Utilizing the SacB-mediated gene editing system in Komagataeibacter xylinus to explore the function of bacterial cellulose synthase.

Bacterial cellulose (BC) is a microbial polysaccharide, which is widely used in biotechnology, food, medicine, and other industries. Although existing genetic toolkits have laid a solid foundation for the genetic manipulation of BC-producing strains, there is still room for improvement in enhancing editing efficiency, simplifying operational procedures, and achieving scarless modifications. In the present study, we developed a SacB-based system, pK18mobsacB, to achieve marker-free gene editing with an efficiency of up to 83.33 %. Gene deletion, insertion, and replacement were successfully performed in Komagataeibacter xylinus CGMCC 2955 using this system. Subsequently, the SacB-based system was used to explore the function of bacterial cellulose synthase in the synthesis and structure of BC. It was found that the bcs I operon played an important role in BC synthesis. The deletion of the bcs II and bcs III operon regions, either individually or in combination, led to an increase in the fiber diameter and crystallinity of the BC films. The SacB-based system and its applications established in this study provide valuable tools and a theoretical foundation for the modification of BC-producing strains using synthetic biology, thereby facilitating the sustainable application of BC and the development of innovative products.