The construction of microbial cell factories is revolutionizing industrial biomanufacturing, enabling the sustainable production of pharmaceuticals, biofuels, and fine chemicals. Building efficient cell factories typically requires introducing heterologous metabolic pathways larger than 10 kilobases (kb), reprogramming host metabolism at the megabase scale, and expanding biosynthetic networks that span hundreds of kilobases. These demands have made large DNA fragment assembly a pivotal enabling technology, and rapid advances in DNA synthesis are now accelerating its methodological development.

Recently, the research teams of Zhejiang University and BGI Research published a review article “Advances in large DNA fragment assembly for microbial cell factory engineering” in Quantitative Biology. The review systematically summarizes recent advances in large DNA fragment assembly, from in vitro and in vivo multigene assembly to genome-scale technologies such as synthetic genomes and neochromosomes, and compares their key features in terms of assembly principle, capacity, and efficiency.

Figure 1 illustrates how large DNA fragment assembly facilitates microbial host engineering: panel (a) shows its roles in pathway construction, metabolism reprogramming, and network expansion together with current development trends, and panel (b) presents a timeline of key milestones in the field. In vitro methods have evolved from restriction digestion based modular cloning (BioBrick, Golden Gate) to homology directed seamless assembly such as Gibson, which reaches a capacity of up to 900 kb, and further to programmable nuclease guided systems. For biosynthetic gene clusters of 10 kb to megabase scale, in vivo homologous recombination in Escherichia coli, Saccharomyces cerevisiae, and other microorganisms offers higher efficiency and stability. Synthetic genome assembly, exemplified by genomically recoded E. coli and the Synthetic Yeast Genome Project (Sc2.0), elevates microbial engineering from local optimization to building purpose-built chassis, while synthetic neochromosomes provide orthogonal, scalable platforms for expanding metabolic networks without disrupting native chromosomes. The authors note that current assembly strategies remain largely confined to model organisms and that cross-species DNA delivery is still inefficient. They highlight future directions including assembly systems for a broader range of hosts, novel delivery technologies such as the synNICE nuclear isolation and transplantation approach, and the integration of high-throughput synthesis, automated platforms, and AI driven design. Together, these advances promise a high-throughput, high-precision Design-Build-Test-Learn cycle that could slash the cost and time of developing microbial cell factories by orders of magnitude.
DOI
10.1002/qub2.70039