An efficient endogenous type I-E CRISPR-Cas genome-editing platform for producing transglutaminase in Streptomyces mobaraensis.

Streptomyces mobaraensis is an industrially important actinomycete capable of producing transglutaminase (TGase), a valuable crosslinking enzyme that is widely used in the food, pharmaceutical, and textile industries. However, its genetic manipulation remains challenging owing to the lack of efficient genome-editing tools. Here, we characterized an endogenous type I-E CRISPR-Cas system in S. mobaraensis IPIO2 through bioinformatics analysis and plasmid interference assays, identifying the protospacer adjacent motif as 5'-AAC-3'. We engineered an artificial editing plasmid, pCRISPR, by inserting a mini-CRISPR array (repeat-spacer-repeat) and homologous recombination repair templates into the replicative plasmid pJTU1278. This system exhibited high editing efficiencies, achieving 70% for single-gene deletions and 75-80% for large DNA fragment deletions ranging from 10 to 40 kb. Based on this system, deletion of four genes consistently downregulated during TGase production, identified through comparative proteomics, enhanced TGase production by 8.5-18.5%. Furthermore, deleting the pseudouridimycin and piericidin A1 biosynthetic gene clusters using this system significantly improved the safety profile of TGase production, resulting in a 17% increase in TGase yield. This study established a robust and efficient endogenous CRISPR-Cas-based genome-editing platform in S. mobaraensis, providing a powerful tool for strain engineering and industrial optimization of TGase production.