effectively produces 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) from phytosterol, but also is capable of slow steroid core degradation. However, the set of the genes with products that are involved in phytosterol oxidation, their organisation and regulation remain poorly understood.ResultsHigh-throughput sequencing of the global transcriptomes of the Mycobacterium sp. VKM Ac-1817D cultures grown with or without phytosterol was carried out. In the presence of phytosterol, the expression of 260 genes including those related to steroid catabolism pathways significantly increased. Two of the five genes encoding the oxygenase unit of 3-ketosteroid-9α-hydroxylase (kshA) were highly up-regulated in response to phytosterol (55- and 25-fold, respectively) as well as one of the two genes encoding its reductase subunit (kshB) (40-fold). Only one of the five putative genes encoding 3-ketosteroid-∆1-dehydrogenase (KstD_1) was up-regulated in the presence of phytosterol (61-fold), but several substitutions in the conservative positions of its product were revealed.Among the genes over-expressed in the presence of phytosterol, several dozen genes did not possess binding sites for the known regulatory factors of steroid catabolism. In the promoter regions of these genes, a regularly occurring palindromic motif was revealed. The orthologue of TetR-family transcription regulator gene Rv0767c of M. tuberculosis was identified in Mycobacterium sp. VKM Ac-1817D as G155_05115.ConclusionsHigh expression levels of the genes related to the sterol side chain degradation and steroid 9α-hydroxylation in combination with possible defects in KstD_1 may contribute to effective 9α-hydroxyandrost-4-ene-3,17-dione accumulation from phytosterol provided by this biotechnologically relevant strain. The TetR-family transcription regulator gene G155_05115 presumably associated with the regulation of steroid catabolism. The results are of significance for the improvement of biocatalytic features of the microbial strains for the steroid industry.
Highlights
Aerobic side chain degradation of phytosterols by actinobacteria is the basis for the industrial production of androstane steroids which are the starting materials for the synthesis of steroid hormones
Bragin et al BMC Biotechnology (2019) 19:39 hydroxyandrost-4-ene-3,17-dione (9-OH-AD). 9-OH-AD is extensively used as a starting compound for the synthesis of various steroid drugs bearing a halogen in position 9, such as dexamethasone, fluoxymesterone, triamcinolone, bethametasone and others [3]
The glycerol-grown cells were harvested in the late active growth stage after 18 h when reaching 1.76 ± 0.05 g/l (d.c.w.) In the induction medium, the culture converted phytosterol mainly to 9-OH-AD as a major product (87% relative abundance (r.a.) after 48 h of incubation, mol/mol) and 9-hydroxylated 3-keto-4-ene steroids with partly, or completely degraded side chain: 9-hydroxy-22-carboxy-23,24-bisnorchol-4-ene-3-one (9-HCBС, r.a 6.5%), 9, 22-dihydroxy-23,24-bisnorchol-4-en-3-one (9,22-DHBC, r.a. 4.4%), 9,24-dihydroxychol-4-en-3-one (9,24-DHC, r.a. 2.2%) and 9-hydroxy-testosterone (9-HT, r.a. 1.9%)
Summary
Aerobic side chain degradation of phytosterols by actinobacteria is the basis for the industrial production of androstane steroids which are the starting materials for the synthesis of steroid hormones. VKM Ac-1817D effectively produces 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) from phytosterol, and is capable of slow steroid core degradation. Steroids are terpenoid lipids that contain a gonane core of four cycloalkane rings (A-D) in their structure This class of organic molecules includes the compounds which fulfil essential vital functions in higher organisms. The molecular mechanisms of sterol degradation have been intensively studied, mainly due to their essential role in the pathogenesis of Mycobacterium tuberculosis [1, 2] and the wide application of non-pathogenic mycobacteria capable of partial catabolism of sterols to produce intermediates for the pharmaceutical industry, such as androst-4-ene-3,17dione (AD), androsta-1,4-diene-3,17-dione (ADD) and 9α-. The groups of putative genes engaged in sterols degradation have been characterised in the genomes of several strains such as Mycobacterium neoaurum VKM Ac-1815D, Gordonia neofelifaecis NRRL B-59395, M. tuberculosis
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