Date of Award
Doctor of Philosophy (PhD)
Cyanobacteria have the potential to be low-cost and sustainable cell factories for bio-products; however, many challenges face cyanobacteria as biorefineries. This dissertation seeks to advance non-model photosynthetic organisms for biotechnology applications by characterizing central carbon metabolism and its regulations. Cyanobacteria phenotypes for bio-production are examined and their intracellular metabolism is quantified. Using isotopic labeling experiments, phenotypic relationships between biomass composition, central carbon fluxes, and metabolite pool sizes are investigated. Metabolic analyses of cyanobacteria led to new investigations of flux regulation mechanisms via protein spatial organizations or metabolite channeling. Metabolite channeling is further explored as a hypothesis to explain enigmatic labeling patterns and as a method to organize and regulate enzymes for robust central metabolisms. The insights reveal strategies for redirecting central metabolic fluxes for value-added chemicals as well as broad impacts for intracellular modeling approaches.
First, Synechococcus UTEX 2973 was probed with isotopic nonstationary metabolic flux analysis under changing growth conditions. Despite similar genetics to Synechococcus 7942, Synechococcus UTEX 2973’s exhibits a fast growth phenotype with greater carbon fixation driven by higher energy charges, optimal ATP/NADPH ratios, low glycogen production during exponential growth, and a central metabolism that reduces CO2 loss. Unusual labeling patterns indicated metabolite channeling as a possible flux regulation mechanism. As cyanobacteria are known to have carboxysomes, a microcompartment that concentrates CO2 for RuBisCO, it was hypothesized that carboxysome mutants may reveal channeling mechanisms. Carboxysome-free mutants (high CO2 requiring phenotypes) were found to accumulate metabolites and reach higher steady state 13C enrichment, indicating more homogenous cytoplasms. Carboxysome-free mutants may provide a method for unlocking cyanobacteria flux constraints, reducing catabolic repression, and providing a way to contain genetically modified cyanobacteria.
To ease the constraints of highly regulated and complex metabolic networks, platform or non-model strains can be used to provide a good starting point for small molecules of interest. To take advantage of cyanobacterial native sugar phosphate metabolisms, Synechococcus was engineered for the photoautotrophic production of a high-value polysaccharide, heparosan, which is an unsulfated polysaccharide important for cosmetic and pharmaceutical applications. Via overexpressing two key enzymes, the recombinant strain improves heparosan production by over 50 folds. Synechococcus was also found to naturally synthesize multiple glycosaminoglycans.
Lastly, to further explore metabolite channeling as evidenced by isotopic labeling patterns, we developed cell-free glycolysis pathways and compared their performance with in vivo glycolysis functions in E. coli and its PTS mutants. Enzyme assays, dynamic metabolite labeling and flux analysis further confirmed the hypothesized channel of EMP enzymes where the PTS may be an anchor point to initiate enzyme assemblies. In summary, the outcomes of this thesis provide new insights into non-model phototrophic microbial chassis, reveal flux control mechanisms beyond genetic or transcriptional regulations, and offer practical guidelines for sustainable bio-production via synthetic biology approaches.
Douglas Allen, Marcus Foston, Tae Seok Moon, Himadri Pakrasi,