Full-length single-stranded PCR product mediated chromosomal integration in intact Bacillus subtilis

► A new and efficient protocol for gene replacement in intact B. subtilis was developed. ► It is based on endogenous HR machinery and full ssDNA constructs as direct substrate. ► We showed that ssDNA is advantageous over dsDNA to stimulate HR in intact B. subtilis. ► Increased HR frequency correlates with lagging-strand bias and 5′ end protection. ► A lipase gene was successfully integrated and expressed by applying this strategy.