Tolypocladium Inflatum Research Articles

Optimization of cyclosporin A production by Beauveria nivea in continuous fed-batch fermentation

To develop the effective control method for fed-batch culture of cyclosporin A production, we chose fructose, L-valine and (NH4)2HPO4 as feeding nutrients and compared their productivities in relation to different concentrations. The feeding rate of three kinds of feeding materials was controlled to maintain the suitable residual concentration. The fed-batch fermentation results indicated that the optimal concentrations of fructose, L-valine and (NH4)2HPO4 were about 20 g/L, 0.5 g/L and 0.6 g/L for cyclosporin A production, respectively. The cultivation of Beauveria nivea could produce cyclosporin A up to 6.2 g/L for 240 hrs through a continuous feeding-rate-controlled-batch process under the optimal feeding conditions.

Targeted Gene Disruption and Functional Complementation of Cytochrome

A cyclic undecapeptide-family natural product, cyclosporin A (CyA), which is one of the most valuable immunosuppressive drugs, is produced nonribosomally by a multifunctional cyclosporin synthetase enzyme complex in a filamentous fungal strain named Tolypocladium niveum. Previously, structural modifications of cyclosporins such as a regionspecific hydroxylation at the 4th N-methyl leucine in a rare actinomycetes called Sebekia benihana were reported to lead to dramatic changes in their bioactive spectra. However, the reason behind this change could not be determined since a system to genetically manipulate S. benihana has not yet been developed. To address this limitation, in this study, we utilized the most commonly practiced gene manipulation techniques including conjugation-based foreign gene transfer-and-expression as well as targeted gene disruption to genetically manipulate S. benihana. Using these optimized genetic manipulation systems, a putative cytochrome P450 hydroxylase (CYP) gene named CYP506, which is involved in CyA hydroxylation in S. benihana, was specifically disrupted and genetically complemented. The S. benihana deltaCYP506 exhibited a significantly reduced CyA hydroxylation yield as well as considerable yield restoration by functional complementation of the S. benihana CYP506 gene, suggesting that the genetically manipulated S. benihana CYP mutant strains may serve as a more efficient bioconversion host for various valuable metabolites including CyA.

Production of fumonisins B2 and B4 in Tolypocladium species

Tolypocladium inflatum is known primarily for its production of the cyclosporines that are used as an immunosuppressive drug. However, we report here the production of the carcinogenic fumonisins B(2) and B(4) by this biotechnologically relevant fungal genus. These mycotoxins were detected in 11 strains tested from three species: Tolypocladium inflatum, T. cylindrosporum, and T. geodes. Production of fumonisins by Fusarium spp. and Aspergillus niger is highly medium- and temperature-dependent, so the effect of these parameters on fumonisin production by three T. inflatum strains was studied. Maximum production was achieved on media with high sugar content incubated at 25-30°C. Since these results demonstrate that fumonisin production could be widespread within the genus Tolypocladium, the potential contamination of commercial cyclosporine preparations with fumonisins needs to be investigated.

Dominating species of entomophilous ascomycetes anamorphs in West Siberia, Primorsky krai, and Kyrgyzstan

The paper studies mycobiota of the dead insects in West Siberia, Primorsky krai, and Kyrgyzstan. Ascomycetes anamorphs of 13 genera are revealed. In all regions Beauveria bassiana (Bals.-Criv.) Vuill. dominated comprising on average 68% of the total number of isolates. The fungus hosts list the insects of 7 orders and 32 families with Coleoptera, Lepidoptera, and Hemiptera dominating. The rarely found entomopathogens include Tolypocladium inflatum Gams (primarily on Lepidoptera), Metarhizium anisopliae (Metschn.) Sorokin (on Coleoptera). The mortality rate of the insects due to micromycetes is observed mainly on enzootic level. The study of the pathogenic properties of the dominating species (B. bassiana) show the absence of specificity of its environmental isolates for a number of representatives of Orthoptera, Lepidoptera, Coleoptera, and Diptera.

Gellan Gum as an Immobilization Matrix for the Production of Cyclosporin A

This study explored the use of gellan gum as an immobilization matrix for production of cyclosporin A (CyA) by immobilized spores and mycelia of Tolypocladium inflatum MTCC 557. Different carriers, such as gellan gum, sodium alginate, celite beads and silica, were tested as immobilization carriers, along with the role of the carrier concentration, biomass weight, number of spore-inoculated beads, and repeated utilization of the immobilized fungus. The maximum CyA production was 274 mg/l when using gellan gum (1 % w/v) and a mycelial weight of 7.5 % w/v supported the maximum production of CyA. Additionally, a combination of L-valine (6 g/l) and L-leucine (5 g/l) after 48 h of fermentation produced 1,338 mg/l when using gellan gum. The immobilized mycelia beads were found to remain stable for four repetitive cycles, indicating their use for semi-continuous CyA production.

Drug/natural product interactions at the blood brain barrier

Drug/natural product interactions at the blood brain barrier

Use of coconut coir fibers as an inert solid support for production of cyclosporin A

In the present study, coconut coir was evaluated as an inert support for the production of cyclosporin A (CyA) using Tolypocladium inflatum MTCC 557 by solid state fermentation. Initially, four different inert supports such as coconut coir, polyurethane foam, polystyrene beads, and sugarcane baggase were screened using different production media as moistening agents for the maximum production of CyA. Different parameters such as fermentation time, carbon sources, moisture content, pH, and inoculum size were optimized. It was observed that coconut coir impregnated with medium modified with glycerol as carbon source, pH 6, at 80% moisture content, and inoculum size of 2.5 mL/2.5 g support produced 2641 mg/kg of CyA after 12 days as compared to 998 mg/kg before optimization. The yields were further increased to 3597 mg/kg substrate with addition of combination of amino acids after 48 h of fermentation.

Combination Strategy to Increase Cyclosporin A Productivity by Tolypocladium niveum Using Random Mutagenesis and Protoplast Transformation

The cyclic undecapeptide cyclosporin A (CyA), one of the most valuable immunosuppressive drugs, is produced nonribosomally by a multifunctional cyclosporin synthetase enzyme complex by the filamentous fungus Tolypocladium niveum. To increase CyA productivity by wild-type T. niveum (ATCC 34921), random mutagenesis was first performed using an antifungal agar-plug colony assay (APCA) selection approach. This generated a mutant strain producing more than 9-fold greater CyA than the wild-type strain. Additionally, a foreign bacterial gene, Vitreoscilla hemoglobin gene (VHb), was transformed via protoplast regeneration and its transcription was confirmed by RT-PCR in the UV-irradiated mutant cell. This led to an additional 33.5% increase of CyA production. Although most protoplastregenerated T. niveum transformants tend to lose CyA productivity, the optimized combination of random mutagenesis and protoplast transformation described here should be an efficient strategy to generate a commercially valuable, yet metabolite low-producing, fungal species, such as CyA producing T. niveum.

A Novel Medium for the Enhanced Production of Cyclosporin A byTolypocladium inflatum MTCC 557 Using Solid State Fermentation

Cyclosporin A (CyA) produced by Tolypocladium inflatum is a promising drug owing to its immunosuppressive and antifungal activities. From an industrial point of view, the necessity to obtain a suitable and economic medium for higher production of CyA was the aim of this work. The present study evaluated the effect of different fermentation parameters in solid state fermentation, such as selection of solid substrate, hydrolysis of substrates, initial moisture content, supplementation of salts, additional carbon, and nitrogen sources, as well as the inoculum age and size, on production of CyA by Tolypocladium inflatum MTCC 557. The fermentation was carried out at 25+/-2 degrees for 9 days. A combination of hydrolyzed wheat bran flour and coconut oil cake (1:1) at 70% initial moisture content supported a maximum production of 3,872+/-156 mg CyA/kg substrate as compared with 792+/-33 mg/kg substrate before optimization. Furthermore, supplementation of salts, glycerol (1%w/w), and ammonium sulfate (1%w/w) increased the production of CyA to 5,454+75 mg/kg substrate. Inoculation of 5 g of solid substrate with 6 ml of 72-h-old seed culture resulted in a maximum production of 6,480+95 mg CyA/kg substrate.

Statistical Optimization for Improved Production of Cyclosporin A in Solid-State Fermentation

This work evaluates the effect of different amino acids on production of CyA production in solid-state fermentation that was previously optimized for different fermentation parameters by one-factor-at-a-time for the maximum production of CyA by Tolypocladium inflatum MTCC 557. Based on the Plackett-Burman design, glycerol, ammonium sulfate, FeCl3, and inoculum size were selected for further optimization by response surface methodology (RSM). After identifying effective nutrients, RSM was used to develop mathematical model equations, study responses, and establish the optimum concentrations of the key nutrients for higher CyA production. It was observed that supplementation of medium containing (% w/w) glycerol, 1.53; ammonium sulfate, 0.95; FeCl3, 0.18; and inoculum size 6.4 ml/5g yielded a maximum of 7,106 mg/kg as compared with 6,480 mg CyA/kg substrate using one factor at a time. In the second step, the effect of amino acids on the production of CyA was studied. Addition of L-valine and L-leucine in combination after 20 h of fermentation resulted in maximum production of 8,166 mg/kg.

Good bugs, bad bugs: learning what we can from the microorganisms that colonize our bodies

Good bugs, bad bugs: learning what we can from the microorganisms that colonize our bodies

The Tolypocladium inflatum CPA element encodes a RecQ helicase‐like gene

Previously, a repetitive CPA element was discovered in the genome of the filamentous fungus Tolypocladium inflatum; however, no further characterization was technically possible at that time. In this study, PCR amplification was used to detect a 4 kb conserved portion of the CPA element that appeared to be present in most, if not all, genomic CPA elements. The amplicons included a large open reading frame that was most similar to a RecQ helicase-like gene from Metarhizium anisopliae. The repetitive nature of the CPA element suggests that it is related to the eukaryotic Helitron class of transposable elements.

Diversity of facultatively anaerobic microscopic mycelial fungi in soils

The numbers of microscopic fungi isolated from soil samples after anaerobic incubation varied from tens to several hundreds of CFU per one gram of soil; a total of 30 species was found. This group is composed primarily of mitotic fungi of the ascomycete affinity belonging to the orders Hypocreales (Fusarium solani, F. oxysporum, Fusarium sp., Clonostachys grammicospora, C. rosea. Acremonium sp., Gliocladium penicilloides, Trichoderma aureoviride, T. harzianum, T. polysporum, T. viride. T. koningii, Lecanicillum lecanii, and Tolypocladium inflatum) and Eurotiales (Aspergillus terreus, A. niger, and Paecilomyces lilacimus), as well as to the phylum Zygomycota, to the order Mucorales (Actinomucor elegans, Absidia glauca, Mucor circinelloides, M. hiemalis, M. racemosus, Mucor sp., Rhizopus oryzae, Zygorrhynchus moelleri, Z. heterogamus, and Umbelopsis isabellina) and the order Mortierellales (Mortierella sp.). As much as 10-30% of the total amount of fungal mycelium remains viable for a long time (one month) under anaerobic conditions.

Protoplast fusion technique in Tolypocladium inflatum for increasing Cyclosporine production

Protoplast fusion technique in Tolypocladium inflatum for increasing Cyclosporine production

Cyclosporine A: A Review of Current Oral and Intravenous Delivery Systems

ABSTRACTAs early as 1978, the immunosuppressive effect of cyclosporine A (CsA), a metabolite of the fungus Tolypocladium inflatum (), was reported to be effective in inhibiting organ rejection in patients receiving kidney transplants from mismatched cadaver donors () and in the treatment of graft-versus-host disease in patients with acute leukemia following bone marrow transplants (). Today, CsA is still indicated to prevent rejection following solid organ transplantations, prevent and treat graft-vs-host disease following bone marrow transplants, and has also been used in the treatment of autoimmune disease such as psoriasis, rheumatoid arthritis, and nephrotic syndrome (). The effectiveness of CsA is derived from its ability to specifically and reversibly inhibit immunocompetent lymphocytes in the G0 and G1 phase of the cell cycle. The T-helper cells are the main target, but suppression of the T‐suppressor cells also occurs. The production and release of lymphokines, including interleukin-2 are also inhibited (). CsA can be administered intravenously as well as orally in the form of a solution or a soft gelatin capsule. The following review will focus on the evolution of the emulsion-based oral formulations from the first generation as Sandimmune® to the second generation Neoral®, both products of Novartis Pharmaceutical, as well as on the Sandimmune® commercial intravenous formulation. The potential of alternative delivery systems, including micelles, micro- and nanoparticles, and liposomes, will also be discussed.

Effect of temperature on the extraction kinetics and diffusivity of Cyclosporin A in the fungus Tolypocladium inflatum

The influence of temperature on the extraction kinetics of Cyclosporin A (CyA) from the mycelia of Tolypocladium inflatum was examined in this study. The extraction of CyA from mycelia was performed in a 2-L stirred, baffled vessel using 30% v/v aqueous methanol. The temperature range used was from 5 to 45 degrees C. A linear relationship was found between the extraction yield of CyA and temperature. As the temperature increased, the yield of CyA increased with a maximum CyA yield of 18.3% obtained at 45 degrees C, which is 21.3% higher than the yield at 25 degrees C. The activation energy for the extraction of CyA from T. inflatum was found to be 36.7 kJ/mol, which indicates that the extraction of CyA from T. inflatum is controlled by both solubilization of CyA and diffusion of CyA through the solid phase of mycelia. The overall mass transfer coefficient, k(L)a(S), was found to increase from 1.02 x 10(-3) to 1.34 x 10(-2) s(-1) as the temperature increased from 5 to 45 degrees C. The effective diffusivity of CyA in the solid matrix of mycelia was found to increase from 1.05 x 10(-15) to 1.43 x 10(-14) m(2)/s as the temperature increased from 5 to 45 degrees C. A mathematical diffusion model was developed and was used to fit the experimental kinetic data of CyA extraction and determination of CyA effective diffusivities at different temperatures. This is the first time CyA diffusivities as a function of extraction temperature are reported in the literature.

Effect of solvent concentration on the extraction kinetics and diffusivity of Cyclosporin A in the fungus Tolypocladium inflatum

The kinetics of solid-liquid extraction and extraction yields of the immunosuppressant drug Cyclosporin A (CyA) from the mycelia of Tolypocladium inflatum were examined in this study. A 2 L stirred, baffled vessel was used to extract CyA from wet mycelia mass. Three different organic solvents were used, namely, methanol, acetone, and isopropanol at different concentrations in aqueous mixtures at room temperature. It was found that the best solvent was acetone at 50% v/v concentration achieving 100% extraction of CyA from the mycelia of T. inflatum. Although acetone proved to be the better solvent for CyA extraction, further studies were performed using methanol. A linear relationship was found between extraction yield of CyA and methanol concentration with 100% CyA extraction at 90% v/v methanol. The partition coefficients of CyA between the solid mycelia phase and the aqueous solvent phase were found to decrease exponentially with increasing methanol concentration. A liquid extraction model was developed based on the diffusion equation to correlate the kinetic data of CyA extraction from the solid mycelia of T. inflatum. Non-linear regression analysis of experimental data was used with the diffusion equation in order to calculate the effective diffusivities of CyA in the mycelia of T. inflatum. For all three organic solvents used, the effective diffusivities of CyA were found to be between 4.41 x 10(-15) and 6.18 x 10(-14) m(2)/s. This is the first time CyA effective diffusivities in T. inflatum are reported in the literature.

Nucleotide excision repair and photoreactivation in the entomopathogenic fungi Beauveria bassiana, Beauveria brongniartii, Beauveria nivea, Metarhizium anisopliae, Paecilomyces farinosus and Verticillium lecanii

To compare the DNA repair capabilities of the entomopathogenic fungus (EPF) bassiana to the EPF Beauveria brongniartii, Beauveria nivea, Metarhizium anisopliae, Paecilomyces farinosus, Verticillium lecanii, and the fungi Aspergillus niger and Neurospora crassa. Germination of B. bassiana conidiospores following ultraviolet (UV) irradiation was used to show that nucleotide excision repair and photoreactivation decrease the post-UV germination delay. These two modes of repair were characterized and compared between the aforementioned EPF, A. niger and N. crassa using a physiological assay where per cent survival post-UV irradiation was scored as colony forming units. The results showed B. bassiana and M. anisopliae are the most UV-tolerant EPF. The DNA repair capabilities indicated that EPF do not have all DNA repair options available to fungi, such as A. niger and N. crassa. A key factor detrimental to the survival of EPF in agro-ecosystems is UV light from solar radiation. The EPF literature pertaining to UV irradiation is varied with respect to methodology, UV source, and dose, which prevented comparisons. Here we have characterized the fungi by a standard method and established the repair capabilities of EPF under optimal conditions.

Morphological and genetic characterization of a cultivated Cordyceps sinensis fungus and its polysaccharide component possessing antioxidant property in H22 tumor-bearing mice

Cordyceps sinensis, one of the most precious traditional Chinese medicines, possesses the antitumor activity, antioxidant activity and the capability of modulating the immune system. In the present study, a fungus strain G1 isolated from wild C. sinensis was identified and initially characterized. A phylogenetic tree was generated based on the sequences of the internal transcribed spacer (ITS) region of related fungi. The analysis of ITS sequence showed that fungus G1 was clustered together with C. sinensis, Tolypocladium cylindrosporum and Tolypocladium inflatum in the phylogenetic tree. Both the morphological character and the ITS sequence analysis establish that fungus G1 is one of the anamorph strains of C. sinensis and belongs to Tolypocladium genus. Furthermore, the polysaccharide (PS) extracted from fungus G1 and its antioxidant activity on H22-bearing mice was investigated. H22 cells were hypodermically injected into the right oxter of each mouse after the ICR mice were treated with PS by means of gavage for 7 days. Then the same administration process continued for 9 days. At the end of the experiments, the tumor weight of each mouse was measured. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in mouse liver, brain and serum, as well as glutathione peroxidase (GSH-Px) activity in mouse liver and brain were assayed. The results showed that the H22 tumor growth was significantly inhibited by PS. Moreover, PS significantly enhanced SOD activity of liver, brain and serum as well as GSH-Px activity of liver and brain in tumor-bearing mice. PS also significantly reduced the level of MDA in liver and brain of tumor-bearing mice.

Microbial Biomass, Community Structure and Metal Tolerance of a Naturally Pb-Enriched Forest Soil

The effect of long-term elevated soil Pb levels on soil microbiota was studied at a forest site in Norway, where the soil has been severely contaminated with Pb since the last period of glaciation (several thousand years). Up to 10% Pb (total amount, w/w) has been found in the top layer. The microbial community was drastically affected, as judged from changes in the phospholipid fatty acid (PLFA) pattern. Specific PLFAs that were high in Pb-enriched soil were branched (especially br17:0 and br18:0), whereas PLFAs common in eukaryotic organisms such as fungi (18:2omega6,9 and 20:4) were low compared with levels at adjacent, uncontaminated sites. Congruent changes in the PLFA pattern were found upon analyzing the culturable part of the bacterial community. The high Pb concentrations in the soil resulted in increased tolerance to Pb of the bacterial community, measured using both thymidine incorporation and plate counts. Furthermore, changes in tolerance were correlated to changes in the community structure. The bacterial community of the most contaminated soils showed higher specific activity (thymidine and leucine incorporation rates) and higher culturability than that of control soils. Fungal colony forming units (CFUs) were 10 times lower in the most Pb-enriched soils, the species composition was widely different from that in control soils, and the isolated fungi had high Pb tolerance. The most commonly isolated fungus in Pb-enriched soils was Tolypocladium inflatum. Comparison of isolates from Pb-enriched soil and isolates from unpolluted soils showed that T. inflatum was intrinsically Pb-tolerant, and that the prolonged conditions with high Pb had not selected for any increased tolerance.