title: The use of T-DNA insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens Y1.
abstract: Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase, and enhanced green fluorescent protein. We optimized transformation efficiency to obtain over 300 transformants/10(6) conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library, and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase activities of T4, measured at the end of fermentation, were 60%, 440%, 320%, 41%, and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned, and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens.
authors: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors: Xinxin
authors: X
authors: Xu
authors: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors: Jinyang
authors: J
authors: Li
authors: Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors: Pengjun
authors: P
authors: Shi
authors: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors: Wangli
authors: W
authors: Ji
authors: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors: Bo
authors: B
authors: Liu
authors: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors: Yuhong
authors: Y
authors: Zhang
authors: Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors: Bin
authors: B
authors: Yao
authors: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors: Yunliu
authors: Y
authors: Fan
authors: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors: Wei
authors: W
authors: Zhang
mesh_terms: D001203:Ascomycota / Q000378:metabolism*; D002480:Cellulase / Q000096:biosynthesis*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D008856:Microscopy, Fluorescence; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction
chemical_list: D004269:DNA, Bacterial; C036483:T-DNA; D002480:Cellulase