Multi‐Targeting Non‐Specific Genome Engineering in Bacteria

ABSTRACT Genome engineering plays a crucial role in the rapidly growing fields of metabolic engineering and synthetic biology. Chromosomal integration and stable expression of functional genes or large metabolic pathways necessitate the development of host‐independent enabling technologies in diverse bacteria. Here, a generalizable genome engineering approach, MNGE (Multi‐targeting Non‐specific Genome Engineering), is developed based on the multi‐targeting integrase (MTI) systems for multi‐copy (at least three copies), highly random (only requiring the core TT dinucleotide) integration of metabolic genes or pathways in both Gram‐positive bacteria (i.e., Streptomyces and Saccharopolyspora ) and Gram‐negative bacteria (i.e., Burkholderia and Chromobacterium ). Using MNGE, the fungicide UK‐2 BGC (41 kb) and the polyether antibiotic salinomycin BGC (106 kb) were randomly integrated into a heterologous host Streptomyces albus , significantly enhancing their fermentation levels based on chromosome position effects. Furthermore, the potent G _q/11 ‐signaling inhibitor FR900359 BGC (66 kb) was successfully expressed in Burkholderia gladioli by the MTI1 system. Together, the MNGE approach exhibits broad applicability for next‐generation genome engineering in diverse bacteria, thereby achieving highly efficient production of high‐value compounds.