Conversion of phytosterols into androstenedione by Mycobacterium neoaurum

Optimum conditions for transformation of phytosterols by Mycobacterium neoaururm, required for selective cleavage of the lateral chain into androstenedione, were shown to differ from the known conditions of animal sterol (cholesterol) transformation. Complete conversion of phytosterols into androstenedione at a substrate load of no less than 20 g/l was achieved on increasing the amount of the inoculum and the concentration of glucose (by 2 and 4 times, respectively, relative to cholesterol) and performing the fermentation under conditions of turbulent mixing. Under these conditions, both the rate of the transformation and the yield of the reaction product were high, due to the saturation of the culture liquid with hydrocarbonate. Data from the literature show that this ion is involved in cleavage of the branched lateral chain at carbon in position 24.     

Androstenedione production by biotransformation of phytosterols

Androstenedione is a key intermediate of microbial steroid metabolism. It belongs to the 17-keto steroid family and is used as starting material for the preparation of different steroids. Androstenedione can be produced by microbial side chain cleavage of phytosterol, which is an alternative to multi-step chemical synthesis. In this review, various methods of biotransformation of sterols to androstenedione are surveyed. It begins with the history and current research status in this field. The existing methods of chemical and biochemical synthesis are examined. Various issues related to these methods and how researchers have addressed them is presented. Among these, the low solubility of sterols in aqueous systems is a critical problem since it limits the product yield. The main content of this review focuses on new methods of biotransformation that are being investigated. Recent biotechnological advances in this field are presented. The review ends with a note on future perspectives.     

Enhancement of androstadienedione production from progesterone by biotransformation using the hydroxypropyl-beta-cyclodextrin complexation technique

The enhanced production of androstadienedione (ADD) from progesterone (P) using the hydroxypropyl-beta-cyclodextrin (HPbetaCD) complexation technique by biotransformation was demonstrated. The microorganisms used were Bacillus sphaericus ATCC 245, B. sphaericus ATCC 7063, B. sphaericus ATCC 13805, Arthrobacter simplex ATCC 6946, B. sphaericus TISTR 670 and those screened from soils in Chiang Mai Province, Thailand which were B. sphaericus SRP I, B. sphaericus SRP II and B. sphaericus SRP III. The complexed (P-complex) and the uncomplexed P at 0.3-1.2mg/ml were investigated. Samples were withdrawn from the bioconversion mixture at various time intervals for 168 h. The ADD and P contents were determined by HPLC. All organisms showed ADD production from either P or P-complex by one-step biotransformation (including side chain cleavage and dehydrogenation). At 0.3mg/ml of P in the systems of B. sphaericus ATCC 13805, A. simplex ATCC 6946 and B. sphaericus ATCC 245, the uncomplexed form showed the highest ADD yield of 2.82, 1.63 and 64.67% at 168, 168 and 144 h, whereas P-complex gave 98.44, 19.58 and 97.10% at 144, 24 and 168 h, respectively. This indicated an increase of ADD production from the P-complex in comparison to P of 35, 12 and 1.5 times, respectively. This study has shown that the complexation of P with HPbetaCD enhanced the ADD production in a novel one-step bioconversion.     

Enhancement of 17alpha-hydroxyprogesterone production from progesterone by biotransformation using hydroxypropyl-beta-cyclodextrin complexation technique

An enhancement of 17α-hydroxyprogesterone (17α-HP) production from progesterone by biotransformation using hydroxypropyl-β-cyclodextrin (HPβCD) complexation together with aeration and sonication technique was demonstrated. The progesterone–hydroxypropyl-β-cyclodextrin complex was prepared by co-evaporation method. The percentage yield of 17α-HP from P of 11.26 ± 0.64% at 24 h was observed in Curvularia lunata ATCC 12017. In the complex form of P, together with sonication at 40 kHz for 5 s and aeration, the yield of 17α-HP was increased to 72.92 ± 4.28% which was about 6.5 and 1.3 times of that from the uncomplexed (P) and the complexed (PC), respectively without sonication and aeration. The increased aqueous solubility of P by complexation with HPβCD was the main factor which increased the yield of 17α-HP, while aeration had more effect on P than PC. Sonication did not significantly increased the yield of the product from both P and PC. When both aeration and sonication were used in the PC system, the product yield was increased significantly more than that from P. The result from this study can be applied for the biotransformation of other poor aqueous soluble precursors.     

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.     

Mycobactin and the competition for iron between Mycobacterium neoaurum and M. vaccae

Two closely related species of mycobacteria, Mycobacterium vaccae and M. neoaurum, were grown under conditions of iron-deficiency (0.02-0.05 microgram Fe ml-1) and iron-sufficiency (2-4 micrograms Fe ml-1) in a simple glycerol/asparagine medium. The strain of M. vaccae used was a nonmycobactin producer whereas M. neoaurum synthesized between 6-8% of its cell biomass as the lipid-soluble siderophore when grown under iron-limitation. The role of mycobactin for iron-acquisition was examined using both pure and mixed cultures, with cell viability determined following growth at various iron concentrations. M. neoaurum, the mycobactin producer, outgrew M. vaccae when iron was readily available. When grown under conditions where iron was limiting, M. neoaurum showed a decline in viable cell number compared with its competitor, highlighting its increased requirement for the metal. Some recovery was observed following mycobactin biosynthesis, this being greatly enhanced by the addition of an iron supplement to the growing cells. Mycobactin biosynthesis allowed M. neoaurum to rapidly acquire any additional iron presented to the bacteria when growing under iron-limitation. However, M. vaccae did not synthesize the lipid-soluble siderophore with its iron-requirement satisfied by production of extracellular exochelin.     

Isolation and identification of Mycobacterium neoaurum from a patient with urinary infection

Mycobacterium neoaurum is a novel species of Mycobacteria, until now only isolated from catheters in immunosuppressed patients. This report describes the isolation and identification of M. neoaurum from urine obtained from a hospitalized patient.     

Purification and Characterization of an l-Amino Amidase from Mycobacterium neoaurum ATCC 25795

An l-amino amidase from Mycobacterium neoaurum ATCC 25795 responsible for the enantioselective resolution of dl-alpha-methyl valine amide was purified and characterized. The purification procedure included ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography, which resulted in a homogeneous preparation of the enzyme with a native molecular mass of 136 kDa and a subunit molecular mass of 40 kDa. The purified enzyme displayed the highest activity at 50 degrees C and at pH 8.0 and 9.5. The enzyme was strongly inhibited by the metal-chelating agent 1,10-phenanthroline, the disulfide-reducing agent dithiothreitol, and the cysteine proteinase inhibitor iodoacetamide. The purified amino amidase showed a unique l-enantioselective activity towards a broad range of both alpha-H- and alpha-alkyl-substituted amino acid amides, with the highest activity towards the cyclic amino acid amide dl-proline amide. No activity was measured with dl-mandelic acid amide nor with the dipeptide l-phenylalanine-l-leucine. The highest catalytic efficiency (k(cat)/K(m) ratio) was measured with dl-alpha-allyl alanine amide, dl-alpha-methyl phenylalanine amide, and dl-alpha-methyl leucine amide.     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria Mycobacterium neoaurum, Pimelobacter simplex, and Rhodococcus erythropolis

Abstract-Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9alpha-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5-15 nm) or the transformation in the presence of randomly methylated beta-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The technical product ADD was obtained in 75% yield, based on phytosterol. It contained as impurity 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment-the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD-no by products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5 AD was combined. The yield of 9-OH-AD (m.p. 218-220 degrees C) based on transformed phytosterol was 56%.     

Biodegradation and biotransformation of groundwater pollutant mixtures by Mycobacterium vaccae.

Mycobacterium vaccae can catabolize a number of major groundwater pollutants. When added singly, acetone, cyclohexane, styrene, benzene, ethylbenzene, propylbenzene, dioxane, and 1,2-dichloroethylene can be catabolized by M. vaccae. Catabolism of a number of these chemicals was monitored by gas-chromatographic analysis. Gas-chromatographic analysis indicated that the products of benzene degradation are phenol and hydroquinone. The products of chlorobenzene and ethylbenzene degradation are 4-chlorophenol and 4-ethylphenol. The extent that some compounds were catabolized when present as mixtures was also investigated. When toluene and benzene were present concomitantly, toluene was catabolized and benzene oxidation was delayed. Although toluene promoted the degradation of styrene, a lower rate of toluene degradation occurred when styrene was present. Both 4-chlorophenol and 4-ethylphenol had an antagonistic effect on the ability of M. vaccae to degrade other aromatic compounds. Studies with [14C]benzene indicated that M. vaccae can mineralize small amounts of this compound. These results suggest that components in mixtures may have a positive or a negative effect on the rates of biodegradation of other pollutants.     

Isolation and partial characterization of a new mutant for sterol biotransformation in Mycobacterium sp.

Summary A new sterol biotransforming mutant was isolated from NRRL-B3683 Mycobacterium sp. after nitrosoguanidine mutagenesis. The mutant showed an enhanced ability to biotransform stigmasterol into 17-ketosteroids compared with the parental strain.     

Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) in Mycobacterium neoaurum

Abstract Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 μg/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 μg/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 μg/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.     

Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) in Mycobacterium neoaurum

Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 micrograms/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 micrograms/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 micrograms/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.     

Oral absorption of phytosterols and emulsified phytosterols by Sprague-Dawley rats

Clinical studies have demonstrated that consumption of phytosterol esters in lipid-based foods decreases serum concentrations of total and LDL cholesterol. These substances represent minimal potential for adverse effects when consumed orally because of their low bioavailability. However, some studies have reported estrogenic and other effects in laboratory animals treated parenterally with phytosterols, demonstrating that these substances may have the potential to cause adverse effects if absorbed. Water-soluble phytosterols have been prepared by formulation with emulsifiers to expand delivery options to include non-lipid-based foods. However, emulsifiers are used as excipients in the formulation of lipophilic pharmaceuticals to increase solubility, thereby increasing their absorption. Therefore, oral consumption of emulsified water-soluble phytosterols could potentially increase their absorption. In the current study, absorption of phytosterols prepared as water-soluble emulsified micelles with two different food-grade emulsifiers was evaluated in Sprague-Dawley rats and compared with absorption of non-micellar free phytosterols and esterified phytosterol mixtures dissolved in a lipophilic vehicle (soybean oil). Rats were dosed via gavage with 42 mg/kg of formulated phytosterol preparations. Blood was collected at 8, 16, 24, and 32 hours, extracted with hexane, derivatized with benzoyl chloride, and analyzed by high-performance liquid chromatography to determine concentrations of beta-sitosterol, and campesterol. Plasma concentrations and AUC(0-32 hours) [microg/mL/h] of beta-sitosterol and campesterol were lower in plasma obtained from rats treated with emulsified phytosterol preparations than in animals treated with free phytosterols dissolved in soybean oil. Because the pharmacokinetic profile of water-soluble phytosterols is similar to that of phytosterols administered in a lipid vehicle, the safety profile is likely to be the same as that of phytosterols and phytosterol esters in currently used applications.     

Genetic characterization of the Guinea-Bissau family of Mycobacterium tuberculosis complex strains

In a previous study of Mycobacterium tuberculosis complex isolates from Guinea-Bissau in West Africa, we identified a unique group of strains, designated here as the Guinea-Bissau family of strains, which, although genotypically closely related, phenotypically demonstrated a considerable heterogeneity. We conducted here a detailed genotypic analysis of a subset (n = 35) of these isolates. Based on the data obtained, and by comparison of known corresponding genes in mycobacteria outside the M. tuberculosis complex, we propose that the Guinea-Bissau strains belong to a unique branch of the M. tuberculosis complex tree in between classical M. tuberculosis and classical M. bovis. These observations are discussed in their significance in M. tuberculosis complex classification.     

Degradation of the multiple branched alkane 2,6,10,14-tetramethyl-pentadecane (pristane) in Rhodococcus ruber and Mycobacterium neoaurum

Pristane, a highly branched hydrocarbon that also contains iso-branched termini, was used as a substrate for several alkane-metabolizing bacteria. Rhodococcus ruber and Mycobacterium neoaurum were able to utilize pristane for growth effectively. The intermediates produced by these bacteria during incubation with pristane were analyzed by gas chromatography (GC) and gas chromatography/mass spectra (GC/MS). The products revealed as products of 4-methyl pentanoic acid; methyl butanedioic acid; 2-methyl pentadioic acid; methyl propanedioic acid; 4-methyl heptanedioic acid; and 2,6,10,14-tetramethyl-pentadecan-3-one were detected in M. neoaurum cultures. In R. ruber, methyl butanedioic acid; 2-methyl pentadioic acid; 4,8-dimethylnonanoic acid, 4-methyl heptanedioic acid; 2,6,10-trimethylundecanoic acid; 3,7-dimethyl decanedioic acid; and 2,6,10,14-tetramethyl-pentadecan-3-one were detected. The occurrence of these intermediates showed that pristane could be catabolized not only via mono- but also by a di-terminal oxidation pathway. Furthermore, the presence of 2,6,10,14-tetramethyl-pentadecan-3-one; 3,7-dimethyldecandioate; and 2-methylbutandioate established a third pathway initiated by sub-terminal oxidation at the third carbon atom of pristane. Novel intermediates detected suggest simultaneous sub-terminal and di-terminal oxidation pathways.     

Preparation and characterization of a single-chain calcineurin-calmodulin complex

Calcineurin (CN), a Ca(2+)/calmodulin (CaM)-dependent serine/threonine protein phosphatase, is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). The activity of CNA is under the control of two functionally distinct, but structurally similar Ca(2+)-regulated proteins, CaM and CNB. The crystal structure of the holoenzyme reveals that the N-terminus and C-terminus of CNB and the N-terminus of CNA each have a long arm not involved in the active site. We constructed a fusion of the genes of CaM, CNB and CNA in that order using linker primers containing six and ten codons of glycine. A single-chain CaM-CNB-CNA (CBA) complex was expressed and purified to near homogeneity. The single-chain complex was fully soluble, and had biochemical properties and kinetic parameters similar to single-chain CNB-CNA (BA) activated by CaM. It was not regulated by CaM and CNB, but was strongly stimulated by Mn2+, Ni2+ and Mg2+. Intrinsic fluorescence spectroscopy of the complex showed a change in the environment of tryptophan in the presence of Ca2+ and circular dichroism (CD) spectropolarimetry revealed an increase in alpha-helical content. Our findings suggest that fusion of CaM, CNB and CNA does not prevent the structural changes required for their functioning; in particular, CaM within the complex could still interact correctly with CN in the presence of Ca2+.     

工业分枝杆菌植物甾醇转化途径及菌种改造研究进展

相比于传统的化学转化法,微生物转化法在甾体药物的生产中显示出了明显的优势.利用分枝杆菌降解植物甾醇可以生成一系列甾体药物的中间体,这极大地方便了甾体药物的生产.通过基因工程、分子生物学和结构生物学等学科的技术手段,人们对甾醇转化菌株进行了深入的探索和改造.本文对工业分枝杆菌植物甾醇转化途径及菌种改造的研究进展进行了综述...     

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.