Systems Metabolic Engineering of Escherichia coli for High-Titer De Novo β-Thymidine Production via Folate Cycle Enhancement and NADPH Regeneration

The demand for β-thymidine primarily stems from its role as a raw material and intermediate in antiviral drug production. To address this, β-thymidine was synthesized de novo using Escherichia coli. We engineered a high-yielding β-thymidine strain based on the orotate high-yielding strain Ora14–4. First, we introduced the pyrFE gene cluster from Bacillus subtilis to direct the metabolic flux toward β-thymidine synthesis. Genes involved in β-thymidine catabolism and precursor branching were knocked out, and key enzymes in the β-thymidine biosynthetic pathway were optimized. Enhancing the folate cycle and glycine cleavage system provided sufficient one-carbon donors for deoxythymidine monophosphate synthesis. The Entner-Doudoroff pathway from Zymomonas mobiliz was introduced to enable NADPH production. Last, a growth-adaptive promoter regulated tmk gene expression to minimize carbon loss. The final β-thymidine titer was 25.4 g/L, with a conversion rate of 0.079 g/g glucose, which is, to the best of our knowledge, the highest titer reported to date for de novo microbial production of β-thymidine.