Advances in metabolic engineering of Bacillus subtilis towards high-efficiency cytidine biosynthesis.

Cytidine is an important nucleotide widely applied in the pharmaceutical and food industries. Conventional chemical synthesis and enzymatic approaches face limitations related to cost, process complexity, and environmental sustainability, motivating the development of microbial fermentation strategies. Bacillus subtilis, a food-grade and genetically tractable microorganism, has emerged as a promising chassis for cytidine biosynthesis due to its well-characterized metabolic framework and versatile genetic engineering tools. Recent progress in systems metabolic engineering and synthetic biology has enabled extensive reprogramming of pyrimidine biosynthesis, optimization of central carbon metabolism, and the application of advanced genome-editing tools. However, this review critically re-evaluates these existing strategies, identifying a major gap: the historical over-reliance on translating E. coli-centric, static pathway disruptions to B. subtilis. To overcome this, we propose a new conceptual framework of host-specific dynamic thermodynamic balancing. We argue that paradigm-shaping biomanufacturing requires shifting from isolated genetic edits to the systemic integration of B. subtilis-specific regulatory logic, multi-omics-constrained modeling, and dynamic spatiotemporal control.