Lactobacillus plantarum is a potential starter and health-promoting probiotic bacterium. Effective, precise, and diverse genome editing of Lactobacillus plantarum without introducing exogenous genes or plasmids is of great importance. In this study, CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering was established in L. plantarum WCFS1 to seamlessly edit the genome, including gene knockouts, 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 L. plantarum WCFS1 toward producing N-acetylglucosamine (GlcNAc). nagB was truncated to eliminate the reverse reaction of fructose-6-phosphate (F6P) to glucosamine 6-phosphate (GlcN-6P). Riboswitch replacement and point mutation in glmS1 were introduced to relieve feedback repression. The resulting strain produced 797.3 mg/liter 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. IMPORTANCE CRISPR/Cas9-assisted recombineering is restricted in lactic acid bacteria because of the lack of available antibiotics and vectors. In this study, a seamless genome editing method was carried out in Lactobacillus plantarum using CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering, and recombination efficiency was effectively improved by endogenous adenine-specific methyltransferase overexpression. L. plantarum WCFS1 produced 797.3 mg/liter N-acetylglucosamine (GlcNAc) through reinforcement of the GlcNAc pathway, without introducing exogenous genes or plasmids. This seamless editing strategy, combined with the potential exogenous GlcNAc-producing pathway, makes this strain an attractive candidate for industrial use in the future.

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N-acetyl-d-neuraminic acid (NeuAc) has attracted considerable attention because of its wide-ranging applications. The use of cheap carbon sources such as glucose without the addition of any precursor in microbial NeuAc production has many advantages. In this study, improved NeuAc production was attained through the optimization of amino sugar metabolism pathway kinetics and reservation of a phosphoenolpyruvate (PEP) pool in Escherichia coli. N-acylglucosamine 2-epimerase and N-acetylneuraminate synthase from different sources and their best combinations were used to obtain optimized enzyme kinetics and expression intensity, which resulted in a significant increase in NeuAc production. Next, after a design was engineered for enabling the PEP metabolic pathway to retain the PEP pool, the production of NeuAc reached 16.7 g/L, which is the highest NeuAc production rate that has been reported from using glucose as the sole carbon source.