Engineering Redox-based Intercellular Communication Channels Towards the Control of Microbial Consortial Behavior

Abstract

Microbial communities exist in many facets of life, such as in the human body, including the microbiomes of the gut, mouth, respiratory tract, as well as that in the environment, including microbiomes of the soil, permafrost, and the ocean. While vastly different in composition, all microbial communities participate in various modes of intercellular communication, a process in which cells relay information across potentially large distances through molecular interactions. The advent of synthetic biology has enabled facile manipulation of the genetic architectures governing these intercellular communication pathways, leading to the bottom-up assembly of designer microbial communities and the top-down studies of natural consortia pared for specific functions. This assembly process requires careful orchestration of individual behaviors to guide the collective behaviors exhibited by the community. Current engineering approaches employ the use of genetic circuits (ie. genetic regulatory networks) in which cells are programmed to sense a molecular input and respond to that input with the transcription of a targeted gene. However, engineering genetic circuits in microbial communities face two inherent challenges: (1) the difficulty in precisely controlling genetic circuitry in a specific cell and its robustness under dynamic conditions and (2) the challenges associated with predicting behavior at the consortial level. In an attempt to address these challenges, our group and others have developed alternative methods of genetic actuation using electrogenetics, wherein precisely encoded electronic signals can be used to elicit a programmed response. Moreover, electrogenetic induction schemes can be coupled with natural intercellular communication pathways (eg. quorum sensing), taking advantage of nature's diversity to increase signaling efficiency. In this dissertation, we first describe a framework for the systematic assembly of a consortium consisting of soil-based microbes wherein molecular signaling is electronically actuated from a transmitting species, transduced and propagated through other consortial members. Then, we employ the same signaling paradigms in a consortium of soil-based Psuedomonas towards the guided biosynthesis of a plant auxin, indole acetic acid in response to oxidative stress signal molecules such as hydrogen peroxide and acetosyringone. Lastly, we further expand this signaling scheme in the development of cross-kingdom intercellular communication structures. In demonstrating redox-based actuation of these non-canonical signaling pathways between Gram-negative Pseudomonas and Gram-positive Bacillus, we showcase the immense potential of bridging synthetic biology tools with redox-based genetic actuation towards guiding novel signaling networks within microbial consortia. This work expands the horizon of electrogenetics into new chassis organisms and draws broad implications in biofilm engineering, biomanufacturing and the development of living therapeutics.