Pathway Engineering in E Coli for Thymidine Production

Thymidine is used as the raw material for the manufacture of 3'- azidothymidine (AZT) and other thymidine analogs that are currently used in the treatment of HIV-infected patients. Part of the high cost of AZT is due to the high cost of thymidine. The goal is to engineer a bacterial strain to enable it to make and excrete high amounts of thymidine; this project will make it possible to produce thymidine by fermentation at much less cost than is currently available. The approach is to engineer the pyrimidine biosynthetic pathway to increase the levels of the enzymes and to eliminate feedback inhibition of the pathway. We propose a method for modifying one of the key enzymes of the pathway, dCTP deaminase (dcd), that is subject to feedback inhibition, to eliminate the inhibition. The cloned dcd will be subjected to in vitro mutagenesis and mutants will be analyzed for their regulatory properties. Phase II will include incorporation of the mutant dCTP deaminase into thymidine production strains in combination with other relevant genes.