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Cited by 172Microbial Metabolic Engineering and BioproductionBiofuel production and bioconversionGene Regulatory Network Analysis

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Characterizing acetogenic metabolism using a genome-scale metabolic reconstruction of Clostridium ljungdahlii

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Nagarajan, Harish, Sahin, Merve, Nogales, Juan, Latif, Haythem, Lovley, Derek R, Ebrahim, Ali, Zengler, Karsten·2013·Microbial Cell Factories
DOI 10.1186/1475-2859-12-118PMC PMC4222884
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Abstract

BACKGROUND: The metabolic capabilities of acetogens to ferment a wide range of sugars, to grow autotrophically on H2/CO2, and more importantly on synthesis gas (H2/CO/CO2) make them very attractive candidates as production hosts for biofuels and biocommodities. Acetogenic metabolism is considered one of the earliest modes of bacterial metabolism. A thorough understanding of various factors governing the metabolism, in particular energy conservation mechanisms, is critical for metabolic engineering of acetogens for targeted production of desired chemicals. RESULTS: Here, we present the genome-scale metabolic network of Clostridium ljungdahlii, the first such model for an acetogen. This genome-scale model (iHN637) consisting of 637 genes, 785 reactions, and 698 metabolites captures all the major central metabolic and biosynthetic pathways, in particular pathways involved in carbon fixation and energy conservation. A combination of metabolic modeling, with physiological and transcriptomic data provided insights into autotrophic metabolism as well as aided the characterization of a nitrate reduction pathway in C. ljungdahlii. Analysis of the iHN637 metabolic model revealed that flavin based electron bifurcation played a key role in energy conservation during autotrophic growth and helped identify genes for some of the critical steps in this mechanism. CONCLUSIONS: iHN637 represents a predictive model that recapitulates experimental data, and provides valuable insights into the metabolic response of C. ljungdahlii to genetic perturbations under various growth conditions. Thus, the model will be instrumental in guiding metabolic engineering of C. ljungdahlii for the industrial production of biocommodities and biofuels.

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Figure 1 Reconstruction workflow. Iterative reconciliatory reconstruction workflow adopted for generating the metabolic model for C. ljungdahlii.
Figure 1 Reconstruction workflow. Iterative reconciliatory reconstruction workflow adopted for generating the metabolic model for C. ljungdahlii.CC BY · PubMed Central
Figure 2 Characterizing the nitrate reduction pathway in C. ljungdahlii. (A) Pathway map of the reconstructed nitrate reduction pathway with the respective genes associated to the reactions. Inset shows the differential expression of the genes in the nitrate reduction pathway under ammonium-free medium with nitrate compared to nitrate-free medium with ammonium as the nitrogen source. Reaction abbreviations: NO3t2 (nitrate transporter), NO2t2r (nitrite transporter), NTRARf (nitrate reductase), NTRIR5 (nitrite reductase), HAMR (hydroxylamine reductase), NH4t (ammonium transporter). (B) Physiological data when C. ljungdahlii is grown on fructose in ammonium-free media with nitrate as the nitrogen source. Purple line represents fructose, green line represents OD600, brown line represents acetate, red line represents nitrate and orange line represents ammonia measurements respectively.
Figure 2 Characterizing the nitrate reduction pathway in C. ljungdahlii. (A) Pathway map of the reconstructed nitrate reduction pathway with the respective genes associated to the reactions. Inset shows the differential expression of the genes in the nitrate reduction pathway under ammonium-free medium with nitrate compared to nitrate-free medium with ammonium as the nitrogen source. Reaction abbreviations: NO3t2 (nitrate transporter), NO2t2r (nitrite transporter), NTRARf (nitrate reductase), NTRIR5 (nitrite reductase), HAMR (hydroxylamine reductase), NH4t (ammonium transporter). (B) Physiological data when C. ljungdahlii is grown on fructose in ammonium-free media with nitrate as the nitrogen source. Purple line represents fructose, green line represents OD600, brown line represents acetate, red line represents nitrate and orange line represents ammonia measurements respectively.CC BY · PubMed Central
Figure 3 Energy conservation mechanisms in C. ljungdahlii. (A) Shown is the Wood-Ljungdahl pathway with an emphasis on the electron bifurcation in the Wood-Ljungdahl pathway at the methylene-tetrahydrofolate deydrogenase small subunit to generate reduced ferredoxin. The detailed Wood-Ljungdahl pathway is shown in Figure 4. Reaction abbreviations are the same as those in Figure 4. (B) Energy conservation through proton gradient established at the Rnf complex. (C) Identification of a gene encoding the electron bifurcating transhydrogenase for the generation of NADPH during autotrophic growth (Nfn complex). (D) Electron bifurcating hydrogenase for the reduction of ferredoxin from hydrogen during autotrophic growth. Yellow nodes indicate the oxidized form of reducing equivalents and blue nodes indicate the reduced form.
Figure 3 Energy conservation mechanisms in C. ljungdahlii. (A) Shown is the Wood-Ljungdahl pathway with an emphasis on the electron bifurcation in the Wood-Ljungdahl pathway at the methylene-tetrahydrofolate deydrogenase small subunit to generate reduced ferredoxin. The detailed Wood-Ljungdahl pathway is shown in Figure 4. Reaction abbreviations are the same as those in Figure 4. (B) Energy conservation through proton gradient established at the Rnf complex. (C) Identification of a gene encoding the electron bifurcating transhydrogenase for the generation of NADPH during autotrophic growth (Nfn complex). (D) Electron bifurcating hydrogenase for the reduction of ferredoxin from hydrogen during autotrophic growth. Yellow nodes indicate the oxidized form of reducing equivalents and blue nodes indicate the reduced form.CC BY · PubMed Central
Figure 4 Wood-Ljungdahl pathway and pathways for acetate and ethanol formation. (A) Shown is the detailed representation of the Wood-Ljungdahl pathway explicitly summarizing the reducing equivalents required for carbon fixation. (B) Pathway showing acetate formation and ethanol formation from acetyl-coA. Reaction abbreviations: FDH7 (formate dehydrogenase), FTHFLi (formate-tetrahydrofolate ligase), MTHFC (methenyltetrahydrofolate cyclohydrolase), MTHFD (methylenetetrahydrofolate dehydrogenase (NADP)), MTHFR5 (5,10-methylenetetrahydrofolate reductase (Ferredoxin) Electron bifurcating), METR (Methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase), CODH_ACS (CODH/Acetyl-CoA synthase), PTAr (phospotransacetylase), ACKr (acetate kinase), ACALD (acetaldehyde dehydrogenase), ALCD2x (ethanol dehydrogenase). Yellow nodes indicate the oxidized form of reducing equivalents and blue nodes indicate the reduced form.
Figure 4 Wood-Ljungdahl pathway and pathways for acetate and ethanol formation. (A) Shown is the detailed representation of the Wood-Ljungdahl pathway explicitly summarizing the reducing equivalents required for carbon fixation. (B) Pathway showing acetate formation and ethanol formation from acetyl-coA. Reaction abbreviations: FDH7 (formate dehydrogenase), FTHFLi (formate-tetrahydrofolate ligase), MTHFC (methenyltetrahydrofolate cyclohydrolase), MTHFD (methylenetetrahydrofolate dehydrogenase (NADP)), MTHFR5 (5,10-methylenetetrahydrofolate reductase (Ferredoxin) Electron bifurcating), METR (Methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase), CODH_ACS (CODH/Acetyl-CoA synthase), PTAr (phospotransacetylase), ACKr (acetate kinase), ACALD (acetaldehyde dehydrogenase), ALCD2x (ethanol dehydrogenase). Yellow nodes indicate the oxidized form of reducing equivalents and blue nodes indicate the reduced form.CC BY · PubMed Central
Figure 5 Insights from modeling genetic perturbation into aspects of energy conservation in C. ljungdahlii. (A) Deletion of acetate kinase during growth on fructose shifts the flux from acetate towards ethanol. (B) Growth is feasible when CO is the electron donor despite the deletion of acetate kinase. (C) Growth is feasible when H2 is the electron donor despite the deletion of acetate kinase if the bifurcating hydrogenase is NADPH dependent. (D) Acetate kinase is essential for autotrophic growth with H2 as the electron donor if the bifurcating hydrogenase is NADH dependent. Modeling analysis reveals an insufficient ATP in the absence of ackA if the hydrogenase is NADH dependent (0.75 ATP) . However, if the bifurcating hydrogenase is NADP dependent, sufficient ATP (1.5 ATP) is generated to enable ethanol production in the absence of ackA.
Figure 5 Insights from modeling genetic perturbation into aspects of energy conservation in C. ljungdahlii. (A) Deletion of acetate kinase during growth on fructose shifts the flux from acetate towards ethanol. (B) Growth is feasible when CO is the electron donor despite the deletion of acetate kinase. (C) Growth is feasible when H2 is the electron donor despite the deletion of acetate kinase if the bifurcating hydrogenase is NADPH dependent. (D) Acetate kinase is essential for autotrophic growth with H2 as the electron donor if the bifurcating hydrogenase is NADH dependent. Modeling analysis reveals an insufficient ATP in the absence of ackA if the hydrogenase is NADH dependent (0.75 ATP) . However, if the bifurcating hydrogenase is NADP dependent, sufficient ATP (1.5 ATP) is generated to enable ethanol production in the absence of ackA.CC BY · PubMed Central

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Authors
Nagarajan, H., Sahin, M., Nogales, J., Latif, H., Lovley, D. R., Ebrahim, A., & Zengler, K.
Affiliations
  • University of California San Diego (US)
  • University of Massachusetts Amherst (US)
Corresponding author
Zengler, Karsten
ORCID
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2013-11-25
Volume
12
Issue
1
Pages
118-118
ISSN
1475-2859 (ISSN-L)
DOI
10.1186/1475-2859-12-118
PMC
PMC4222884
OpenAlex
W2038304602
Cited by
170
References
52
Citations by year
2026: 8 · 2025: 8 · 2024: 10 · 2023: 9 · 2022: 23 · 2021: 11 · 2020: 13 · 2019: 13 · 2018: 19 · 2017: 19 · 2016: 11 · 2015: 13 · 2014: 13
FWCI
5.52
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Microbial Cell Factories (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • National Science Foundation (1144086)
  • U.S. Department of Energy (DEAR0000159, DEAR0000087, DESC0004917)
  • Advanced Research Projects Agency - Energy (DE-AR0000159)
  • Office of Science
  • University of California, San Diego
  • Advanced Research Projects Agency
  • National Institute of General Medical Sciences (U01GM102098)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Physical Sciences › Engineering › Biomedical Engineering › Biofuel production and bioconversion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
Keywords
Clostridium ljungdahlii, genome-scale metabolic models, autotrophic growth, energy conservation, CO2 fixation, metabolic engineering, biofuels
MeSH terms
Acetates, Clostridium, Genome, Metabolic Networks and Pathways, Biofuels, Metabolic Engineering

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