Engineering of Lactobacillus Reuteri As a Biotherapeutic Delivery System

Advances in the fields of synthetic biology and the human microbiome are enabling the use of biologically relevant microorganisms to fight human diseases. Lactobacillus reuteri 6475 (LR) is a probiotic strain with desirable features for therapeutic delivery purposes due to its remarkable safety profile, its ability to survive in the gastrointestinal tract (GIT), and the availability of genetic tools that facilitate editing of its genome. Our goal is to generate tools that allow precise control of protein production and secretion by LR to develop a robust platform for efficient delivery of therapeutic proteins to the GIT. As a proof of concept, we are engineering LR to secrete the human cytokine interleukin-22 (IL-22), which plays a key role in maintaining the integrity of epithelial barriers such as the skin and the intestinal epithelium. IL-22 has proven to confer colonization resistance against Vancomycin Resistant Enterococci in mice and significantly improve wound healing in murine diabetic models; therefore, engineering of LR to secrete IL-22 would be an effective strategy for the in situ delivery of this therapeutic cytokine from a probiotic strain that is well adapted to the human GIT.

In order to develop gene expression tools for the control of protein production, we designed promoter and ribosomal binding site (RBS) libraries by randomly mutating a strong promoter and a canonical RBS that drives the expression of green fluorescence protein (GFP). We identified a library of promoters and RBSs that have a dynamic range for the expression of GFP displaying five orders and four orders of magnitude of range, respectively. To achieve efficient secretion of proteins we evaluated the activity of several signal peptides (SPs) and found a SP that resulted in the secretion of more than 80% of the total protein yield. Lastly, to explore the ability of LR to deliver IL-22, we built strains capable of inducible and constitutive expression (up to 2µg/ml) in order to evaluate their biological activity in vitro and in vivo.  The bioactivity of the secreted IL-22 has been confirmed in vitro through the induction of IL-10 and phosphorylation of STAT3 in colonic cells, as well as in vivo by stimulating the production of Reg3γ in animals gavaged with LR secreting IL-22. Preliminary results suggest that IL-22-secreting LR improves healing and resolution of wounds in a murine traumatic wound model. In summary, we have developed strains and genetic tools that will facilitate and enhance programming of LR as a therapeutic delivery system. We have also engineered LR to secrete biologically active IL-22 and we are currently working on optimizing IL-22 production along with evaluating its therapeutic value in a number of disease models.