Genome-scale metabolic analysis of Clostridium thermocellum for bioethanol production

Abstract

Background: Microorganisms possess diverse metabolic capabilities that can potentially be leveraged for efficient production of biofuels. Clostridium thermocellum (ATCC 27405) is a thermophilic anaerobe that is both cellulolytic and ethanologenic, meaning that it can directly use the plant sugar, cellulose, and biochemically convert it to ethanol. A major challenge in using microorganisms for chemical production is the need to modify the organism to increase production efficiency. The process of properly engineering an organism is typically arduous. Results: Here we present a genome-scale model of C. thermocellum metabolism, iSR432, for the purpose of establishing a computational tool to study the metabolic network of C. thermocellum and facilitate efforts to engineer C. thermocellum for biofuel production. The model consists of 577 reactions involving 525 intracellular metabolites, 432 genes, and a proteomic-based representation of a cellulosome. The process of constructing this metabolic model led to suggested annotation refinements for 27 genes and identification of areas of metabolism requiring further study. The accuracy of the iSR432 model was tested using experimental growth and by-product secretion data for growth on cellobiose and fructose. Analysis using this model captures the relationship between the reduction-oxidation state of the cell and ethanol secretion and allowed for prediction of gene deletions and environmental conditions that would increase ethanol production.

Atypical Glycolysis in Clostridium thermocellum

“…Of these three, the evidence for pyruvate kinase is the weakest. Although no standard pyruvate kinase has been found (15,20), in one study, where the presence of a pyruvate kinase gene was presumed, a nonstandard pyruvate kinase was identified (13). PPDK has been shown to allow PEP-to-pyruvate conversion in Thermoproteus tenax (21) and E. histolytica (22).…”

“…There is a broad consensus that glycolysis is the predominant pathway for conversion of intracellular glucose and glucose-1-phosphate to pyruvate. Based on analysis of the genome, all of the genes in the glycolysis pathway except pyruvate kinase have been reported to exist in C. thermocellum (12,13). A variety of key glycolytic enzymes have been detected in cell extracts (8,11).…”

Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose

“…While these microorganisms are capable of efficiently hydrolyzing cellulose, their biofuel productivities are significantly lower than those of existing industrial strains. In addition to improving biofuel productivity (22), research efforts are also focused on increasing ethanol yields (31), eliminating competing pathways (26), and improving ethanol tolerance (30).…”

Innovative Biological Solutions to Challenges in Sustainable Biofuels Production

“…The desirable traits of microorganisms for biofuel production include high substrate utilization and processing capacities, fast and deregulated pathways for sugar transport, good tolerance to inhibitors and product, and high metabolic fluxes (Alper & Stephanopoulos, 2009). With beneficial traits for biofuel-related applications, some native microorganisms, such as Clostridium acetobutylicum for the ABE process, have become the unambiguous organisms of choice for biofuel production in industry (Inui et al, 2008;Alper & Stephanopoulos, 2009;Roberts et al, 2010). However, since the properties required for industrial processing are very different from the features evolved in the native biomes, the transformation from an innate capacity of environmental isolates into an industrially relevant performance can sometimes be strenuous (Alper & Stephanopoulos, 2009).…”