Deciphering The Function Of Cellulase Related Gene Ncw1

Biomass degradation focuses on how to depolymerize complicated lignocellulose into monosaccharides, as well as how to convert simple sugars into bio-based products efficiently. However, the high cost of cellulase lags this conversion. Considering how to lower the production costs of cellulase, it is necessary to decipher the mechanism of how the vital model fungus Neurospora crassa degrade cellulose and to implement genetic modification for cellulase high-yielding strain Trichoderma reesei RL-P37.As an important model fungus, Neurospora crassa can be readily used to study the mechanism of cellulase expression and secretion. The Ancwl mutant can promote the expression and secretion of cellulase, so in this study the molecular and system biology methods were employed to decipher the mechanism under the surface. Firstly, the boosting cellulase phenotype was affirmed by shake flask fermentation and RT-qPCR assay. Then the growth test confirmed that the mutant is consistent with the sensitivity of the wild type when grown on MM with temperature or Congo Red. And the mycelia induced transferring experiment showed no difference on cellulase secretion compared with spores inoculated into Avicel medium directly. The cold-field scanning electron microscopy result indicates deletion of new 1 can give rise to changes in cell wall. When complementing newl ORF with eGFP expressing gene under different promoters into △ncwl.his-strain, the fluorescent signal focused in the septum of mature mycelium. The expressing strain with ccgl promoter shows similar phenotype with wild type. By crossing the Ancwl mutant with the carbon metabolism repressor factor mutant Acrel, the dual mutant with enhanced cellulase was got, demonstrating NCW1 doesn’t affect carbon catabolite reaction directly. And when crossed with △3βG, the quadruple mutant S22 was got. This strain can accelerate cellobiose consumption and cellalse induction compared with △3βG, implying NCW1 correlates with cellobiose utilization and cellulases induction. From the present study, we elucidate the basic roles of the ncwl in cellulose expression and secretion in N. crassa.As a cellulase high-yielding strain, Trichoderma reesei RL-P37 has been widely used to produce large quantities of enzymes and proteins for industrial processes. It is well known that the properties of industrial strains could be improved by genetic manipulation. Thus the pyrimidine-auxotrophic mutant of T. reesei RL-P37 needed to be constructed. This system could be employed to introduce or delete desired genes without leaving any undesired DNA. In this paper, hygromycin was used as a selective marker to disrupt the pyr4 gene in T. reesei RL-P37. And the pyr4 disrupted strain was verified by southern blotting. The mutant could grow well in MM containing 1.5 mg/mL 5-FOA and 2 mg/mL uridine, but could not grow on MM medium without uridine. In addition, the mutant restored to prototrophy with pyr4 gene from Neurospora crassa. The system constructed here could be used as a host strain for genetic operation of targeted gene (ncwl included) as well as expression of exogenous proteins.