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Modification of the glycolytic pathway in Pyrococcus furiosus and the implications for metabolic engineering

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Abstract

The key difference in the modified Embden–Meyerhof glycolytic pathway in hyperthermophilic Archaea, such as Pyrococcus furiosus, occurs at the conversion from glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) where the typical intermediate 1,3-bisphosphoglycerate (1,3-BPG) is not present. The absence of the ATP-yielding step catalyzed by phosphoglycerate kinase (PGK) alters energy yield, redox energetics, and kinetics of carbohydrate metabolism. Either of the two enzymes, ferredoxin-dependent glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) or NADP+-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), responsible for this “bypass” reaction, could be deleted individually without impacting viability, albeit with differences in native fermentation product profiles. Furthermore, P. furiosus was viable in the gluconeogenic direction (growth on pyruvate or peptides plus elemental sulfur) in a ΔgapnΔgapor strain. Ethanol was utilized as a proxy for potential heterologous products (e.g., isopropanol, butanol, fatty acids) that require reducing equivalents (e.g., NAD(P)H, reduced ferredoxin) generated from glycolysis. Insertion of a single gene encoding the thermostable NADPH-dependent primary alcohol dehydrogenase (adhA) (Tte_0696) from Caldanaerobacter subterraneus, resulted in a strain producing ethanol via the previously established aldehyde oxidoreductase (AOR) pathway. This strain demonstrated a high ratio of ethanol over acetate (> 8:1) at 80 °C and enabled ethanol production up to 85 °C, the highest temperature for bio-ethanol production reported to date.

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Acknowledgements

CT Straub acknowledges support from a US Dept of Education GAANN Fellowship (P200A160061). This work was also supported by grants to RMK and MWA (DE-PS02-06ER64304) from the Office of Biological and Environmental Research, Office of Science, US Department of Energy, and to MWA (DE-FG05-95ER20175) from the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the Department of Energy.

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, North Carolina State University, EB-1, 911 Partners Way, Raleigh, NC, 27695-7905, USA

    Christopher T. Straub, Jonathan K. Otten, Lisa M. Keller & Robert M. Kelly

  2. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA

    Gerritt Schut & Michael W. W. Adams

Authors
  1. Christopher T. Straub
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  2. Gerritt Schut
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  3. Jonathan K. Otten
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  4. Lisa M. Keller
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  5. Michael W. W. Adams
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  6. Robert M. Kelly
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Correspondence to Robert M. Kelly.

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Communicated by H. Atomi.

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Figure S1.

During growth on pyruvate, P. furiosus catabolizes glucose via the ATP consuming phosphorylation of 3-phosphoglycerate (3-PG) followed by the NADPH-dependent reduction of 1,3-bisphosphoglycerate (1,3-BPG). The GAPN activity could potentially re-oxidize glyceraldehyde-3-phosphate (GAP) back to 3-phosphoglycerate (3-PG), creating an energy burning futile cycle (PDF 53 kb).

Figure S2.

Fermentation profile of P. furiosus strain RK304. Analysis from 50-mL defined 5 g/L cellobiose media in sealed serum bottle for 72 h (PDF 27 kb).

Figure S3.

Genetic construction of strains utilized in this study (PDF 47 kb).

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Straub, C.T., Schut, G., Otten, J.K. et al. Modification of the glycolytic pathway in Pyrococcus furiosus and the implications for metabolic engineering. Extremophiles 24, 511–518 (2020). https://doi.org/10.1007/s00792-020-01172-2

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  • DOI: https://doi.org/10.1007/s00792-020-01172-2

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