Genes encoding uridine phosphorylase (UP) and thymidine phosphorylase (TP) from Escherichia coli K12 were cloned and recombined respectively into plasmids pET-21a(+) and pET-28a(+). The recombinant plasmids BL21/pET21a-UP and BL21/pET28a-TP were co-transformed into E. coli BL21(DE3) to construct highly effective BTU strain (BL21/pET28a-TP, pET21a-UP) overexpressing both of UP and TP. BTU was cultivated in ZYM-Fe-5052 medium for 10 h and used as catalyst to synthesize 2'-deoxyuridine (dUR). It was found to increase the productivity of dUR by 8-9 fold when compared to wild E. coli I(12 and E. coli BL21(DE3) strains. A series of experiments were carried out to find out the optimal conditions for synthesis of dUR. At 50 degrees C, with 0.25 parts per thousand. dry wt./v to catalyze the reaction of 2:1 beta thymidine: uracil (60 mM beta-thymidine, 30 mM uracil), the conversion rate of dUR would reach 61.6% at 1 h, which was much higher than the rates obtained by BTU strain cultured in LB medium and induced by IPTG. This result proved co-expression and auto-induction were efficient methods in enhancing the expression quantity and activity of nucleoside phosphorylases, and they also had significant implications for large-scale industrial production of dUR and synthesis of other nucleoside derivatives. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.