Measurement of Cellulase and Xylanase Activities in Trichoderma reesei.

The microbial cellulase system is responsible for the generation of glucose from cellulose. Cellulases are comprised of at least three major groups of enzymes, namely endoglucanases, exoglucanases, and ß-glucosidases. On the other hand, xylanases function in the degradation of hemicellulose and work synergistically with cellulases for the degradation of lignocellulosic biomass. Here, we describe the most commonly used methods for the activity measurement of cellulases and xylanases.

Engineering the Effector Domain of the Artificial Transcription Factor to Improve Cellulase Production by Trichoderma reesei.

Filamentous fungal strains of Trichoderma reesei have been widely used for cellulase production, and great effort has been devoted to enhancing their cellulase titers for the economic biorefinery of lignocellulosic biomass. In our previous studies, artificial zinc finger proteins (AZFPs) with the Gal4 effector domain were used to enhance cellulase biosynthesis in T. reesei, and it is of great interest to modify the AZFPs to further improve cellulase production. In this study, the endogenous activation domain from the transcription activator Xyr1 was used to replace the activation domain of Gal4 of the AZFP to explore impact on cellulase production. The cellulase producer T. reesei TU-6 was used as a host strain, and the engineered strains containing the Xyr1 and the Gal4 activation domains were named as T. reesei QS2 and T. reesei QS1, respectively. Compared to T. reesei QS1, activities of filter paper and endoglucanases in crude cellulase produced by T. reesei QS2 increased 24.6 and 50.4%, respectively. Real-time qPCR analysis also revealed significant up-regulation of major genes encoding cellulase in T. reesei QS2. Furthermore, the biomass hydrolytic performance of the cellulase was evaluated, and 83.8 and 97.9% more glucose was released during the hydrolysis of pretreated corn stover using crude enzyme produced by T. reesei QS2, when compared to the hydrolysis with cellulase produced by T. reesei QS1 and the parent strain T. reesei TU-6. As a result, we proved that the effector domain in the AZFPs can be optimized to construct more effective artificial transcription factors for engineering T. reesei to improve its cellulase production.

Identification of a novel repressor encoded by the putative gene ctf1 for cellulase biosynthesis in Trichoderma reesei through artificial zinc finger engineering.

Strains from Trichoderma reesei have been used for cellulase production with a long history. It has been well known that cellulase biosynthesis by the fungal species is controlled through regulators, and elucidation of their regulation network is of great importance for engineering T. reesei with robust cellulase production. However, progress in this regard is still very limited. In this study, T. reesei RUT-C30 was transformed with an artificial zinc finger protein (AZFP) library, and the mutant T. reesei M2 with improved cellulase production was screened. Compared to its parent strain, the filter paper activity and endo-ß-glucanase activity in cellulases produced by T. reesei M2 increased 67.2% and 35.3%, respectively. Analysis by quantitative reverse transcription polymerase chain reaction indicated significant downregulation of the putative gene ctf1 in T. reesei M2, and its deletion mutants were thus developed for further studies. An increase of 36.9% in cellulase production was observed in the deletion mutants, but when ctf1 was constitutively overexpressed in T. reesei RUT-C30 under the control of the strong pdc1 promoter, cellulase production was substantially compromised. Comparative transcriptomic analysis revealed that the deletion of ctf1 upregulated transcription of gene encoding the regulator VIB1, but downregulated transcription of gene encoding another regulator RCE1, which consequently upregulated genes encoding the transcription factors XYR1 and ACE3 for the activation of genes encoding cellulolytic enzymes. As a result, ctf1 was characterized as a gene encoding a repressor for cellulase production in T. reesei RUT-C30, which is significant for further elucidating molecular mechanism underlying cellulase biosynthesis by the fungal species for rational design to develop robust strains for cellulase production. And in the meantime, AZFP transformation was validated to be an effective strategy for identifying functions of putative genes in the genome of T. reesei.

Engineering Trichoderma reesei Rut-C30 with the overexpression of egl1 at the ace1 locus to relieve repression on cellulase production and to adjust the ratio of cellulolytic enzymes for more efficient hydrolysis of lignocellulosic biomass.

Cellulose hydrolysis is a synergetic process performed sequentially by different cellulolytic enzymes including endoglucanases, exoglucanases and glucosidases. Trichoderma reesei has been acknowledged as the best cellulase producer, but cellulase production by T. reesei through submerged fermentation is costly due to intensive energy consumption associated with the process for mixing and aeration, since non-Newtonian fluid properties are developed with mycelial growth. Therefore, engineering the ratio of cellulolytic enzymes in the cocktail for more efficient cellulose hydrolysis is an alternative strategy for reducing cellulase dosage and thus saving cost in enzyme consumption for cellulose hydrolysis. In this study, T. reesei QS305 with high endoglucanase activity was developed from T. reesei Rut-C30 by replacing the transcription repressor gene ace1 with the coding region of endoglucanase gene egl1. Compared to T. reesei Rut-C30, T. reesei QS305 showed 90.0% and 132.7% increase in the activities of total cellulases and endoglucanases under flask culture conditions. When cellulase production by T. reesei QS305 was performed in the 5-L fermentor, cellulases activity of 10.7 FPU/mL was achieved at 108 h, 75.4% higher than that produced by T. reesei Rut-C30. Moreover, cellulases produced by T. reesei QS305 were more efficient for hydrolyzing pretreated corn stover and Jerusalem artichoke stalk.

A Newly Designed Fiber-Optic Based Earth Pressure Transducer with Adjustable Measurement Range.

A novel fiber-optic based earth pressure sensor (FPS) with an adjustable measurement range and high sensitivity is developed to measure earth pressures for civil infrastructures. The new FPS combines a cantilever beam with fiber Bragg grating (FBG) sensors and a flexible membrane. Compared with a traditional pressure transducer with a dual diaphragm design, the proposed FPS has a larger measurement range and shows high accuracy. The working principles, parameter design, fabrication methods, and laboratory calibration tests are explained in this paper. A theoretical solution is derived to obtain the relationship between the applied pressure and strain of the FBG sensors. In addition, a finite element model is established to analyze the mechanical behavior of the membrane and the cantilever beam and thereby obtain optimal parameters. The cantilever beam is 40 mm long, 15 mm wide, and 1 mm thick. The whole FPS has a diameter of 100 mm and a thickness of 30 mm. The sensitivity of the FPS is 0.104 kPa/µÎµ. In addition, automatic temperature compensation can be achieved. The FPS's sensitivity, physical properties, and response to applied pressure are extensively examined through modeling and experiments. The results show that the proposed FPS has numerous potential applications in soil pressure measurement.

[XPS analysis of beads formed by fuse breaking of electric copper wire].

The in-depth composition of beads formed by fuse breaking of the electric copper wire in different circumstances was studied by XPS with Ar+ ion sputtering. In addition, the measured Auger spectra and the calculated Auger parameters were compared for differentiation of the substances of Cu and Cu2O. Corresponding to the sputtering depth, the molten product on a bead induced directly by fuse breaking of the copper wire without cover may be distinguished as three portions: surface layer with a drastic decrease in carbon content; intermediate layer with a gentle change in oxygen content and gradually diminished carbon peak, and consisting of Cu2O; transition layer without Cu2O and with a rapid decrease in oxygen content. While the molten product on a bead formed by fuse breaking of the copper wire after its insulating cover had been burned out may be distinguished as two portions: surface layer with carbon content decreasing quickly; subsurface layer without Cu2O and with carbon and oxygen content decreasing gradually. Thus, it can be seen that there was an obvious interface between the layered surface product and the substrate for the first type of bead, while as to the second type of bead there was no interface. As a result, the presence of Cu2O and the quantitative results can be used to identify the molten product on a bead induced directly by fuse breaking of the copper wire without cover and the molten product on a bead formed by fuse breaking of the cupper wire after its insulating cover had been burned out, as a complementary technique for the judgments of fire cause.