Thermodynamic analysis of the pathway for ethanol production from cellobiose in Clostridium thermocellum

Satyakam Dash, Daniel G. Olson, Siu Hung Joshua Chan, Daniel Amador-Noguez, L. R. Lynd, Costas D. Maranas

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Clostridium thermocellum is a candidate for consolidated bioprocessing by carrying out both cellulose solubilization and fermentation. However, despite significant efforts the maximum ethanol titer achieved to date remains below industrially required targets. Several studies have analyzed the impact of increasing ethanol concentration on C. thermocellum's membrane properties, cofactor pool ratios, and altered enzyme regulation. In this study, we explore the extent to which thermodynamic equilibrium limits maximum ethanol titer. We used the max-min driving force (MDF) algorithm (Noor et al., 2014) to identify the range of allowable metabolite concentrations that maintain a negative free energy change for all reaction steps in the pathway from cellobiose to ethanol. To this end, we used a time-series metabolite concentration dataset to flag five reactions (phosphofructokinase (PFK), fructose bisphosphate aldolase (FBA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH)) which become thermodynamic bottlenecks under high external ethanol concentrations. Thermodynamic analysis was also deployed in a prospective mode to evaluate genetic interventions which can improve pathway thermodynamics by generating minimal set of reactions or elementary flux modes (EFMs) which possess unique genetic variations while ensuring mass and redox balance with ethanol production. MDF evaluation of all generated (336) EFMs indicated that, i) pyruvate phosphate dikinase (PPDK) has a higher pathway MDF than the malate shunt alternative due to limiting CO2 concentrations under physiological conditions, and ii) NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) can alleviate thermodynamic bottlenecks at high ethanol concentrations due to cofactor modification and reduction in ATP generation. The combination of ATP linked phosphofructokinase (PFK-ATP) and NADPH linked alcohol dehydrogenase (ADH-NADPH) with NADPH linked aldehyde dehydrogenase (ALDH-NADPH) or ferredoxin: NADP ​+ ​oxidoreductase (NADPH-FNOR) emerges as the best intervention strategy for ethanol production that balances MDF improvements with ATP generation, and appears to functionally reproduce the pathway employed by the ethanologen Thermoanaerobacterium saccharolyticum. Expanding the list of measured intracellular metabolites and improving the quantification accuracy of measurements was found to improve the fidelity of pathway thermodynamics analysis in C. thermocellum. This study demonstrates even before addressing an organism's enzyme kinetics and allosteric regulations, pathway thermodynamics can flag pathway bottlenecks and identify testable strategies for enhancing pathway thermodynamic feasibility and function.

Original languageEnglish (US)
Pages (from-to)161-169
Number of pages9
JournalMetabolic engineering
Volume55
DOIs
StatePublished - Sep 1 2019

Fingerprint

Clostridium thermocellum
Cellobiose
Clostridium
Thermodynamics
Ethanol
NADP
Adenosinetriphosphate
Phosphofructokinases
Aldehyde Dehydrogenase
Alcohol Dehydrogenase
Metabolites
Adenosine Triphosphate
Aldehydes
Glyceraldehyde-3-Phosphate Dehydrogenases
Phosphates
Alcohols
Thermoanaerobacterium
Orthophosphate Dikinase Pyruvate
Ferredoxin-NADP Reductase
Allosteric Regulation

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Dash, Satyakam ; Olson, Daniel G. ; Joshua Chan, Siu Hung ; Amador-Noguez, Daniel ; Lynd, L. R. ; Maranas, Costas D. / Thermodynamic analysis of the pathway for ethanol production from cellobiose in Clostridium thermocellum. In: Metabolic engineering. 2019 ; Vol. 55. pp. 161-169.
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Thermodynamic analysis of the pathway for ethanol production from cellobiose in Clostridium thermocellum. / Dash, Satyakam; Olson, Daniel G.; Joshua Chan, Siu Hung; Amador-Noguez, Daniel; Lynd, L. R.; Maranas, Costas D.

In: Metabolic engineering, Vol. 55, 01.09.2019, p. 161-169.

Research output: Contribution to journalArticle

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AU - Lynd, L. R.

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