Sunday, April 29, 2007

Over-expression of bgl1 under the control of multiple copies promoter to improve cellulolytic activity of T. reesei

Tianhong Wang, Xiena Zhao, Ti Liu, and Yinbo Qu. State Key Laboratory of Microbial Technology, Shandong University, 27 Shandanan road, Jinan, 250100, China

Trichoderma reesei is known as an industrially important cellulolytic filamentous fungus for its capacity to hydrolyze cellulose, but the amount of β-glucosidase secreted by T. reesei is insufficient for effective cellulose degradation. So, overproducing β-glucosidase (BGL I) is a valuable way to improve the cellulolytic activity of T. reesei. To improve heterologous gene expression in T. reesei, a high expression vector pTHP was constructed in our laboratory in the basis of cbh1 promoter by deletion of -677`-724 region with three potential glucose repressor binding sites, and insertion of 4 copies of -620`-820 region including the CCAAT box and Ace2 binding sites. The bgl1 gene from T. reesei was guided by this high expression promoter, co-transformed with plasmid pAB4-1 which contains the orotidine-5'-phosphate decarboxylase gene (pyrG) from Aspergillus niger into the pyrG deficient T. reesei strain M23. PCR result with special primers identified that additional bgl1 copy had integrated into chromosome DNA of two positive transformants 1-17 and 2-6. β-glucosidase and filter paper activities of the transformants were examined. The strain 1-17 and 2-6 show a 3.8-fold and 3.2-fold increase in the rate of production of glucose from cellobiose, and a 33% and 56% increase from filter paper, respectively. The data testified that over-expression of bgl1 could obviously improve the cellulolytic activity of T. reesei.