Multivariate Modular Metabolic Engineering for the Enhanced Biosynthesis of Cytidine in Bacillus subtilis

As a pyrimidine nucleoside, cytidine is widely used in the medicine and food fields. Therefore, it is important to construct a microbial cell factory for efficient and sustainable cytidine production. Here, we perform modular metabolic engineering modifications on Bacillus subtilis 168 to achieve efficient synthesis of cytidine. First, the cytidine titer reached 0.88 g/L by blocking cytidine degradation and enhancing the cytidine de novo synthesis pathway. Next, the central carbon metabolism was modulated by knocking down CcpA, but the cytidine titer decreased instead. Transcriptome analysis revealed that differential genes were mainly enriched in PTS, glycolysis, TCA cycle, PP pathway, pyrimidine metabolism, aspartate metabolism, and glutamate metabolism. Then, by enhancing the l-aspartate and glutamine synthesis pathways, the cytidine titer was increased to 3.83 g/L. By strengthening the PP pathway to increase PRPP synthesis, the cytidine titer was further increased to 7.03 g/L. Finally, the cytidine titer reached 31.41 g/L by fed-batch fermentation in a 5 L fermenter, which was 4.47-fold that of shake flask fermentation. Overall, the efficient production of cytidine was accomplished through modular metabolic engineering, opening new pathways for the production of cytidine and other nucleosides.