Double Knock-out Of Cellobiohydrolase Gene Cbh1and Cbh2in Trichoderma Reesei

Trichoderma reesei (anamorph:Hypocrea jecorina), known as a very important filamentous fungus, has the ability to secrete series cellulolytic enzymes known to participate synergistically in degradation of biomass. The production of cellobiohydrolases is perfect and60percent of total extracellular proteins are cellobiohydrolases. Trichoderma reesei has the advantages of simple structure, fast growth, and easy to manipulate and culture. And as eukaryote, it also has the similar eukaryotic protein modification apparatus and post-translation modification mechanism. Trichoderma reesei has enjoyed a long history of producing many types of enzymes, such as cellulases, hemicellulases and proteinases in the biotechnology industry. Trichoderma reesei has already been used as the host of heterologous protein. All the excellent characters encourage the researchers to study the molecular biological and genetic modification of Trichoderma reesei.In filamentous fungus multiple gene deletions are limited by the number of readily available selection markers. Therefore the development of a versatile transformation system independent on the number of available markers would be beneficial. With high transformation frequency and easy to obtain transformants, the uridine auxotrophy is widely used in the genetic transformation systems of filamentous fungus. According to the wild type phenotype screening the transformation strains after using the marker gene pyrG of uridine auxotrophy transformed the corresponding auxotrophy strain. When constructing blaster cassette, the marker gene pyrG encoding the orotidine-5-decarboxylase is flanked by two direct repeats. The transformants with the excision of the pyrG marker by recombination between the flanking direct repeats are obtained by selection on5-fluoroorotic acid (5-FOA). This strategy can realize a successful re-use of the marker gene pyrG in multiple gene deletions while does not lead to the accumulation of antibiotic resistance marker gene.The factors that affect the production of heterologous protein include the promoter and its copy number, signal peptide and the stability of protein in genetic engineering. In addition, the efficiently expressing of endogenous gene may affect the production of heterologous protein. It has been reported that the production of heterologous protein was improved via suppressing expression of an endogenic highly expressed gene. Therefore the expression of the main endogenous genes cbhl,cbh2,egl and eg2certainly will affect the expression of the exogenous genes in Trichoderma reesei. In this study, we carry on research depending on the idea that the main endogenous genes deletion mutant may be contribute to improving the production of heterologous protein.The blaster cassettes for cbhl gene and cbh2gene with the pyrG marker were constructed by recombinant PCR. In order to reuse the selection markers, the pyrG gene was flanked by two direct repeats. T. reesei△tku70strain was transformed with the cbh1blaster cassette and the cbh2blaster cassette respectively. Both cbhl-disrupted mutant Acbhl:;pyrG+and cbh2-disrupted mutant△cbh2::pyrG+were obtained via screening the mutants on MM. Then the cbhl-disrupted mutant△cbhl::pyrG+was selected on the selective media with5-fluoroorotic acid (5-FOA).Thus, the cbhl gene deletion mutant Acbhl without any selection marker was also obtained.The double cbhl/cbh2deletion mutant△cbh1△cbh2::pyrG+was constructed based on the T. reesei Acbhl strain. For excision of the pyrG blaster cassette, the mutant△cbh1△cbh2::pyrG+was selected for growth in the presence of5-fluoroorotic acid (5-FOA). And the double Acbh1Acbh2deletion mutant without pyrG marker was obtained. The T. reesei transformants were detected in PCR, Southern and SDS-PAGE analysis. These results indicated that gene deletion mutants△cbhl,△cbh2::pyrG+and Acbhl Acbh1were constructed successfully. SDS-PAGE analysis of culture supernatants indicated the secretion of cellobiohydrolase Ⅰ of the mutant strain Acbhl decreased markedly while secretion of cellobiohydrolase Ⅱ was a bit higher than that of the parental strain△tku70; the secretion of cellobiohydrolase I of the mutant strain△cbh2::pyrG+was similar to the parental strain△tku70while secretion of cellobiohydrolase Ⅱ had a little decrease; both the secretion of cellobiohydrolase I and cellobiohydrolase Ⅱ of the mutant strain△cbh1△cbh2were lowered compared with that of the parental strain△tku70.In this study, the cbhl deletion strain, the cbh2deletion strain and the double cbh1/cbh2deletion strain were successfully constructed in T. reesei△tku70using modern molecular biology technology. Functionality of this pyrG blaster cassette via the excision of the pyrG marker by recombination between the flanking direct repeats was a valuable pathway in filamentous fungus multiple gene deletions. In addition, T. reesei multiple gene deletions strains that constructed by genetic engineering will play a great role in industries of cellulose enzyme production, food and chemistry.