Multi‐Targeting Non‐Specific Genome Engineering in Bacteria

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 Gq/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.