Development Of Genome Editing Method And Modification Of N-acetylglucosamine Metabolic Pathway In Lactobacillus Plantarum

Lactobacillus plantarum is a broad member of fermented foods and lactic acid bacteria(LAB)found in the gastrointestinal tract.It is commonly used as a potential probiotic in the food industry liking many other strains of LAB.In this study,Red(RecT)-mediated recombinant engineering of dsDNA and ssDNA was combined with the CRISPR/Cas9 system to develop a genome-free editing of Lactobacillus plantarum WCFS1,including gene knockout,insertion and point mutation.It was also found that adenine-specific methyltransferase significantly improved ssDNA recombination efficiency.This method is applied to the metabolic engineering of GlcNAc production by Lactobacillus plantarum WCFS1.We knock out nagB to reduce the reverse reaction of F6P to GlcN6P.Endogenous strong promoter 3a was introduced to replace the glmS riboswitch and the point mutations in glmS1(G472S)can mitigate feedback inhibition.The resulting strain was able to produce 797.3 mg/L of GlcNAc without introducing any foreign genes and plasmids.This strategy contributes to the precise genetic engineering of Lactobacillus plantarum WCFS1 and promotes its application in more fields.A seamless genome knockout method was developed based on the prophage recombinase Lp₀640,Lp₀641 and Lp₀642-mediated double-strand recombination in Lactobacillus plantarum WCFS1,which are the potential analogs of Gam,Beta and Exo.The CRISPR/Cas9 system was introduced as a screening tool.The wild-type genome is altered by recombination with dsDNA under the action of recombinase,and recombinant successful strains can survive under the screening of CRISPR/Cas9 system.The introduction of the CRISPR/Cas9 system without the use of the loxP/Cre system and antibiotics avoids the introduction of exogenous DNA into the genome while reducing experimental time.Gene insertion is difficult to achieve,so the two-step method was developed.First,the antibiotic was used as a screening condition to achieve gene insertion.and then the CRISPR/Cas9-assisted knockout method was used to knock out the loxP site.Double-stranded recombination is not effective in point mutations,so we attempt to introduce RecT-mediated single-strand recombination in conjunction with the CRISPR/Cas9 system.The pSIP411 plasmid was used to express the endogenous RecT of Lactobacillus plantarum WCFS1,and single-strand recombination was used to achieve point mutation of single amino acid of the genome while changing the adjacent PAM site,so that the engineered strain could survive in the presence of CRISPR/Cas9 system.When designing point mutation sites,five consecutive base mismatches are designed to avoid mismatch repair,resulting in limited use of single-strand recombination.We try to introduce DNA adenine methylase(Dam)to improve single-strand recombination efficiency while three bases continuous mismatch achieves the same efficiency as the five base continuous mismatches,thereby realizing the mutation of any single amino acid in the genome,which greatly expands the application range of the point mutation.We chose to engineered the N-acetylglucosamine(GlcNAc)synthetic pathway to validate the validity of our non-marker genome editing method.GlcNAc is a precursor of glycosaminoglycans that plays an important role in maintaining healthy cartilage and joint tissue function.Therefore,it is widely used as a food supplement for the treatment of osteoarthritis as well as in the fields of cosmetics and pharmaceuticals.The production of GlcNAc by the fermentation process has accumulated some technology in Escherichia coli and Bacillus subtilis.We used the developed genome-free editing method to knock out the nagB gene encoding glucosamine-6-phosphate(GlcN-6P)isomerase/deaminase in Lactobacillus plantarum WCFS1 and the glmS ribozyme that regulates the production of GlcN-6P.The ribozyme inhibits the accumulation of GlcN6P resulting in a decrease in GlcNAc production.In addition,insertion of the endogenous strong promoter 3 a(P3a)in front of the glmSl gene increased the expression level of the key enzyme GlmS1.Finally,the G472 position of the glmS1 gene in the genome was mutated to serine to relieve the negative feedback inhibition of GlmS1.The above genomically engineered strain was named WG3.and GlcNAc was produced by fermentation to 456 mg/L.Production of GlcNAc will consume pyruvate to produce acetyl-coenzyme A resulting in N ADH accumulation,and excessive NADH accumulation disrupts energy balance.The addition of heme and menaquinone during fermentation can activate the respiratory chain and consume NADH.By balancing NADH in this way,the growth state of the strain is improved and the yield is increased.The engineered strain can produce 797.3 mg/L GlcNAc under optimized fermentation conditions.