Thermodynamics shape the in vivo enzyme burden of glycolytic pathways

Khana, Daven B.; Jen, Annie; Shishkova, Evgenia; Thusoo, Eashant; Williams, Jonathan; Henkel, Alex; Stevenson, David M.; Coon, Joshua J.; Amador-Noguez, Daniel

doi:10.1101/2025.01.31.635972

PreprintCited by 5Microbial Metabolic Engineering and BioproductionGene Regulatory Network Analysisthermodynamics and calorimetric analyses

Thermodynamics shape the in vivo enzyme burden of glycolytic pathways

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Khana, Daven B., Jen, Annie, Shishkova, Evgenia, Thusoo, Eashant, Williams, Jonathan, Henkel, Alex, Stevenson, David M., Coon, Joshua J. +1 more·2025·mBio

DOI 10.1101/2025.01.31.635972PMC PMC11838459

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Abstract

Thermodynamically constrained reactions and pathways are hypothesized to impose greater protein demands on cells, requiring higher enzyme amounts to sustain a given flux compared to those with stronger thermodynamics. To test this, we quantified the absolute concentrations of glycolytic enzymes in three bacterial species — Zymomonas mobilis , Escherichia coli , and Clostridium thermocellum — which employ distinct glycolytic pathways with varying thermodynamic driving forces. By integrating enzyme concentration data with corresponding in vivo metabolic fluxes and ΔG measurements, we found that the highly favorable Entner-Doudoroff (ED) pathway in Z. mobilis requires only one-fourth the amount of enzymatic protein to sustain the same flux as the thermodynamically constrained pyrophosphate-dependent glycolytic pathway in C. thermocellum , with the Embden-Meyerhof-Parnas (EMP) pathway in E. coli exhibiting intermediate thermodynamic favorability and enzyme demand. Across all three pathways, early reactions with stronger thermodynamic driving forces generally required lower enzyme investment than later, less favorable steps. Additionally, reflecting differences in glycolytic strategies, the highly reversible ethanol fermentation pathway in C. thermocellum requires 10-fold more protein to maintain the same flux as the irreversible, forward-driven ethanol fermentation pathway in Z. mobilis . Thus, thermodynamic driving forces constitute a major in vivo determinant of the enzyme burden in metabolic pathways.

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The coordinated upregulation of these already abundant enzymes represents a large increase in cellular protein investment in Z. mobilis.
“Given that these enzymes are already among the most abundant proteins in Z. mobilis (45), their coordinated upregulation represents a large increase in cellular protein investment (Fig. 10).”
From Physiological and metabolic responses of Zymomonas mobilis to lignocellulosic hydrolysate (2025) · cites this paper as [45]

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Research works printed but not linked: 1

[110]Heinonen J. K. & Drake H. L. Comparative assessment of inorganic pyrophosphate and pyrophosphatase levels of Escherichia coli, Clostridium pasteurianum, and Clostridium thermoaceDcum. FEMS Microbiology Letters 52, 205– 208 (1988).

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Physiological and metabolic responses of Zymomonas mobilis to lignocellulosic hydrolysate
Rivera Vazquez, J., Trujillo, E., Wenger, Z., Botts, M., Coon, J. J., & Amador-Noguez, D. (2025) · Microbiology Spectrum · doi:10.1128/spectrum.00610-25 · Cited by 3 · see citing works
“10 are calculated using data from (45).”
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Authors: Khana, D. B., Jen, A., Shishkova, E., Thusoo, E., Williams, J., Henkel, A., Stevenson, D. M., Coon, J. J., & Amador-Noguez, D.
Affiliations: University of Wisconsin–Madison (US)
Oak Ridge National Laboratory (US)
ORCID: Khana, Daven B.
Jen, Annie
Shishkova, Evgenia
Thusoo, Eashant
Henkel, Alex
Coon, Joshua J.
Amador-Noguez, Daniel
Venue: mBio
Publisher: openRxiv
Published: 2025-02-06
Volume: 16
Issue: 10
Pages: e0183725-e0183725
ISSN: 2150-7511 (ISSN-L)
DOI: 10.1101/2025.01.31.635972
PMC: PMC11838459
OpenAlex: W4414242478
Cited by: 5
References: 129
Citations by year: 2026: 3 · 2025: 2
FWCI: 1.50
Type: journal article
Language: en
Open access: gold
doi.org
Licence: cc-by
creativecommons.org/licenses/by-nc-nd/4.0/ (vor)
Locations: mBio (publishedVersion, cc-by)
bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
Europe PMC (PubMed Central) (publishedVersion, cc-by)
PubMed (publishedVersion)
PubMed Central (publishedVersion, cc-by)
PubMed Central (submittedVersion, cc-by-nc-nd)
DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders: U.S. Department of Energy (DESC0018998, ERKP886, DESC0018409)
National Institutes of Health (GM118110)
Office of Science (DESC0018409)
Great Lakes Bioenergy Research Center (DE-SC0018409)
National Institute of General Medical Sciences (R35GM118110, P41GM108538)
Biological and Environmental Research (DE‐SC0018409, ERKP 886, SC0018409)
Center for Bioenergy Innovation (ERKP886)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
Physical Sciences › Chemistry › Physical and Theoretical Chemistry › thermodynamics and calorimetric analyses
Keywords: glycolysis, thermodynamic driving force, Entner-Doudoroff pathway, Zymomonas mobilis, Escherichia coli, Clostridium thermocellum, alcoholic fermentation, metabolic flux, enzyme concentration, resource allocation, metabolic engineering

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Thermodynamics shape the in vivo enzyme burden of glycolytic pathways

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