Mutagenesis and evaluation of cellulase properties and cellulose hydrolysis of Talaromyces piceus.

A fungal species with a high yield of ß-glucosidase was isolated and identified as Talaromyces piceus 9-3 (anamorph: Penicillium piceum) by morphological and molecular characterization. Through dimethyl sulphate mutagenesis, the cellulase over-producing strain T. piceus H16 was obtained. The FPase activity and ß-glucosidase activity of T. piceus H16 were 5.83 and 53.12 IU ml(-1) respectively--a 5.34- and 4.43-times improvement from the parent strain T. piceus 9-3. The optimum pH and temperature for enzyme activity were pH 5.0 and 50 °C for FPase activity and pH 5.0 and 55 °C for ß-glucosidase activity, respectively. The cellulase were quite stable at 37 °C, only losing <10% of their initial activity after 24 h of incubation. Hydrolysis analysis results showed that a highly efficient synergistic effect was achieved by combining cellulase from T. piceus H16 with that from Trichoderma reesei RUT C30 on hydrolyzing different substrates due to the high ß-glucosidase activity of T. piceus H16. These data suggest that T. piceus H16 can be used as a potential cellulase producer with good prospects.

Effect of earlier unfolded protein response and efficient protein disposal system on cellulase production in Rut C30.

Trichoderma reesei (T. reesei) has been widely used in production of cellulolytic enzymes and heterologous proteins because of its high secretion capacity. The lack of knowledge on protein secretion mechanisms, however, still hinders rational improvement on cellulase production. The transcript levels of cellulases and components involved in post-transcriptional procedures were compared in this study between two mutants, QM9414 and Rut C30 for evaluating the effects of modification and secretion upon cellulase production. The results showed that cellulase induction by cellulose drastically up-regulated expressions of the sensor of unfolded protein, chaperone and folding-assisted enzymes in endoplasmic reticulum and resulted in unfolded protein response (UPR) and low-grade increase in secretory transporters' expression similar to that of chemical treatment. Rut C30 demonstrated earlier and more sustainable expressions of elements involved in UPR and lower amount of cellular retained cellulase compared to QM9414, indicating that Rut C30 had hypercellulolytic property partially for its earlier and enhanced UPR to more efficiently dispose of protein. Modifying post-translational peptides and enhancing protein flux to avoid protein accumulation during cellulase production may be a feasible approach for strain improvement.

Duckweed (Lemna minor) is a novel natural inducer of cellulase production in Trichoderma reesei.

An inducer is crucial for cellulase production. In this study, duckweed was used as an inducer of cellulase production by Trichoderma reesei RUT C30. In a reaction induced by 50 g/L duckweed in shake flasks, the filter-paper activity (FPA) reached 6.5 FPU/mL, a value comparable to that induced by avicel. The enzyme-hydrolysis rate induced by steam-exploded corn stalk was 54.2%, representing a 28% improvement over that induced by avicel. The duckweed starch was hydrolyzed to glucose, which was subsequently used for biomass accumulation during the fermentation process. Furthermore, to optimize the control of the fermentation process, a combined substrate of avicel and duckweed was used to induce cellulase production by T. reesei RUT C30. The cellulase production and hydrolysis rates of the combined substrate, compared with avicel alone, were 39.6% and 36.7% higher, respectively. The results of this study suggest that duckweed is a good inducer of cellulase production in T. reesei, and it might aid in decreasing the cost of lignocellulosic materials hydrolysis.

Construction of an efficient RNAi system in the cellulolytic fungus Trichoderma reesei.

An improved RNA interference method was developed in Trichoderma reesei, using convergent dual promoters for efficient and high-throughput RNA silencing. This new vector allowed for the silencing of the eGFP gene and target genes to occur simultaneously, significantly facilitating the rapid screening of the transformants using eGFP as a reporter.

Purification and characterization of a new ß-glucosidase from Penicillium piceum and its application in enzymatic degradation of delignified corn stover.

A new ß-glucosidase (Cel3B) was first isolated from cellulytic fungi, designated as PpCel3B. Although PpCel3B was classified to GH family 3 based on the homology sequence, PpCel3B had different biological functions in cellulose degradation and signaling molecules production. PpCel3B was constitutive and could form multiple soluble lignocellulose inducers for cellulase and hemicellulase synthesis via high tranglycosylation activity and new enzymatic activity. Moreover, PpCel3B showed apparent synergism with cellulases by removing several inhibitors. Supplementing low doses of PpCel3B (52 µg/g substrate) increased saccharification efficiency of cellulase produced by Trichoderma reesei and Penicillium piceum by 15% and 35%, respectively on delignified corn stover. PpCel3B had important application in boosting cellulase yield and efficiency.