CRISPR/Cas9-Assisted Seamless Genome Editing in Lactobacillus plantarum and Its Application in N -Acetylglucosamine Production

Abstract

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.

Genome Editing in Bacteria: CRISPR-Cas and Beyond

“…Among them, recombinase T (RecT), has been established successfully to enhance the CRISPR-Cas-mediated genome editing in Corynebacterium glutamicum [ 34 ], Lactoccocus lactis [ 35 ], Lactobacillus plantarum , and Lactobacillus brevis [ 36 ]. RecT binds to ssDNA and protects it from degradation, fulfilling a similar function to gamma protein in the lambda Red system [ 37 ].…”

Novel Strategies for Efficient Production and Delivery of Live Biotherapeutics and Biotechnological Uses of Lactococcus lactis: The Lactic Acid Bacterium Model

“…This recombinant L. plantarum WCFS1 was able to produce 797.3 mg/L N -acetylglucosamine (GlcNAc) by inducing the GlcNAc pathway. This genetic edition facilitated industrial production of this protein and demonstrated the potential of this genetic engineering technique for application in other organisms ( Zhou et al, 2019 ).…”

“… Zhou et al (2019) , in an attempt to increase protein production without inserting exogenous genes, used CRISPR/Cas9 to engineer L. plantarum strain WCFS1 to produce N-acetylglucosamine (GlcNAc). This was achieved by truncating the gene nag B, which eliminates the reversion of fructose-6-phosphate (F6P) back to glucosamine-6P.…”

Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V

“…The plasmid system developed here could potentially be transportable to other Gram-positive bacteria, as the used origin of replication was shown to be functional in Lactococcus lactis and Bacillus subtilis ( 20 ). While similar approaches have recently been undertaken to perform genome engineering in certain Gram-positive organisms such as Enterococcus faecium ( 21 ), Clostridium ( 22 ), Lactobacillus reuteri ( 23 ), Lactobacillus plantarum ( 24 ), and Lactococcus lactis ( 25 ), a concise CRISPR sgRNA:Cas9 gene editing system was not yet available for S. pneumoniae , and the vector described here has the advantage of being readily curable due to its temperature-sensitive origin of replication.…”