Thesis Committee

Lee Lynd, Ph.D. (Chair)

Margie Ackerman, Ph.D.

Karl Griswold, Ph.D.

Terry Papoutsakis, Ph.D. 

Abstract

Cellulosic ethanol provides an alternative source for transportation fuel. It has less greenhouse gas emissions compared to fuel derived from petroleum and can help us reduce the dependency on fossil fuels. The conversion of lignocellulose to ethanol is hampered by high processing costs, due to the recalcitrant nature of the feedstock. Thermophilic anaerobic bacteria potentially can overcome this recalcitrance since they produce hydrolytic enzymes. Clostridium thermocellum, Thermoanaerobacterium saccharolyticum and Thermoanaerobacter ethanolicus are thermophilic anaerobic bacteria. C. thermocellum can efficiently degrade cellulose and use the products directly for fermentation. However, wild type C. thermocellum produces ethanol at a relative low level and previous attempts to engineer it for high ethanol production have not been very successful. T. saccharolyticum and T. ethanolicus are closely related phylogenetically, can use a broad range of C5 and C6 sugars and break down hemicellulose. T. saccharolyticum has been successfully engineered for producing ethanol at high yield and titer. However, the central metabolism of these three organisms has not been well understood.

In this thesis, an atypical glycolysis was discovered in C. thermocellum and the cofactor specificities of key enzymes involved in it were investigated.  It was found that the phosphofructokinase in C. thermocellum is pyrophosphate dependent instead of ATP dependent and that C. thermocellum does not have a pyruvate kinase for conversion from phosphoenolpyruvate to pyruvate.  Based on this discovery, C. thermocellum was engineered to produce ethanol over 60% of theoretical yield with a homogeneous NADH dependent ethanol producing pathway. Meanwhile, efforts were also made towards understanding the central metabolism of T. saccharolyticum and T. ethanolicus. The conversion of pyruvate to acetyl-CoA was examined in T. saccharolyticum. It was observed that pyruvate ferredoxin oxidoreductase primarily plays the catabolic role, carrying most flux from pyruvate to acetyl-CoA, while pyruvate formate lyase was shown to be essential for biosynthesis of T. saccharolyticum. This work contributed to the identification of two different ethanol producing pathways in T. saccharolyticum. The roles of different alcohol dehydrogenases in T. ethanolicus during ethanol formation were determined as well. It indicated that even with the existence of bifunctional alcohol dehydrogenase (AdhE), other alcohol dehydrogenases (AdhA or AdhB) were important for producing ethanol at high yield. In the end, a close comparison of central metabolism between C. thermocellum and T. saccharolyticum was conducted. The result of this thesis lays a foundation for understanding the central metabolism of C. thermocellum, T. saccharolyticum and T. ethanolicus and provides insights for engineering C. thermocellum with either a homogeneous ethanol producing pathway or a transferable high ethanol producing pathway from T. saccharolyticum and T. ethanolicus.