基于酵母表面展示技术的胸苷磷酸化酶全细胞催化剂的构建

Construction of Pichia pastoris Surface Display Technology as Wholecell Biocatalyst for Thymidine Phosphorylase

胸苷磷酸化酶(TP)在核苷类代谢通路中发挥重要作用,可催化生成多种核苷类似物。构建了TP的酵母表面展示系统作为全细胞催化剂。从大肠杆菌K12菌株中克隆编码TP的deo A基因,利用酵母表达质粒p KFS构建重组质粒,电击转化毕赤酵母GS115菌株。高拷贝阳性转化子经甲醇诱导96 h后,免疫荧光结果显示TP在酵母细胞表面成功展示。利用β-胸苷为底物,重组酵母细胞作为全细胞催化剂,经HPLC检测,结果表明展示在酵母表面的TP有催化活性,可以催化β-胸苷生成产物胸腺嘧啶。

Thymidine phosphorylase（TP）extensively involves in nucleoside metabolism and catalyzes the formation of many nucleoside analogs（NA）. A yeast cell surface display system for TP was firstly constructed in this study as whole-cell biocatalyst. A deo Agene encoding TP was cloned from Escherichia coli K12 strain and inserted into the yeast expression vector p KFS. Recombinant vector was linearized and electro-transformed into Pichia pastoris GS115 cells. High copy transformant was induced by methanol for 96 h, and the results of immunofluorescence indicated that TP successfully displayed on the surface of P. pastoris. β-thymidine was used as substrate and recombinant GS115 cells as whole-cell biocatalyst, HPLC analysis demonstrated that TP on the surface of P. pastoris had catalytic activity, and catalyzed the production from β-thymidine to thymine.