Construction And Characterization Of Endoplasmic Reticulum Protein Folding Factor Mutants In Trichoderma Reesei

Life on Earth relys on photosynthesis, which results in production of plant biomass having cellulose as the major component. With no doubt, cellulose is the largest renewable resource that can supply energy and materials persistently for hunman beings on the earth. With the exhaustion of oil resources and the proposed requirements for sustainable development of the environment, the development and utilization of clean energy arouses wide attention and research.Filamentous fungi include species characterized by efficient synthesis and secretion of hydrolytic enzymes and thus play an important role in decomposition of biological material in their natural habitats. Trichoderma reesei produces a variety of cellulolytic enzymes in large quantities and in a strictly controlled manner. After the complement of genomic sequencing in2008, the genome content of the carbohydrate active enzyme categories of T. reesei has been analysed. It was observed that on average it has lower numbers of glycosyl hydrolases, carbohydrate esterases, polysaccharide lyases and carbohydrate-binding module-containing enzymes than the Ascomycete species with sequenced genomes. T. reesei has200genes encoding glycosyl hydrolases, and the glycosyl hydrolase families involved in cellulose or hemicellulose degradation clearly have less members in T. reesei than in the other filamentous fungi with sequenced genomes. And also the kinetics of protein secretion and processing in the secretory pathway has been addressed. According to this study, the average synthesis time of the CBHI is4min, and its average secretion time is11min. The secretion time is actually longer than that measured with similar methodology in Saccharomyces cerevisiae and another filamentous fungus. Thus, it was concluded that the reason for the good secretion capacity of T. reesei is not the speed of secretion; it may rather be the large capacity of the secretion pathway. It was also found that if the protein can not effectivily enter into endoplasmic reticulum or correctly folded, it will induce the unfolded protein response (UPR) in the endoplasmic reticulum which can coordinate protein folding factors to participate in protein folding and secretion, and also it may inhibites the transcription of the protein. So in comparation with transcription, the effective folding and secretion may be the limiting factor of the celluases expressed in large quantity. Based on the above, our research work mainly includes the following aspects:1. Construction and characterization of T. reesei mutant strains defect on protein folding factors-Calnexin and PrpACalnexin is a transmembrane lectin in the endoplasmic reticulum which interacts with monoglucosylated, trimmed intermediates of the N-linked core glycans on newly synthesized glycoproteins; PrpA (PDI related protein A) is a PDI related peotein in the endoplasmic reticulum which also includes in protein folding. The transcription level of both of the two genes have an obvious increase when induced. We have knocked out the gene cne1and prpA in Trichoderma reesei first and analysed the properties of the mutant strains. The deletion cassette of cne1and prpA was constructed using Gateway system, and tansformed into T.reesei. Deletion of cne1and prpA had no effect on growth of mycelia. Compared to the original strain, the extracellular protein concentration and enzymes activities of ΔprpA were unconspicuous while the pNPC and pNPG activities of Δcne1were significantly increased.2. The formation analysis and purification of CBHI in Δcne1strainSince Calnexin is an important folding chaperone in the endoplasmic reticulum, its deficiency may has effect on two aspects:(1) the stringency of the endoplasmic reticulum quality control system is damaged and the unfolded protein can be secreted to the out space;(2) the uncorrectly folded protein can not be secreted and retained in the endoplasmic reticulum,which can cause the UPR. To investigate the phenotype of the mutant strain, we try to anlyse the formation, secretion and the stability of cellulases and separate and purify CBHI from fermentation liquor.Without Calnexin, the major cellulase component CBHI can be detected in intracellular at12h and with the extended induction time, the signal became weakened. However, the amount of the extracellular CBHI and the stability of it share no distinction between the Δcne1and WT strain.The cellulases in the fermentation liquor was first been concentrated and then through two step ion-exchange column chromatography to be purified. When we got the purified CBHI, we found that the pNPC activity of the mutant strain was not higher than the wide type strain any more. And also we found that there was an obvious differential protein in the fermentation liquor between wide type and the mutant strains. Through mass spectrum identification, we found the differential protein was an extracellular β-glucosidase named Cel3b.In the meanwhile, we try to isolate the possible protein folding factors that assist the cellulases in folding and secretion efficiently through co-immunoprecipitation. The work is still needed to be further.