Advancing Tools for Quantifying and Engineering Microbial Consortia

Abstract

From biosynthesis to bioremediation, microbes have been engineered to address a variety of biotechnological applications. A promising direction in these endeavors is harnessing the power of designer microbial consortia that consist of multiple populations with well-defined interactions. Consortia can accomplish tasks that are difficult or potentially impossible to achieve using monocultures. Despite their potential, the rules underlying microbial community maintenance and function are not well defined, though rapid progress is being made. This limited understanding is in part due to the greater challenges associated with increased complexity when dealing with multi-population interactions. For example, although metabolic pathways are often engineered in single microbial populations, the introduction of heterologous circuits into the host can create a substantial metabolic burden that limits the overall productivity of the system. This limitation could be overcome by metabolic division of labor (DOL), whereby distinct populations perform different steps in a metabolic pathway, reducing the burden each population will experience. While conceptually appealing, the conditions when DOL is advantageous have not been rigorously established. In my dissertation, I analyzed 24 common architectures of metabolic pathways in which DOL can be implemented. My analysis revealed general criteria defining the conditions that favor DOL, accounting for the burden or benefit of the pathway activity on the host populations as well as the transport and turnover of enzymes and intermediate metabolites. These criteria can help guide engineering of metabolic pathways and has implications for understanding evolution of natural communities. I next investigated utilizing horizontal gene transfer (HGT) as a tool to engineer microbial consortia. Due to the role of HGT in spreading and maintaining diverse functional traits such as metabolic functions, virulence factors, and antibiotic resistance, suppressing plasmid transfer in microbial communities has profound implications for consortia engineering. However, existing tools for inhibiting HGT are limited in their modes of delivery, efficacy, and scalability. I demonstrated a generalizable denial-of-spread (DoS) strategy that can target and eliminate specific conjugative plasmids from communities. My strategy exploits retrotransfer, whereby an engineered DoS plasmid is introduced into host cells containing a target plasmid via the target’s own conjugative machinery. Within the same host, DoS eliminates the target plasmid through a combination of transfer competition and plasmid incompatibility, after which DoS can be removed via induced suicide. DoS’s design is highly tunable and scalable to various conjugative plasmids, different plasmid curing mechanisms, or environmental contexts. Together, my findings contribute to a greater understanding of consortia stability and establish a potential new tool for precision engineering of said consortia.