Multicopy Chromosome Integration and Deletion of Negative Global Regulators Significantly Increased the Heterologous Production of Aborycin in .

Aborycin is a type I lasso peptide with a stable interlocked structure, offering a favorable framework for drug development. The aborycin biosynthetic gene cluster from marine sponge-associated sp. HNS054 was cloned and integrated into the chromosome of hosts with different copies. The three-copy -integration strain M1346::3 showed superior production compared to the one-copy or two-copy -integration strains, and the total titer reached approximately 10.4 mg/L, i.e., 2.1 times that of the native strain. Then, five regulatory genes, (), (), , () and (), which reportedly have negative effects on secondary metabolism, were further knocked out from the M1346::3 genome by CRISPR/Cas9 technology. While the mutant showed a significant decrease (4.6 mg/L) and the mutant showed no significant improvement (12.1 mg/L) in aborycin production, the , and mutations significantly improved the aborycin titers to approximately 23.6 mg/L, 56.3 mg/L and 48.2 mg/L, respectively, which were among the highest heterologous yields for lasso peptides in both systems and systems. Thus, this study provides important clues for future studies on enhancing antibiotic production in systems.