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

• Published in: Extremophiles : life under extreme conditions Vol. 24; no. 4; pp. 511 - 518
• Main Authors: Straub, Christopher T., Schut, Gerritt, Otten, Jonathan K., Keller, Lisa M., Adams, Michael W. W., Kelly, Robert M.
• Format: Journal Article
• Language: English
• Published: Tokyo Springer Japan 01.07.2020; Springer Nature B.V; Springer
• Subjects: Acetates; Acetic acid; Alcohol dehydrogenase; Alcohols; Aldehydes; Archaea; ATP; Biochemistry; Biochemistry & Molecular Biology; Biofuels; Biomedical and Life Sciences; Biotechnology; Butanol; Carbohydrate metabolism; Carbohydrates; Dehydrogenase; Dehydrogenases; Ethanol; Fatty acids; Fermentation; Ferredoxin; Glyceraldehyde-3-phosphate dehydrogenase; Glycolysis; Hyperthermophilic archaea; Kinases; Kinetics; Life Sciences; Metabolic engineering; Metabolism; Microbial Ecology; Microbiology; NAD; NADP; Original Paper; Oxidoreductase; Oxidoreductases; Oxidoreductions; Peptides; Phosphates; Phosphoglycerate kinase; Profiles; Pyrococcus furiosus; Pyruvic acid; Reaction kinetics; Space life sciences; Sulfur; Sulphur; Glycolysis; Thermophile; Central metabolism; Pyrococcus
• DOI: 10.1007/s00792-020-01172-2
• ISSN: 1431-0651

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.