Engineering Bacteroides Thetaiotaomicron to Secrete Heterologous Therapeutic Protein

Protein drugs, including engineered antibodies, present many advantages, including high specificity, high potency, and high biocompatibility. To prevent degradation in the stomach, past studies have attempted to engineer probiotic bacteria to produce protein drugs in situ, within the gastrointestinal tract. Lactococcus lactis has been the most commonly used species for this purpose because of its “generally recognized as safe” (GRAS) status and the availability of tools for genetic manipulation of this organism. However, L. lactis only transiently colonizes the human gut and therefore requires repetitive dosing to maintain a therapeutic effect. Our research aims to engineer a prevalent and abundant permanently colonizing gut commensal bacterium, Bacteroides thetaiotaomicron, to secrete small antibody variants for the treatment of intestinal diseases. To facilitate engineering of B. thetaiotaomicron, we created a Golden Gate Assembly system to enable rapid construction of plasmids with a variety of genetic parts. We identified eleven signal peptides by bioinformatic analysis and three therapeutic antibodies against various targets of gastrointestinal disease (two targeting Clostridioides difficile infection and one targeting chronic colitis), which we have engineered into smaller variants to accommodate bacterial expression and increase stability in the GI tract. To screen for secretion activity, we will use our plasmid assembly system to pair each of the signal peptides with each of the different antibody variants, and then measure antibody accumulation in culture media. Preliminary results have revealed two signal peptides supporting 15-50 times higher levels of antibody secretion from B. thetaiotaomicron than previously reported in another Bacteroides speices. Future work will include engineering an improved secretory pathway to optimize secretion. Ultimately, we aim to establish engineered B. thetaiotaomicron strains as self-replicating drug production factories for in situ delivery of active biotherapeutic proteins with the goal of developing a platform for single-dose, low-cost, long-term therapy for chronic intestinal diseases.