Construction The Saccharomyces Cerevisiae Expression Vector That Containing The Cellulase Genes And Expression The Cellulase Genes In Pichia Pastoris

Alcohol is one of the fastly promoted renewable energies. Cellulosic biomass is the most abundant renewable resource. So fuel alcohol production from raw cellulosic biomass materials is an important aspect of cellulose resource utilization. The cellulose of the cellulosic biomass materials can be hydrolisised to glucose by cellolase. And the glucose can be fermented to alcohol.Cellulase contains three main components: endo-1,4-β-D-glucanase (EC 3 .2.1.4),exo-1,4-β-D-glucanase (EC3.2.1.91) andβ-glucosidase (EC3.2.1.21). Endo-1,4-β-D- glucanase is short for EG, is also called Cx or CMCase. Exo-1,4-β-D- glucanase can cut cellobiose from the linear end of cellulose by hydrolysisβ-1, 4-glucoside. So it is also called cellobiohydrolase, short for CBH.β-glucosidase is short for BG.In this research, three main components of cellulose (bg1,cbh2 and eg3) were amplified from Trichoderma reesei QM9414 (Trichoderma sp.) by cross-over PCR method ,introns and signal peptides were also deleted. And three genes were cloned into E.coli DH5αto amplification. Then, Pichia induced expressing vectors of three genes were constructed by ligating three genes with pPIC9K respectively. And the AOX1 promoter, which was the promoter of pPIC9K, was replaced by strong and constitutive expression promoter GAP to construct a Pichia constitutive expressing vector pGAP9K. Three genes also were ligated in this expressing vector.Linearized pPIC9K-CBH and pGAP9K-CBH were transformed into P. pastoris GS115 with the method of electroporation, and thereby the cbh2 was inserted into the Pichia genome with different promoters. The most efficient constitutive expression recombinant Pichia strain, which was designated as P. pastoris GS115(pGAP9K-CBH), was fermented in the medium which the glycerin was the unique carbon source. By SDS-PAGE analysis, 50mg/L of CBHⅡcan be detected in the supernatant of the fermentation product. And by the CMCase activity assay, 2.045U/mL of CMCase activity can be detected in the supernatant of the fermentation product.In this research, a vector that contains three expression boxes of the cellulase genes also was constructed by a series transformation of pSP72.