Electrofusion of Trichoderma reesei protoplasts

Protoplasts of Trichoderma reesei were fused according to the method of Zimmermann. For optimizing the fusion parameters the central composite design was used. Genetic evidence for fusion has been obtained by segregation of the auxotrophic markers in the haploid conidia. The parameters which were optimized were: pulse voltage, pulse duration and number of pulses. The optimal parameters for the fusion of T. reesei protoplasts are 90 V pulse voltage, 37 μS pulse duration and six pulses at intervals of 1-0 s.     

Cellulase secretion by immobilized protoplasts of Trichoderma reesei

Protoplasts from Trichoderma reesei were immobilized in alginate and induced to produce cellulase (endoglucanase and β-glucosidase) enzymes. The specific activities of the synthesized enzymes were higher in immobilized protoplasts than in both free and immobilized mycelia. Immobilized protoplasts show an enhanced rate of exocellular β-glucosidase production compared to intact mycelia due to the lack of cell wall. The ratio of the exocellular/intracellular β-glucosidase was 5.9 for immobilized protoplasts and 0.32 for free mycelia.     

Effects of Antioxidants on Regeneration of Protoplasts of the Filamentous Fungus Trichoderma reesei6/16

The relative content of antioxidants in the mycelium of Trichoderma reesei 6/16 obtained by propagation of fungal protoplasts was shown to decrease (as compared to the initial culture taken for preparation of protoplasts) and restored only in the second generation of regenerated mycelium. In this respect, the effects of various antioxidants (-carotene, ascorbic acid, -tocopherol, and ionol) on the frequency of regeneration of T. reesei 6/16 protoplasts were studied. -Carotene increased the viability of fungal protoplasts to the greatest extent. The effect of ascorbic acid depended on the presence of Fe ions. Ionol did not cause any measurable protective effect.     

The formation and regeneration of mycelial protoplasts in hyper lignolytic fungus,strain IZU-154

Over 2 × 10⁷/ml protoplasts were obtained from mycelia of hyper lignolytic fungus (nomenclatured as strain IZU-154) by treatment with the lytic enzyme NovoZym 234 in the presence of 0.05 M maleic acid buffer (pH 5.6) containing 0.6 M MgSO₄. The protoplasts regenerated at more than 10% of frequency on solid 2% agar medium containing 0.6 M sucrose as an osmotic stabilizer overlaid with 0.5% agar containing the stabilizer. In the determination of the lignolytic activities of 50 regenerants from protoplasts, 2 strains which degraded more than 56% of the lignin during incubation for 30 d and showed activity higher than the parent were found. The regeneration from protoplasts of this fungus was suggested to be useful for the breeding of strains having higher lignolytic activity than this fungus.     

Improved technique on reversion of mycelial protoplast of Trichoderma reesei into mycelia

Important parameters in the regeneration of protoplasts are viability, the capacity to synthesize cell walls and the retention of properties of the parent cell. Mycelial protoplasts of Trichoderma longibrachiatum (Trichoderma reesei) have been regenerated. Factors influencing the regeneration of protoplasts of T. longibrachiatum QM 9414 were found to be, the nature of osmotic stabilizer, the concentration of osmotic stabilizer, pH, temperature, and the composition of regeneration medium. With glucose-mineral regeneration medium, the optimum conditions for protoplasts regeneration were 0.5 M KCl, pH 6.0 and temperature 30°C. With Czapek-Dox medium, the optimum conditions were 0.7 M mannitol, pH 6.0 and temperature 30°C. Maximum regeneration frequency of T. longibrachiatum protoplasts were obtained using glucose-mineral medium.     

Production of protoplasts from the fungi Curvularia inaequalis and Trichoderma reesei

Summary Protoplast formation in Curvularia inaequalis was achieved using non-commercial and commercial snail gut enzymes or Trichoderma harzianum enzymes. The cells were grown for enzyme treatment on cellophane sheets or in liquid cultures for varying periods of time. The production of T. harzianum enzymes is discussed. The highest protoplast yields were 2.6x10⁷ protoplasts/ml enzyme solution. Protoplasts were shown to have zero to four nuclei. Protoplast regeneration was succesfully carried out in semisolid agar.     

Immobilization of the cellulolytic fungus Trichoderma reesei on the polymeric sorbent - Sorfix

Summary The immobilization of T. reesei mycelium on activated polymeric sorbent was investigated with respect to the intended flow-trough cellulase production. The retention of extracellular production of cellulolytic enzymes was monitored in a packed-column recycle reactor. Some factors affecting cellulase elution pattern are described.     

Transfer of a benomyl resistance marker by heat-inactivated Trichoderma reesei protoplasts

Summary Protoplasts from a benomyl resistant Trichoderma reesei mutant were heat inactivated at 60°C for 8 min and fused with viable protoplasts from an osmosensitive, non-sporulating T. reesei strain. Fusants recovered on 50 g/ml benomyl containing potato dextrose agar plates grew and sporulated well. Cellulolytic enzyme activities produced in liquid culture by selected fusants were higher than those produced by parental strains.     

Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei.

Basic features of regulation of expression of the genes encoding the cellulases of the filamentous fungus Trichoderma reesei QM9414, the genes cbh1 and cbh2 encoding cellobiohydrolases and the genes egl1, egl2 and egl5 encoding endoglucanases, were studied at the mRNA level. The cellulase genes were coordinately expressed under all conditions studied, with the steady-state mRNA levels of cbh1 being the highest. Solka floc cellulose and the disaccharide sophorose induced expression to almost the same level. Moderate expression was observed when cellobiose or lactose was used as the carbon source. It was found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels. These carbon sources thus provide a useful means to study cellulase regulation without significantly affecting the growth of the fungus. RNA slot blot experiments showed that no expression could be observed on glucose-containing medium and that high glucose levels abolish the inducing effect of sophorose. The results clearly show that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression.     

A new appraisal of the endoglucanases of the fungus Trichoderma reesei.

The properties and enzymic activity of endoglucanases (EC 3.2.1.4) of the fungus Trichoderma reesei were studied by means of immunological methods and by using polyglycosidic substrates. Endoglucanases exist in the culture liquid as a series of immunologically related components. The most active endoglucanase component has an Mr of 43 000 and pI value of 4.0. The most abundant components have a value of pI about 5.0, an Mr of 56 000-67 000 and specific activity only one-fifth of that of the pI-4.0 component. During purification and storage the endoglucanases are spontaneously modified; the relative proportion of components having greater Mr values, more alkaline pI values and lower specific activities is increased. The hexose content of the endoglucanase components is 2-7%. Endoglucanases hydrolyse soluble beta-1,4 glycans. The enzymes described here differ from endoglucanase preparations described previously in not showing activity towards insoluble substrates. The role of endoglucanases in wood hydrolysis is consequently limited to the stage where wood constituents are already in soluble form.     

A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei

An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per microgram of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia coli beta-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter.     

A comparison of genetically improved strains of the cellulolytic fungus Trichoderma reesei

Summary Production of cellulases by three different genetically improved strains of Trichoderma reesei: MCG 77, RUT-C30 and CL-847, has been assessed on various fermentation media. The three strains produce high levels of enzymes when grown on purified cellulose as the main carbon energy source; when grown on lactose, decrease in enzyme yield and productivity, differs significantly from strain to strain.     

Ultra-high-throughput picoliter-droplet microfluidics screening of the industrial cellulase-producing filamentous fungus Trichoderma reesei

Journal of Industrial Microbiology & Biotechnology - The selection of improved producers among the huge number of variants in mutant libraries is a key issue in filamentous fungi of industrial...     

Flow cytometric sorting of the filamentous fungus Trichoderma reesei for improved strains

Metabolic measurements and screening of Trichoderma reesei have conventionally been performed during the hyphal stage of fungal development. To determine if flow cytometric measurements of protein expression could be made on germinating spores we created a gene construct, placing the Renilla reniformis green fluorescent protein gene under control of the cellobiohydrolase I (cbh1) promoter and terminator of T. reesei. This vector was transformed into T. reesei and GFP expression was measured in germlings by flow cytometry. Fluorescence associated with GFP expression was observed in germlings grown under conditions known to induce cellulases in Trichoderma. Spores were mutated using UV light and germinating spores were screened for increased GFP expression using high-speed cell sorting, to select for strains with genetic changes associated with increased protein expression. Secondary screens for cellulase production were conducted in microtitre plates. Flow cytometric screening of germinating spores expressing GFP yielded a mutant with improved ability to hydrolyse biomass.     

Repeat-induced point (RIP) mutation in the industrial workhorse fungus Trichoderma reesei

Trichoderma reesei (syn. Hypocrea jecorina) is a filamentous ascomycete. Due to its capability of producing large amounts of lignocellulolytic enzymes and various heterologous proteins, this fungus has been widely used for industrial applications for over 70 years. It is also a model organism for lignocellulosic biomass degradation and metabolic engineering. Recently, we experimentally and computationally demonstrated that Trichoderma reesei exhibits high homology pairing and repeat-induced point (RIP) mutation activities at a premeiotic stage, i.e., between fertilization and karyogamy or premeiotic DNA replication. The discovery of RIP in Trichoderma reesei not only reveals significant impacts of sexual reproduction on evolution and chromosome architecture but also provides intriguing perspectives for industrial strain improvement. This review emphasizes two major points about RIP and RIP-like processes in Pezizomycotina fungi. First, the molecular mechanisms of RIP and RIP-like processes in Trichoderma reesei and other Pezizomycotina fungi are apparently distinct from those originally described in the model fungus Neurospora crassa. Second, orthologs of the rid1 (deficient in RIP-1) DNA methyltransferase gene were shown to be essential for sexual development in at least four Pezizomycotina fungi, including Trichoderma reesei. In contrast, rid1 is dispensable for Neurospora crassa sexual development. We suggest that the rid1-like gene products and/or their DNA methyltransferase activities play critical roles in promoting fungal sexual development. The Neurospora crassa rid1 gene might have lost this evolutionarily conserved function.

     

Preparation and regeneration of mycelial protoplasts ofPleurotus florida andP. ostreatus

A method for isolation of higher frequency of regenerated protoplast fromPleurotus florida andP. ostreatus is reported.