Developing the Limosilactobacillus reuteri Chassis through an Endogenous Programmable Endonuclease-Based Genome Editing Tool.

Using genetically tractable probiotics to engineer live biotherapeutic products (LBPs) for disease treatment is urgently needed. Limosilactobacillus reuteri is an important vertebrate gut symbiont, which has great potential for developing LBPs. However, in L. reuteri, synthetic biology work is largely limited by the long editing cycle. In this study, we identified a subtype II-A CRISPR-Cas9 system in L. reuteri 03 and found the endogenous Cas9 (LrCas9) recognizing a broad protospacer-adjacent motif (PAM) sequence (3'-NDR; N = A, G, T, C; D = A, G, T; R = A, G). We reprogrammed the LrCas9 for efficient gene deletion (95.46%), point mutation (86.36%), large fragment deletion (40 kb), and gene integration (1743 bp, 73.9%), which uncovered the function of the repeated conserved domains in mucus-binding protein. Moreover, we analyzed the distribution of endogenous endonucleases in 304 strains of L. reuteri and found the existence of programmable endonucleases in 98.36% of L. reuteri strains suggesting the potential to reprogram endogenous endonucleases for genetic manipulation in the majority of L. reuteri strains. In conclusion, this study highlights the development of a new probiotic chassis based on endogenous endonucleases in L. reuteri 03, which paves the way for the development of genome editing tools for functional genetic studies in other L. reuteri. We believe that the development of an endogenous endonuclease-based genetic tool will greatly facilitate the construction of LBPs.

Postbiotics from Pichia kudriavzevii promote intestinal health performance through regulation of Limosilactobacillus reuteri in weaned piglets.

Postbiotics are attractive as alternatives to antibiotics for use against post-weaning diarrhea. However, their beneficial mechanisms are largely unknown. In the current study, we first demonstrated that supplementation with 0.5% Pichia kudriavzevii FZ12 postbiotics in the diet significantly reduced diarrhea incidence, promoted growth performance, improved gut health performance, and significantly enriched beneficial bacteria, particularly Lactobacillus spp., in the intestines of weaned piglets. Importantly, we identified a heat- and proteinase K-sensitive component, cytochrome c, of the postbiotics that significantly promoted the growth and biofilm formation of Limosilactobacillus reuteri FP13. We demonstrated the importance of P. kudriavzevii FZ12 postbiotics in improving the intestinal health of a model animal and revealed that cytochrome c is one of the important components of yeast postbiotics. These findings may provide new insights into microbe-postbiotics interplay that can be applied to guidelines for dietary modulation to alleviate weaning-induced diarrhea.