Improving Cellulase Secretion Of Trichoderma Reesei By Modulating The Expression Of Transcriptional Factors

As the most commonly used microbe in cellulase production,Trichoderma reesei not only has a strong ability of protein expression but also can secrete extracellular cellulase.This character is helpful to reduce the cost of industrial application.However,the enzyme production ability of wild-type strains is very weak and commonly cannot meet the industrial production demand.Hence,this study sought to improve cellulase production by strain improvement for industrial manufacture.The RNA interference(RNAi)technique was used to repress the expression of the main transcriptional repressor ere1 in order to improve the cellulase production of Trichoderma reesei.The TU6?KU70 strain was selected as the research object,because it has improved efficiency for homologous recombination of exogenous genes.The cbhl promoter,a reversed cre1 gene fragment(568-963 bp),an ordinary cre1 gene fragment(655-961 bp),and the cbh2 terminator were all amplified from the genomic DNA of T.reesei by polymerase chain reaction(PCR).The DNA assembler method was used to assemble all these gene fragments in pRS424 to obtain the pCre1-i plasmid.Two fragments encompassing the RNAi cassette were amplified from the plasmid and transformed simultaneously into T.reesei.PCR was used to verify the positive colonies.Five positive transformants were obtained.In the flask batch culture,compared with the parent strain,the filter paper activity of one transformant increased by 30%.RT-qPCR revealed that the transcript level of cre1 in this transformant reduced by 43%in comparison with the parent strain.To further explore how simultaneous control of transcription activation and repression factor of T.reesei would effect on the cellulase production,we overexpressed xyr1 while repressed crel expression by RNAi as mentioned above.A selected strong promoter was used to drive xyr1 gene expression and the RNAi technique was used to repress the expression of cre1.Four positive transformants were obtained.In the flask batch culture,the celluase of all transformants were increased.One of the transformants had 2-fold increased filter paper cellulase activity higher than the parent strain.Compared with the strain only under regulation of cre1 RNA interference,the cellulase of this transformant was increased by 70%.This technique will be beneficial to cellulase production by T.reesei in the field of food.