Date of Award
Doctor of Philosophy (PhD)
In the 1970s, spikes in oil prices sparked early interests into first-generation biofuels (i.e., the production of ethanol from corn). Four decades later, biofuel manufacturing has under-performed as developed industrial strains suffer from poor performance and high costs of feedstocks. While the advancement in molecular biology has greatly benefited pathway engineering, synthetic biology hosts still cannot overcome the obstacles for profitable microbial bio-production. One approach may be to study non-model organisms and employ their metabolic features towards an improved bio-production pipeline. Here, this thesis aims to develop 13C-metabolism analysis tools to characterize novel organisms and re-program cell metabolism using novel pathways for better biosynthesis.
First, the thesis introduces fast 13C-metabolism analysis via parallel 13C-fingerprinting for pathway delineation as well as quantitative 13C-MFA using WUflux or other published software. Second, genome-to-phenome mapping was performed for an oil-rich and inhibitor-tolerant bacterium, Rhodococcus opacus PD630, to improve conversion of lignocellulose to biodiesel. One major discovery was the simultaneous use of the Entner-Doudoroff pathway (EDP) and gluconeogenesis for co-utilizing of phenolic compounds and sugar without catabolic repression.
Third, we analyzed the fastest-growing cyanobacterial strain isolated, Synechococcus elongatus UTEX 2973, through isotopically non-stationary 13C-Metabolic Flux Analysis. The flux results indicate that Synechococcus 2973 has efficient catabolic pathways with minimal carbon loss after CO2 fixation comparing to the model cyanobacterial chassis Synechocystis 6803. This inspired the engineering of Synechocystis 6803 for improved carbon fixation efficiency by overexpression of EDP and knockout of CO2 re-generating pathways.
Fourth, the thermodynamically favorable EDP pathway was also engineered in E. coli for improving carbon utilization and biomass growth. However, the EDP engineering only re-directed a small portion of its flux, but through a downregulation of Embden-Meyerhof-Parnas Pathway via pfkA knockout the flux was significantly elevated. Also, the _pfkA mutants showed co-utilization of acetate and xylose without glucose catabolic repression. Furthermore, 13C-pulse experiments suggested a possible presence of glycolysisosome, in which sugar phosphate metabolites can be passed between enzymes without mixing with the bulk phase.
Costas Maranas, Fuzhong Zhang, Tae Seok Moon, Himadri Pakrasi