The improvement of natural transformation procedure and the development of genome manipulation methods for Streptococcus thermophilus B-6

Background Streptococcus thermophilus is invaluable in both of dairy factory and scientific research, and different S. thermophilus strains have different advantages in industry. As a result, fast, easy, biosafe, and widely applicable genome manipulation methods for S. thermophilus are of high demands. The traditional temperature-sensitive plasmid-based homologous recombination method is neither fast nor easy, the current natural transformation method is not widely applicable, and the recently established endogenous CRISPR-Cas system-assisted method is not easy in plasmid construction. Results In this project, we characterized the features of the natural transformation of S. thermophilus B-6 strain, and for the first time found that the DNA transport pathway activated at natural competent stage might be bidirectional. We optimized the natural transformation protocol to upgrade the transformation ratio of S. thermophilus B-6 from ~ 10 ^− 5 to ~ 10 ^− 2 . With the improved natural transformation procedure, we developed a fast, easy, biosafe, and plasmid-independent method for the genome manipulation of this strain. We also established a novel native CRISPR-Cas system-assisted genome manipulation pathway with a higher efficiency, which did not require any new plasmid construction. By the novel genome manipulation methods, we created different CRISPR-Cas system mutant strains and a recA overexpressing strain. Conclusions Our finding contributes to better understanding the features of natural competence. Our convenient and biosafe genome manipulation methods will be valuable for most of the S. thermophilus strains, and will contribute to the germplasm improvement of dairy industry. Besides, the S. thermophilus mutants we generated in this project will be useful in the future cellular metabolism investigations.