Abstract
Clostridium thermocellum is a cellulolytic thermophile that is considered for the consolidated bioprocessing of lignocellulose to ethanol. Improvements in ethanol yield are required for industrial implementation, but the incompletely understood causes of amino acid secretion impede progress. In this study, amino acid secretion was investigated via gene deletions in ammonium-regulated, nicotinamide adenine dinucleotide phosphate (NADPH)-supplying and NADPH-consuming pathways as well as via physiological characterization in cellobiose-limited or ammonium-limited chemostats. First, the contribution of the NADPH-supplying malate shunt was studied with strains using either the NADPH-yielding malate shunt (Dppdk) or a redox-independent conversion of PEP to pyruvate (Dppdk DmalE::Peno-pyk). In the latter, branched-chain amino acids, especially valine, were significantly reduced, whereas the ethanol yield increased from 46 to 60%, suggesting that the secretion of these amino acids balances the NADPH surplus from the malate shunt. The unchanged amino acid secretion in Dppdk falsified a previous hypothesis on an ammonium-regulated PEP-to-pyruvate flux redistribution. The possible involvement of another NADPH-supplier, namely, NADH-dependent reduced ferredoxin:NADP1 oxidoreductase (nfnAB), was also excluded. Finally, the deletion of glutamate synthase (gogat) in ammonium assimilation resulted in the upregulation of NADPH-linked glutamate dehydrogenase activity and decreased amino acid yields. Since gogat in C. thermocellum is putatively annotated as ferredoxin-linked, a claim which is supported by the product redistribution observed in this study, this deletion likely replaced ferredoxin with NADPH in ammonium assimilation. Overall, these findings indicate that a need to reoxidize NADPH is driving the observed amino acid secretion, likely at the expense of the NADH needed for ethanol formation. This suggests that metabolic engineering strategies that simplify the redox metabolism and ammonium assimilation can contribute to increased ethanol yields.
| Original language | English |
|---|---|
| Journal | Applied and Environmental Microbiology |
| Volume | 89 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 2023 |
Funding
Funding for J.Y., T.K., A.K., and A.J.A.M. was provided by the Formas grant 2017-00973 and by the Novo Nordisk Foundation grant NNF20OC0064164. D.J.H. was funded by the Swedish Foundation for Strategic Research (SSF) (ITM17-0236). T.R. and A.M.G. were supported by the Center for Bioenergy Innovation, U.S. Department of Energy (DOE) Bioenergy Research Center, supported by the Office of Biological and Environmental Research in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. Funding for J.Y., T.K., A.K., and A.J.A.M. was provided by the Formas grant 2017-00973 and by the Novo Nordisk Foundation grant NNF20OC0064164. D.J.H. was funded by the Swedish Foundation for Strategic Research (SSF) (ITM17-0236). T.R. and A.M.G. were supported by the Center for Bioenergy Innovation, U.S. Department of Energy (DOE) Bioenergy Research Center, supported by the Office of Biological and Environmental Research in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. We thank Gustav Sjöberg, Jeroen G. Koendjbiharie, and Chonticha Phongsawat for their insightful comments on the data analysis as well as for their help in setting up and sampling the chemostats. We are also thankful to Amparo Jiménez Quero for the experimental help with the metabolite analysis.
Keywords
- Acetivibrio thermocellus
- Clostridium thermocellum
- amino acids
- ammonium assimilation
- chemostat cultures
- glutamate synthase
- malate shunt
- redox-cofactors