(633e) Tracking Pyrophosphate Metabolism and Evaluating Its Significance in the Bioprocessing of Lignocellulosic Biomass By Clostridium Thermocellum

Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is difficult (and cost prohibitive) to use in conventional processes. A promising candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its natural competence to produce ethanol, amino acids, and hydrogen gas as well as its ability to grow at high substrate loading. This organism has an atypical glycolytic pathway in that pyrophosphate drives it, being used as an energy source in phosphofructokinase (pfk) and pyruvate phosphate dikinase (ppdk) reactions. The identification of pyrophosphatase sources is key to the engineering of C. thermocellum. Multiple in vivo efforts to identity the primary pyrophosphate source have resulted only in the exclusion of various processes from being the primary pyrophosphate source. To better understand these experiments and closely track pyrophosphate metabolism, an existing Genome-Scale Model (GSM) of metabolism was carefully curated for pyrophosphate metabolism into a new model, iCTH659. Particularly focuses included transport, nucleotide, energy, vitamin, and amino acid metabolism in addition to addressing reaction imbalances, infeasible cycling, and removing the soluble pyrophosphatase assumption from biomass synthesis. The iCTH659 model agrees with fermentation and biomass yield metrics from the most recent investigations of C. thermocellum metabolism. From this model, stoichiometrically feasible pyrophosphate sources are explored. In addition, alternatives to pyrophosphate-utilizing reactions are proposed through the model curation process and are evaluated for feasibility. This shows stoichiometrically viable candidates as yet uninvestigated for providing the driving C. thermocellum glycolysis, including alternative pfk phosphate donors.