Basic Research On Mechanism Of Cellulase Biosynthesis By T.reesei Under PEG-induced Osmotic Stress

Trichoderma reesei is the main strain for production of cellulase and hemicellulase,and possesses excellent ability of cellulase biosynthesis.Preliminary research showed that certain stress conditions could promote cellulase synthesis in T.reesei.In this work,influence of osmotic stress mediated by polyethylene glycol(PEG)on cellulase biosynthesis was investigated using the strain T.reesei CICC2626.The DEGs related to cellulase biosynthesis were analyzed by transcriptome,and the regulatory mechanism of cellulase biosynthesis in T.reesei under PEG8000-induced osmotic stress was preliminarily revealed.The works in this thesis mainly were listed as following:First,effects of different nitrogen and carbon sources on cellulase production by T.reesei were investigated.The results showed that using corn steep liquor as nitrogen source and pretreated rice straw as carbon source,the maximal cellulase activities 3.83U/m L(FPase),4.02 U/m L(CMCase),2.50 U/m L(?-glucosidase)were observed,respectively.The fermentation conditions of cellulase production were optimized using response surface method(RSM).The result showed that the optimal conditions for enzyme production were nitrogen source concentration of 4.5 g/L,carbon source concentration of 9.5 g/L,p H 6,30oC.The peak of cellulase activity was up to 4.53(FPase),9.9(CMCase),and 4.15 U/m L(?-glucosidase)under the optimal condition,which was increased twice than that of the pre-optimization.Then,the thermal stability,p H stability and hydrolytic characteristics of the crude enzyme in the fermentation solution were investigated,respectively.It was found that cellulase could maintain relatively high activity within the range of p H(5-7)and temperature(30-50°C),the maximal cellulase activity was obtained at temperature of 50°C and p H of 7.The maximal reducing sugar concentration of 55.5 mg/m L was obtained when the pretreated rice straw had been hydrolyzed for 36 h by crude enzyme in the fermentation solution.The characteristics of cellulase biosynthesis in T.reesei CICC2626 under osmotic stress condition was investigated.The results showed that cellulase activity was significantly enhanced under osmotic stress induced by PEG8000,and the maximal cellulase activity was obtained at PEG8000 concentration of 1.5%(w/v,229 m Osm/kg),which was increased 2-fold than that of the control(0%PEG8000).Moreover,the cellulase-encoding genes cbh,cel3b,egl,and transcription factor xyr1 at different concentrations of PEG8000 were detected by q PCR.The result showed that the transcriptional levels of cellulase-encoding genes were significantly upregulated,especially,the transcriptional level of the cellulase-encoding gene cbh was increased about 50-fold.Thus,it could be considered that the improvement of cellulase activity under osmotic stress induced by PEG8000 was achieved by up-regulation expression of cellulase-encoding genes.To further reveal the regulatory mechanism of cellulase biosynthesis by T.reesei CICC2626 under osmotic stress induced by PEG8000,transcriptomics of the T.reesei CICC2626 subjected to 1.5%PEG8000(229 m Osm/kg)stress was performed.3850DEGs were obtained,including 2081 up-regulation genes and 1769 down-regulation genes.Functional annotation of these DEGs were performed,and it was found that there were 79 up-regulation genes in the glycoside hydrolase family,the genes of encoding cellobiohydrolase cel7a and cbh were up-regulated 5.51-fold and 6.20-fold,respectively.Also,the transcriptome data demonstrated that these genes related to Ca²⁺transporter and Ca²⁺metabolic pathways were up-regulated.The results showed that the up-regulation expression of these genes related to Ca²⁺signaling pathway in T.reesei subjected to PEG8000-induced osmotic stress caused a significant improvement of cellulase activity.