Grant Abstract: Advancing synthetic biology tools for engineering gut commensals

Grant Number: 1R21EB037892-01A1
PI Name: Sonnenburg
Project Title: Advancing synthetic biology tools for engineering gut commensals

Abstract: The human gut microbiota, a complex and dynamic community of microorganisms, plays a crucial role in regulating host biology, influencing many processes and tissues, such as immunity, metabolism, and the nervous system. The relationship between gut microbes and humans offers opportunities to develop therapeutic strategies that harness or modify microbial functions. However, current efforts to deliver engineered bacterial therapies have encountered significant challenges, particularly in achieving stable colonization and the high microbial densities required for therapeutic efficacy. We have overcome these limitations with key technologies engineered into Bacteroides, the dominant genus in the US gut microbiome. This project aims to further advance synthetic biology approaches for the therapeutic application of gut- resident bacteria, focusing on two key challenges. Aim 1 seeks to expand the therapeutic capabilities of the Bacteroides platform. To date, engineered bacteria have been limited in their ability to secrete large protein complexes due to size constraints (~80 kDa). In this aim, we propose to develop a synthetic scaffolding system that will enable the production of larger protein complexes capable of multivalent binding or more sophisticated biological functions. By creating a protease-resistant scaffold, we will coordinate the assembly and secretion of therapeutic proteins directly in the gut environment. This innovation will significantly broaden the range of therapeutic applications for Bacteroides-based systems. As proof of concept, we will demonstrate the effectiveness of this platform by treating Clostridium difficile infection in a mouse model using multimeric nanobodies. The development of this synthetic scaffolding technology will provide a foundation for future therapies involving complex protein secretion and localization in the gut. Aim 2 focuses on expanding the repertoire of bacterial chassis available for therapeutic engineering by developing Bifidobacterium infantis as a platform for developing microbial medicines for infants. Bifidobacterium is a key member of the infant gut microbiota and is ideally suited for therapeutic applications in early life. We will build on recent advances in molecular genetics and phage genome research to create the necessary genetic tools for engineering Bifidobacterium, including systems for genome integration, gene expression, and protein secretion. This platform will be used to address diseases prevalent in infants, such as necrotizing enterocolitis and metabolic disorders. With over a decade of experience in microbial synthetic biology, our team has successfully developed technologies that have application in basic research and preclinical models and have been validated in Phase 2A human trials. The proposed project will further advance the therapeutic potential of engineered gut microbes, paving the way for innovative treatments for a range of diseases across different age groups.

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