The Genetic Construction Of The Cellulase-overproducing Strain Of Filamentous Fungi And Functional Study On P24Proteins

With the massive depletion of fossil fuels, biofuels based on lignocellulose have received much attention. The major barrier to realize the industrial lignocellulosic biorefinery is the high cost of Jignocellulolytic enzymes. The study on filamentous fungi is a research hotspot because of their protein-hypersecreting ability. It is generally accepted that the most effective strategy of improving cellulase production is to elevate cellulase transcription expression in filamentous fungi. Therefore, many research focus on cellulase transcription regulation in filamentous fungi. The secretory pathway is also very important, however, the reports on this aspect are fewer in comparison with cellulase transcription regulation. It is necessary to strengthen the study of secretory pathway for the purpose of construction of cellulase-hypersecreting strains in the further.The study in this dissertation is designed into two parts. One aspect is genetic engineering of cellulase-hypersecreting strain based on the knowledge of cellulase transcription regulation in filamentous fungi; the other is the function study on p24proteins which are predicted to locate in the secretory pathway of filamentous fungi.1. The rational reconstruction of P. oxalicum JUA10-1extracellular lignocellulolytic enzyme systemThe cellulase transcription repression factor creA and acel were identified by homologous alignment in P. oxalicum JUA10-1. The gel shift analysis experiment was shown:the CreA binding domain could bind to the cel7a promoter from-216to-446region; and Acel binding domain could bind to cell a promoter from-434to-643region. A new transcription regulation element was constructed as follows:creA binding domain+xlnR effect domain+acel binding domain. The new transcription regulation element was transformed into P. oxalicum JUA10-1to obtain the P. oxalicum CXA. The FPase,pNPCase, pNPGase and extracellular protein secretion were improved1.14,1.12,1.64and1.39fold in P. oxalicum CXA after feeding glucose into synthetic medium. And major cellulase and hemicellulase genes were significantly upregulated in P. oxalicum CXA. When they were cultivated in medium for industrial production, pNPCase,pNPGase and CMCase were improved1.31,1.55, and1.10fold in P. oxalicum CXA. The transcriptional expression of the major cellulase genes was also upregulated in P. oxalicum CXA. These results are indicated that the P. oxalicum CXA shows a promising prospect in industrial application.2. In comparison with physiological roles of p24homologous Erp between Trichoderma reesei and Penicillium oxalicump24family members located in the eukaryotic cell endomembrane system are small transmembrane proteins. The main roles of p24homologs in Saccharomyce cerevisiae and Drosophila melanogaster are cargo selective transportation and quality control. It is the first report on function study of the p24homologous Erp in filamentous fungi and a new cellulase transcription activation mechanism is discovered. Although Erp homologs in two filamenous fungi share above>75%sequence identity, the physiological pathways of two Erp homologs involved are apparently different. From the aspect of morphology. tErp in T. reesei takes part in the conidiospore formation, cell membrane maintenance, and hypha polarity formation; pErp in P. oxalium involves in cell membrane maintenance and lateral branching maturation. From the aspect of protein secretion:both strains suffered from secretion stress if erp was deleted in two filamentous fungi. But the two strains showed different transcription response mechanisms:the corresponding genes were down regulated in T. reesei, but their homologs in P. oxalicum were upregulated.3. The function study of a p24heterodimer in P. oxalicumThere are four p24members in P. oxalicum genome through bidirectional blastP best-hit search with S. cerevisiae p24proteins as queries. They belong to four different p24subfamilies. It is inferred that the most possible combination pattern of the four p24proteins in P. oxalicum is heterodimers formed by each other through intracellular protein SDS-PAGE analysis. Erp and Pδ were chosen for further study. They were confirmed to form a heterodimer in vivo by BiFC assay. The heterodimer takes part in many important morphological events. For example, conidiospore formation, hypha growth and lateral branching maturation. The quantitative proteomics revealed that the strain suffered from secretion stress after deletion of erp and pδ. It is indicated that the p24heterodimer involves in cargo transportation. Meanwhile, the strain activated many response mechanisms to survive:many proteins involved in clearing secreted protein jammed in the endomembrane system were upregulated; proteins involved in DNA replication and biosynthesis were downregulated. The p24heterodimer may possible take part in vesicle formation. There is minor effect on morphology and cargo transportation in S. cerevisiae Δp248strain, however, the P. oxalicum ΔpErppδ strain exhibits major defect on cargo transportation and morphology. This also reflects the function divergence of p24proteins among species.