Functional Characterization of the Exopolysaccharide Gene Clusters of Lactiplantibacillus plantarum YZH81 Using CRISPR/Cas9 Technology

The exopolysaccharide (EPS) gene clusters are the basis for understanding the structure-function relationship of EPS in lactic acid bacteria. In our earlier work, we had constructed the cps1 deletion strain (YZH81∆cps1), with a decreased capacity to produce EPS, from Lactiplantibacillus plantarum YZH81, a strain with high EPS production ability, which had been found to possibly contain two capsular polysaccharide syntheis (cps) gene clusters, cps1 and cps2. In this study, the cps2 gene cluster of strain YZH81 was knocked out using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9) technology. Compared with YZH81, the EPS production of YZH81∆cps1 and YZH81∆cps2 decreased by 22.88% and 35.42%, respectively, and the monosaccharide compositions of EPS from the two cps strains were altered considerably. The monosaccharide rhamnose was not detected in any of the mutant strains, and the percentages of mannose and glucose increased dramatically. Proteomics identified a total of 29 and 22 differential proteins in YZH81∆cps1 and YZH81∆cps2 versus YZH81 after fermentation for 10 and 32 h, respectively, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses showed that the differential proteins were involved in the biological processes, the metabolism and processing of cellular components in YZH81, but those resulting from knockout of the cps2 cluster had richer functions such as lipid metabolism, nucleotide metabolism, and glycan biosynthesis and metabolism. In conclusion, the present study provides theoretical support for future research on the structure-probiotic function relationship of EPS from L. plantarum YZH81.