Over-expression Of β-Glucosidase To Improve Cellulolytic Activity Of Trichoderma Reesei

Filamentous fungi have the advantages of simple structure and fast growth, so it is easy to be manipulated and cultured. Being a kind of lower eukaryotes, it also has eukaryotic protein modification apparatus and the post translation modification mechanism. So molecular biological research on filamentous fungus has been one of the hotspots. Saprophytic fungus Trichoderma reesei has a long history of industrial production of different plant material hydrolyzing enzymes due to its extraordinary ability to secrete cellulases and hemicellulases.The cellulase complex isolated from Trichoderma reseei comprises at least three different enzymes that together hydrolyze cellulose to oligosaccharides and glucose. Of these, the endoglucanases and cellobiohydrolases synergistically hydrolyze cellulose into small cellooligosaccharides, mainly cellobiose. Subsequently, cellobiose is hydrolyzed to glucose byβ-glucosidase. The accumulation of cellobiose could slow down the hydrolysis process significantly by inhibiting both endoglucanase and cellobiohydrolase activities.β-Glucosidases can break down cellobiose into glucose and then relieve the inhibition of endoglucanase and cellobiohydrolase by it. The amount ofβ-glucosidase secreted by T. reesei is insufficient for effective cellulose degradation. Therefore, construction of BGLI-overproducing strain is a valuable pathway to improve the efficiency of cellulose hydrolysis.The bgl1 gene encoding the most important extracellularβ-glucosidase from T, reesei was inserted into a high-expression vector pTHP, which was constructed in our laboratory on the basis of cbh1 promoter by deletion of -677～-724 region containing three potential glucose repressor binding sites and insertion of 4 copies of -620～-820 region including the CCAAT box and ACEII binding sites. Then the recombinant plasmid pTHB was co-transformed with plasmid pAB4-l, which contains the orotidine-5'-phosphate decarboxylase (pyrG) gene from Aspergillus niger, into the pyrG-deficient strain T. reesei M23. 96 transforrnants were selected from the minimal media plate without uridine. Meanwhile, PCR amplification was carried out with the special primers to identify the transformants and the result showed that additional bgl1 copies had integrated into chromosome DNA of 6 transformants. Theβ-glucosidase and filter paper activities of the 6 transformants were examined and the transformant 2-6 showed the strongest increases in the rate of production of glucose from cellobiose (2.8-fold), and filter paper (1.48-fold). The analyzing of bgl1 gene copies on chromosome in transformant 2-6 by semiquantitative PCR indicated that the copies of bgl1 gene in transformant 2-6 were increased comparing to M23. Over-expression of BGLI by increasing the copies of bgl1 gene can enhance the ability of T. reesei to hydrolyze cellulose. The result indicated that the gene engineering method was a valuable pathway to reconstruct the strain for breeding.To observe the secretory mechanism of BGLI, gfp gene was fused to bgl1 gene as a reporter. The fusion fragment was cloned into the vector pTHP. The recombinant plasmid pTHR1 and pTHR2 was co-transformed with plasmid pAB4-1 into T. reesei M23. 110 transformants were selected from the minimal media plate without uridine and 7 positive transformants were isolated by PCR amplification. Lactose was used as carbon source to induce the expression of the fusion protein. Theβ-glucosidase and filter paper activities of transformant 1-17 showed 3.3-fold and 1.28-fold increase comparing to the strain M23, respectively. According to semiquantitative PCR analysis, the copy number of bgl1 gene in transformant 1-17 was increased comparing to M23. It demonstrated that the bgl1 gene transformed into the T. reesei had integrated into its chromosome successfully. A band about 100kD was detected by SDS-PAGE analysis of protein from 1-17 and it was estimated to be BGLI/GFP fusion protein. The fluorescence of transformants was detected but nothing was found. The possible reason was that GFP couldn't fold to the correct structure in fusion form with BGLI or its fluorescence was so weak that it couldn't be observed under the background fluorescence of T. reesei.The bgl1 gene containing a fragment coding 6×His tag at 3' end was cloned into the vector pTHP. Then the recombinant plasmid pTHB2 was co-transformed with the plasmid pAN7-1 containing hph gene or with p3SR2 containing amdS gene into Penicillium decumbens JU-A10. 40 transformants were selected by hygromycin B or acetamide. PCR amplification was carried out and the result showed that additional bgl1 copies had integrated into chromosome DNA of 3 transformants. Then the transformant, which showed enhanced β-glucosidase and filter paper enzyme activities, was selected for further research.