Restructuring a Complex Genetic Function on Episomal Vectors in <i>Escherichia coli</i>

Genetic functions have evolved over long timescales and can be encoded by multiple genes dispersed in different locations in genomes, and although contemporary molecular biology enables control over single genes, more complex genetic functions remain challenging. Here, we study the restructuring and mobilization of a complex genetic function encoded by 10 genes, originally expressed from four operons and two loci on the <i>Escherichia coli</i> genome. We observe subtle phenotypic differences and reduced fitness when expressed from episomal DNA and demonstrate that mutations in the transcriptional machinery are necessary for successful implementation in different bacteria. The work provides new approaches for advanced genome editing and constitutes a first step toward modularization and genome-level engineering of complex genetic functions.