A mutant form of 3-ketosteroid-Δ<Superscript>1</Superscript>-dehydrogenase gives altered androst-1,4-diene-3, 17-dione/androst-4-ene-3,17-dione molar ratios in steroid biotransformations by <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis> ST-095

Mycobacterium neoaurum ST-095 and its mutant M. neoaurum JC-12, capable of transforming phytosterol to androst-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (AD), produce very different molar ratios of ADD/AD. The distinct differences were related to the enzyme activity of 3-ketosteroid-Δ¹-dehydrogenase (KSDD), which catalyzes the C_1,2 dehydrogenation of AD to ADD specifically. In this study, by analyzing the primary structure of KSDD_I (from M. neoaurum ST-095) and KSDD_II (from M. neoaurum JC-12), we found the only difference between KSDD_I and KSDD_II was the mutation of Val³⁶⁶ to Ser³⁶⁶. This mutation directly affected KSDD enzyme activity, and this result was confirmed by heterologous expression of these two enzymes in Bacillus subtilis. Assay of the purified recombinant enzymes showed that KSDD_II has a higher C_1,2 dehydrogenation activity than KSDD_I. The functional difference between KSDD_I and KSDD_II in phytosterol biotransformation was revealed by gene disruption and complementation. Phytosterol transformation results demonstrated that ksdd _I and ksdd _II gene disrupted strains showed similar ADD/AD molar ratios, while the ADD/AD molar ratios of the ksdd _I and ksdd _II complemented strains were restored to their original levels. These results proved that the different ADD/AD molar ratios of these two M. neoaurum strains were due to the differences in KSDD. Finally, KSDD structure analysis revealed that the Val³⁶⁶Ser mutation could possibly play an important role in stabilizing the active center and enhancing the interaction of AD and KSDD. This study provides a reliable theoretical basis for understanding the structure and catalytic mechanism of the Mycobacteria KSDD enzyme.     

Single nucleotide polymorphism analysis for the production of valuable steroid intermediates in <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis>

Objectives

To investigate single nucleotide polymorphism (SNP) in the transformation process of phytosterol to valuable steroid intermediates in three steroid-producing Mycobacterium neoaurum strains using deep sequencing and bioinformation analysis.

Results

The assembled contig sequences from RNA sequencing of strains producing 9α-hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-androstadiene-3,17-dione (ADD), and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (1,4-BNA) were analyzed for the presence of putative SNPs for steroid catabolism. 413, 375, and 491 SNPs were detected in the coding domain sequences and non-coding domain sequences of RNA sequencing reads of M. neoaurum strains producing 9OHAD, ADD, and BNA, respectively. Special attention was focused on SNPs associated with genes showing differential expression at proteome level, including the genes for sterol catabolism, glycerol catabolic process, signal transduction systems, transport system and energy metabolism.

Conclusions

The work facilitates the understanding of underlying genetic changes that may be responsible for steroid accumulation in M. neoaurum and is useful for its targeted genetic engineering.

     

Site-directed mutagenesis under the direction of in silico protein docking modeling reveals the active site residues of 3-ketosteroid-Δ<Superscript>1</Superscript>-dehydrogenase from <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis>

3-Ketosteroid-Δ¹-dehydrogenases (KsdD) from Mycobacterium neoaurum could transform androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione. This reaction has a significant effect on the product of pharmaceutical steroid. The crystal structure and active site residues information of KsdD from Mycobacterium is not yet available, which result in the engineering of KsdD is tedious. In this study, by the way of protein modeling and site-directed mutagenesis, we find that, Y122, Y125, S138, E140 and Y541 from the FAD-binding domain and Y365 from the catalytic domain play a key role in this transformation. Compared with the wild type, the decline in AD conversion for mutants illustrated that Y125, Y365, and Y541 were essential to the function of KsdD. Y122, S138 and E140 contributed to the catalysis of KsdD. The following analysis revealed the catalysis mechanism of these mutations in KsdD of Mycobacterium. These information presented here facilitate the manipulation of the catalytic properties of the enzyme to improve its application in the pharmaceutical steroid industry.     

Preparation of androsta-1,4-diene-3,17-dione from sterols using Mycobacterium neoaurum VKPM As-1656 strain

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM An-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI3 in situ.     

Overexpression of cytochrome p450 125 in <Emphasis Type="Italic">Mycobacterium</Emphasis>: a rational strategy in the promotion of phytosterol biotransformation

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD⁺/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD⁺ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD⁺ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD⁺/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.     

Identification and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum

Mycobacteria have been modified to transform sterols to produce valuable steroids. Here, we demonstrated that the oxidation of sterols to sterones is a rate-limiting step in the catabolic pathway of sterols in Mycobacterium neoaurum. Two cholesterol oxidases ChoM1 and ChoM2 involved in the step were identified in M. neoaurum and the ChoM2 shared up to 45% identity with other cholesterol oxidases. We demonstrated that the combination of ChoM1 and ChoM2 plays a significant role in this step. Accordingly, we developed a strategy to overcome this rate-limiting step by augmenting the activity of cholesterol oxidases in M. neoaurum strains to enhance their transformation productivity of sterols to valuable steroids. Our results indicated that the augmentation of ChoM2 achieved 5.57 g/l androst-1,4-diene-3,17-dione in M. neoaurum NwIB-01MS and 6.85 g/l androst-4-ene-3,17-dione in M. neoaurum NwIB-R10, greatly higher than the original yield, 3.87 g/l androst-1,4-diene-3,17-dione and 4.53 g/l androst-4-ene-3,17-dione, respectively.     

Biotransformation of progesterone to 14 alpha-hydroxypregna-1,4-diene-3,20-dione, a novel fungal metabolite, by Colletotrichum antirrhini

The fermentation of progesterone by Colletotrichum antirrhini SC 2144 was examined. Instead of 15 alpha-hydroxyprogesterone, the reported product, this fungus converted progesterone to androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, 14 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 11 alpha-hydroxypregn-4-ene-3,20-dione, 14 alpha-hydroxypregn-4-ene-3,20-dione, and a hitherto undescribed compound, 14 alpha-hydroxypregna-1,4-diene-3,20-dione.     

The biotransformation of hyodeoxycholic acid by Pseudomonas sp. NCIB 10590 under anaerobic conditions

The bacterial degradation of hyodeoxycholic acid under anaerobic conditions was studied. The major acidic product has been identified as 6 alpha-hydroxy-3-oxochol-4-ene-24-oic acid whilst the major neutral product has been identified as 6 alpha-hydroxyandrosta-1,4-diene-3,17-dione. The minor acidic products were 3,6-dioxochola-1,4-diene-24-oic acid, 3-oxochol-5-ene-24-oic acid, 3-oxochol-4-ene-24-oic acid, 3-oxochola-1,4-diene-24-oic acid and 6 alpha-hydroxy-3-oxochola-1,4-diene-24-oic acid and the minor neutral products were androst-4-ene-3,17-dione, androst-4-ene-3,6,17-trione, androsta-1,4-diene-3,6,17-trione, androsta-1,4-diene-3,17-dione, 17 beta-hydroxyandrosta-1,4-diene-3-one and 6 alpha-hydroxyandrost-4-ene-3,17-dione. Evidence is presented which suggests that under aerobic conditions, one pathway of hyodeoxycholic acid metabolism exists whilst under anaerobic conditions an extra biotransformation pathway becomes operative involving the induction of a 6 alpha-dehydroxylase enzyme. A biochemical pathway of hyodeoxycholic acid metabolism by bacteria under anaerobic conditions is discussed incorporating a scheme involving such an enzyme.     

Mycobacterium sp. mutant strain producing 9α-hydroxyandrostenedione from sitosterol

Mycobacterium sp. VKM Ac-1815D and its derivatives with altered resistance to antibacterial agents were able to produce androst-4-ene-3,17-dione (AD) as a major product from sitosterol. In this study, those strains were subjected to subsequent mutagenization by chemical agents and UV irradiation in combination with sitosterol selection pressure. The mutant Mycobacterium sp. 2-4 M was selected, being capable of producing 9-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol, with a 50% molar yield. Along with 9-OH-AD, both AD and 9-hydroxylated metabolites with a partially degraded side-chain were formed from sitosterol by the mutant strain. The strain was unable to degrade 9-OH-AD, but degraded androsta-1,4-diene-3,17-dione (ADD), thus indicating a deficiency in steroid 1(2)-dehydrogenase and the presence of 9-hydroxylase activity.     

Lithocholic Acid Side-chain Cleavage to Produce 17-Keto or 22-Aldehyde Steroids by Pseudomonas putida strain ST-491 Grown in the Presence of an Organic Solvent,Diphenyl Ether

We devised a method to screen for microorganisms capable of growing on bile acids in the presence of organic solvents and producing organic solvent-soluble derivatives. Pseudomonas putida biovar A strain ST-491 isolated in this study produced decarboxylated derivatives from the bile acids. Strain ST-491 grown on 0.5% lithocholic acid catabolized approximately 30% of the substrate as a carbon source, and transiently accumulated in the medium androsta-1,4-diene-3,17-dione in an amount of corresponding to 5% of the substrate added. When 20% (v/v) diphenyl ether was added to the medium, 60% of the substrate was converted to 17-keto steroids (androst-4-ene-3,17-dione-like steroid, androsta-1,4-diene-3,17-dione) or a 22-aldehyde steroid (pregna-1,4-dien-3-on-20-al). Amounts of the products were responsible for 45, 10, and 5% of the substrate, respectively. In the presence of the surfactant Triton X-100 instead of diphenyl ether, 40% of the substrate was converted exclusively to androsta-1,4-diene-3,17-dione.     

Synthesis and Biochemical Evaluation of the Novel Steroid Androsta-4,6,8(9)-Triene-3,17-Dione

Abstract

According to a proposed aromatisation mechanism by which estrogens are biosynthesized from androgens, the novel steroid androsta-4,6,8(9)-triene-3,17-dione (FCE 24918) should behave as a suicide substrate for aromatase. The synthesis of this triene steroid has been accomplished starting from androsta-4,7-diene-3,17-dione (4) by the acid-catalysed cleavage of the corresponding 7,8α-epoxide, 5, and it was obtained together with androsta-4,6,8(14)-triene-3,17-dione (FCE 24917) as a side product. The time-dependent inactivation of placental aromatase by the two isomers was studied comparatively and showed that the 4,6,8(9)-triene moiety acts as a latent alkylating group.     

Microbial transformation of sterols to C19-steroids by Rhodococcus equi

A wild-type strain of Rhodococcus equi, isolated from soil, degraded cholesterol, -sitosterol, stigmasterol and mixed sterois to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). A definite preference for a relatively simply structured cholesterol side chain was observed. Highest specific cholesterol side-chain cleavage was associated with active growth of the culture. Maximum yield of ADD was obtained when sodium acetate and cholesterol were incorporated together in the medium. Specific side-chain cleavage required the presence of 2,2-dipyridyl, an inhibitor of ring cleavage.S. Ahmad and B.N. Johri are with the Department of Microbiology, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantriagar 263 145, Nainital, UP, India. P.K. Roy, A.W. Khan and S.K. Basu are at Fermentation Technology Division, Central Drug Research Institute, Lucknow, India.     

Synthesis and aromatase inhibitory activity of some new 16E-arylidenosteroids

A new series of 16E-arylidene androstene derivatives has been synthesized and evaluated for aromatase inhibitory activity. The impact of various aryl substituents at 16 position of the steroid skeleton on aromatase inhibitory activity has been observed. The 16E-arylidenosteroids 6, 10 and 11 exhibited significant inhibition of the aromatase enzyme. 16-(4-Pyridylmethylene)-4-androstene-3,17-dione (6, IC₅₀: 5.2 μM) and 16-(benzo-[1,3]dioxol-5-ylmethylene)androsta-1,4-diene-3,17-dione (11, IC₅₀: 6.4 μM) were found to be approximately five times more potent in comparison to aminoglutethimide.     

The degradation of beta-sitosterol by Pseudomonas sp. NCIB 10590 under aerobic conditions

The bacterial degradation of beta-sitosterol by Pseudomonas sp NCIB 10590 has been studied. Major biotransformation products included 24-ethylcholest-4-en-3-one, androsta-1,4-diene-3,17-dione, 3-oxochol-4-en-3-one-24-oic acid and 3-oxopregn-4-en-3-one-20-carboxylic acid. Minor products identified were 26-hydroxy-24-ethylcholest-4-en-3-one, androst-4-ene-3,17-dione, 3-oxo-24-ethylcholest-4-en-26-oic acid, 3-oxochola-1,4-dien-3-one-24-oic acid, 3-oxopregna-1,4-dien-3-one-20 carboxylic acid and 9 alpha-hydroxyandrosta-1,4-diene-3,17-dione. Studies with selected inhibitors have enabled the elucidation of a comprehensive pathway of beta-sitosterol degradation by bacteria.     

Bioconversion of Lithocholic Acid Under Anaerobic Conditions by Pseudomonas sp. Strain NCIB 10590

The biotransformation of lithocholic acid by Pseudomonas sp. strain NCIB 10590 under anaerobic conditions was studied. The major products were identified as androsta-1,4-diene-3,17-dione and 3-oxochol-4-ene-24-oic acid. The minor products included 17β-hydroxyandrost-4-ene-3-one, 17β-hydroxyandrosta-1,4-diene-3-one, 3-oxo-5β-cholan-24-oic acid, 3-oxochola-1,4-diene-24-oic acid, 3-oxopregn-4-ene-20-carboxylic acid, and 3-oxopregna-1,4-diene-20-carboxylic acid. Anaerobiosis increases the number of metabolites produced by Pseudomonas sp. NCIB 10590 from lithocholic acid.     

Genetic diversity of the house mouse <Emphasis Type="Italic">Mus musculus</Emphasis> and geographic distribution of its subspecies-specific RAPD markers on the territory of Russia

Genetic diversity and geographic distribution of taxon-specific RAPD markers was examined in ten local populations of the house mouse Mus musculus (n = 42). The house mice were generally characterized by moderate genetic variation: polymorphism P ₉₉ = 60%, P ₉₅ = 32.57%; heterozygosity H = 0.12; the observed allele number n _a = 1.6; the effective allele number n _e = 1.18; the within-population differentiation ?_s = 0.388; and Shannon index I = 0.19. The degree of genetic isolation of individual local populations was greatly variable. The genetic subdivision index G _st varied from 0.162 to 0.770 at the gene flow of Nm = 2.58?0.149, while the among-population distances D _N varied from 0.026 to 0.178. The largest part of the genetic diversity was found among the populations (H _T = 0.125), while the within-population diversity was twice lower (H _S = 0.06). The samples examined were well discriminated relative to the sets of RAPD markers. The character distribution pattern provided conditional subdivision of the mice into the “western” and the “eastern” groups with the putative boarder along the Baikal Lake. The first group was characterized by the prevalence of the markers typical of M. m. musculus and M. m. domesticus. The second group was characterized by the prevalence of the markers typical of M. m. musculus, M. m. gansuensis, M. m. castaneus, M. m. domesticus, and M. m. wagneri. The genotype of the nominative subspecies M. m. musculus was background for all populations. In the populations examined some of earlier described subspecies-specific molecular markers were found at different frequencies, pointing to the involvement of several subspecies of M. musculus in the process of hybridization.     

Terpenoid bioactive compound from <Emphasis Type="Italic">Streptomyces rochei</Emphasis> (M32): taxonomy,fermentation and biological activities

The present study emphasized the production of biologically active terpenoid compound from Streptomyces rochei M32, which was isolated from Western Ghats ecosystem, South India. The presence of resistant genes like mecA, vanA of Staphylococcus aureus and bla _SHV, bla _TEM of Pseudomonas aeruginosa was confirmed by molecular studies. The isolated compound from Streptomyces rochei M32 inhibited wide range of standard and clinical drug resistant pathogens and enteric pathogens. The rice bran supplemented basal medium influenced the active compound production on 8th day of fermentation and yielded 1875 mg of crude extract from 10 g of rice bran substrate. Purification and characterization of crude ethyl acetate extract was achieved by preparative thin layer chromatography. The active fraction was identified as terpenoid class compound by chemical screening. Based on the results of spectral studies (NMR, LC–MS, FTIR, etc.), the active compound was tentatively identified as 1, 19-bis (3-hydroxyazetidin-1-yl) nonadeca-5, 14-diene-1, 8, 12, 19-tetraone with molecular weight 462.41 g/mol. Minimum inhibitory concentration value ranges between 7.6 and 31.2 µg/mL against test organisms was observed. The cytotoxicity results on cervical cancer (HeLa) cell line showed IC₅₀ value of 2.034 µg/mL. The corresponding compound is not previously reported from any microbial resources.     

Identification of a New 1,4-beta-D-xylosidase <Emphasis Type="Italic">Pae</Emphasis>1263 from the Whole Genome Sequence of <Emphasis Type="Italic">Paenibacillus terrae</Emphasis> HPL-003

The gene of Pae1263 (2,196 bp, 732 aa) was found from the full-length sequence analysis of bacterium Paenibacillus terrae HPL-003 isolated from soil on Gara Mountain in Korea (CP003107, our previous study). Among the 20 open reading frames (ORFs) related with the xylose substrate, only the recombinant enzyme of ORF Pae1263 showed a 1,4-beta-D-xylosidase activity when all of the ORFs were transformed into E. coli. This gene is considered to be a new 1,4-beta-D-xylosidase because it has up to 93% similarity with other genes of ZP_10240221.1 from Lactococcus raffinolactis 4877 and ZP_11237858.1 from Paenibacillus peoriae in the GenBank blast search. The enzyme activity was confirmed by HPLC in which xylose was produced from xylobiose as a substrate by this recombinant enzyme. Mass production of the recombinant enzyme was done with the construction of the pET22(+)- Pae1263-6H expression vector system from E. coli. This new 1,4-beta-D-xylosidase was highly active at 50°C in a pH range between 6.0 and 8.0 and had thermo-stability for at least 24 h at 50°C and a K _m and V _max of 6.42 mg/mL and 75.76 U/mg on a xylobiose substrate, respectively.     

1,4-Dioxane degradation potential of members of the genera <Emphasis Type="Italic">Pseudonocardia</Emphasis> and <Emphasis Type="Italic">Rhodococcus</Emphasis>

In recent years, several strains capable of degrading 1,4-dioxane have been isolated from the genera Pseudonocardia and Rhodococcus. This study was conducted to evaluate the 1,4-dioxane degradation potential of phylogenetically diverse strains in these genera. The abilities to degrade 1,4-dioxane as a sole carbon and energy source and co-metabolically with tetrahydrofuran (THF) were evaluated for 13 Pseudonocardia and 12 Rhodococcus species. Pseudonocardia dioxanivorans JCM 13855^T, which is a 1,4-dioxane degrading bacterium also known as P. dioxanivorans CB1190, and Rhodococcus aetherivorans JCM 14343^T could degrade 1,4-dioxane as the sole carbon and energy source. In addition to these two strains, ten Pseudonocardia strains could degrade THF, but no Rhodococcus strains could degrade THF. Of the ten Pseudonocardia strains, Pseudonocardia acacia JCM 16707^T and Pseudonocardia asaccharolytica JCM 10410^T degraded 1,4-dioxane co-metabolically with THF. These results indicated that 1,4-dioxane degradation potential, including degradation for growth and by co-metabolism with THF, is possessed by selected strains of Pseudonocardia and Rhodococcus, although THF degradation potential appeared to be widely distributed in Pseudonocardia. Analysis of soluble di-iron monooxygenase (SDIMO) α-subunit genes in THF and/or 1,4-dioxane degrading strains revealed that not only THF and 1,4-dioxane monooxygenases but also propane monooxygenase-like SDIMOs can be involved in 1,4-dioxane degradation.