An analysis of E. coli thymidine producing strains developed with patented ChemGen technology strongl 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 phosphoribosyltransferase, 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 supphed to fermentations as an inexpensive feed stock for bioconversion into thymidine.Awardee's statement of the potential commercial applications of the research: AZT (Retrovir/R) is the main drug treatment used for AIDS patients and the deoxyribonucleoside, thymidine, is its key precursor. This new fermentation process is expected to reduce the full cost of thymidine from its current @ $400/kg to less than $100/kg. The fermentation process will lead to a much lower production cost and hopefully price of AZT. Other AIDS drugs, fluorothymidine (FT) and dihydrodideoxythymidine (D4T), are also produced from thymidine.National Institute of Allergy and Infectious Diseases (NIAID)
