Genome-Scale Characterization of Metabolic Interactions in a Clostridial Co-culture for Consolidated Bioprocessing.

摘要

The co-culture of Clostridium acetobutylicum and Clostridium cellulolyticum can offer a potential CBP approach for producing commodity chemicals from cellulosic biomass. This thesis examines the nature of interactions between these two species in this co-culture. An expanded genome-scale metabolic model of C. acetobutylicum, which incorporates thermodynamic and metabolomic data, was presented. Flux variability analysis showed the presence of alternate carbon and electron sinks that allows for different carbon assimilation patterns and ranges of product formation rates. The genome-scale metabolic model of C. cellulolyticum was presented and validated against experimental data, and was able to predict the metabolism of C. cellulolyticum on cellulose and cellobiose. A genome-scale model of the clostridial co-culture metabolism was developed by integrating C. cellulolyticum and C. acetobutylicum metabolic models, and was used to analyze the integrated physiology of this co-culture; the simulation results showed that at high cellulose concentrations, the model is not able to capture the C. cellulolyticum growth arrest, suggesting that the removal of cellobiose inhibition by C. acetobutylicum is not the main factor that improves cellulose degradation in the co-culture. Experimental methods were developed to characterize the cellulolytic activity, the co-culture metabolism and the metabolic interactions in this co-culture. Comparative qPCR analyses and characterization of the metabolism in this clostridial co-culture along with the mono-cultures revealed that significant increase in the rate of cellulose hydrolysis can be achieved using the co-culture due to the synergy between the two clostridial species. It is likely that C. acetobutylicum improves the cellulolytic activity of C. cellulolyticum in the co-culture through exchange of metabolites, such as pyruvate, enabling it to grow and metabolize cellulose under suboptimal co-culture conditions. An in vivo metabolite analysis of C. cellulolyticum suggested a limitation on the lactate transportation pathway that leads to intracellular lactate accumulation and the growth arrest.