Lactobacillus casei has drawn increasing attention as a health-promoting probiotic, while effective genetic manipulation tools are often not available, e.g., the single-gene knockout in L. casei still depends on the classic homologous recombination-dependent double-crossover strategy, which is quite labor-intensive and time-consuming. In the present study, a rapid and precise genome editing plasmid, pLCNICK, was established for L. casei genome engineering based on CRISPR-Cas9 ^D10A . In addition to the P ₂₃ -Cas9 ^D10A and P _ldh -sgRNA (single guide RNA) expression cassettes, pLCNICK includes the homologous arms of the target gene as repair templates. The ability and efficiency of chromosomal engineering using pLCNICK were evaluated by in-frame deletions of four independent genes and chromosomal insertion of an enhanced green fluorescent protein (eGFP) expression cassette at the LC2W_1628 locus. The efficiencies associated with in-frame deletions and chromosomal insertion is 25 to 62%. pLCNICK has been proved to be an effective, rapid, and precise tool for genome editing in L. casei , and its potential application in other lactic acid bacteria (LAB) is also discussed in this study. IMPORTANCE The lack of efficient genetic tools has limited the investigation and biotechnological application of many LAB. The CRISPR-Cas9 ^D10A nickase-based genome editing in Lactobacillus casei , an important food industrial microorganism, was demonstrated in this study. This genetic tool allows efficient single-gene deletion and insertion to be accomplished by one-step transformation, and the cycle time is reduced to 9 days. It facilitates a rapid and precise chromosomal manipulation in L. casei and overcomes some limitations of previous methods. This editing system can serve as a basic technological platform and offers the possibility to start a comprehensive investigation on L. casei . As a broad-host-range plasmid, pLCNICK has the potential to be adapted to other Lactobacillus species for genome editing.

Polysaccharides and polyphenols coexist in many plant-based food products. Polyphenol–polysaccharide interactions may affect the physicochemical, functional, and physiological properties, such as digestibility, bioavailability, and stability, of plant-based foods. In this review, the interactions (physically or covalently linked) between the selected polysaccharides and polyphenols are summarized. The preparation and structural characterization of the polyphenol–polysaccharide conjugates, their structural–interaction relationships, and the effects of the interactions on functional and physiological properties of the polyphenol and polysaccharide molecules are reviewed. Moreover, potential applications of polyphenol–polysaccharide conjugates are discussed. This review aids in a comprehensive understanding of the synthetic strategy, beneficial bioactivity, and potential application of polyphenol–polysaccharide complexes.