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Abstract
The human microbiome is intrinsically linked to human health and the progression of various disease states. Until recently, we have been limited by molecular and computational tools to probe many of the bacteria that reside in our intestinal tracts. Advancements in ‘omics approaches and anaerobic culture techniques have significantly de-risked harnessing native microbiota members for synthetic biology implementations. A dearth of molecular tools limits our understanding of how many of these microbes affect host-facing mechanisms. Here we lay the foundation for advanced manipulation and genetic manipulation in one such family of bacteria, Lachnospiraceae. Known for their role in maintaining human health through the production of unique metabolites including short-chain fatty acids (SCFAs), we propose a molecular toolkit to reveal their role in host physiology and to harness Lachnospiraceae biology to secrete recombinant protein cargoes. We then apply this toolkit to program Lachnospiraceae species to produce biologically relevant protein targets important for treating and maintaining health for in vivo applications. In this thesis, I will outline the foundational steps we have taken towards understanding Lachnospiraceae species as well as generating engineered live biotherapeutic systems from native microbial chassis. First, I will outline the state of the field of engineering non-model bacteria, adapting work I have published as a co-first author with Joshua Glazier (Arnold 2023). Second, we begin by taking empirical and computational approaches to generating novel molecular tools to control gene transcription and translation. Then we investigate controllable gene expression by implementing inducible promoter systems. We work to stabilize these expression systems by co-opting mechanisms for integration into the Lachnospiraceae genome (Enyeart 2013, Cerisy 2019), and to evaluate the functionality of Lachnospiraceae expression systems in vivo. Third, I will describe our efforts to discover and develop systems for efficient recombinant protein secretion in Lachnospiraceae hosts. These tools will enable the testing and characterization of bioactive therapeutic protein targets, with a focus on interleukin 10-family cytokines. Bacteria engineered to produce these recombinant proteins will be vetted for in vitro and in vitro bioactivity, before preliminary tests in an in vivo model of diet-induced metabolic disease (Oh 2020). Finally, I will summarize the contributions I have made in advancing the field of engineering non-model bacteria and developing synthetic biological systems, as well as an outlook towards future projects.