Systematic rewiring of Bacillus subtilis for efficient de novo biosynthesis of the neuroprotectant cytidine-5′-diphosphocholine

Cytidine-5′-diphosphocholine (CDP-choline) is a crucial neuroprotective agent. Current industrial production relies on chemical and enzymatic methods that face inherent sustainability challenges and share a dependence on the costly precursor, cytidine monophosphate (CMP). Here, we report the systems metabolic engineering of Bacillus subtilis for the efficient, de novo biosynthesis of CDP-choline from glucose, completely obviating the need for CMP. A synthetic pathway was first established by introducing heterologous choline kinase and phosphocholine cytidylyltransferase. Subsequently, a multi-module engineering strategy was implemented, focusing on enhancing precursor supply and redirecting carbon metabolism. This involved systematically optimizing choline uptake by overexpressing the transporter OpuD and deleting the transcriptional repressor opcR, fortifying the cytidine triphosphate (CTP) pool by overexpressing feedback-resistant CTP synthase gene pyrG^E156K, and deleting the transcriptional repressor pyrR along with other pyrimidine nucleotide consumption genes, and channeling carbon flux towards the TCA cycle by reducing pyruvate and malate consumption. The final engineered strain achieved a titer of 4.79 ± 0.24 g/L CDP-choline in a 5 L fed-batch bioreactor, with a high specific yield of 149.0 ± 5.8 mg/g DCW. Notably, the process exhibited a highly advantageous intracellular accumulation of 92.7 %, which simplifies downstream purification. This study represents the first successful demonstration of CDP-choline production from simple sugars in a microbial host, establishing a robust and economically competitive platform for its industrial manufacture.