is a potential starter and health-promoting probiotic bacterium. Effective， precise and diverse genome-editing of without introducing exogenous genes or plasmids is of great importance. In this study， CRISPR/Cas9-assisted dsDNA and ssDNA recombineering were established in WCFS1 to seamlessly edit the genome， including gene knockout， insertions and point mutations. To optimize our editing method， phosphorothioate modification was used to improve the dsDNA insertion， and adenine-specific methyltransferase was used to improve the ssDNA recombination efficiency. These strategies were applied to engineer WCFS1 towards producing N-acetylglucosamine (GlcNAc). was truncated to eliminate the reverse reac... More
is a potential starter and health-promoting probiotic bacterium. Effective， precise and diverse genome-editing of without introducing exogenous genes or plasmids is of great importance. In this study， CRISPR/Cas9-assisted dsDNA and ssDNA recombineering were established in WCFS1 to seamlessly edit the genome， including gene knockout， insertions and point mutations. To optimize our editing method， phosphorothioate modification was used to improve the dsDNA insertion， and adenine-specific methyltransferase was used to improve the ssDNA recombination efficiency. These strategies were applied to engineer WCFS1 towards producing N-acetylglucosamine (GlcNAc). was truncated to eliminate the reverse reaction of fructose-6-phosphate (F6P) to GlcN-6P. Riboswitch replacement and point mutation in 1 were introduced to relieve feedback repression. The resulting strain produced 797.3 mg/L GlcNAc without introducing exogenous genes or plasmids. This strategy may contribute to the available methods for precise and diverse genetic engineering in lactic acid bacteria and boost strain engineering for more applications. CRISPR/Cas9-assisted recombineering is restricted in lactic acid bacteria because of the lack of available antibiotics and vectors. In this study， seamless genome editing method was carried out in using CRISPR/Cas9-assisted dsDNA and ssDNA recombineering and was effectively improved on the recombination efficiency by endogenous adenine-specific methyltransferase overexpression. WCFS1 produced 797.3 mg/L N-acetylglucosamine (GlcNAc) through reinforcing the GlcNAc pathway without introducing exogenous genes or plasmids. This seamless editing strategy， combined with the potential exogenous GlcNAc producing pathway， made this strain an attractive candidate for industrial use in the future.
