Genome editing system for S. thermodiastaticus K5

Streptomyces thermodiastaticus K5, a thermophilic chitinase producer isolated from compost, has shown a strong ability to degrade cellulose and xylan in Ca(OH)2-pretreated rice straw at 50 ºC. To enhance or modify the cellulose and xylan metabolite pathways of this strain, a genome editing system was developed. The high-copy plasmid pL99 successfully transformed the K5 strain via conjugation. However, the introduction of Cas9 likely failed due to cell toxicity. Consequently, a genome editing plasmid for K5 (pGEK5) was constructed using nCas9 instead of Cas9. The pGEK5 plasmid could easily be lost from the transformed clone through spore formation and cultivation on apramycin-free medium, allowing its reuse for subsequent genome editing. Stop codons were introduced to uracil–DNA glycosylases Udg1 and Udg2, resulting in a double mutant clone with higher base editing efficiency than original K5 strain.