Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

Abstract

Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a k cat of 45.8 s ؊1 or 14.9 s ؊1 , respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a k cat of 520.8 s ؊1 . The malic enzyme used NADP ؉ as a cofactor along with NH 4 ؉ and Mn 2؉ as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a K i of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH 4 ؉ based on the characterization of the malate dehydrogenase and malic enzyme.C lostridium thermocellum is a Gram-positive, anaerobic thermophile capable of reaching one of the highest growth rates on crystalline cellulose (1, 2). Furthermore, given its native ability to produce ethanol and H 2 , C. thermocellum is seen as an attractive microorganism for the production of biofuels via consolidated bioprocessing of lignocellulosic biomass. Unfortunately, current yields and production rates of ethanol and/or H 2 are low due to branched product pathways (3-5) which redirect carbon and electron flux away from a desired biofuel. These unwanted products include lactate, formate, and/or acetate (6-8), as well as secreted amino acids (9, 10). Thus, redirecting carbon and electron flux away from these secreted products toward either ethanol or H 2 may improve the economic viability of biofuels production using C. thermocellum.A key pathway node involved in the interconversion of phosphoenolpyruvate (PEP) and pyruvate, which may catalyze either substrate level phosphorylation or transhydrogenation reactions between NADH to NADP ϩ , has been revisited with C. thermocellum (10, 11). The interconversion of PEP and pyruvate may be catalyzed using a number of different putative enzymes. In contrast to most clostridia and other fermentative ethanol and/or H 2 -producing organisms (7), C. thermocellum ATCC 27405 (GenBank accession number NC_009012.1) does not encode a pyruvate kinase, which catalyzes PE...

Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism

“…Electrons can be directly transferred from ferredoxin to generate NADH using Rnf [27], while electrons from ferredoxin and NADH together can be used to reduce two NADP + to NADPH [28, 29]. Furthermore, a carbon pathway, the malate shunt [30, 31], can functionally result in transhydrogenation during the conversion of phosphoenolpyruvate to pyruvate by oxidation of NADH by malate dehydrogenase and reduction of NADP + by malic enzyme. The ability to utilize NADPH as a reductant for ethanol production would presumably provide additional flexibility in redox balancing, but the fact that sulfite reduction to sulfide is predicted to be ferredoxin-dependent makes the relevance of the mutant AdhE for sulfur metabolism tenuous.…”

Glycolysis without pyruvate kinase in Clostridium thermocellum

“…shown to be reversible in several organisms (Varela-Gomez et al, 2004) (South and Reeves, 1975) and is 50 expressed at high levels in C. thermocellum (Taillefer et al, 2015). Since there are no known growth 51 conditions in C .thermocellum under which one might expect gluconeogenesis to occur,it could 52 participate in glycolysis.…”

CO 2 -fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

“…There is limited information on pathways relating to CO 2 uptake thus far. One proposed pathway involving CO 2 utilization in C. thermocellum is the malate shunt and its variant pathway (7,14), in which CO 2 is first incorporated by phosphoenolpyruvate carboxykinase (PEPCK) to form oxaloacetate and then is released either by malic enzyme or via oxaloacetate decarboxylase complex, yielding pyruvate. However, other pathways capable of carbon fixation may also exist but remain uncharacterized.…”

“…To determine which pathway(s) may also lead to 13 C-labeled pyruvate at the carboxylic carbon, we examined the fate of carbon atoms among all possible candidate pathways, including the malate shunt (7,14) which is known to assimilate and release CO 2 in C. thermocellum. As shown in Fig.…”