Conversion of phosphoenolpyruvate to pyruvate in Thermoanaerobacterium saccharolyticum

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.