Multi-gene Co-expression systems in E. coli: From single-vector designs to programmable expression platforms

Escherichia coli ( E. coli ) has long served as a versatile workhorse for recombinant protein production. As synthetic biology expands the demand for coordinated expression of multiple genes, co-expression systems in E. coli have evolved from basic dual-gene constructs to programmable, polygenic expression platforms. This review critically examines the major strategies enabling multigene co-expression in E. coli, including internal ribosome entry sites (IRES), 2A self-cleaving peptides, dual-promoter cassettes, multicistronic operons, and multi-plasmid configurations. We highlight the mechanistic principles, design trade-offs, and regulatory bottlenecks associated with each approach, such as translational imbalance, inclusion body formation, and plasmid compatibility. Real-world applications in metabolic engineering, complex protein assembly, and biomanufacturing are analyzed to demonstrate the functional advantages of these systems. Finally, we explore emerging programmable toolkits that integrate modular architecture, expression modeling, and AI-assisted design, paving the way for next-generation synthetic expression control in microbial chassis. This review offers a comprehensive and strategic roadmap for researchers engineering multi-gene systems in E. coli and beyond.