Functional Study Of A New Factor MtTRC-1 For Regulation Of Cellulase Expression In Myceliophthora Thermophila

The thermophilic fungus Myceliophthora thermophila has been used to produce various industrial enzymes and biobased chemicals because of its characteristics of high temperature fermentation,biomass degradation ability and strong protein secretion ability.In contrast to the research on the mesophilic fungi,there have been relatively few studies on the mechanism regulating cellulase production as well as the ER stress-triggered UPR pathway in thermophilic fungi.In this study,we identified and characterized a novel regulator of cellulase production,MtTRC-1（Cys₂His₂ transcription factor,Mycth＿90344）,in the thermophilic fungus M.thermophila by conducting functional phenotypic assays and applying genetic tools.The deletion of MtTRC-1 significantly decreased the cellulase production induced by crystalline cellulose（Avicel）.The secreted protein amount and enzyme activities were restored to MtWT levels via native and interspecies complementation.The MtTRC-1 was exclusively located in the M.thermophila nucleus.The transcription levels of 83 CAZyme genes were significantly down-regulated inΔMtTrc-1.The initial transcriptome analysis ofΔMtTrc-1 also showed that the expression of Mthac-1,which encodes a regulator of the UPR pathway,decreased significantly,indicating MtTRC-1 may contribute to MtHAC-1-dependent UPR signaling.ER stress-induced splicing removed a 23-nt intron from the Mthac-1 m RNA.Moreover,protein secretion was dramatically impaired by the deletion of MtHAC-1.The Mthac-1transcription level was markedly lower in theΔMtTrc-1 mutant than in the wild-type strain under cellulose and ER stress conditions.Electrophoretic mobility shift assays（EMSA）and chromatin immunoprecipitation assays（Ch IP）verified that MtTRC-1 regulates the transcription of Mthac-1 and the major cellulase gene Mtcbh-1 by binding directly to the promoters.Furthermore,DNase I footprinting assays identified the putative consensus binding site（5′-GNG/C-3′）.In conclusion,this study reveals the importance of MtTRC-1 for regulating cellulase production through a novel two-dimensional mechanism involving the MtHAC-1-mediated secretory pathway and direct transcriptional regulation.This two-dimensional regulatory mechanism precisely controls cellulase production in M.thermophila.The conservation of MtTRC-1 in filamentous fungi suggests this mechanism may be exploited to engineer filamentous fungal cell factories capable of producing proteins on an industrial scale.