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Identification of metabolic constraints in clostridium phytofermentans using experimental evolution and metabolic flux analysis
Identifying microbes possessing a synergistic combination of plant biomass breakdown and biofuel production are essential to develop effective strategies for producing next-generation liquid fuels. Clostridium phytofermentans is a novel, anaerobic soil microbe that produces ethanol as a primary product of fermentation during growth on plants such as switchgrass, corn stover and Brachypodium distachyon as well as on plant components such as cellulose, cellobiose, xylan, starch and glucose. A comprehensive understanding of its nutritional diversity, carbohydrate specificity along with identification of specific bottlenecks limiting growth and ethanol production is essential to the development of C. phytofermentans as a model biofuel organism. The work in this dissertation represents the first laboratory evolution experiment with a cellulolytic microorganism. For my dissertation research, I have used adaptive evolution as a tool to generate improved populations of C. phytofermentans with faster growth rate and ethanol producing capabilities when cultured on cellulose, cellobiose and xylan, as compared to the native strain. Whole-genome resequencing of the evolved populations was effective in identifying beneficial mutations in carbohydrate modules at varying levels of frequency. Analysis of mutations and protein modeling of mutated ABC transporter complexes detected mechanisms that may help to overcome constraints on carbohydrate metabolism in C. phytofermentans. These results reveal novel strategies for evolving and engineering cellulosic microorganisms for faster growth on plant biomass substrates. Based on the genome sequence of the native strain, known physiology and available experimental data, I also reconstructed the first genome-scale metabolic model for C. phytofermentans. Constraints on substrate uptake and ATP production were applied along with extensive manual curation and comparison with published clostridial models to bring model growth predictions closer to those observed under experimental conditions. The current model provides a systems-level view of C. phytofermentans metabolism and lays the groundwork for designing strategies to engineer a novel microbe to convert a broad range of biomass substrates to targeted biofuels of commercial interest.^
Mukherjee, Supratim, "Identification of metabolic constraints in clostridium phytofermentans using experimental evolution and metabolic flux analysis" (2013). Doctoral Dissertations Available from Proquest. AAI3603128.