Effects of heterologous expression of glycolysis enzymes on product formation in Clostridium thermocellum.

Glycolysis is central to Clostridium thermocellum metabolism; however, strains engineered for high ethanol titer exhibit a decrease in the conversion of cellobiose to ethanol (i.e., cellobiose-to-ethanol yield), suggesting the presence of glycolytic bottlenecks. We expressed heterologous triosephosphate-isomerases (tpi), fructose-1,6-bisphosphate aldolases (fba), and glyceraldehyde-3-phosphate dehydrogenase (gapDH) genes from Thermoanaerobacterium saccharolyticum and Zymomonas mobilis, along with 26 non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases (gapN) variants, to identify limiting reactions. We demonstrated functional expression and increased activity for several Fba and Tpi enzymes in the PPi-free glycolysis engineered strain of C. thermocellum (LL1711). Despite C. thermocellum's low native FBA activity compared to other industrial strains, increasing Fba or Tpi activity via heterologous expression had no significant effect on cellobiose uptake or ethanol titers in high-substrate fermentations. Furthermore, 25 of 26 tested gapN genes had toxic effects on C. thermocellum upon transformation. In conclusion, none of the tested glycolytic enzyme modifications improved product titers. These results suggest that the primary metabolic limitation is not at the FBA or TPI reactions, supporting a shift in future engineering efforts toward downstream fermentation pathways.