Genetic investigation of hydrogenases in Thermoanaerobacterium thermosaccharolyticum suggests that redox balance via hydrogen cycling enables high ethanol yield

Article

Author: Lynd, Lee R. ; Herring, Christopher D. ; de Souza, Layse C.

ABSTRACTThermoanaerobacterium thermosaccharolyticum is an anaerobic and thermophilic bacterium that has been genetically engineered for ethanol production at very high yields. However, the underlying reactions responsible for electron flow, redox equilibrium, and how they relate to ethanol production in this microbe are not fully elucidated. Therefore, we performed a series of genetic manipulations to investigate the contribution of hydrogenase genes to high ethanol yield, generating evidence for the importance of hydrogen-reacting enzymes in ethanol production. Our results indicate that a high ethanol yield, >85% of the theoretical maximum, only occurs when the hfsD, hydAB , and nfnAB genes are all present together, while the hfsB gene is absent. We propose that the products of these three gene clusters facilitate an NADPH-generating reaction via hydrogen cycling, with a stoichiometry comparable with a canonical ferredoxin:NADP + oxidoreductase (FNOR; EC 1.18.1.2) reaction. The hypothesized mechanism provides a balance of nicotinamide cofactors and facilitates ferredoxin recycling, leading to progress in optimizing the energy conversion of biomass-derived sugars to ethanol. IMPORTANCEOur study elucidates the crucial role of electron flow and redox balancing mechanisms in improving ethanol yields from renewable biomass. We delve into the mechanism of electron transfer, highlighting the potential of key genes to be leveraged for enhanced ethanol production in anaerobic microbial species. We suggest by genetic investigation the existence of a novel Ferredoxin:NADP+ Oxidoreductase (FNOR) reaction, comprising HfsD, HydAB, and NfnAB enzymes, as a promising avenue for achieving balanced stoichiometry and efficient ethanol synthesis. Our findings not only advance the understanding of microbial metabolism but also offer practical insights for developing strategies to improve bioenergy production and sustainability.

Biotechnology for Biofuels and Bioproducts

Pyrophosphate-free glycolysis in Clostridium thermocellum increases both thermodynamic driving force and ethanol titers

Article

Author: Olson, Daniel G ; Lynd, Lee R ; Sharma, Bishal Dev ; Stevenson, David M ; Amador-Noguez, Daniel ; Hon, Shuen ; Guss, Adam M ; Thusoo, Eashant

BACKGROUNDClostridium thermocellum is a promising candidate for production of cellulosic biofuels, however, its final product titer is too low for commercial application, and this may be due to thermodynamic limitations in glycolysis. Previous studies in this organism have revealed a metabolic bottleneck at the phosphofructokinase (PFK) reaction in glycolysis. In the wild-type organism, this reaction uses pyrophosphate (PP_i) as an energy cofactor, which is thermodynamically less favorable compared to reactions that use ATP as a cofactor. Previously we showed that replacing the PP_i-linked PFK reaction with an ATP-linked reaction increased the thermodynamic driving force of glycolysis, but only had a local effect on intracellular metabolite concentrations, and did not affect final ethanol titer.RESULTSIn this study, we substituted PP_i-pfk with ATP-pfk, deleted the other PP_i-requiring glycolytic gene pyruvate:phosphate dikinase (ppdk), and expressed a soluble pyrophosphatase (PPase) and pyruvate kinase (pyk) genes to engineer PP_i-free glycolysis in C. thermocellum. We demonstrated a decrease in the reversibility of the PFK reaction, higher levels of lower glycolysis metabolites, and an increase in ethanol titer by an average of 38% (from 15.1 to 21.0 g/L) by using PP_i-free glycolysis.CONCLUSIONSBy engineering PP_i-free glycolysis in C. thermocellum, we achieved an increase in ethanol production. These results demonstrate that optimizing the thermodynamic landscape through metabolic engineering can enhance product titers. While further increases in ethanol titers are necessary for commercial application, this work represents a significant step toward engineering glycolysis in C. thermocellum to increase ethanol titers.

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