Phosphorylation of uridine and cytidine by uridine–cytidine kinase

Abstract

Uridine 5′-monophosphate (5′-UMP) and cytidine 5′-monophosphate (5′-CMP) were biosynthesized by recombinant uridine–cytidine kinase (UCK) and acetate kinase (ACK). The ack and uck genes from Escherichia coli K12 and the uck1, uck2 and ack genes from Lactobacillus bulgaricus ATCC 11842 were cloned and inserted into the plasmid pET-28a. All of the recombinant E. coli strains were capable of overexpressing UCK and ACK, which catalyzed the reaction using guanosine 5′-triphosphate (GTP) as a phosphate intermediate that was regenerated by ACK from acetyl phosphate. The effect of several parameters, including the substrate concentration, the GTP concentration, the temperature and the reaction pH, were optimized. High efficiency was achieved if uridine or cytidine was phosphorylated by UCK encoded by uck from E. coli and ACK encoded by ack from L. bulgaricus. The maximum conversion yield of 5′-UMP and 5′-CMP was 97% at 37°C and pH 7.5 when 30mM uridine/cytidine and 0.5mM GTP in a total of 1mL were used. In addition, the 5′-UMP and 5′-CMP products were very stable in the reaction system and did not undergo significant degradation.