Advances in large DNA fragment assembly for microbial cell factory engineering

The efficient, rapid, and reliable assembly of DNA fragments is essential for advancing metabolic engineering and synthetic biology. With the rapid advancement of DNA synthesis and assembly technologies, the scale of DNA assembly has expanded from single genes to metabolic pathways and even genomes. Large DNA fragment assembly, in particular, has developed into a pivotal tool for microbial cell factory engineering, significantly contributing to the understanding and construction of biological systems. This review summarizes recent advancements in large DNA fragment assembly methods, encompassing in vitro and in vitro methods for multiple‐gene assemblies, as well as genome‐scale technologies, including synthetic genome and neochromosome construction. We systematically compare their key features in terms of assembly principle, capacity, and efficiency. Especially, we highlight their applications in microbial cell factory engineering, including heterologous pathway construction, reprogramming host metabolism, and expanding complex biosynthetic networks. Finally, we discuss the challenges and prospects of applying large DNA fragment assembly to advance cell factories. In summary, this review provides a theoretical and technical framework for engineering high‐performance microbial cell factories, contributing to the advancement of industrial biomanufacturing.