Abstract
Thermoanaerobacterium saccharolyticum is an anaerobic thermophile that can ferment hemicellulose to produce biofuels, such as ethanol. It has been engineered to produce ethanol at high yield and titer. T. saccharolyticum uses the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis. However, the genes and enzymes used in each step of the EMP pathway in T. saccharolyticum are not completely known. In T. saccharolyticum, both pyruvate kinase (PYK) and pyruvate phosphate dikinase (PPDK) are highly expressed based on transcriptomic and proteomic data. Both enzymes catalyze the formation of pyruvate from phosphoenolpyruvate (PEP). PYK is typically the last step of EMP glycolysis pathway while PPDK is reversible and is found mostly in C4 plants and some microorganisms. It is not clear what role PYK and PPDK play in T. saccharolyticum metabolism and fermentation pathways and whether both are necessary. In this study we deleted the ppdk gene in wild type and homoethanologen strains of T. saccharolyticum and showed that it is not essential for growth or ethanol production.
Highlights
Lignocellulosic biomass is a sustainable feedstock for biofuels, it is not currently commercially viable due to its recalcitrance (Wyman, 2007)
Since one PEP is converted to pyruvate per glucose transport event, but glucose is converted to two PEP molecules by glycolysis, the phosphotransferase system (PTS) system can account for at most half of the PEP → pyruvate flux
Both pyruvate kinase (PYK) and phosphate dikinase (PPDK) are expressed at high levels in T. saccharolyticum, independent of substrate, based on transcriptomic and proteomic analysis (Currie et al, 2014), suggesting the possibility that one or both enzymes play a significant role in T. saccharolyticum metabolism
Summary
Lignocellulosic biomass is a sustainable feedstock for biofuels, it is not currently commercially viable due to its recalcitrance (Wyman, 2007). Since one PEP is converted to pyruvate per glucose transport event, but glucose is converted to two PEP molecules by glycolysis, the PTS system can account for at most half of the PEP → pyruvate flux (assuming negligible carbon leaves at intermediate stages of glycolysis) Both PYK and PPDK are expressed at high levels in T. saccharolyticum, independent of substrate, based on transcriptomic and proteomic analysis (Currie et al, 2014), suggesting the possibility that one or both enzymes play a significant role in T. saccharolyticum metabolism. Genes encoding typical gluconeogenic enzymes such as PEP synthase and fructose 1,6-biosphosphatase are absent in T. saccharolyticum genome, and there are no known conditions under which gluconeogenesis occurs, PPDK likely participate in catabolism (glycolysis) instead of gluconeogenesis Our aim for this project is to answer the question of whether PYK and PPDK are both required in the glycolysis of T. saccharolyticum and their role in strains engineered for high-titer ethanol production. We attempted to delete the genes encoding PYK and PPDK in both wild type and homoethanologen (Herring et al, 2016) (i.e. engineered to produce only ethanol as a fermentation product) strains of T. saccharolyticum
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