Enzymatic synthesis of 2′-deoxyuridine by whole cell catalyst co-expressing uridine phosphorylase and thymidine phosphorylase through auto-induction system

• Published in: Journal of bioscience and bioengineering 2014-12, Vol.118 (6), p.723-727
• Main Authors: Xiong, Shuli, Wang, Yingbin, Wang, Xi, Wang, Jie, Li, Jie, Zhang, Guiyou, Zhang, Rongqing, Xie, Liping, Wang, Hongzhong
• Format: Article
• Language: English
• Subjects: 2′-Deoxyuridine; Auto-induction; Biocatalysis; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Co-expression; Deoxyuridine - metabolism; Enzyme Induction; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Methods. Procedures. Technologies; Nucleoside phosphorylase; Plasmids - genetics; Thymidine - metabolism; Thymidine Phosphorylase - biosynthesis; Thymidine Phosphorylase - genetics; Thymidine Phosphorylase - metabolism; Uridine Phosphorylase - biosynthesis; Uridine Phosphorylase - genetics; Uridine Phosphorylase - metabolism
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2014.05.005

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 K12 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°C, with 0.25‰ dry wt./v to catalyze the reaction of 2:1 β-thymidine: uracil (60 mM β-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.