Engineering Modular Synthetic Microbial Consortia for Sustainable Bioproduction From CO2
Carruthers, David
2020
Abstract
Engineering of synthetic microbial consortia has emerged as a new and powerful biotechnology platform with enormous potential for the production of biobased commodity chemicals. In this dissertation, I have designed, constructed, and optimized a tripartite system in which three microbes of differentiated specializations can convert sunlight, carbon dioxide, and atmospheric nitrogen into desired molecules or materials. Specifically, Synechococcus elongatus, a photosynthetic cyanobacterium that exports sucrose, and Azotobacter vinelandii, a nitrogen-fixing bacterium that secretes ammonia, form a symbiotic foundation hypothesized to support a third producer specialist. The tripartite consortia were implemented using a novel experimental set-up for continuous culture and extensive optimization was carried out with insights and guidance from computational modeling of the system dynamics. As a clear and strong proof of concept, I demonstrated various realizations of this tripartite platform, employing producer specialist strains ranging from model microorganism Escherichia coli to widely used industrial chassis such as Corynebacterium glutamicum and Bacillus subtilis. This versatile and modular technology platform offers potential for bioproduction without environmentally or monetarily expensive nutrient inputs thereby a pathway towards sustainable manufacturing of a wide range of bio-products. As an important component of the effort of engineering the tripartite system described above, I also carried out genetic modifications of E. coli K-12, the most widely used microbial chassis in synthetic biology, to enable efficient utilization of sucrose. A multigene csc operon encoding non-PTS sucrose catabolism was randomly transposed into E. coli K-12 using Tn5 transposase. Isolates from the transposon library yielded a range of growth rates on sucrose, including some that were comparable to that of E. coli K-12 on glucose. Narrowness of the growth rate distributions, improved gene expression conferring faster growth compared to that of plasmids, and enhanced growth rate upon transduction into strains that underwent adaptive laboratory evolution indicate that efficient csc expression is attainable and not limiting to cellular growth. Transduction of a csc fast-growth locus into an isobutanol production strain also yielded high titer with significant sustainability benefits. This work demonstrated that random integration is a viable and effective strategy for optimizing heterologous expression within the context of cellular metabolism for certain desirable phenotypes. In the last part of my thesis, through life cycle assessment, I investigated multi-species algal polycultures, which are different yet related CO2-fixing microbial communities. Experimental studies have previously shown that algal polycultures can be designed to enhance biomass production, stability, and nutrient recycling compared to monocultures. However, it remains unclear whether these impacts of biodiversity make polycultures more sustainable than monocultures. I have conducted a comparative life cycle assessment which showed that when algae were grown in outdoor experimental ponds, certain bicultures improved the energy return on investment and greenhouse gas emissions substantially, compared to the best monoculture. Bicultures outperformed monocultures by performing multiple functions simultaneously (e.g., improved stability, nutrient efficiency, biocrude characteristics), which outweighed the higher productivity attainable by a monoculture. These results demonstrated that algal polycultures with optimized multi-functionality lead to enhanced life cycle metrics, highlighting the significant potential of ecological engineering for enabling future environmentally sustainable algal bio-refineries. Collectively, this dissertation demonstrates how CO2-fixing microbial communities may be engineered to enhance sustainability metrics compared to monocultures. By successfully engineering more sustainable bioproduction platforms, we move closer to a society with lower dependence on petrochemicals.Subjects
microbial consortia synthetic biology bioproduction sustainability
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