Abstract
Streptomyces fungicidicus, an industrial strain for enduracidin production, shows significant potential as a cellular chassis for the synthesis of novel small peptides. Targeted deletion of secondary metabolite gene clusters offers a promising strategy to enhance strain performance, but is often hampered by the lack of efficient gene editing tools. In this study, we optimized the traditional homologous recombination method by integrating selection and counter-selection markers to streamline the gene editing process, and successfully deleted gene clusters of up to 54.4 kb. Recognizing the significant potential of CRISPR/Cas-based systems in Streptomyces, we evaluated the base editing efficiency of the CRISPR/cBEST system in S. fungicidicus, which enabled stop codon insertions in the targeted gene with different mutation rates depending on the applied sgRNA. Additionally, we established a CRISPR/Cas9 system in S. fungicidicus while incorporating a counter-selection marker for efficient screening, which greatly shortened the gene editing cycle. The resulting mutants with single and cumulative gene cluster deletions exhibited improved growth characteristics, including a prolonged logarithmic phase and increased biomass. Although cumulative deletions did not result in consistent yield improvements, the mutants with improved growth characteristics show potential for further strain optimization in the future. The optimized gene editing systems developed in this study provide a valuable foundation for engineering other Streptomyces species.