The Role Of Transcription Factors CRE1 And ACE1 In Regulation Of Cellulase Gene Expression Of Myceliophthora Thermophila ATCC42464

In nature, the widespread lignocellulose is the most suitable for the production of energy of cheap raw materials. Lignocellulose can be degraded by many kinds of cellulase produced by microorganism.The enzymes of thermophilic fungi take advantage of high temperature resistance, strong stability and not easily polluted, which makes a high potential applications in industrial. In this study, RNAi technology was used to investigate the roles of CRE1 and ACE1 on gene regulation of lignocellulose degradation enzyme in Myceliopthora thermophila.The main contents of this study are as follows:(1)The role of carbon catabolite repressor 1(CRE1)is widely investigated in filamentous fungi. However, its role has not been reported in thermophilic fungi. This paper verifies the role of CRE1 in a thermophilic fungus, Myceliophthora thermophila ATCC42464, by using the method of RNA interference. In the cre1-silenced strain C88, the filter paper activity and β- 1, 4-endoglucanase activity was 3.76, and 1.31-fold higher, respectively than that in the parental strain when strains were cultured in inducing medium for 144 h. The activities of β-1, 4-exoglucanase and cellobiase were 2.64 and 5.59-fold higher, respectively than that in the parental strain when strains were cultured for 120 h. Quantitative reverse-transcription polymerase chain reaction showed that the gene expression of egl3, cbh1, and cbh2 was significantly increased in transformant C88 compared to the wild-type strain. Therefore, the result suggests CRE1 regulatory factor had a significant inhibition of cellulase genes in Myceliopthora thermophila.(2) ACE1 is also an important regulatory factor in Myceliopthora thermophila. Its application has not been reported in Myceliopthora thermophila. This paper verifies the role of ACE1 in a thermophilic fungus, Myceliophthora thermophila ATCC42464. Compared with the origin strain, in the ace1-silenced strain A98, the filter paper activity, β- 1, 4-endoglucanase activity, β-1, 4-exoglucanase, cellobiase and xylanase were 4.23, 3.43, 2.30, 7.99 and 3.33-fold higher. Therefore, the result suggested that ACE1 regulatory factor had a significant inhibition of cellulase genes in Myceliopthora thermophila.(3)What is more, another recombinant vector p UC19-Cre1 can be transformated into the strain A98 for improving cellulase production furtherly. The single colonies AC8 were screened from hygromycin B-containing(100μ g/m L) selection plates. Finally, in the co-transformation strain AC8, filter paper activity, β- 1, 4-endoglucanase activity, β-1, 4-exoglucanase and cellobiase were 11.47%、24.67%, 2.44% and 254% higher, respectively than that in the ace1-silenced strain A98. However, xylanase rarely changes in the result.The present study verified that it is a convenient and effective way to improve cellulase production by silencing genes cre1 and ace1. Interfering with two transcription factors in the same strain at the same time can further enhance cellulase activity and total protein production in the thermophilic fungus.