SBIR-STTR Award

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 HlV-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.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 deoxyribonucleotide, thymidine, is its key precursor. This new fermentation process is expected to reduce the full cost of thymidine. The fermentation process may significantly lead to a much lower production cost and, hopefully, price of AZT. A new AIDS drug, d4T, in climcal trials and available to the public under the Parallel Track Program is also produced from thymidine.National Institute of Allergy and Infectious Diseases (NIAID)

The objective of this Phase II proposal is to complete the genetic development for a low cost, fermentation based, production method for thymidine. Thymidine, currently produced by chemical synthesis, is an important raw material for several anti-viral pharmaceuticals and other biomedical applications. Previous work at ChemGen to engineer E. coli for thymidine production established the technical feasibility. Several enzymatic steps of the de novo pathway for thymidine production have been engineered resulting in inducible non-inhibited thymidine production by dividing cells. In small scale fermentation, one strain produced 5.2 g/Liter thymidine from glucose. ChemGen proposes to complete the strain development by deregulating the first steps of the biosynthetic pathway utilizing Bacillus genes (cpa and pyrB) that code for enzymes not feed-back inhibited by pyrimidine nucleotides. Once regulated in concert with the rest of the system, this modification should effectively uncouple thymidine production from cell growth to allow commercial production. The technical innovation in this proposal lies in the first demonstration that an essential, long biosynthetic pathway, that is normally highly regulated, can be successfully modified for a commercial production process using a rational design. The final strain will contain at least seven genes foreign to the host. These genes code for enzymes with allosteric properties predicted to be favorable for thymidine production. The design includes genes from five sources, as well as a number of engineered host genes and chromosomal mutations. The investigators believe success will set a precedent for synthesis of worthwhile biochemical pharmaceutical intermediates by use of a rational approach to genetic modification and engineering of complex biosynthetic pathways. PROPOSED COMMERCIAL APPLICATION: AZT (Retrovir) is an important drug treatment for AIDS patients. The deoxyribonucleoside thymidine is the key chemical precursor, and single most expensive component of AZT manufacture. Thymidine currently sells for $500 to $600/Kg, but this new fermentation process is expected eventually to reduce the production cost of thymidine to less that $100/Kg. Other AIDS drugs, d4T and fluorothymidine, currently in trials are also produced from thymidine