The Functional Characterization Of Cellulase Regulators CER-3 And CER-4 In Neurospora Crassa

The development of sustainable biomass-based chemicals and fuels has been attracting extensive attentions and synergistic action of many lignocellulytic enzymes on the degradation of the complex structure of biomass is the research focus in the biorefineries.Expression and secretion of lignocellulose hydrolyase genes in filamentous fungi are mainly regulated at the transcriptional level in the large-scale production of industrial enzymes.Therefore,the identification of the key cellulases regulators would play an important role in exploring the mechanism and promoting the cellulases production.In order to uncover the key regulatory genes which were important for cellulases expression and secretion,the cellulase secretion levels of 53 knock-outs of single zinc finger transcription factor respectively in Neurospora crassa were screened using the 2%crystalline cellulose as the sole carbon.By measuring secreted protein,endo-beta-1,4-glucanase activity,cellobiohydrolase activity,beta-glucosidase activity and xylansae activity,that the protein concentration and the cellulase production were significantly decreased in the strains with deletion of cer-3 and cer-4,which revealed that CER-3 and CER-4 presented an important influence on the regulation of the cellulases expression and secretion in N.crassa.For further study of transcriptional regulation of new transcription factors CER-3 and CER-4,we constructed complement strains for cer-3 and cer-4 mutants.And the results revealed the lack of the cer-3 and cer-4 devoted to the decrease of the protein concentration and cellulases production in N.crassa.Meanwhile,through the analysis of the transcriptome of the Acer-3 and ?cer-4,we found that CER-3 and CER-4 belonged to the upstream regulators,ownig the regulation and control function for CLR-2,a positive regulator of cellulases and Cre-1,a regulator of carbon catabolic repression.The cellulases were increased on the level of secretion and the level of transcriptome in the N.crassa of overexpressing cer-3 and cer-4 respectively,which proved that CER-3 and CER-4 serviced the positive regulators in the expression of cellulases.In addition,we found the homologous genes of cer-3 and cer-4 in Myceliophthora thermophila via the analysis of evolutionary tree.In order to identify the function conservation of cer-3 and cer-4,we developed the genetic operation system for M.thermophila and we respectively expressed the cer-3 and cer-4 from N.crassa.The results revealed the expression of cer-3 and cer-4 enhanced the expression and secretion levels of cellulases in M.thermophila,which presented the function conservation.In a word,the CER-3 and CER-4 played an important role in the expression and secretion of cellulases,of which the discovery would make the regulatory network of cellulases more perfect and promoted the industrial strain improvement for commercial cellulases production.