Precursor-Directed Biosynthesis Mediated Amplification of Minor Aza Phenylpropanoid Piperazines in an Australian Marine Fish-Gut-Derived Fungus, Chrysosporium sp. CMB-F214.

Chemical analysis of an M1 agar plate cultivation of a marine fish-gut-derived fungus, Chrysosporium sp. CMB-F214, revealed the known chrysosporazines A-D (11-14) in addition to a suite of very minor aza analogues 1-6. A microbioreactor (MATRIX) cultivation profiling analysis failed to deliver cultivation conditions that significantly improved the yields of 1-6; however, it did reveal that M2 agar cultivation produced the new natural product 15. A precursor-directed biosynthesis strategy adopting supplementation of a CMB-F214 M1 solid agar culture with sodium nicotinate enhanced production of otherwise inaccessible azachrysposorazines A1 (1), A2 (2), B1 (3), C1 (4), C2 (5) and D1 (6), in addition to four new chrysosporazines; chrysosporazines N-P (7-9) and spirochrysosporazine A (10). Structures inclusive of absolute configurations were assigned to 1-15 based on detailed spectroscopic and chemical analyses, and biosynthetic considerations. Non-cytotoxic to human carcinoma cells, azachrysosporazies 1-5 were capable of reversing doxorubicin resistance in P-glycoprotein (P-gp)-overexpressing human colon carcinoma cells (SW620 Ad300), with optimum activity exhibited by the C-2' substituted analogues 3-5.

A Fasciclin Protein Is Essential for Laccase-Mediated Selective Phenol Coupling in Sporandol Biosynthesis.

The biaryl scaffold, often showing axial chirality, is a common feature of various fungal natural products. Their biosynthesis requires an oxidative phenol-coupling reaction usually catalyzed by laccases, cytochrome P450 enzymes, or peroxidases. The combination of a laccase and a fasciclin domain-containing (fas) protein is encoded in many biosynthetic gene clusters of biaryls from ascomycetes. However, such phenol-coupling systems including their regio- and stereoselectivity have not been characterized so far. Elucidating the biosynthesis of the antiparasitic binaphthalene sporandol from Chrysosporium merdarium, we demonstrate the combination of a laccase and a fas protein to be crucial for the dimerization reaction. Only the heterologous coproduction of the laccase and the fas protein led to a functional phenol-coupling system, whereas the laccase alone showed no coupling activity. Thus, the laccase/fas protein combination forms an independent group of phenol-coupling enzymes that determines the coupling activity and selectivity of the reaction concurrently and applies to the biosynthesis of many fungal natural products with a biaryl scaffold.

Comparative characterization of extracellular enzymes secreted by Phanerochaete chrysosporium during solid-state and submerged fermentation.

Influence of water content on the expression of lignocellulolytic enzymes by Phanerochaete chrysosporium remains unclear. This work compares the enzyme production profiles of P. chrysosporium during solid-state and submerged fermentation. There were 110 and 64 extracellular carbohydrate-active enzymes identified in solid-state and submerged fermentation respectively, among which 57 enzymes were common to both of the secretomes. P. chrysosporium secreted more cellulases (especially lytic polysaccharide monooxygenase) and hemicellulases during solid-state fermentation while the proportion of enzyme containing carbohydrate-binding module was higher for submerged fermentation. Although its activities were weaker, the enzyme cocktail from submerged fermentation was surprisingly more effective in hydrolysis at low substrate loading. This advantage of enzymes from submerged fermentation was mainly attributed to carbohydrate-binding module because more xylanases bound with substrate at the beginning of hydrolysis. These results reveal the influence of fermentation conditions on enzyme produced by P. chrysosporium for the first time and show the importance of carbohydrate-binding module in the hydrolysis process of lignocellulose.

Impact of the Cultivation Technique on the Production of Secondary Metabolites by Chrysosporium lobatum TM-237-S5, Isolated from the Sponge Acanthella cavernosa.

The fungi Chrysosporium lobatum TM-237-S5 was isolated from the sponge Acanthella cavernosa, collected from the mesophotic coral ecosystem of the Red Sea. The strain was cultivated on a potato dextrose agar (PDA) medium, coupling solid-state fermentation and solid-state extraction (SSF/SSE) with a neutral macroreticular polymeric adsorbent XAD Amberlite resin (AMBERLITE XAD1600N). The SSF/SSE lead to high chemodiversity and productivity compared to classical submerged cultivation. Ten phenalenone related compounds were isolated and fully characterized by one-dimensional and two-dimensional NMR and HRMS. Among them, four were found to be new compounds corresponding to isoconiolactone, (-)-peniciphenalenin F, (+)-8-hydroxyscleroderodin, and (+)-8-hydroxysclerodin. It is concluded that SSF/SSE is a powerful strategy, opening a new era for the exploitation of microbial secondary metabolites.

Biological activity of terpene compounds produced by biotechnological methods.

CONTEXT: Biotransformation systems are profitable tools for structural modification of bioactive natural compounds into valuable biologically active terpenoids. OBJECTIVE: This study determines the biological effect of (R)-(+)-limonene and (-)-α-pinene, and their oxygenated derivatives, (a) perillyl alcohol and (S)-(+)- and (R)-(-)-carvone enantiomers and (b) linalool, trans-verbenol and verbenone, respectively, on human colon tumour cells and normal colonic epithelium. MATERIALS AND METHODS: Biotransformation procedures and in vitro cell culture tests were used in this work. Cells were incubated for 24 h with terpenes at concentrations of 5-500 µg/mL for NR, MTT, DPPH, and NO assays. IL-6 was determined by ELISA with/without 2 h pre-activation with 10 µg/mL LPS. RESULTS: trans-Verbenol and perillyl alcohol, obtained via biotransformation, produced in vitro effect against tumour cells at lower concentrations (IC50 value = 77.8 and 98.8 µg/mL, respectively) than their monoterpene precursors, (R)-(+)-limonene (IC50 value = 171.4 µg/mL) and (-)-α-pinene (IC50 value = 206.3 µg/mL). They also showed lower cytotoxicity against normal cells (IC50 > 500 and > 200 µg/mL, respectively). (S)-(+)-Carvone was 59.4% and 27.1% more toxic to tumour and normal cells, respectively, than the (R)-(-)-enantiomer. (R)-(+)-limonene derivatives decreased IL-6 production from normal cells in media with or without LPS (30.2% and 13.9%, respectively), while (-)-α-pinene derivatives induced IL-6 (verbenone had the strongest effect, 60.2% and 29.1% above control, respectively). None of the terpenes had antioxidative activity below 500 µg/mL. DISCUSSION AND CONCLUSIONS: Bioactivity against tumour cells decreased in the following order: alcohols > ketones > hydrocarbons. (R)-(+)-limonene, (-)-α-pinene, and their derivatives expressed diverse activity towards normal and tumour cells with noticeable enantiomeric differences.

Bioconversion of α-pinene by a novel cold-adapted fungus Chrysosporium pannorum.

The psychrotrophic fungus Chrysosporium pannorum A-1 is reported for the first time as a novel biocatalyst for O2-promoted oxidation of α-pinene. GC-MS analysis indicated that the main products of the reaction were compounds of a high commercial value, verbenol (1) and verbenone (2). Exponentially growing cells (days 2-3) were about twice as active as cells in the late stationary phase in terms of the total concentration of products. The highest yields of 1 and 2 were obtained using three-day and two-day-old mycelia and a medium containing 1.5 and 1 % (v/v) of the substrate, respectively. The optimal time for the bioconversion of α-pinene varied from 1 to 3 days, and depended on the kind of product desired. Most of 1 was produced at a relatively high concentration of 360 mg/L after the first six hours of α-pinene bioconversion [with an average yield of 69 mg/(g dry cell L aqueous phase)]. The oxidative activity of C. pannorum was identified across a wide temperature range of 5-25 °C, 10 °C being the optimum for the production of 1 and 20 °C for the production of 2. Sequential addition of the substrate during 3 days of the biotransformation resulted in a significant increase in 1 and 2 up to 722 and 176 mg/L, respectively, and a 2-fold enhancement of product yield as compared to bioconversion with a single supply of α-pinene. The concentration of total conversion products in the culture medium reached 1.33 g/L [which corresponded product yield of 225 mg/(g dry cell L)]. This represents probably the most promising result reported to date for oxidative biotransformation of α-pinene by a wild-type microorganism.

Fungal-mediated nano silver: an effective adulticide against mosquito.

Fungi as such are known to be an effective mosquito control agent. In the present investigation, the effect of silver nanoparticles synthesized with Chrysosporium keratinophilum, Verticillium lecanii, and Fusarium oxysporum f.sp. pisi has been evaluated against the adult mosquito of filariasis vector Culex quinquefasciatus. The silver nanoparticles were characterized by using the UV-Vis spectrophotometer and X-ray diffraction techniques. The micrographs of silver nanoparticles were obtained by transmission electron microscope and scanning electron microscope. Elemental analysis on single particle was carried out by EDX analysis. The characterization study confirmed different shapes and sizes of silver nanoparticles. The efficacy test was performed at five different concentrations for a period of 24 h by the probit analysis. The C. quinquefasciatus has shown higher efficacy against the silver nanoparticles synthesized with C. keratinophilum and V. lecanii (lethal concentration (LC)(50) 0.19 and 0.4 µl/cm(2); LC(90) 2.4 and 3.2 µl/cm(2); and LC(99) 4.0 and 5.6 µl/cm(2)) after 22 h of exposure. While the silver nanoparticles synthesized with F. oxysporum f.sp. pisi were found to be less effective against the C. quinquefasciatus, the silver nanoparticles synthesized by C. keratinophilum and V. lecanii were found to be more effective than those generated with the help of F. oxysporum f.sp. pisi and C. quinquefasciatus. The use of fungus-mediated silver nanoparticles is a rapid, environmentally safer, and greener approach for vector control strategy and is adaptable globally.

Efficacy of fungus mediated silver and gold nanoparticles against Aedes aegypti larvae.

Chrysosporium tropicum is a pathogenic fungus. It is known to be an effective mosquito control agent. In the present study, we have synthesized the silver and gold nanoparticles using C. tropicum. These nanoparticles have been characterized through Microscan reader, X-ray diffractometer, transmission electron microscopy, and further confirmed by scanning electron microscopy. The characterization study confirmed the spherical shape and size (2-15 and 20-50 nm) of gold and silver nanoparticles. These silver and gold nanoparticles have been tested as a larvicide against the Aedes aegypti larvae. The larvicidal efficacy was noted when performed against all instars of A. aegypti at six different log concentrations, and significant results could be observed. The gold nanoparticles used as an efficacy enhancer have shown mortality at three times higher concentration than the silver nanoparticles. The larval mortality was observed after different time of exposures. The mortality values were obtained using the probit analysis. The larvae of A. aegypti were found to be highly susceptible for the silver nanoparticles. The second instar larvae have shown 100% mortality against the silver nanoparticles after 1 h, whereas the first, third, and fourth instars have shown efficacy (LC(50) = 3.47, 4, and 2; LC(90) = 12.30, 8.91, and 4; LC(99) = 13.18, 13.18, and 7.58, respectively) after 1 h. The results could suggest that the use of fungus C. tropicum, silver, and gold nanoparticles is a rapid, environmentally safer, and greener approach for mosquito control. This could lead us to a new possibility in vector control strategy.

Isolation, optimization, and partial purification of amylase from Chrysosporium asperatum by submerged fermentation.

A potent fungus for amylase production, Chrysosporium asperatum, was isolated from among 30 different cultures obtained from wood samples collected in the Junagadh forest, India. All of the isolated cultures were screened for their ability to produce amylase by submerged fermentation. Among the selected cultures, C. asperatum (Class Euascomycetes; Onygenales; Onygenaceae) gave maximum amylase production. In all of the different media tested, potato starch was found to be a good substrate for production of amylase enzyme at 30 degrees C and pH 5.0. Production of enzyme reached the maximum when a combination of starch and 2% xylose, and organic nitrogen (1% yeast extract) and ammonium sulfate were used as carbon and nitrogen sources, respectively. There was no significant effect of metal ions on enzyme activity. The enzyme was relatively stable at 50 degrees C for 20 min, and no inhibitory effect of Ca+2 ions on amylase production was observed.

Microbial mannosidation of bioactive chlorogentisyl alcohol by the marine-derived fungus Chrysosporium synchronum.

The biological transformation of the biologically active chlorogentisyl alcohol (1), isolated from the marine-derived fungus Aspergillus sp., was studied. Preparative-scale fermentation of chlorogentisyl alcohol with marine-derived fungus Chrysosporium synchronum resulted in the isolation of a new glycosidic metabolite, 1-O-(α-D-mannopyranosyl)chlorogentisyl alcohol (2). The stereostructure of the new metabolite obtained was assigned on the basis of detailed spectroscopic data analyses, chemical reaction, and chemical synthesis. Compounds 1 and 2 exhibited significant radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) with IC(50) values of 1.0 and 4.7 µM, respectively. The compounds 1 and 2 were more active than the positive control, L-ascorbic acid (IC(50), 20.0 µM).

PRP8 intein in Ajellomycetaceae family pathogens: sequence analysis, splicing evaluation and homing endonuclease activity.

Inteins are intervening sequences that are transcribed and translated with flanking host protein sequences and then self-excised by protein splicing. Bi-functional inteins also contain a homing endonuclease responsible for their genetic mobility. The PRP8 intein, the most widespread among fungi, occurs in important pathogens such as Histoplasma capsulatum and Paracoccidioides brasiliensis, from the Ajellomycetaceae family. Herein, we describe the bi-functional PRP8 intein in two other Ajellomycetacean pathogens, Blastomyces dermatitidis and Emmonsia parva. Sequence analysis and experimental evidence suggest that the homing endonuclease from PbrPRP8 is inactive. The splicing activity of the PRP8 intein from the B. dermatitidis, E. parva and P. brasiliensis species complex was demonstrated in a non-native protein context in Escherichia coli. Since the PRP8 intein is located in a functionally essential nuclear protein, it can be considered a promising therapeutic target for anti-fungal drugs, because inhibition of intein splicing should inhibit proliferation of intein-containing pathogens.

Effect of Chrysosporium keratinophilum metabolites against Culex quinquefasciatus after chromatographic purification.

Chrysosporium keratinophilum is known to be a keratinophilic fungus and an effective mosquito control agent. This fungus was grown on Sabauraud dextrose broth in the laboratory at 25°C, while the relative humidity was maintained at 75 ± 5% for 15 days. Filtration process of metabolites was done using whatman-1 filter paper, column chromatography and flash, chromatography. Larvicidal efficacy was performed against all instars of Culex quinquefasciatus. Larvicidal efficacy was performed at six different concentrations with different effective ratios (ethanol/metabolites: 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9). The mortality values were then subjected by the probit analysis. The larval mortalities were observed for a period of 24, 48, and 72 h, respectively. The first and second instars were highly susceptible to 2:8 ratio. In the first instar after column chromatography, LC(50) =26.66 ppm, LC(90) =121.96 ppm, LC(99) =231.86 ppm were observed after 72 h, while after flash chromatography the LC(50) =20 ppm, LC(90) =123.02 ppm, LC(99) =281.83 ppm were observed after 48 h. In the second instar after column chromatography, LC(50) =18.19 ppm, LC(90) =102.32 ppm, LC(99) =162.18 ppm were observed after 72 h, while doing flash chromatography 100% mortality could be recorded after 24 h. In the third instar after column chromatography, the LC(50) =38.01 ppm, LC(90) =131.82 ppm, LC(99) =245.47 ppm were observed after 72 h, while after flash chromatography the LC(50) =17.78 ppm, LC(90) =100 ppm, LC(99) =151.35 ppm. In the fourth instar, LC(50) =61.65 ppm, LC(90) =181.97 ppm, LC(99) =436.51 ppm, while after flash chromatography LC(50) =40 ppm, LC(90) =120 ppm, and LC(99) =223.87 ppm were observed after 72 h. The extracellular metabolites of C. keratinophilum could be a fungal based larvicides resource for the control of C. quinquefasciatus larvae. This could be another agent for biotechnological exploitation, if found suitable in field trials.

Chrysosporium pseudomerdarium produces gibberellins and promotes plant growth.

We isolated 10 endophytic fungi from the roots of drought stressed soybean cultivar Hwangkeumkong and bioassayed on waito-c rice and soybean seedlings, in order to identify plant growth-promoting fungi. The fungal isolate D-2-1 provided the best result for plant height and biomass promotion as compared to wild type Gibberella fujikuroi. The D-2-1 culture filtrate (CF) was analyzed for the presence of gibberellins (GAs) and it was observed that all physiologically active GAs, especially gibberellic acid, were present in higher amounts (GA1, 0.24 ng/ml; GA3, 8.99 ng/ml; GA4, 2.58 ng/ml and GA7, 1.39 ng/ml) in conjunction with physiologically inactive GA5, GA9, GA15, GA19, and GA24. The fungal isolate D-2-1 was identified as a new strain of Chrysosporium pseudomerdarium through phylogenetic analysis of 18S rDNA sequence. Plant growth promotion and GAs production capacity of genus Chrysosporium have been reported for the first time in this study.

Effects of culture media on larvicidal property of secondary metabolites of mosquito pathogenic fungus Chrysosporium lobatum (Moniliales: Moniliaceae).

In order to determine the potential role of secondary metabolites of Chrysosporium lobatum as a biological control agent for mosquitoes, effects of culture media on the larvicidal property of secondary metabolites was evaluated. The secondary metabolites of C. lobatum released in the Sabouraud's dextrose broth (SDB) and chitin broth (CB) were collected by filtering through Whatman No. 3 chr filter after 7 days of growth. First, second and third instars of Anopheles stephensi and Culex quinquefasciatus were exposed to six different concentrations of secondary metabolites for the calculation of LC(50) and LC(90) values. C. quinquefasciatus were more susceptible than A. stephensi to secondary metabolites in SDB and CB. The LC(50) and LC(90) values for first instars of both the mosquitoes were significantly lower than second and third instars. A significantly higher mortality was recorded in both the mosquitoes to the secondary metabolites in CB than SDB. In the field trial, secondary metabolites in CB reduced 56.3% of A. stephensi and 66.42% of C. quinquefasciatus populations after 5 days of application. The LT(50) of first instars of both the mosquitoes were lower than the second and third instars. The precipitates of the secondary metabolites were more effective to both the mosquitoes than the secondary metabolite and heat-shocked secondary metabolites. In addition, the insecticidal effect of the secondary metabolites was reduced after exposure for 5 min at 120 degrees C, suggesting that the bioactive compounds were proteinaceous. The secondary metabolites in CB contained proteases which may cause more mortality than the secondary metabolites in SDB.

Role of Chrysosporium keratinophillum in the parasitic evolution of dermatophytes.

Anti-dermatophytic activity of Chrysosporium keratinophillum against species of the genera Trichophyton, Microsporum and Epidermophyton floccosum was tested in vitro. When C. keratinophillum and different species of dermatophytes were inoculated on Sabouraud's dextrose agar plates 2 cm apart, no antagonistic effect of C. keratinophillum on the mycelial growth of dermatophytes was observed. However, conidia production was not observed on the hyphae of Trichophyton rubrum, Trichophyton tonsurans and E. floccosum grown near C. keratinophillum. The secretory substances released by C. keratinophillum inhibited the growth of T. rubrum, T. tonsurans, Trichophyton mentagrophytes var. interdigitale and E. floccosum at a concentration of 2,000 microg ml(-1) when tested by broth dilution technique. No inhibition of the growth was observed for Microsporum gypseum and Microsporum nanum. The anti-fungal activity of secretory substances released by C. keratinophillum was recorded to be heat stable. Results of the present study suggest that the anti-dermatophytic activity of the secretory substances of C. keratinophillum on T. rubrum, T. mentagrophytes var. interdigitale, T. tonsurans and E. floccosum may be responsible in part, for the absence of these dermatophyte species in soil. Considering the global prevalence of C. keratinophillum in soil one may speculate that the anti-dermatophytic activity of C. keratinophillum is one of the early events for the evolutionary divergence of saprophytic archi-dermatophytes to obligate parasitic dermatophyte species.

Keratinophilic fungi recovered from muddy soil in Cairo vicinities, Egypt.

One hundred samples of muddy soil were collected from seven areas in the vicinity of Cairo and screened for the presence of keratinophilic fungi by using hair baiting isolation technique. Forty isolates of keratinophilic fungi were recovered and identified by recognition of their cultures, macro- and micromorphological features. Their physiological and molecular characteristics were studied by determination of their ubiquinone (Coenzyme Q) composition and DNA sequences of (ITS1-5.8S-ITS2) and 18S rRNA region sequences. The Keratinophilic isolates were identified as Chrysosporium carmichaelii, C. queenslandicum, C. zonatum, C. indicum, Aphanoascus mephitalis, and Uncinocarpus reesii. Chrysosporium zonatum was the most prevalent species and represented 42.5% of the total number of isolates. Each of C. carmichaelii and C. queenslandicum were equal in their prevalence and represented 15%. C. indicum comes next constituting 12.5%; followed by Uncinocarpus reesii which represented 10%. The least prevalent species in our study was Aphanoascus mephitalis, which was represented only 5% of the total keratinophilic isolates.

Morphological characterization of in-vitro human hair keratinolysis, produced by identified wild strains of Chrysosporium species.

Chrysosporium species were isolated from soil and keratinized material. Primary isolation was performed following the general method of hair baiting on modified Czapek-agar media with washed, defated and sterilized human hair fragments added. Strains were maintained in test tubes of potato dextrose agar at 29 degrees C and cultivated on phytone yeast extract agar at 28 degrees C for 14 days for identification. Isolates were characterized using Van Oorschot's key. Keratinolytic activity was expressed following a subjective scale representing degree/severity of attack upon hair surface and presence of fungal structures observed in substrate. Culture results and characterization methods were effective for soil Chrysosporium strain isolation. A new hair attack mode is described. Of 71 keratinolytic fungal isolates, eight (12%) Chrysosporium species were identified. One keratinolytic Chrysosporium sp. isolate is yet to be identified.