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.

Meng QS ，Liu CG ，Zhao XQ ，Bai FW ... -  《-》

Improvement of cellulase production in Trichoderma reesei Rut-C30 by overexpression of a novel regulatory gene Trvib-1.

Trichoderma reesei is a widely used cellulase producer, and development of robust strains for improved cellulase production is of great interest. In this study, the gene Trvib-1 encoding a putative transcription factor was overexpressed in T. reesei Rut-C30, and effects on cellulase production by the manipulation as well as corn stover degradation by the crude enzyme were investigated. Cellulase production and protein secretion were significantly improved in the culture of the recombinant T. reesei Vib-1, which were 200% and 219%, respectively, higher than that produced by the parent strain. Cellulase induction was enhanced in the presence of pure cellulose as well as various soluble inducers. Glucose released from the pretreated corn stover hydrolyzed by the crude enzyme in the recombinant strain was improved 40%. These results indicate that the overexpression of Trvib-1 is a feasible strategy for producing cellulase to enhance bioconversion efficiency of lignocellulosic biomass.

Zhang F ，Zhao X ，Bai F 《-》

Enhanced cellulase production in Trichoderma reesei RUT C30 via constitution of minimal transcriptional activators.

Zhang J ，Zhang G ，Wang W ，Wang W ，Wei D ... -  《Microbial Cell Factories》

Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator.

The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72IU/mL, respectively, were achieved using glucose as sole carbon source within 48h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved.

Zhang X ，Li Y ，Zhao X ，Bai F ... -  《-》

Enhanced cellulase production from Trichoderma reesei Rut-C30 by engineering with an artificial zinc finger protein library.

Trichoderma reesei Rut-C30 is a well-known cellulase producer, and improvement of its cellulase production is of great interest. An artificial zinc finger protein (AZFP) library is constructed for expression in T. reesei Rut-C30, and a mutant strain T. reesei U3 is selected based on its enhanced cellulase production. The U3 mutant shows a 55% rise in filter paper activity and 8.1-fold increased β-glucosidase activity, when compared to the native strain T. reesei Rut-C30. It is demonstrated that enhanced β-glucosidase activity was due to elevated transcription level of β-glucosidase gene in the U3 mutant. Moreover, significant elevation in transcription levels of several putative Azfp-U3 target genes is detected in the U3 mutant, including genes encoding hypothetical transcription factors and a putative glycoside hydrolase. Furthermore, U3 cellulase shows 115% higher glucose yield from pretreated corn stover, when compared to the cellulase of T. reesei Rut-C30. These results demonstrate that AZFP can be used to improve cellulase production in T. reesei Rut-C30. Our current work offers the establishment of an alternative strategy to develop fungal cell factories for improved production of high value industrial products.

Zhang F ，Bai F ，Zhao X 《-》