Construction And Application Of Two Yeast Surface Display Systems Of Thymidine Phosphorylase

Nucleosides and their modified derivatives are most widely used as antiviral and anticancer prodrugs. Nucleosides are catalyzed in vivo by nucleoside phosphorylases, such as thymidine phosphorylase(TP), which extensively involves in nucleoside metabolism and catalyzes the formation of many nucleoside analogs(NA). Yeast surface display system, which has been developed as a novel tenique in biocatalysis field in recent years, has an advantage of reusability and high efficiency. In this study, two yeast cell surface display systems for TP were firstly constructed as whole-cell biocatalysts.The deoA gene encoding TP from E. coli K12 strain was amplified and sequenced. Then, the deoA gene was inserted into the yeast expression vector pKFS. Recombinant vector pKFS-LA was linearized and electro-transformed into Pichia pastoris GS115 cells. Positive clones were selected by genomic PCR analysis and cultured in BMMY medium, induced by 1% methanol. Transformant cells at 96 h were detected with immunofluorescence and showed green color under FITC filter. !-thymidine was used as substrate to test the catalytic activity of GS115 cells displaying TP. HPLC analysis indicated the whole-cell catalyst could catalyze !-thymidine to produce thymine and the conversion rate was 7.5%.Besides, deoA gene cloned from E. coli K12 strain was also inserted into pYD1 vector. pYD1 acted as an integration vector for transformation into Saccharomyces cerevisiae yeast cells and was used to construct the pYD1-deoA recombinant vector. TP was displayed on the cell surface of Saccharomyces cerevisiae EBY100 strain with two "-agglutinin subunits, aga1 p from yeast cells and aga2 p from pYD1 as an anchor system. Transformants were selected by MD/–trp plates and induced for 48 h in 2% galactose-containing YNB-CAA medium. Approximately 80% of the transformant cells were detected with green under a FITC filter by immunofluorescence. Results from western blotting showed that the TP- aga2 p fusion protein was approximately 62 kDa. Transformant EBY100 cells were used as whole-cell catalysts to catalyze the conversion of !-thymidine to thymine. HPLC analysis indicated the conversion rate of !- thymidine was 10.87%. Then, the S. cerevisiae transformants were used to detect the substrate specificity of TP. The result showed that TP could not accept 2'-deoxycytidine as substrates.Thus, two different novel yeast cell-surface display systems of TP from microbial origin were successfully developed as TP whole-cell catalysts. Considering the low recovery of purification, whole-cell catalyst is efficient as well as convenient. Moreover, the restoration of cells is a lot easier than that of proteins. Our work lays the foundation for research on producing nucleoside analogs using the TP whole-cell catalysts.