Screening Of Cellulase-producing Mutants By T-DNA Insertional Mutagenesis And Functional Analysis Of Vacuolar Protein Sorting Receptor In Trichoderma Reesei

Trichoderma reesei (anamorph:Hypocrea jecorina), known as an economically important filamentous fungus, has enjoyed a long history of producing many types of enzymes, such as cellulases, hemicellulases and proteinases in the biotechnology industry. Also, T. reesei has been used as an efficient producer of heterologous proteins due to its excellent ability to secrete proteins and the mechanism of eukaryotic protein modification and transportation. At present, the relatively low activity and production of cellulases is one of the major bottlenecks in the conversion of biomass into ethanol so that screening of mutants with high level of cellullase production is still highly regarded. In addition, the process of protein transportation in a fungal cell plays an important role in the secretion of a large amount of cellulases. Therefore, establishment of efficient molecular mutagenesis methods will facilitate the rapid screening of mutants with high-yield cellulase production, while functional analysis of specific target genes involved in protein secretion will help to identifiy the essential factors regulating the cellulases transfortation and secretion.Agrobacterium-mediated T-DNA insertional mutagenesis has proven to be a simple and reproducible filamentous fungal transformation method, whose advantages contain high efficiency, genetic stability and single-copy integration of T-DNA. Gene targeting has become a promising approach for gene function analysis and strain improvement. Therefore, development of convenient and efficient method for Agrobacterium-mediated T-DNA insertional mutagenesis and obtaining the mutant library in T. reesei would promote the rapid screening of mutants with higher ability to degrade cellulose. And, application of gene targeting to study the important genes involved in the protein secretion will contribute to understanding of the mechanism of cellulase transportation. This study used a new antibiotic resistance marker to develop T. reesei T-DNA insertion mutant library to select cellulase-producting mutants and applied gene targeting method to assay the vacuolar protein sorting receptor gene vps10 to understand the mechanism of cellulase transportation during secretion passway. T. reesei Rut-C30 was successfully transformed with Agrobacterium tumefaciens EHA105 using pyrithiamine resistance gene (ptrA) as an antibiotic marker. At first, the expression ptrA cassette derived from the plasmid T-ptrA was inserted into the Ti plasmid pCAMBIA0390 to construct the binary vector pCTA. Then, A. tumefaciens EHA105 was transformed by this vector, co-cultured with the spores of T. reesei Rut-C30, and screened on the plates containing pyrithiamine for the putative transformants. PCR assay showed that ptrA gene was integrated into the genomes of the selected transformants. After primary screening of transformants on the cellulose plates, over 30 cellulase-producing mutants were selected. Then the mutants were used for submerged fermentation for examination of the specific cellulase activities and 3 mutants with higher cellulase production than the parental strain were confirmed. These results indicated that Agrobacterium-mediated T-DNA insertional mutagenesis is a promising strategy to produce the cellulase-producing mutants.With the aim to study the function of the vacuolar protein sorting receptor gene vps10 in the cellulase secretion, the sequece of vps10 gene was retrieved from the T. reesei genome database and used to construct the gene disruption vector Pvps10-ptrA-Tvps10. This vector was transformed into the T. reesei by protoplast-based transformation and the vps10-disrupted strain QM-△VPS10 was constructed. Characterizaiton of the mutant strain QM-△VPS10 showed that its growth curve was similar to the parental strain. The biomass of the mutant strain displayed a littled bit higher than that of the parental strain, and vps10 disruption didn't influence the cellulase production in the mutant strain. vps10 disruption did not have significant effect on QM9414 strain, but it is reported that the vps10 gene knockout had enhanced protein production by heterlogous protein expression strain.In addition, a xylanase gene xynl-disrupted mutant△XYN1 was constructed based on the T. reesei Atku70 strain to assay the influence of absence of xylanase 1 on the cellulase production. It was found that the growth rate of the mutant strain△XYN1 was lower than that of the parental strain. The xylanase activity produced by△XYN1 decreased markedly and the FPA activity showed a little bit lower than that of the parental strain. This strain may be helpful in clarifying the interaction mechanism between the expression of hemicellulase and cellulose.