Construction of a genome-editing system for the thermophilic actinomycete Streptomyces thermodiastaticus K5 strain

Abstract

Thermophilic actinomycetes significantly contribute to the terrestrial carbon cycle via the rapid degradation of lignocellulosic polysaccharides in composts. In this study, a genome-editing system was constructed for the thermophilic actinomycete Streptomyces thermodiastaticus K5 strain, which was isolated from compost. The genome-editing plasmid (pGEK5) harboring nickase Cas9 was derived from the high-copy plasmid pL99 and used for the K5 strain. It was found that pGEK5 could easily be lost from the transformed clone through cultivation on apramycin-free medium and spore formation, enabling its reuse for subsequent genome-editing cycles. With the aid of this plasmid, mutations were sequentially introduced to 2 uracil-DNA glycosylase genes (Udg1 and Udg2) and 1 β-glucosidase gene (Bgl1). Thus, the genome-editing system using pGEK5 enables us to start the functional modification of this thermophilic actinomycete, especially for improved conversion of lignocellulosic biomass.