Cunninghamella Elegans Research Articles

The impact of biofertilizers with diazotrophic bacteria and fungi chitosan on melon characteristics and nutrient uptake as an alternative for conventional fertilizers

Fertilization is one of the most important factors to improve plant characteristics and nutrient uptake. Biological N2 Fixation (BNF) is a process of great importance in crop production systems, to enhance sustainability and prevent land degradation in modern agriculture. The impact of the biofertilizer (NPKB) produced from PK rock biofertilizer mixed with earthworm compound enriched in N by free living diazotrophic bacteria and of the bioprotector (NPKP) with chitosan from Cunninghamella elegans were investigated in a field experiment with melon. The study compared the influence of biofertilizers with soluble fertilizers (NPKF) on the melon characteristics and nutrient uptake. The experiment was conducted (February–April 2012) in the Bahia state, Brazil, and the treatments were NPKP (4tha−1); NPKP (8tha−1); NPKP (12tha−1); NPKB (8tha−1); NPKB (12tha−1); and NPKF (soluble mineral fertilizers) at the recommended rate for irrigated melon. A control with an earthworm compound (20tha−1) that was not inoculated with diazotrophic bacteria was added as a comparison. The results demonstrated a positive effect of NPKP and NPKB, especially when applied at a higher rate (12tha−1), which increased the commercial characteristics of melon compared with the soluble fertilizer applied in the recommended rate. Nutrient uptake in the melon fruits indicated a significant difference among the different fertilizer sources and the best results were displayed with NPKB and NPKP applied at the highest rate (12tha−1), especially for total S-SO4−2. Therefore, NPKP and the NPKB are potential alternatives to soluble fertilizers (NPKF).

Biotransformation of Salpichrolides A, C, and G by Three Filamentous Fungi.

Incubation of salpichrolide A (1) with Rhizomucor miehei produced hydroxylation in rings B and C (C-7 and C-12) and led to C-5-C-6 epoxide opening, while incubation of salpichrolides C (2) and G (3) with R.miehei led to epoxide opening at the C-24-C-25 and C-5-C-6 positions, respectively. Biotransformation of salpichrolide A (1) with Cunninghamella elegans produced stereoselective hydroxylated, oxidized, and reduced derivatives in different positions of the A, B, and C rings and C-5-C-6 epoxide opening. In addition, selective epoxide opening at the C-5-C-6 or C-24-C-25 positions was obtained from the incubation of salpichrolide A (1) with Curvularia lunata.

New insight into microbial diversity and functions in traditional Vietnamese alcoholic fermentation

The roles of microorganisms in traditional alcoholic fermentation are often assumed based on abundance in the starter and activity in pure culture. There is a serious lack of hard evidence on the behavior and activity of individual microbial species during the actual fermentation process. In this study, microbial succession and metabolite changes during 7days of traditional Vietnamese alcoholic fermentation were monitored. Special attention was devoted to starch degradation. In total, 22 microbial species, including 6 species of filamentous fungi (Rhizopus microsporus, Rhizopus arrhizus, Mucor indicus, Mucor circinelloides, Cunninghamella elegans, Aspergillus niger), 1 yeast-like fungus (Saccharomycopsis fibuligera), 7 yeasts (Saccharomyces cerevisiae, Clavispora lusitaniae, Wickerhamomyces anomalus, Lindnera fabianii, Pichia kudriavzevii, Candida rugosa, Candida tropicalis), and 8 bacteria (Stenotrophomonas maltophilia, Lactobacillus brevis, Lactobacillus helveticus, Acinetobacter baumannii, Staphylococcus hominis, Bacillus megaterium, Enterobacter asburiae, Pediococcus pentosaceus) were identified. Despite the presence of a complex microbiota in the starter, the fermentation process is consistent and involves a limited number of functional species. Rapid change in microbial composition of fermentation mash was observed and it was correlated with ethanol content. Microbial biomass reached maximum during first 2days of solid state fermentation. Acidification of the medium took place in day 1, starch degradation in days 2, 3, 4, and alcohol accumulation from day 3. Although Sm. fibuligera dominated by cell count amongst potential starch degraders, zymography indicated that it did not produce amylase in the fermentation mash. In mixed culture with Rhizopus, amylase production by Sm. fibuligera is regulated by the moisture content of the substrate. Rhizopus was identified as the main starch degrader and S. cerevisiae as the main ethanol producer. Bacterial load was high but unstable in species composition and dominated by acid producers. M. indicus, Sm. fibuligera, W. anomalus and bacteria were regarded as satellite microorganisms. Their possible influence on organoleptic quality of fermentation product was discussed.

English

Microbial biotransformation of coumarin (I) was undertaken using a battery of twenty five microorganisms. Among them, the fungus Cunninghamella elegans NRRL 1392 was the sole microbe that efficiently metabolized coumarin (I) into umbelliferone (II), 3, 4-dihydrocoumarin (V) and transcinnamic acid (VI). Umbelliferone was also biotransformed into 6, 7-dihydroxycoumarin (aesculetin, VII) and p-coumaric acid (VIII). On the other hand, warfarin (III) was biotransformed into 4`-hydroxywarfarin (IX) and also dicoumarol (IV) was transformed into 4-hydroxycoumarin (X) by the same microorganism. The structures of the metabolites were established using only one property and spectroscopic techniques including melting points, 1H NMR, 13C NMR and mass spectroscopy. The cytotoxic effect of coumarin substrates and their metabolites was also investigated. Key words: Biotransformation, coumarins, Cunninghamella elegans, cytotoxic.

The potentiality of cross-linked fungal chitosan to control water contamination through bioactive filtration

Water contamination, with heavy metals and microbial pathogens, is among the most dangerous challenges that confront human health worldwide. Chitosan is a bioactive biopolymer that could be produced from fungal mycelia to be utilized in various applied fields. An attempt to apply fungal chitosan for heavy metals chelation and microbial pathogens inhibition, in contaminated water, was performed in current study. Chitosan was produced from the mycelia of Aspergillus niger, Cunninghamella elegans, Mucor rouxii and from shrimp shells, using unified production conditions. The FT-IR spectra of produced chitosans were closely comparable. M. rouxii chitosan had the highest deacetylation degree (91.3%) and the lowest molecular weight (33.2kDa). All chitosan types had potent antibacterial activities against Escherichia coli and Staphylococcus aureus; the most forceful type was C. elegans chitosan. Chitosan beads were cross-linked with glutaraldehyde (GLA) and ethylene-glycol-diglycidyl ether (EGDE); linked beads became insoluble in water, acidic and alkaline solutions and could effectively adsorb heavy metals ions, e.g. copper, lead and zinc, in aqueous solution. The bioactive filter, loaded with EGDE- A. niger chitosan beads, was able to reduce heavy metals’ concentration with >68%, and microbial load with >81%, after 6h of continuous water flow in the experimentally designed filter.

Evaluation of fluorinated biphenyl ether pro-drug scaffolds employing the chemical-microbial approach

Incorporation of fluorine in a drug can dramatically affect its metabolism and methods to assess the effect of fluorine substitution on drug metabolism are required for effective drug design. Employing a previously developed chemical-microbial method the metabolism of a series of fluorinated biphenyl ethers was determined. The substrates were synthesized via Ullmann-type condensation reactions between bromotoluene and fluorophenol. The ethers were incubated with the fungus Cunninghamella elegans, which oxidises xenobiotics in an analogous fashion to mammals, generating a number of hydroxylated biphenyl ethers and acids. The propensity of the fluorinated ring to be hydroxylated depended upon the position of the fluorine atom, and the oxidation of the methyl group was observed when it was meta to the oxygen. The experiments demonstrate the applicability of the method to rapidly determine the effect of fluorine substitution on CYP-catalysed biotransformation of pro-drug molecules.

Data on individual metabolites of synthetic cannabinoids JWH-018, JWH-073 and AM2201 by Cunninghamella elegans.

Synthetic cannabinoids JWH-018, JWH-073 and AM2201 were metabolised by the fungus Cunninghamella elegans. In this article, data on individual metabolites of their retention times, mass accuracies, major product ions and structures indicated by product ions are presented. The data in this article is related to “Biotransformation of synthetic cannabinoids JWH-018, JWH-073 and AM2201 by Cunninghamella elegans” [1].

Quinoline biodegradation by filamentous fungus Cunninghamella elegans and adaptive modifications of the fungal membrane composition.

Quinoline, which belongs to N-heterocyclic compounds, occurs naturally in the environment and is used in numerous industrial processes. The structures of various chemicals, such as dyes and medicines, are based on this compound. Due to that fact, quinoline and its derivatives are widely distributed in environment and can exert toxic effects on organisms from different trophic levels. The ability of the filamentous fungus Cunninghamella elegans IM 1785/21Gp to degrade quinoline and modulate the membrane composition in response to the pollutant was studied. C. elegans IM 1785/21Gp removes quinoline with high efficiency and transforms the pollutant into two novel hydroxylated derivatives, 2-hydroxyquinoline and 3-hydroxyquinoline. Moreover, due to the disruption in the membrane stability by quinoline, C. elegans IM 1785/21Gp modulates the fatty acid composition and phospholipid profile.

Enantioselective Conversion of Racemic Felodipine to S(-)-Felodipine by <i>Aspergillus niger</i> and Lipase AP6 Enzyme

The present study involves the enantioselective resolution of racemic Felodipine by using free and immobilized forms of microbial cultures as well as an enzyme (Lipase AP6). Among the microbial cultures employed in the present study, Aspergillus niger, Sphingomonas paucimobilis, Cunninghamella elegans, Escherichia coli, Pseudomonas putida and Cunninghamella blakesleeana were found to possess capability of enantioselective resolution of racemic Felodipine. The enantiomeric excess (ee%) of Felodipine after reaction catalyzed by whole-cell A. niger and S. paucimobilis was found as 81.59 and 71.67%, respectively. Immobilization enhanced the enantioselectivity (enantiomeric ratio (E)) of the biocatalysts and hence this led to enhanced enantiomeric purity of the drug. The ee% values were found to be enhanced in reactions catalyzed by A. niger and S. paucimobilis cultures after immobilization as 98.27 and 93.56%, respectively. Enantiomeric ratio (E) of the reactions catalyzed by all the biocatalysts has been improved after immobilization. E value of the reaction catalyzed by immobilized A. niger was found to be excellent (E > 100) and hence the drug showed high enantiomeric purity. In lipase AP6 catalyzed study, the enantioselectivity was enhanced after immobilization with excellent E value, which led to enhanced enantiomeric purity of the drug (99.21% ee%).

Microbial transformation of methyl cyperenoate by Cunninghamella elegans AS 3.2028 and the antithrombotic activities of its metabolites

Microbial transformation is a remarkable tool for the structural modification of bioactive natural compounds converting them into more valuable biologically active derivatives.

Biotransformation of synthetic cannabinoids JWH-018, JWH-073 and AM2201 by Cunninghamella elegans

Being marketed as “legal” smoking blends or mixtures, synthetic cannabinoids are abused widely owing to its cannabis-like effect. Due to the rapid introduction of new generation analogues of synthetic cannabinoids to escape from legislative/judicial control, the investigation of the metabolic pathways of these substances is of particular importance for drug control, abstinence and forensic toxicology purposes. In this study, the in vitro metabolism of JWH-018, JWH-073 and AM2201 by the fungus Cunninghamella elagans has been investigated with the purpose of validating its potential as a complementary model for investigating synthetic cannabinoid metabolism. JWH-018, JWH-073 and AM2201 were incubated for 72h with C. elegans. Detection of metabolites was based on liquid chromatography–tandem mass spectrometry and high resolution mass spectrometry analysis. C. elegans was found capable of producing the majority of the phase I metabolites observed in earlier in vitro and in vivo mammalian studies as a result of monohydroxylation, dihydroxylation, carboxylation, dehydrogenation, ketone formation, dihydrodiol formation, dihydrodiol formation with N-dealkylation and combinations thereof. C. elegans can thus be a useful and economic model for studying synthetic cannabinoid metabolism.

Bio-clarification of water from heavy metals and microbial effluence using fungal chitosan

Water pollution is among the most hazardous problems that threaten human health worldwide. Chitosan is a marvelous bioactive polymer that could be produced from fungal mycelia. This study was conducted to produce chitosan from Cunninghamella elegans and to use it for water pollutants elimination, e.g. heavy metals and waterborne microorganisms, and to investigate its antibacterial mode of action against Escherichia coli. The produced fungal chitosan had a deacetylation degree of 81%, a molecular weight of 92.73kDa and a matched FT-IR spectrum with standard shrimp chitosan. Fungal chitosan exhibited remarkable antimicrobial activity against E. coli, Staphylococcus aureus and Candida albicans. Chitosan was proved as an effective metal adsorbent, toward the examined metal ions, Cu2+, Zn2+ and Pb2+, and its adsorption capacity greatly increased with the increasing of metal concentration, especially for Cu and Zn. The scanning electron micrographs, of treated E. coli cells with fungal chitosan, indicated that the cells began to lyse and combine after 3h of exposure and chitosan particles attached to the combined cells and, after 12h from exposure, the entire bacterial cell walls were fully disrupted and lysed.Therefore, fungal chitosan could be recommended, as a bioactive, renewable, ecofriendly and cost effective material, for overcoming water pollution problems, from chemical and microbial origins.

Identification and formation pattern of metabolites of cyazofamid by soil fungus Cunninghamella elegans

This study was performed to investigate the formation of microbial metabolites from cyazofamid by the soil fungus Cunninghamella elegans. The incubation of cyazofamid with C. elegans was conducted for 10 days. Cyazofamid disappeared after 7 days of incubation, producing three metabolites. Metabolites identified by liquid chromatography–tandem mass spectrometry were 4-chloro-5-(4-(hydroxymethyl)phenyl)-imidazole-2-carbonitrile (CHCN), 4-(4-chloro-2-cyanoimidazole-5-yl)benzoic acid (CCBA) and 4-chloro-2-cyano-5-(4-(hydroxymethyl)phenyl)N,N-dimethyl-1H-imidazole-1-sulfonamide (CCHS). A new metabolite, CCHS, was further confirmed by 1H-13C HSQC (heteronuclear single-quantum correlation) using nuclear magnetic resonance. As a possible metabolic pathway, cyazofamid could be oxidized to CCHS, degraded to CHCN and further oxidized to CCBA. The metabolic system of C. elegans would be a powerful tool for predicting and identifying phase I metabolites that could be formed in mammalian systems.

Cowpea resistance induced against Fusarium oxysporum f. sp. tracheiphilum by crustaceous chitosan and by biomass and chitosan obtained from Cunninghamella elegans

Pathogenic microorganisms increase the production of reactive oxygen species (ROS), and the response can cause oxidative damage and even death in a plant. Plant resistance inducers activate antioxidant enzymes such as catalase (CAT) and peroxidase (POX). The aims of this study were to evaluate the antimicrobial activity and the potential to induce the CAT and POX activities by the biopolymer chitosan in cowpea plants inoculated with the phytopathogen Fusarium oxysporum f. sp. tracheiphilum. The antimicrobial activity of crustaceous chitosan (CrCh) was tested in a laboratory assay and analyzed by optic and scanning electron microscopy. In a greenhouse experiment, the effectiveness of CrCh, fungal chitosan obtained from Cunninghamella elegans (C.eCh), and C. elegans biomass (C.eCh source) plus NPK biofertilizer, named bioprotector (C. elegans+NPKB), were evaluated in relation to the disease severity and the CAT and POX activities in cowpea plants infected with F. oxysporum f. sp. tracheiphilum. The higher rates of CrCh (4.0–6.0mgmL−1) showed the total fungicide ability, and the other concentrations (0.5–3.0mgmL−1) promoted a greater fungistatic effect and morphological changes on the growth of F. oxysporum f. sp. tracheiphilum during the antimicrobial test in vitro. Chitosan effectively protected cowpea plants against this pathogen. The higher chitosan concentrations (4.0 and 6.0mgmL−1) were directly related to CAT and POX activity, which controlled the ROS equilibrium for plant resistance and resulted in a significant reduction of the disease severity in cowpea. These findings are important for the establishment of a sustainable agriculture and to avoid the use of pesticides.

Carbazole hydroxylation by the filamentous fungi of the Cunninghamella species.

Nitrogen heterocyclic compounds, especially carbazole, quinolone, and pyridine are common types of environmental pollutants. Carbazole has a toxic influence on living organisms, and the knowledge of its persistence and bioconversion in ecosystems is still not complete. There is an increasing interest in detoxification of hazardous xenobiotics by microorganisms. In this study, the ability of three filamentous fungi of the Cunninghamella species to eliminate carbazole was evaluated. The Cunninghamella elegans IM 1785/21Gp and Cunninghamella echinulata IM 2611 strains efficiently removed carbazole. The IM 1785/21Gp and IM 2611 strains converted 93 and 82 % of the initial concentration of the xenobiotic (200 mg L−1) after 120 h incubation. 2-Hydroxycarbazole was for the first time identified as a carbazole metabolite formed by the filamentous fungi of the Cunninghamella species. There was no increase in the toxicity of the postculture extracts toward Artemia franciscana. Moreover, we showed an influence of carbazole on the phospholipid composition of the cells of the tested filamentous fungi, which indicated its harmful effect on the fungal cell membrane. The most significant modification of phospholipid levels after the cultivation of filamentous fungi with the addition of carbazole was showed for IM 1785/21Gp strain.

Microbial transformaton of bavachin by Absidia coerulea

Bavachin is one of the major bioactive flavonoid constituents in Fructus Psoraleae (Buguzhi in Chinese), and it displays a broad range of biological activities such as antibacterial, antifungal, anti-inflammatory, antioxidant, antitumor, phytoestrogenic, α-glucosidase inhibitory, and osteoblastic proliferation stimulating activities. In order to improve its bioavailability, we investigated the application of microorganisms as biocatalysts for the transformation of bavachin. Screening studies using twenty two microbes indicated that bavachin could be transformed by several fungal strains including Absidia coerulea, Mucor hiemalis, and Cunninghamella elegans. And our subsequent scale-up fermentation studies of bavachin using A. coerulea resulted in the identification of two novel microbial metabolites together with a recently reported compound bavachinone A. Chemical structures of the three compounds were elucidated on the basis of spectroscopic analysis. This is the first report on the biotransformation of bavachin by microbes, which suggested a potential process to prepare novel derivatives from plant secondary metabolites through microbial enzymes.

Production of drug metabolites by immobilised Cunninghamella elegans: from screening to scale up.

Cunninghamella elegans is a fungus that has been used extensively as a microbial model of mammalian drug metabolism, whilst its potential as a biocatalyst for the preparative production of human drug metabolites has been often proposed, little effort has been made to enable this. Here, we describe a workflow for the application of C. elegans for the production of drug metabolites, starting from well-plate screening assays leading to the preparative production of drug metabolites using fungus immobilised either in alginate or as a biofilm. Using 12- and 96-well plates, the simultaneous screening of several drug biotransformations was achieved. To scale up the biotransformation, both modes of immobilisation enabled semi-continuous production of hydroxylated drug metabolites through repeated addition of drug and rejuvenation of the fungus. It was possible to improve the productivity in the biofilm culture for the production of 4'-hydroxydiclofenac from 1 mg/l h to over 4 mg/l h by reducing the incubation time for biotransformation and the number of rejuvenation steps.

Microbial transformations of betulinic and betulonic acids

Enzymatic transformation of betulinic acid by growing cells of microorganisms provided several hydroxylated and oxidized products. Bacillus megaterium SC16644 gave 7β,15α-dihydroxybetulinic acid, 7β,15α-dihydroxybetulonic acid, and a new compound 7β,15α,23-trihydroxybetulinic acid [3β,7β,15α,23-tetrahydroxy-lup-20(29)en-28-oic acid]. Another strain of B. megaterium SC6394 produced 30-oxobetulonic acid, and a mixture of 30-hydroxybetulonic acid and a new compound 7β-hydroxy-30-oxobetulonic acid [7β-hydroxy-3,30-dioxo-lup-20(29)en-28-oic acid]. Three products were obtained from the biotransformation of betulinic acid by Streptomyces fragilis SC16401: 7β-hydroxybetulonic acid, and two new compounds 2α,7β-dihydroxybetulinic acid [2α,3β,7β-trihydroxy-lup-20(29)en-28-oic acid] and 2-oxo-7β-hydroxy-betulinic acid [2-oxo-3β,7β-dihydroxy-lup-20(29)en-28-oic acid]. Cunninghamella elegans SC16025 gave 25-hydroxybetulinic acid from betulinic acid. Oxidation of betulinic acid by Aspergillus terreus SC16513 led to two A-ring fission products: 4-hydroxy-3,4-seco-lup-20(29)-en-3,28-dioic acid and 3,4-seco-lup-20(29), 4(23)-dien-3,28-dioic acid. B. megaterium SC16644 catalyzed transformation of betulonic acid provided 7β-hydroxybetulonic acid, 7β,15α-dihydroxybetulonic acid, and a new compound 7β,15α,30-trihydroxybetulonic acid [3-oxo-7β,15α,30-trihydroxy-lup-20(29)en-28-oic acid].

Characterization of a non-approved selective androgen receptor modulator drug candidate sold via the Internet and identification of in vitro generated phase-I metabolites for human sports drug testing.

Potentially performance-enhancing agents, particularly anabolic agents, are advertised and distributed by Internet-based suppliers to a substantial extent. Among these anabolic agents, a substance referred to as LGD-4033 has been made available, comprising the core structure of a class of selective androgen receptor modulators (SARMs). In order to provide comprehensive analytical data for doping controls, the substance was obtained and characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography/electrospray ionization high resolution/high accuracy tandem mass spectrometry (LC/ESI-HRMS). Following the identification of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile, the substance was subjected to in vitro metabolism studies employing human liver microsomes and Cunninghamella elegans (C. elegans) preparations as well as electrochemical metabolism simulations. By means of LC/ESI-HRMS, five main phase-I metabolites were identified as products of liver microsomal preparations including three monohydroxylated and two bishydroxylated species. The two most abundant metabolites (one mono- and one bishydroxylated product) were structurally confirmed by LC/ESI-HRMS and NMR. Comparing the metabolic conversion of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile observed in human liver microsomes with C. elegans and electrochemically derived metabolites, one monohydroxylated product was found to be predominantly formed in all three methodologies. The implementation of the intact SARM-like compound and its presumed urinary phase-I metabolites into routine doping controls is suggested to expand and complement existing sports drug testing methods.

A new and fast DLLME-CE method for the enantioselective analysis of zopiclone and its active metabolite after fungal biotransformation

Zopiclone (ZO) is a chiral drug that undergoes extensive metabolism to N-desmethylzopiclone (N-Des-ZO) and zopiclone-N-oxide (N-Ox-ZO). Pharmacological studies have shown (S)-N-Des-ZO metabolite presents anxiolytic activity and a patent for this metabolite was requested for anxiety treatment and related disorders. In this context, biotransformation employing fungi may be a promising strategy to obtain N-Des-ZO. To perform the biotransformation study in this work, an enantioselective method based on capillary electrophoresis (CE) and dispersive liquid–liquid microextraction (DLLME) was developed. CE analyses were carried out in sodium phosphate buffer (pH 2.5; 50mmolL−1) containing 0.5% (w/v) carboxymethyl-β-CD, at a constant voltage of +25kV. DLLME was conducted using 2mL of liquid culture medium pH 9.5. Chloroform (100μL) and methanol (300μL) were employed as extraction and disperser solvent, respectively. After CE and DLLME optimization, the analytical method was fully validated. The method was linear over a concentration range of 90–6000ngmL−1 for each ZO enantiomer (r>0.999) and 50–1000ngmL−1 for each N-Des-ZO enantiomer (r>0.998). Absolute recovery of 51 and 82% was achieved for N-Des-ZO and ZO, respectively. The accuracy and precision results agreed with the EMA (European Medicines Agency) guideline, and so did the stability study. Application of the developed method in a biotransformation study was conducted in order to investigate the ability of fungi, belonging to the genus Cunninghamella, in metabolizing ZO chiral drug. Fungi Cunninghamella elegans ATCC 10028B and Cunninghamella echinulata var elegans ATCC 8688A demonstrated to be able to enantioselectively biotransform ZO to its active metabolite, N-Des-ZO. Therefore, the proposed goals of this work, i.e. a fast DLLME-CE method and an outstanding strategy to obtain N-Des-ZO, were successfully attained.