(257g) Redirecting Carbon Flux in Clostridium Thermocellum to Increase Ethanol Yield

In Clostridium thermocellum, a thermophilic anaerobic bacterium able to rapidly ferment cellulose to ethanol and organic acids, pyruvate kinase (EC 2.7.1.40) appears to be absent based on both the genome sequence as well as enzymatic assays. Lamed and Zeikus hypothesized that a pyridine nucleotide transhydrogenation pathway through oxaloacetate and malate. We further hypothesize that there is a conversion of phosphoenolpyruvate (PEP) to pyruvate may occur via a three step pathway involving conversion of PEP to oxaloacetate(OAA) by phosphoenolpyruvate carboxykinase, OAA to malate by malate dehydrogenase, and conversion of malate to pyruvate by malic enzyme. In this work we examined the impact of targeted modification of enzymes associated with this pathway, termed the “malate shunt”, including exogenous expression of the pyruvate kinase gene from Thermoanaerobacterium saccharolyticum, mutation of the phosphoenolpyruvate carboxykinase and deletion of malic enzyme gene. Cellobiose-grown cultures of strain YD01, in which phosphoenolpyruvate carboxykinase expression was diminished by modifying the start codon from ATG to GTG, exhibited an ethanol yield of 1.33 moles ethanol/mole-glucose equivalent, 3.25-fold higher than the wild-type strain. A second strain, YD02, in which the gene for malic enzyme and part of malate dehydrogenase were deleted, exhibited an ethanol yield of 1.28 mole-ethanol/mole-glucose equivalent, also over 3-fold higher than the wild-type strain. In addition to increased ethanol yields, other consequences of routing flux away from the malate shunt were observed including increased recovery of substrate carbon in documented fermentation products, decreased production of amino acids, and increased tolerance to added ethanol. We hypothesize that these may be consequences of relieving NADPH oversupply when flux is directed away from the malate shunt, an interpretation supported by the measured cofactor specificities of malate dehydrogenase and malic enzyme.