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Olson, Daniel G.

Publications and source records attributed to Olson, Daniel G..

4 records
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DOE OSTI2026

Effects_of_heterologous_expression_of_glycolysis_enzymes_on_product_formation_in_Clostridium_thermocellum

Glycolysis is central toClostridium thermocellummetabolism; however, engineered strains for high ethanol titer exhibit a decrease in yield, suggesting the presence of glycolytic bottlenecks. We expressed heterologoustpi,fba, andgapDHgenes fromThermoanaerobacterium saccharolyticumandZymomonas mobilis, along with 26 non-phosphorylatinggapNvariants, to identify limiting reactions. We demonstrated functional expression and increased activity for several Fba and Tpi enzymes in the engineered strain LL1711. DespiteC. thermocellum'slow 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 proved highly toxic toC. thermocellumupon transformation. In conclusion, none of the tested glycolytic enzyme modifications improved fermentation profiles. 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.

6-bisphosphate aldolase; glyceraldehyde-3-phosphat↗
DOE OSTI2026

Adaptive_Evolution_2026

Thermoanaerobacterium saccharolyticum, an anaerobic and thermophilic bacterium capable of metabolizing sugar monomers and soluble oligomers into ethanol, has been proposed for use in consolidated bioprocessing in coculture with compatible cellulolytic bacteria such asClostridium thermocellum. Although the mixed acid fermentation of both of these strains has been engineered to produce ethanol as the major fermentation product, the maximum titer produced thus far byC. thermocellum, about 3% (w/v) ethanol, is half that produced byT. saccharolyticum. There is thus motivation to understand the mechanistic basis of the robust ethanol pathway inT. saccharolyticumso that key features can be recapitulated inC. thermocellumand other organisms.Previously, we characterized theindividualrole of the main genes responsible for electron transfer in the ethanol production ofT. saccharolyticum. However, the consequences of thecombinedloss of function of all these genes have not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. In this work, we combined knockouts of ferredoxin nicotinamide oxidoreductase (fnor) genes (nfnAandnfnB) and hydrogenase genes (hydAandhfsD) and studied their effects on fermentation and grow. We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth, and most notably, we observed a single nucleotide variation in the alcohol dehydrogenaseadhAgene. Through molecular dynamics simulations and enzymatic assays, we determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings consolidate our understanding of the functioning of electron transfer pathways in this organism.

alcohol dehydrogenase↗