Analysis Of The Cellobiohydrolase I Promoter Mutation As Well As Functional Study On The Gene Ppo Encoding Psi Factor Producing Oxygenase In Trichoderma Reesei

The cellulolytic fungus Trichoderma reesei is widely used in industry to produce cellulases. It can secretes a set of extracellular cellulases to degrade cellulase synergistically, among which exoglucanase CBHⅠ is the most abundant component. Inducible expression of cellulase genes is under the influence of carbon source, transcription regulators, as well as many other factors related to secondary metabolism.Many5’-GGC(A/T)3-3’motifs were found in the1.5-kbp5’-upstream region of cbhl. EMSA assay indicated Xyr1can bind five individual motifs with the sequence5’-GGC(A/T)3-3’present in the upstream region of cbhl. In the present study, we investigated the the effect of the individual mutation in the five5’-GGC(A/T)3-3’ motifs on Xyr1-cbh1promoter interaction by combinational employment of yeast-one-hybridization in vitro and construction of mutants of cbh1promoter in vivo. We also investigated the roles of psi factor producing oxygenases that are involved in synthesis of many secondary metabolites in cellulase induction by disrupting their two encoding genes ppoa and ppob respectively.To sum up, the research in this paper mainly includes the following three aspects:1. Site-directed mutagenesis in cbh1promoter affected transcriptional regulator Xyr1-cbh1promoter interactions.Yeast-one-hybridization system was employed to investigate the effect of the individual mutation at position2in the five5’-GGC(A/T)3-3’motifs on the interaction between Xyr1DBD and cbh1promoter. β-galactosidase activities of yeast transformants reflects interaction strength between Xyr1DBD and cbh1promoter. Analysis of β-galactosidase activities indicated that, yeast strains harboring mutation at position510displayed similar activities with that carrying wild type cbh1promoter. However, individual mutation at position187,320,733and778decreased the activity by23%,15%,26%and24%respectively, indicating the four5’-GGC(A/T)3-3’motifs containing the above four sites are probably involved in the interaction between Xyrl DBD and cbhl promoter.2. Analysis of the effect of site-directed mutagenesis in cbhl promoter on its in vivo activity by using uidA as reporter gene indicates that four5’-GGC(A/T)3-3’ motifs are probably involved in the interaction between Xyrl and cbhl promoter.Cbhl promoter fragments with mutations described above were introduced into T. reesei strain, and their activities were tested by measurement of GUS activities. Mutation at position510did not cause any change in GUS activity. However, individual mutation at position-187R,-320,-733R and-778results in significant decrease by50%,43%,45%and43%respectively, indicating these mutations affect activity of cbhl promoter in vivo. It is assumed the alteration in the interaction between transcriptional activator Xyrl and chbl promoters was responsible for this remarkable decreased promoter activity and in accordance with the result obtained by yeast-one-hybridization, these four5’-GGC(A/T)3-3’motifs containing the above four sites are probably involved in the interaction between Xyrl and cbhl promoter.3. Study on the role of psi factor producing oxygenases by deleting their coding genes ppoa and ppob respectively indicates that ppo are involved in the mycelium growth.Psi factor producing oxygenases (Ppo) are invovled in synthesis of many secondary metabolites. In order to investigate the role of Ppo in cellulase biosynthesis in T. reesei, we disrupted their two encoding genes ppoa and ppob to construct Appoa and Δppob. The absence of Ppo compromised the mycelium growth under several carbon sources, promoting aging and production of secondary metabolite such as pigment. Measurement of pNPC activity as well as western blot detection of CBH1of the supernatant from Avicel culture indicted that disruption of Ppo hardly has any effect on cellulase production.