Fermentation Condition Optimization And Transcriptome Analysis Of A Efficient Cellulose Degradation Fungus Aspergillus Fumigatus YC2

Cellulose is the most abundant renewable resource in agricultural waste,and it is of great significance if it can be transformed and utilized efficiently.Because the structure of cellulose is complex and not easy to be degraded,hydrolysis with cellulase is the key to this transformation process.However,at present,the production cost of commercial cellulase is high and the thermal stability of the enzyme is poor.with the development of the enzyme industry,the demand for cellulase is also increasing day by day.it is necessary to explore and discover new potential cellulose-degrading microorganisms and their cellulases.Domestic pig feed contains a high proportion of indigestible dietary fiber,which will become a potential source of cellulose-degrading microorganisms with fecal excretion.In this study,two strains of cellulose-degrading fungi were isolated from pig manure samples from pig farms in Northeast China and identified by morphology and molecular biology.by comparing the CMCase enzyme activity of the two strains,YC2 strain with higher CMCase enzyme activity was selected as the research target,and the fermentation conditions were optimized by PB design and CCD response surface analysis.The enzymatic properties of CMCase in YC2 crude enzyme solution were studied.The transcriptome of YC2 strain was sequenced and analyzed by RNA-seq technology,and the differentially expressed endoglucanase gene in the transcriptome was verified by q RT-PCR.This is the first report on the transcriptomics study of Aspergillus fumigatus on soluble substrate carboxymethylcellulose sodium(CMCNa).The main results are as follows:1.Two strains of fungi,named JC1 and YC2,were isolated from pig manure samples by dilution plate method.By morphological and molecular biological identification,they were identified as Geotrichum candidum and Aspergillus fumigatus,respectively.According to the preliminary determination of enzyme activity,it was found that the maximum FPase of strain JC1and YC2 was 0.281 U/m L and 1.74 U/m L,and the maximum CMCase activity was 0.541 U/m L and 3.89 U/m L,respectively.YC2 with higher FPase and CMCase activity was selected for further study.2.Optimize the fermentation conditions of YC2 on the initial production medium.First,select the two significant influencing factors of temperature and time through the PB design,and then conduct the central combination design(CCD)optimization analysis to determine that the strain YC2 produces cellulase(The best conditions for FPase are:when the temperature is 34.39?and the time is 2.04d,the maximum predicted value of FPase is 1.833 U/m L.The enzyme production verification was carried out under the optimized conditions,and the FPase activity was 1.803U/m L,which was 43.67%higher than the pre-optimized 1.255 U/m L.3.According to the study on the properties of YC2 crude enzyme solution CMCase,it was found that the optimum temperature was 60?,and the relative enzyme activity was more than70%in the range of 30??70?.The temperature stability was the best at 50?,and the residual activity could be maintained in the temperature range of 10?to 60?for 1 h.The optimum pH of CMCase of YC2 was 4.5,which could maintain more than 60%of CMCase activity in the range of pH 2-6,and the stability of pH was the best in pH=4.5,and the residual activity could be maintained in the range of pH 2-6 for 1 h.Metal ion Fe²⁺,Ni²⁺,Ca²⁺,Ba²⁺significantly promoted the activity of CMCase,while Fe³⁺,Cu²⁺,NH₄⁺,Zn²⁺,Mn²⁺,Al³⁺inhibited the activity of CMCase,while the chelating agent EDTA had no significant effect on the activity of CMCase.4.YC2 cultured with glucose and CMCNa as sole carbon sources were sequenced and analyzed by RNA-seq technique,and the biological processes and metabolic pathways related to the degradation of cellulose substrate by YC2 were analyzed at the transcriptional level.Compared with glucose group,1920 genes in CMCNa group were up-regulated and 2045 genes were down-regulated.Comparing these differential genes to GO,KOG and KEGG databases,we found a key GOterm"GO:0030248(cellulosebinding)"for cellulose degradation,an important KOG category G(Carbohydratetransportandmetabolism)related to cellulose degradation.Ko00500(starchandsucrosemetabolism),a key pathway for cellulose degradation,was significantly up-regulated by exoglucanase,endoglucanase and?-glucosidase genes related to cellulose decomposition in CMCNa group.Further annotation analysis of all the differentially expressed carbohydrate active enzyme(CAZymes)genes in CMCNa group showed that a total of 62CAZymes genes were up-regulated,which is inferred that they are closely related to the growth and metabolism of YC2 with CMCNa as the sole carbon source.5.Nine endoglucanase genes were selected for q RT-PCR verification,with glucose as the control group and CMCNa substrate as the treatment group.The results showed that the relative expression ratio of AFUA?8G01490 gene in CMCNa was the highest,up to 194.48 folds,followed by AFUA?7G06740(34.04 folds)?AFUA?6G11600(17.32 folds)?AFUA?7G06150(8.92 folds)and AFUA?6G01800(3.48 folds).They may be endoglucanases that play a major role in the degradation of CMCNa by YC2.