Design, Construction, and Application of Synthetic Microbial Consortia.

Abstract

Mixtures of interacting microbes, or microbial consortia, may be a key part of the solution to overcoming current environmental and technological challenges, such as a dearth of renewable fuel sources. Microbial consortia have various advantages over single species, or “superbugs”, such as efficiency, robustness, and modularity. The goal of this dissertation is to develop tools for making use of microbial consortia and to demonstrate their utility through practical applications. Specifically, our efforts in technology development and application include: a tunable, programmable cross-feeding circuit; production of isobutanol (a next-generation biofuel); and sensing/screening of metabolite secretion. First, we designed and constructed a programmable genetic circuit based on engineered symbiosis between two E. coli auxotrophs. By regulating and tuning the export or production of the cross-fed metabolites we were able to tune the exchanges and achieve a wide range of growth rates and strain ratios. In addition, we created two-dimensional design space plots by inverting the relationship of growth/ratio vs. inducer concentrations. Using the plots, we could “program” the co-culture for pre-specified outcomes. This proof-of-concept circuit can be applied to more complex systems where precise tuning of the consortium would facilitate the optimization of specific objectives. Next, we engineered a consortium of E. coli specialist strains fermenting either hexose or pentose sugars into isobutanol, and demonstrated that this co-culture exhibits improved isobutanol production over a diauxic monoculture under several growth conditions. Notably, the co-culture outperformed the monoculture on an enzymatically-hydrolyzed lignocellulosic biomass, producing up to almost 3 g/L isobutanol without detoxification or supplementation. Lastly, we demonstrated the utility of a microbial consortium for detecting highly-secreting L-valine (and subsequently isobutanol) production strains. We designed a secretor/sensor pair that can be used to detect increased L-valine secretion by the “secretor” via the changes in growth of the “sensor”, a fluorescently-tagged L-valine auxotroph. This will be part of a larger effort to develop a new method for high-throughout screening of microbial over-production strains. This dissertation presents the design, construction, and/or application of three synthetic microbial consortia. Through tool development and biofuel applications, our work demonstrates the potential, utility, and benefits of microbial consortia in synthetic biology.