Elucidating central metabolic redox obstacles hindering ethanol production in Clostridium thermocellum

Thompson, R. Adam; Layton, Donovan S.; Guss, Adam M.; Olson, Daniel G.; Lynd, Lee R.; Trinh, Cong T.

doi:10.1016/j.ymben.2015.10.004

Elucidating central metabolic redox obstacles hindering ethanol production in Clostridium thermocellum

Journal Article · Wed Oct 21 00:00:00 EDT 2015 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2015.10.004· OSTI ID:1327638

Thompson, R. Adam ^[1]; Layton, Donovan S. ^[2]; Guss, Adam M. ^[1]; Olson, Daniel G. ^[3]; Lynd, Lee R. ^[3]; Trinh, Cong T. ^[1]

The Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); The Univ. of Tennessee, Knoxville, TN (United States)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Dartmouth College, Hanover, NH (United States)

Clostridium thermocellum is an anaerobic, Gram-positive, thermophilic bacterium that has generated great interest due to its ability to ferment lignocellulosic biomass to ethanol. However, ethanol production is low due to the complex and poorly understood branched metabolism of C. thermocellum, and in some cases overflow metabolism as well. In this work, we developed a predictive stoichiometric metabolic model for C. thermocellum which incorporates the current state of understanding, with particular attention to cofactor specificity in the atypical glycolytic enzymes and the complex energy, redox, and fermentative pathways with the goal of aiding metabolic engineering efforts. We validated the model s capability to encompass experimentally observed phenotypes for the parent strain and derived mutants designed for significant perturbation of redox and energy pathways. Metabolic flux distributions revealed significant alterations in key metabolic branch points (e.g., phosphoenol pyruvate, pyruvate, acetyl-CoA, and cofactor nodes) in engineered strains for channeling electron and carbon fluxes for enhanced ethanol synthesis, with the best performing strain doubling ethanol yield and titer compared to the parent strain. In silico predictions of a redox-imbalanced genotype incapable of growth were confirmed in vivo, and a mutant strain was used as a platform to probe redox bottlenecks in the central metabolism that hinder efficient ethanol production. The results highlight the robustness of the redox metabolism of C. thermocellum and the necessity of streamlined electron flux from reduced ferredoxin to NAD(P)H for high ethanol production. The model was further used to design a metabolic engineering strategy to phenotypically constrain C. thermocellum to achieve high ethanol yields while requiring minimal genetic manipulations. Furthermore, the model can be applied to design C. thermocellum as a platform microbe for consolidated bioprocessing to produce ethanol and other reduced metabolites.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1327638

Alternate ID(s):: OSTI ID: 1250937

Journal Information:: Metabolic Engineering, Journal Name: Metabolic Engineering Journal Issue: C Vol. 32; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables isobutyl acetate production directly from cellulose by Clostridium thermocellum at elevated temperatures Seo, Hyeongmin; Lee, Jong-Won; Garcia, Sergio Biotechnology for Biofuels, Vol. 12, Issue 1 https://doi.org/10.1186/s13068-019-1583-8	journal	October 2019
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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
Clostridium thermocellum
elementary mode analysis
energy metabolism
ethanol
minimal metabolic functionality
redox metabolism

Elucidating central metabolic redox obstacles hindering ethanol production in Clostridium thermocellum

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