title: Expression of Talaromyces thermophilus lipase gene in Trichoderma reesei by homologous recombination at the cbh1 locus.
abstract: CBH1 (cellobiohydrolase) comprises the majority of secreted proteins by Trichoderma reesei. For expression of Talaromyces thermophilus lipase gene in T. reesei, a self-designed CBH1 promoter was applied to drive the lipase gene expression cassette which was bracketed by flanking sequences of cbh1 gene for homologous recombination. Protoplast and Agrobacterium-mediated plasmid transformations were performed and compared, resultantly, transformation mediated by Agrobacterium was overall proved to be more efficient. Stable integration of lipase gene into chromosomal DNA of T. reesei transformants was verified by PCR. After shaking flask fermentation, lipase activity of transformant reached 375 IU mL-1, whereas no cellobiohydrolase activity was detected. SDS-PAGE analysis further showed an obvious protein band about 39 kDa and no CBH1 band in fermentation broth, implying lipase gene was successfully extracellularly expressed in T. reesei via homologous recombination at cbh1 locus. This study herein would benefit genetic engineering of filamentous fungi and industrial application of thermo-alkaline lipase like in paper making and detergents addition.
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China. xialm@zju.edu.cn.
authors.forename: Liming
authors.initials: L
authors.lastname: Xia
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics; D005800:Genes, Fungal; D005818:Genetic Engineering; D056426:Genetic Loci; D059765:Homologous Recombination; D007218:Industrial Microbiology; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D000074041:Microorganisms, Genetically-Modified; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D032901:Talaromyces / Q000201:enzymology / Q000235:genetics*; D014242:Trichoderma / Q000235:genetics*
chemical_list: D008049:Lipase; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: Agrobacterium tumefaciens; Homologous recombination; Lipase; Protoplast; Trichoderma reesei