And 1,4-androstadiene-3,17-dione Research Articles

Whole-Genome Analysis of Mycobacterium neoaurum DSM 1381 and the Validation of Two Key Enzymes Affecting C22 Steroid Intermediates in Sterol Metabolism.

Mycobacterium neoaurum DSM 1381 originated from Mycobacterium neoaurum ATCC 25790 by mutagenesis screening is a strain of degrading phytosterols and accumulating important C22 steroid intermediates, including 22-hydroxy-23, 24-bisnorchola-4-en-3-one (4-HP) and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (HPD). However, the metabolic mechanism of these C22 products in M. neoaurum DSM 1381 remains unknown. Therefore, the whole-genome sequencing and comparative genomics analysis of M. neoaurum DSM 1381 and its parent strain M. neoaurum ATCC 25790 were performed to figure out the mechanism. As a result, 28 nonsynonymous single nucleotide variants (SNVs), 17 coding region Indels, and eight non-coding region Indels were found between the genomes of the two strains. When the wild-type 3-ketosteroid-9α-hydroxylase subunit A1 (KshA1) and β-hydroxyacyl-CoA dehydrogenase (Hsd4A) were overexpressed in M. neoaurum DSM 1381, the steroids were transformed into the 4-androstene-3, 17- dione (AD) and 1,4-androstadiene-3,17-dione (ADD) instead of C22 intermediates. This result indicated that 173N of KshA1 and 171K of Hsd4A are indispensable to maintaining their activity, respectively. Amino acid sequence alignment analysis show that both N173D in KshA1 and K171E in Hsd4A are conservative sites. The 3D models of these two enzymes were predicted by SWISS-MODEL and AlphaFold2 to understand the inactivation of the two key enzymes. These results indicate that K171E in Hsd4A may destroy the inaction between the NAD+ with the NH3+ and N173D in KshA1 and may disrupt the binding of the catalytic domain to the substrate. A C22 steroid intermediates-accumulating mechanism in M. neoaurum DSM 1381 is proposed, in which the K171E in Hsd4A leads to the enzyme's inactivation, which intercepts the C19 sub-pathways and accelerates the C22 sub-pathways, and the N173D in KshA1 leads to the enzyme's inactivation, which blocks the degradation of C22 intermediates. In conclusion, this study explained the reasons for the accumulation of C22 intermediates in M. neoaurum DSM 1381 by exploring the inactivation mechanism of the two key enzymes.

Biocatalysis of Steroids by Mycobacterium sp. in Aqueous and Organic Media.

Mycobacterium sp. can convert steroids such as β-sitosterol, campesterol, and cholesterol, by selective side-chain cleavage and oxidation of the C3 hydroxyl group to a ketone, into key intermediates that can be easily functionalized to yield commercially interesting pharmaceutical products. In aqueous systems, the biocatalysis is limited by the low solubility of the steroids in water. Several strategies have been introduced to tackle this limitation, e.g., formation of cyclodextrin-steroid complexes and generation of aqueous microdispersions with steroid particle size in the range of hundreds of nanometers. Still, the introduction of an organic phase acting as a substrate and/or product reservoir is a well-established and relatively easy to implement strategy to overcome the sparing water solubility of steroid molecules. However, the organic phase has to be carefully chosen to prevent tampering with the activity/viability of microbial cells.In this chapter, we describe the methodology for the biocatalysis of β-sitosterol to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD), both in aqueous and organic:aqueous systems. In the latter case, both traditional organic solvents and green solvents are proposed.

Engineering the Steroid Hydroxylating System from Cochliobolus lunatus in Mycolicibacterium smegmatis.

14α-hydroxylated steroids are starting materials for the synthesis of contraceptive and anti-inflammatory compounds in the steroid industry. A synthetic bacterial operon containing the cytochrome P450 CYP103168 and the reductase CPR64795 of the fungus Cochliobolus lunatus able to hydroxylate steroids has been engineered into a shuttle plasmid named pMVFAN. This plasmid was used to transform two mutants of Mycolicibacterium smegmatis named MS6039-5941 and MS6039 that accumulate 4-androstene-3,17-dione (AD), and 1,4-androstadiene-3,17-dione (ADD), respectively. The recombinant mutants MS6039-5941 (pMVFAN) and MS6039 (pMVFAN) were able to efficiently express the hydroxylating CYP system of C. lunatus and produced in high yields 14αOH-AD and 14αOH-ADD, respectively, directly from cholesterol and phytosterols in a single fermentation step. These results open a new avenue for producing at industrial scale these and other hydroxylated steroidal synthons by transforming with this synthetic operon other Mycolicibacterium strains currently used for the commercial production of steroidal synthons from phytosterols as feedstock.

Identification of bottlenecks in 4-androstene-3,17-dione/1,4-androstadiene-3,17-dione synthesis by Mycobacterium neoaurum JC-12 through comparative proteomics

Intermediates such as 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) have extensive clinical applications in the production of steroid pharmaceuticals. The present study explores the effect of two factors in the production of these intermediates in Mycobacterium neoaurum JC-12: the precursor, phytosterol and a molecule that increases AD/ADD solubility, hydroxypropyl-β-cyclodextrin (HP-β-CD). Differentially expressed proteins were separated and identified using 2D gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS). In total, 31 proteins were identified, and improved expression levels of ten proteins involved in metabolism was induced by phytosterol and/or HP-β-CD, which strengthened the stress resistance of the strain. In the presence of phytosterol and/or HP-β-CD, five proteins involved in the synthesis of AD/ADD, acetyl-CoA acetyltransferase (AAT), alcohol dehydrogenase (ADH), enoyl-CoA hydratase (EH) and short-chain dehydrogenase 1 and 2, increased their expression levels. Reverse transcription-quantitative PCR (RT-qPCR) was used to verify the 2-DE results and the transcriptional level of these five proteins. This analysis identified AAT, ADH, EH, and electron transfer flavoprotein subunit α/β as the possible bottlenecks for AD/ADD synthesis in M.neoaurum JC-12, which therefore are suggested as targets for strain modification.

Production of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione from rice germ and wheat germ extracts by Mycobacterium sp.

To study the biotransformation of phytosterol and phytosterol-containing rice germ and wheat germ ethanolic extracts to produce 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by Mycobacterium sp. DSM 2966 using phytosterol to hydroxypropyl-β-cyclodextrin (2:1, 1:1 and 1:2mol/mol) and 2% (w/v) Tween 80 as solubilizing agents. A maximum yield of 180±27mgADl(-1) and 31±11.4mgADDl(-1) with a total conversion of 65% (day 12) was obtained using 1g phytosterol l(-1) and hydroxypropyl-β-cyclodextrin (2 : 1mol/mol) with 2% (w/v) Tween 80 in the fermentation medium. The most appropriate conditions for rice germ extract and wheat germ extract which gave the maximum conversion of 22 and 43% (day 14) were obtained by using 2% (w/v) Tween 80. Phytosterol and wheat germ are effective sources for AD and ADD production while rice germ required further development. Hydroxypropyl-β-cyclodextrin (2 :1mol/mol) and/or 2% (w/v) Tween 80 in the biotransformation process could improve AD and ADD yields, depending on substrates and biotransformation conditions.

Microbial side-chain cleavage of phytosterols by mycobacteria in vegetable oil/aqueous two-phase system.

Microbial side-chain cleavage of natural sterols to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by Mycobacteria has received much attention in pharmaceutical industry, while low yield of the reaction owing to the strong hydrophobicity of sterols is a tough problem to be solved urgently. Eight kinds of vegetable oils, i.e., sunflower, peanut, corn, olive, linseed, walnut, grape seed, and rice oil, were used to construct oil/aqueous biphasic systems in the biotransformation of phytosterols by Mycobacterium sp. MB 3683 cells. The results indicated that vegetable oils are suitable for phytosterol biotransformation. Specially, the yield of AD carried out in sunflower biphasic system (phase ratio of 1:9, oil to aqueous) was greatly increased to 84.8 % with 10 g/L feeding concentration after 120-h transformation at 30 °C and 200 rpm. Distribution coefficients of AD in different oil/aqueous systems were also determined. Because vegetable oils are of low cost and because of their eco-friendly characters, there is a great potential for the application of oil/aqueous two-phase systems in bacteria whole cell biocatalysis.

Accumulation of androstadiene-dione by overexpression of heterologous 3-ketosteroid Δ1-dehydrogenase in Mycobacterium neoaurum NwIB-01

Mycobacterium neoaurum NwIB-01 exhibits powerful ability to cleave the side chain of soybean phytosterols to accumulate 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). The difficulty in separation of AD from ADD is one of the key bottlenecks to the microbial transformation of phytosterols in the industry. To enhance ADD quantity in products, 3-ketosteroid Δ(1)-dehydrogenase genes (kstD M and kstD(A)) were obtained from M. neoaurum NwIB-01 and Arthrobacter simplex respectively. Using replicating vector pMV261, kstD(M) and kstD(A) were overexpressed in M. neoaurum NwIB-01. For foreign gene stable expression, the integration vector pMV306 was used for kstD M/kstD(A) overexpression and the relevant sequences of promoter and kanamycin antibiotic resistance gene sequences were amplified by PCR to verify plasmid integrity. The resultant plasmid and mutant strain were verified and the kstD augmentation mutants were good ADD-producing strains. The ADD producing capacity of NwIB-04 and NwIB-05 was 0.1401 and 0.1740 g/l (cultured in shake bottles with 0.4 g/l phytosterols), and the molar ratio of ADD in products was 98.34 and 98.60%, respectively. This study on the manipulation of the main kstDM gene in Mycobacterium sp. provides a feasible way to achieve excellent phytosterol-transformation strains with high product purity.

Effect of inoculation strategies, substrate to biomass ratio and nitrogen sources on the bioconversion of wood sterols by Mycobacterium sp.

4-Androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) are the main precursors in the production of steroidal drugs from phytosterols. To carry out the bioconversion, different inoculation strategies have been proposed. We compared the use of whole fermented broth and of free resting cells of two mutant strains of Mycobacterium sp. (DSMZ2966 and DSMZ2967) in shake flasks. Also the effect of the nitrogen source (ammonium sulfate, ammonium chloride and ammonium nitrate) and the sterol to biomass ratio at high substrate concentrations (19.2 g/l and 48.1 g/l) was evaluated. We found that the bioconversion with free resting cells (cell pellets) is more efficient than that with whole fermented broth, increasing both AD and ADD production. The use of ammonium nitrate in the culture medium and low substrate to biomass ratios (close to 1.0) increased the production yield. We also found that the bioconversion can be run at high substrate concentration under non-sterile conditions.

A New Steroid-Transforming Strain of Mycobacterium neoaurum and Cloning of 3-Ketosteroid 9α-Hydroxylase in NwIB-01

Using enrichment procedures, five strains that can utilize soybean phytosterols as the sole carbon source were isolated from steroids-contaminated soil samples. Among the isolated strains, the strain NwIB-01 with the highest steroid degradation ability was identified as Mycobacterium neoaurum by morphological, physiological, biochemical tests and 16S rRNA sequence analysis. Meanwhile, the key enzyme gene, which was involved in steroid metabolism and encoding 395-amino acid 3-ketosteroid 9alpha-hydroxylase (KSH), was obtained from M. neoaurum NwIB-01 with the assistance of homology analysis and chromosome walking. To our best knowledge, this is the first report to the gene of key enzyme KSH from M. neoaurum. Strain NwIB-01 exhibited powerful ability of cleaving the side chain specifically from soybean phytosterols to accumulate 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). It was showed that when cultured in 15 g/l phytosterols, the yield of ADD reached 4.23 g/l while accompanied by 1.76 g/l AD in 96-h-old culture (the molar yield of AD + ADD is 64.7%). The strain NwIB-01 can be applied as excellent phytosterols-transformation strains in potential industrial applications.

Methyl-β-cyclodextrin alters growth, activity and cell envelope features of sterol-transforming mycobacteria

Modified beta-cyclodextrins have been shown previously to enhance sterol conversion to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by growing Mycobacterium spp. The enhancement effect was mainly attributed to steroid solubilization by the formation of inclusion complexes with modified cyclodextrins. In this work, the influence of randomly methylated beta-cyclodextrin (MCD) on the growth, AD- and ADD-producing activity, cell wall (CW) composition and ultrastructure of sterol-transforming Mycobacterium sp. VKM Ac-1816D was studied. The specific growth rate of the strain on glycerol increased in the presence of MCD (20-100 mM). Washed cells grown in the presence of MCD (20-40 mM) expressed 1.6-fold higher ADD-producing activity than did the cells grown without MCD, and their adhesiveness differed. Electron microscopy showed MCD-mediated CW exfoliation and accumulation of membrane-like structures outside the cells, while preserving cells intact. The analysis of CW composition revealed both a decrease in the proportion of extractable lipids and a considerable shift in fatty acid profile resulting from MCD action. The MCD-mediated enhancement of mycolic and fatty acids content was observed outside the cells. The total secreted protein level rose 2.4-fold, and the extracellular 3-hydroxysteroid oxidase activity 3.2-fold. The composition of the CW polysaccharide was not altered, while the overall proportion of the carbohydrates in the CW of the MCD-exposed mycobacteria increased. The results showed that the multiple mechanisms of MCD-mediated intensification of sterol to AD(D) conversion by mycobacteria include not only solubilization of steroids, but also the increase of CW permeability for both steroids and soluble nutrients, disorganization of the lipid bilayer and the release of steroid-transforming enzymes weakly associated with the CW.

Molecular and functional characterization of kshA and kshB, encoding two components of 3-ketosteroid 9alpha-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1.

9 alpha-Hydroxylation of 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) is catalysed by 3-ketosteroid 9 alpha-hydroxylase (KSH), a key enzyme in microbial steroid catabolism. Very limited knowledge is presently available on the KSH enzyme. Here, we report for the first time the identification and molecular characterization of genes encoding KSH activity. The kshA and kshB genes, encoding KSH in Rhodococcus erythropolis strain SQ1, were cloned by functional complementation of mutant strains blocked in AD(D) 9 alpha-hydroxylation. Analysis of the deduced amino acid sequences of kshA and kshB showed that they contain domains typically conserved in class IA terminal oxygenases and class IA oxygenase reductases respectively. By definition, class IA oxygenases are made up of two components, thus classifying the KSH enzyme system in R. erythropolis strain SQ1 as a two-component class IA monooxygenase composed of KshA and KshB. Unmarked in frame gene deletion mutants of parent strain R. erythropolis SQ1, designated strains RG2 (kshA mutant) and RG4 (kshB mutant), were unable to grow on steroid substrates AD(D), whereas growth on 9 alpha-hydroxy-4-androstene-3,17-dione (9OHAD) was not affected. Incubation of these mutant strains with AD resulted in the accumulation of ADD (30-50% conversion), confirming the involvement of KshA and KshB in AD(D) 9 alpha-hydroxylation. Strain RG4 was also impaired in sterol degradation, suggesting a dual role for KshB in both sterol and steroid degradation.

Isolation of a biodegradable sterol-rich fraction from industrial wastes

Several industrial waste materials were screened for their sterol content. The possibility of using these industrial by-products as sterol sources for the microbiological production of 4-androsten-3,17-dione (AD) and 1,4-androsta-diene-3,17-dione (ADD) was investigated. Two methods of obtaining the sterol fraction from wastes were developed. Sterol-rich (96–98%) fractions were isolated in a yield above 70%, from a tall-oil effluent of a paper pulp industry and from edible-oil deodorizates. These fractions were subsequently used as a substrate for microbial degradation by a Mycobacterium sp. strain and proved to be easily converted to AD and ADD.

Whole-cell bioconversion of β-sitosterol in aqueous–organic two-phase systems

The selective cleavage of the β-sitosterol side-chain by free Mycobacterium sp. NRRL B-3805 cells was used as a model system for the study of solvent effects in a whole-cell bioconversion in two phase aqueous–organic media. This multi-step degradation pathway leads to the production of 4-androstene-4,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) as a minor product. In an attempt to correlate the substrate and cell partition effects and solvent hydrophobicity (log P) with biocatalytic activity, 15 carboxylic acid esters with log P values between 3 and 10 were screened. The results indicated that the toxicity of the tested solvents in this system could not be correlated to their log P, but seemed to depend on their ability to accumulate in the cells, as these showed a strong affinity towards the organic phase. Different solvent/aqueous ratios and hydrodynamic conditions were further tested in the solvent systems (phthalates) showing significant biodegradation activity. The bioconversion rate was generally not much affected by the stirring speed in the employed range (150–300 rpm) but was strongly influenced by the aqueous/organic phase ratio. Results suggest that the bioconversion takes place at the interphase, its rate being possibly limited by mass transport inside the organic phase.

Isolation of plasmid pRLI from Arthrobacter oxydans 317 and demonstration of its role in steroid 1 (2)‐dehydrogenation

AbstractArthrobacter oxydans 317 capable of complete degradation of β‐sitosterol harbours two plasmids ‐pJLI and pRLI. Standard plasmid isolation methods can easily detect pJLI but not pRLI. The latter plasmid was isolated by a mild procedure using α,α′‐dipyridyl as lytic agent and separated from pJLI by agarose gel electrophoresis using the technique of electro elution into throughs. It's molecular weight was found to be about 3.5 kilo base (kb) pRLI was introduced into the plasmid cured A. oxydans 317 AL. Through genetic transformation using protoplasts of the recepient strain, a transformant strain A. oxydans 317 ALT bearing only pRLI plasmid was obtained. The transformant strain was unable to grow on 4‐androstene‐3, 17‐dione (AD) and 1,4‐androstadiene‐3,17‐dione (ADD) as sole carbon source but a modest growth was observed on 9α‐hydroxy‐4‐androstene‐3, 17‐dione (9α‐hydroxy AD) indicating the ability for steriod 1 (2)‐dehydrogenation and a lack of the capacity for α,α‐hydroxylation. The strain was able to form ADD from AD, and 1 (2)‐dehydrogenation of 3‐oxochol‐4‐en24‐oic acid from β‐sitosterol. Direct estimation of steroid (1 (2)‐dehydrogenase activity showed that the transformant strain and parent strain containing pRLI had high enzyme activity where as strains lacking in this plasmid had negligible enzyme activity. It was concluded that pRLI is responsible for steroid 1 (2)‐dehydrogenation in A. oxydans 317.

Role of plasmid pJL1 of Arthrobacter oxydans 317 in the degradation of β‐sitosterol

AbstractArthrobacter oxydans 317 is a plasmid (pJL1)‐harbouring organism capable of complete degradation of β‐sitosterol, 4‐androstene‐3,17‐dione (AD) and 1,4‐androstadiene‐3,17‐dione (ADD). Plasmid‐cured strain A. oxydans 317 A1 obtained by treatment of A. oxydans 317 with sodium dodecylsulphate (DUTTA et al. 1989) is capable of only partial degradation of β‐sitosterol side chain and incapable of utilizing AD and ADD. Conjugation experiments were carried out using A. oxydans 317 A1 as the recipient and A. oxydans 317 as the donor of pJL1 plasmid. A transconjugant strain A. oxydans 317 TC containing pJL1 plasmid was obtained. The transconjugant was able to form 9 α‐hydroxy‐4‐androstene‐3,17‐dione (9α‐hydroxy AD) from β‐sitosterol and AD, and to utilize ADD for growth but it could not convert AD to ADD. It was proposed that pJL1 is involved in the conversion of 3‐oxochol‐4‐en‐24‐oic acid to AD and in the 9α‐hydroxylation of the steroid nucleus but it is not connected with 1,2‐dehydrogenation, a critical reaction step in steroid ring opening.

Spectrophotometric estimation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153

A spectrophotometric method for simultaneously estimating 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) in a binary mixture has been developed using sulphuric acid chromogens. The method has been used to estimate both AD and ADD during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153.

Bioconversion of lipophilic compounds in non-aqueous solvent. Effect of gel hydrophobicity on diverse conversions of testosterone by gel-entrapped Nocardia rhodocrous cells

The bioconversion of testosterone (TS) in water-saturated benzene-n-heptane (4:1 by volume) was mediated by Nocardia rhodocrous cells whose steroid Δ1-dehydrogenase and 17β-hydroxysteroid dehydrogenase were induced by TS. TS was transformed into 4-androstene-3,17-dione (4-AD), dehydrotestosterone (DTS) and 1,4-androstadiene-3, 17-dione (ADD) by incubating with the cell suspensions in the presence of phenazine methosulfate (PMS). Time-courses of TS transformation revealed that DTS and 4-AD were produced initially and further oxidized to ADD. Thus, the final product, ADD; was formed via two different pathways: TS→4-AD→ADD and TS→DTS→ADD. In these routes, Δ1-dehydrogenation required PMS, while 17β-dehydrogenation could proceed without any exogenous electron acceptor. N. rhodocrous cells entrapped in hydrophilic gels (H-gel) and lipophilic gels (L-gel) prepared by photo-crosslinkable resin prepolymers and urethane prepolymers were useful for effective dehydrogenations of TS. The cells entrapped in L-gels produced 4-AD as the major product, whereas DTS was the main product by the cells in H-gel. The difference in the profiles of dehydrogenation products can be explained by low affinity of PMS for L-gel-entrapped cells and of TS for H-gel-entrapped cells. Inhibitory effect of DTS on 17β-hydroxysteroid dehydrogenase also would be responsible for the accumulation of DTS in the latter case. Thus, different routes for product formation could be selected by using resin prepolymers of appropriate hydrophilicity or hydrophobicity for entrapment of biocatalysts.