The filamentous fungus Trichoderma reesei has been widely used for cellulase production that has extensive applications in green and sustainable development. Increasing costs and depletion of fossil fuels provoke the demand for hyper-cellulase production in this cellulolytic fungus. To better manipulate T. reesei for enhanced cellulase production and to lower the cost for large-scale fermentation, it is wise to have a comprehensive understanding of the crucial factors and complicated biological network of cellulase production that could provide new perspectives for further exploration and modification. In this review, we summarize recent progress and give an overview of the cellular process of cellulase production in T. reesei, including the carbon source-dependent cellulase induction, complicated transcriptional regulation network, and efficient protein assembly and trafficking. Among that, the key factors involved in cellulase production were emphasized, shedding light on potential perspectives for further engineering.

The methylotrophic yeast, Pichia pastoris, is widely used as a useful experimental tool in protein engineering and production. It is common for proteins expressed in P. pastoris to exhibit N-glycosylation. In recent years, glycosylation studies in P. pastoris have attracted increasing attention from scholars. Rhizopus chinensis lipase (RCL) is one of the most important industrial lipases, and it has four potential N-linked glycosylation sites. The aim of the present study was to determine whether RCL undergoes asparagine-linked (N-linked) glycosylation and to examine the role of this modification in RCL expression and function. In this study, we demonstrated that RCL expressed in Pichia pastoris was N-glycosylated at the sites N-14, N-48 and N-60. The majority of the sites N-14 and N-60 were glycosylated, but the glycosylation degree of the site N-48 was only a very small portion. The glycan on N-60 played a key role in the expression and secretion of RCL. RT-PCR results showed that the mRNA level of proRCLCN60Q remained unchanged even though the protein secretion was hampered. Although the N-glycan on N-14 had no effect on the secretion of RCL, this glycan was beneficial for the lipase catalytic activity. On the other hand, the little amount of N-glycan on N-48 had no effect both on the secretion and activity of RCL in P. pastoris. Moreover, the thermostability analysis of RCL revealed that the lipase with more N-glycan was more thermostable. RCL was N-glycosylated when expressed in P. pastoris. The N-glycans of RCL on the different sites had different functions for the secretion and enzymatic properties of the lipase. Our report may also provide theoretical support for the improvement of enzyme expression and stability based on the N-linked glycosylation modification to meet the future needs of the biotechnological industry.