Insights into the metabolism of Clostridium thermocellum for cellulosic ethanol production
Sammanfattning: The societal goal of reaching net-zero CO2 emissions requires development of integrated biorefineries to produce biomass-derived fuels and chemicals. For sustainable second-generation bioethanol production, consolidated bioprocessing with the thermophile Clostridium thermocellum is regarded as a promising concept in view of the microorganism’s native ability to efficiently degrade plant cell wall material. However, for industrial implementation, improvements in ethanol titer and yield are needed. The aim of this thesis was to increase knowledge on the metabolism of C. thermocellum and thereby guide future metabolic engineering strategies to maximize the ethanol yield and titer.Yield improvements and fundamental studies into the metabolism of C. thermocellum would benefit from higher utilization of hexose monomers as well as minimized byproduct formation. To investigate underlying mechanisms for growth on glucose and fructose, laboratory evolution in chemostats together with genome sequence-based reverse engineering was applied. This successfully revealed two underlying mutations with (regulatory) roles in metabolism or transport of the monosaccharides. Together, these mutations enable reproducible and constitutive growth and are relevant for follow-up studies into transport and upper glycolysis. Separately, the mechanism behind the surprising byproduct formation of secreted amino acids was investigated by knock-out studies in NADPH-supplying and -consuming pathways. Physiological characterization in cellobiose- or ammonium-limited chemostats of mutant strains, with deletions in the NADPH-forming malate shunt or in the putatively ferredoxin-dependent ammonium assimilation, demonstrated a central role of NADPH in driving amino acid secretion. The findings indicated that electron availability will be crucial for further yield improvements in the NADH-dependent ethanol pathway.Fundamental mechanisms that might contribute to improved ethanol titer were addressed by studying thermodynamic and biophysical limitations. The pyrophosphate (PPi)-dependent glycolysis of C. thermocellum has been hypothesized to increase the overall ATP yield at the expense of the overall driving force. Knock-out studies combined with functional annotation of potential PPi-sources questioned this trade-off and increased knowledge of the PPi metabolism. The chaotropic effect (biophysical toxicity) of ethanol is commonly counteracted by lowering the cultivation temperature. Here, physiological characterization at varying ethanol titers demonstrated improved growth and fermentation at lower temperature. Comparisons to a non-ethanol producing mutant indicated both thermodynamic and biophysical limitations specifically in the ethanol pathway.Overall, these findings suggest that improvements in ethanol yield and titer would benefit from a simplified glycolysis that is engineered for a high driving force. While this work is beneficial for second-generation ethanol production, these findings can also be broadly applicable in the research and development of C. thermocellum as a cell factory for sustainable production of other fuels and chemicals.
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