High NADPH/NADP+ ratio improves thymidine production by a metabolically engineered Escherichia coli strain

Thymidine is a commercially important precursor in the production of antiviral drugs, including azidothymidine for the treatment of AIDS. In order to produce thymidine in a large scale, we previously developed a thymidine-overproducing Escherichia coli strain BLdtug24 by engineering pathways. To further enhance thymidine yield, we increased the availability of a cofactor (NADPH) in thymidine biosynthesis by disrupting phosphoglucose isomerase in BLdtug24 to construct BLdtugp24. Additionally, NAD(+) kinase or soluble transhydrogenase was overexpressed in BLdtugp24, which can reroute glucose metabolic flow from the EMP pathway to the PP pathway, to construct BLdtugp34N or BLdtugp34U, respectively. In chemostat cultures, BLdtugp24 had an increased NADPH availability and a 4-fold enhancement in thymidine yield for glucose compared with BLdtug24. BLdtugp34N and BLdtugp34U had increased thymidine yields for glucose by 1.2- and 2-fold compared with BLdtugp24, respectively. The NADPH/NADP(+) ratios at steady-state had overall positive correlations with thymidine yields in these strains. Real-time RT-PCR analysis revealed that the transcriptional regulations of NAD(P)H-related enzymes and transhydrogenases affected the redox balance and shifted reaction equilibrium toward increasing NADPH. In fed-batch fermentation, BLdtugp34U with the highest NADPH/NADP(+) ratio in chemostat experiment produced 1.9gl(-1) of thymidine with 29.7mgl(-1)h(-1) of thymidine productivity.