A Tn5 Transposase-Based System for High-Efficiency Genome-Wide Gene Activation in <i>Escherichia coli</i>

Deciphering gene function to understand the genetic basis of microbial phenotypes in a high-throughput manner is crucial for bacterial engineering. However, efficient tools for generating genome-wide gene activation mutant libraries to enable gain-of-function analyses remain limited. Here, we developed a Tn5 transposase-based system for efficient genome-wide gene activation in <i>Escherichia coli</i>. The cargo DNA incorporated a tetracycline-inducible promoter Ptet and a kanamycin resistance gene, enabling selective growth and conditional gene activation. The system achieved near-random integration with an insertion frequency of approximately 2.83 × 10⁷ cfu/μg DNA. Both in vitro and in vivo transposition results demonstrated the effective activation of specific and nonspecific genes. Using this system, we identified three putative transporters that, despite being unrelated to glycine export, significantly enhanced glycine resistance in <i>E. coli</i>. These results highlight the utility of this method for genotype-phenotype mapping and strain optimization, offering a powerful tool for synthetic biology and functional genomics.