The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase.

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C precursors (such as ribose) during growth on other (non-C) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PP-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PP-PFKs of and C indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of , which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PP-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.