In Vitro Gut Modeling as a Tool for Adaptive Evolutionary Engineering of Lactiplantibacillus plantarum.

Research and marketing of probiotics demand holistic strain improvement considering both the biotic and abiotic gut environment. Here, we aim to establish the continuous in vitro colonic fermentation model PolyFermS as a tool for adaptive evolutionary engineering. Immobilized fecal microbiota from adult donors were steadily cultivated up to 72 days in PolyFermS reactors, providing a long-term compositional and functional stable ecosystem akin to the donor's gut. Inoculation of the gut microbiota with immobilized or planktonic Lactiplantibacillus plantarum NZ3400, a derivative of the probiotic model strain WCFS1, led to successful colonization. Whole-genome sequencing of 45 recovered strains revealed mutations in 16 genes involved in signaling, metabolism, transport, and cell surface. Remarkably, mutations in LP_RS14990, LP_RS15205, and intergenic region LP_RS05100<LP_RS05095 were found in recovered strains from different adaptation experiments. Combined addition of the reference strain NZ3400 and each of those mutants to the gut microbiota resulted in increased abundance of the corresponding mutant in PolyFermS microbiota after 10 days, showing the beneficial nature of these mutations. Our data show that the PolyFermS system is a suitable technology to generate adapted mutants for colonization under colonic conditions. Analysis thereof will provide knowledge about factors involved in gut microbiota colonization and persistence.IMPORTANCE Improvement of bacterial strains in regard to specific abiotic environmental factors is broadly used to enhance strain characteristics for processing and product quality. However, there is currently no multidimensional probiotic strain improvement approach for both abiotic and biotic factors of a colon microbiota. The continuous PolyFermS fermentation model allows stable and reproducible continuous cultivation of colonic microbiota and provides conditions akin to the host gut with high control and easy sampling. This study investigated the suitability of PolyFermS for adaptive evolutionary engineering of a probiotic model organism for lactobacilli, Lactiplantibacillus plantarum, to an adult human colonic microbiota. The application of PolyFermS controlled gut microbiota environment led to adaptive evolution of L. plantarum strains for enhanced gut colonization characteristics. This novel tool for strain improvement can be used to reveal relevant factors involved in gut microbiota colonization and develop adapted probiotic strains with improved functionality in the gut.