Genetic Toolbox Expansion Enables Constitutively Fluorescent Lacticaseibacillus rhamnosus for Functional Microbiome Research

ABSTRACT Lacticaseibacillus rhamnosus strains are widely recognized for their probiotic potential and relevance in urogenital and gut health. However, their genetic tractability and genetic tools remain limited, hindering functional microbiome research and synthetic biology applications. In this study, we expanded the genetic toolbox for the widely used probiotic strains, L. rhamnosus GR‐1 and L. rhamnosus GG, by implementing direct plasmid cloning and testing of a set of genetic elements earlier validated in Lactiplantibacillus plantarum . Among five constitutive promoters (P tlpA , P tec , P cpg , P 48 and P 23 ), P tlpA showed strong promoter activity in L. rhamnosus GR‐1. We further characterized this promoter's functionality by incorporating a repressor and assessing its native thermo‐responsiveness and stability over time, enhancing its potential for industrial applications. Using these tools, we engineered L. rhamnosus GR‐1 with constitutive fluorescence of mCherry, mScarlet3 and sfGFP. The functionality of these fluorescent L. rhamnosus GR‐1 strains was shown in a proof‐of‐concept growth competition experiment with a fluorescent pathogenic Staphylococcus aureus strain. These constitutively fluorescent L. rhamnosus strains, along with the expanded genetic toolkit, offer valuable resources for studying functional properties, such as adhesion, microbe–microbe and host–microbe interactions, and advancing Lactobacillaceae as a chassis for synthetic biology.