Analysis of cellulase synthesis mechanism in Trichoderma reesei using red fluorescent protein

Abstract:

Comprehensive analysis of the mechanism of metabolite synthesis in filamentous fungi is important for optimization of the filamentous fungus related industrial fermentation processes. In this work, the mechanism of cellulase synthesis in Trichoderma reesei was analyzed with red fluorescent protein (DsRed) as the reporting protein. The expression cassette for heterologous protein expression in T. reesei was constructed, through which the DsRed gene was inserted into the chromosomal DNA of T. reesei. The recombinant T. reesei strain, in which expression of DsRed was controlled by the promoter of cellobiohydrolase gene, was designated as T. reesei TR2.Expression of DsRed in T. reesei TR2 under different culture conditions was analyzed by using a fluorescent microscopy, and thereby the mechanism of cellulase gene expression in T. reesei could be interpreted. With induction of lactose, the pattern of change of red fluorescence in T. reesei TR2 was similar to that of the cellulase activity in the cultivation supernatant. As the culture aged, the red fluorescence in the mycelial increased. This was followed by a reduction in the end of the culture period because of death and autolysis of the mycelial. In the spatial aspect, the red fluorescence was distributed uniformly in the whole hypha after induction, indicating that all the three morphology including apical compartment, subapical compartment and hyphal compartment played a same role in cellulase synthesis. When T. reesei TR2 was cultivated without induction, faint red fluorescence appeared after a relative long period of cultivation, indicating that a small amount of cellulase was still synthesized without induction. This result was useful in explaining the mechanism of cellulase induction by insoluble cellulose.

Key words:Filamentous fungus;Trichoderma reesei;Red fluorescent protein;Cellulase;Cellobiohydrolase