De novo synthesis of large-scale metabolic pathways for natural products and their optimization strategies.

Natural products constitute a vital source for drug discovery, yet extraction from producers is inefficient and chemical synthesis involves complex, low yield routes, limiting sustainable supply. Reconstructing large scale metabolic pathways (≥10 enzymatic steps) in microbial cell factories using synthetic biology offers a promising solution for efficient and sustainable production of high value natural products. This review systematically analyzes core strategies and cutting edge technologies for reconstructing such pathways, including advanced cloning methods, modular pathway design, self assembly approaches (protein/DNA scaffolds and metabolic channeling), division of labor optimization in co culture systems, and dynamic regulation mechanisms (promoter engineering, riboswitches, biosensor based feedback control). We further explore the emerging roles of systems biology modeling and machine learning in retrosynthetic pathway design, rational enzyme engineering, and precise metabolic flux regulation. By integrating intelligent algorithms with multidisciplinary tools, these approaches hold promise for overcoming bottlenecks in the biomanufacturing of complex natural products, thereby accelerating drug development and enabling green, sustainable production. This review highlights the importance of combining rational design, dynamic control, and modular co culture strategies to address challenges such as metabolic burden, pathway imbalance, and host toxicity, ultimately paving the way for scalable and cost effective biosynthesis of natural product pharmaceuticals.