Metabolic Engineering Of Escherichia Coli For Thymidine Production

Thymidine is a commercially useful precursor for production of antiviralcompounds such as stavudine and azidothymidine (AZT). Biosynthesis of thymidineby Escherichia coli BL21(DE3) was investigated using metabolic engineeringmethods. And a series of metabolically engineered E.coli BL21(DE3) mutants wereconstructed that could efficiently produce thymidine. The salvage pathway and denovo pathway of pyrimidine synthesis were aborative analyzed; the impacts ofNADPH and ribose-5-phosphate on the thymidine were studied; we also tried toincrease the the thymidine production by adding glycine(Gly) or aspartic acid(Asp) toculture; at last, the feasibility of aerobic production of thymidine at large scale wasevaluated by fed-batch fermentation. The main results were shown as follows:First?The salvage pathway and de novo pathway of pyrimidine synthesis werestudied. The deoA, tdk and udp of the salvage pathway which encoded thymidinephosphorylase (deoA), thymidine kinase (tdk) and uridine phosphorylase (udp)respectively, were disrupted from E. coli to construct BS01(?deoA), BS02(?deoA?tdk), BS03(?deoA?tdk?udp) respectively, and BS03which produced21.6mg thymidine per liter.Then in order to increase the supply the NADPH and ribose-5-phosphate, the pgigene of EMP pathway was deleted on the base of BS03, resulting BS04. BS04produced78.7mg thymidine per liter which was the3.6-fold compared to BS03. Theresult showed that knocking out pgi, a gene required for the EMP pathway, preventsthe isomerization of glucose6-phosphate to fructose6-phosphate, leading to themetabolism of glucose only via the PP pathway and supplied more precursors.To eliminate the feedback repression and make the pyrBI constitutive expression,the pyrL sequence was deleted totally to construct BS05.BS05produced90.5mgthymidine/L.At last, six genes from UDP to thymidine(ushA?thyA?dut?ndk?nrdA and nrdB)were explored. ushA?thyA?dut and ndk were co-expressed to construct pLS4based onp5C; nrdA and nrdB were co-expressed to construct pLM2based on pTRC99A. BS06?pLS4??BS07?pLM2? and BS08?pLS4pLM2?were constructed on the base ofBS05. BS06produced100.7mg/L thymidine, and the BS07produced169.2mg/L thymidine. While BS08containing two plasmids produced272mg thymidine/L. Infed-batch fermentation, BS08accumulated1248.8mg thymidine/L with a yield of12.5mg/(g·glucose) and a productivity of15.61mg/(L·h). The experimental resultsshowed that metabolically engineered strain E.coli has potential applications forthymidine production.