Improved Thymidine Production with a PyrFEH Operon

An analysis of E. coli thymidine producing strains developed with patented ChemGen technology strongly suggests that intracellular production of UDP (or UMP) is now rate limiting for thymidine production. Previous work by ChemGen created a non-feed back inhibited pathway for the efficient synthesis of thymidine from UDP in five steps through the introduction of phage genes. In order to deregulate and improve UDP synthesis, an operon will be constructed with the coding sequences of pyrE, pyrF, and pyrH genes that code for orotate phosphoribosyltransfer- ase, orotidine 5'- phosphate decarboxylase and UMP kinase. This will provide efficient temperature inducible expression of these non-feed back inhibited enzymes. Introduction of the constructed operon into current thymidine production strains will provide deregulated synthesis of thymidine from orotic acid precursor and the basis for a commercial process. Orotic acid can be supplied to fermentations as an inexpensive feed stock for bioconversion into thymidine.