Enzymatic Synthesis Of Deoxythymidine-5'-monophosphate And Deoxythymidine-5'-triphosphate

Deoxythymidine-5'-monophosphate (dTMP) and deoxythymidine-5'-triphosphate (dTTP) have very important use in medicine, food and scientific research. Here, we developed an one-pot process of enzymatic synthesis of dTMP form dTR, with permeable intact cells of E.coli coexpressing thymidine kinase (TKase) and acetate kinase (ACKase) as catalysts. And synthesis of dTTP from dTMP catalyzed by permeable intact cells of E.coli coexpressing thymidylate kinase (TMKase) and acetate kinase.Gene tk, ack and tmk from E.coli were cloned into vector pET-28a, yielding three kinds of recombinant strains. Then gene tk and ack were inserted into the same expression vector pET-28a to construct recombinant bacterias p28AT and p28TA that coexpressed TKase and ACKase. As the same way, we obtained recombinant bacterias pAT and pTA that coexpressed TMKase and ACKase, and recombinant bacterias that expressed TKase, TMKase and ACKase. All of these recombinant strains obtained in the above had good protein expression.Several measures were taken to increase the cell permeability of recombinant bacterials, and recombinant bacterials treated with EDTA were most efficient and had least loss of enzyme activity. In our study, the multi-enzymatic synthesis of dTMP catalyzed by permeable intact cells p28AT was achieved. The yield of dTMP was 89% after 8 hours. The synthesis of dTTP from dTMP was catalyzed by permeable intact cells pAT and the yield of dTTP was 94% after 4 hours. In both of these reactions, ATP was used as the phosphate donors. In order to reduce costs, ATP was generated from acetyl phosphate by the ATP regeneration system, which was catalyzed by ACKase, and only a slight amount of ATP in the reaction system could provide all of the energy required for the reaction.