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ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(10): 667–672 CN 31-1940/Q

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Novel Cellulase Profile of Trichoderma reesei Strains Constructed by cbh1 Gene Replacement with eg3 Gene Expression Cassette

Tian-Hong WANG*, Ti LIU, Zhi-Hong WU, Shi-Li LIU, Yi LU, and Yin-Bo QU

The State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China

Abstract To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases while high endoglucanase activity, the Pcbh1-eg3-Tcbh1 cassette was constructed and the coding sequence of the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous recombination. Disruption of the cbh1 gene was confirmed by PCR, Southern dot blot and Western hybri-dization analysis in two transforments denoted as L13 and L29. The filter paper-hydrolyzing activity of strain L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively modest increase suggested that the eg3 cDNA under the control of the cbh1 promoter was not efficiently transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such strains are of interest from the basic science perspective and also have potential industrial applications.

Key words Trichoderma reesei; gene replacement; gene disruption; cbh1; eg3

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Received: June 14, 2004 Accepted: August 14, 2004

This work was supported by the grants from the National Natural Science Foundation of China (No. 30070014), the Natural Science Foundation of Shandong Province (No. Y2000D12) and the Major State Basic Research Development Program of China (No. 2003CB716006)

*Corresponding author: Tel, 86-531-8364384-8109; Fax, 86-531-8565234; E-mail, [email protected]