Engineering the hyperthermophilic archaeon Pyrococcus furiosus for 1-propanol production

O'Quinn, Hailey C.; Vailionis, Jason L.; Tanwee, Tania N. N.; Holandez-Lopez, Katherine S.; Bing, Ryan G.; Poole, Farris L.; Zhang, Ying; Kelly, Robert M.; Adams, Michael W. W.; Atomi, ed., Haruyuki

doi:10.1128/aem.00471-25

Engineering the hyperthermophilic archaeon Pyrococcus furiosus for 1-propanol production

Journal Article · Wed May 21 00:00:00 EDT 2025 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/aem.00471-25· OSTI ID:2549462

; ; Tanwee, Tania N. N.; Holandez-Lopez, Katherine S.; Bing, Ryan G.; Poole, Farris L.; ; ; ; Atomi, ed., Haruyuki

ABSTRACT Society relies heavily on chemicals traditionally produced through the refinement of fossil fuels. The conversion of renewable biomass to value-added chemicals by microbes, particularly hyperthermophiles (T opt ≥80°C), offers a renewable alternative to this traditional approach. Herein, we describe the engineering of the hyperthermophilic archaeon <italic toggle='yes'>Pyrococcus furiosus</italic> , which grows optimally (T opt ) at 100°C, for the conversion of sugar to 1-propanol. This was accomplished by constructing a hybrid metabolic pathway consisting of two native and seven heterologously produced enzymes to convert acetyl-CoA from carbohydrate metabolism to 1-propanol. A total of eleven foreign genes from two other organisms were utilized, one from the thermophilic bacterium <italic toggle='yes'>Thermoanaerobacter</italic> sp. strain X514 and 10 from the thermoacidophilic archaeon <italic toggle='yes'>Metallosphaera sedula,</italic> both of which grow optimally near 70°C. The recombinant <italic toggle='yes'>P. furiosus</italic> strain produced 1-propanol at similar concentrations (up to ~1 mM) when incubated at 75°C to activate the gene products of <italic toggle='yes'>Thermoanaerobacter</italic> sp. strain X514 and <italic toggle='yes'>M. sedula</italic> and by initially incubating at 95°C for <italic toggle='yes'>P. furiosus</italic> growth and then subsequently returning to 75°C to promote 1-propanol formation. Note that 1-propanol was not produced if the culture was grown only at 95°C. This work has the potential for future optimization through harnessing the genome-scale metabolic model of <italic toggle='yes'>P. furiosus</italic> that was used herein to identify engineering targets to increase 1-propanol titer. <sec> <title>IMPORTANCE

As petroleum reserves become increasingly strained, the development of renewable alternatives to traditional chemical synthesis becomes more important. In this work, a high-temperature biological system for sugar to 1-propanol conversion was demonstrated by metabolic engineering of the hyperthermophilic archaeon Pyrococcus furiosus (T _opt 100°C). The engineered strain produced 1-propanol by temperature shifting from 75°C to 95°C and then back to 75°C to accommodate the temperature ranges for native and foreign proteins associated with 1-propanol biosynthesis. Genome-scale metabolic modeling informed the carbon and reductant flux in the system, identified potential factors limiting 1-propanol production, and revealed potential optimization targets.

View Journal Article

Sponsoring Organization:: USDOE

Grant/Contract Number:: SC0022192

OSTI ID:: 2549462

Journal Information:: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 5 Vol. 91; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

References (49)

Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO ₂ -based 3-hydroxypropionate production : 3-Hydroxypropionate Production by Hawkins, Aaron B.; Lian, Hong; Zeldes, Benjamin M. Biotechnology and Bioengineering, Vol. 112, Issue 8 https://doi.org/10.1002/bit.25584	journal	June 2015
Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO ₂ incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus : Accessory Enzymes Improve 3HP Titers in Lian, Hong; Zeldes, Benjamin M.; Lipscomb, Gina L. Biotechnology and Bioengineering, Vol. 113, Issue 12 https://doi.org/10.1002/bit.26033	journal	June 2016
Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100°C Fiala, Gerhard; Stetter, Karl O. Archives of Microbiology, Vol. 145, Issue 1, p. 56-61 https://doi.org/10.1007/BF00413027	journal	June 1986
Metabolic engineering of Propionibacterium freudenreichii for n-propanol production Ammar, Ehab Mohamed; Wang, Zhongqiang; Yang, Shang-Tian Applied Microbiology and Biotechnology, Vol. 97, Issue 10 https://doi.org/10.1007/s00253-013-4861-6	journal	April 2013
Modification of the glycolytic pathway in Pyrococcus furiosus and the implications for metabolic engineering Straub, Christopher T.; Schut, Gerritt; Otten, Jonathan K. Extremophiles, Vol. 24, Issue 4 https://doi.org/10.1007/s00792-020-01172-2	journal	May 2020
The synthesis of propanol from ethylene over homogeneous Rh/amine catalytic system via auto-tandem reductive hydroformylation Gorbunov, Dmitry; Nenasheva, Maria; Maximov, Anton Applied Catalysis A: General, Vol. 670 https://doi.org/10.1016/j.apcata.2023.119538	journal	January 2024
One-carbon substrate-based biohydrogen production: Microbes, mechanism, and productivity Rittmann, Simon K. -M. R.; Lee, Hyun Sook; Lim, Jae Kyu Biotechnology Advances, Vol. 33, Issue 1 https://doi.org/10.1016/j.biotechadv.2014.11.004	journal	January 2015
Microbial production of propanol Walther, Thomas; François, Jean Marie Biotechnology Advances, Vol. 34, Issue 5 https://doi.org/10.1016/j.biotechadv.2016.05.011	journal	September 2016
A novel one-step synthesis of 1-propanol from hydrogenolysis of glycerol using a Ni-HSiW/Al2O3 catalyst – The impact of H2 pressure on catalyst performance Mai, Chau T. Q.; Ye, Yinmei; Rempel, Garry L. Catalysis Today, Vol. 407 https://doi.org/10.1016/j.cattod.2022.09.017	journal	January 2023
Extremely thermophilic microorganisms for biomass conversion status and prospects Blumer-Schuette, Sara E.; Kataeva, Irina; Westpheling, Janet Current Opinion in Biotechnology, Vol. 19, Issue 3, p. 210-217 https://doi.org/10.1016/j.copbio.2008.04.007	journal	June 2008
Metabolic engineering of Escherichia coli for 1-butanol and 1-propanol production via the keto-acid pathways Shen, C. R.; Liao, J. C. Metabolic Engineering, Vol. 10, Issue 6 https://doi.org/10.1016/j.ymben.2008.08.001	journal	November 2008
Metabolic engineering of Thermobifida fusca for direct aerobic bioconversion of untreated lignocellulosic biomass to 1-propanol Deng, Yu; Fong, Stephen S. Metabolic Engineering, Vol. 13, Issue 5 https://doi.org/10.1016/j.ymben.2011.06.007	journal	September 2011
Metabolic engineering of Escherichia coli for the production of 1-propanol Jun Choi, Yong; Hwan Park, Jin; Yong Kim, Tae Metabolic Engineering, Vol. 14, Issue 5 https://doi.org/10.1016/j.ymben.2012.07.006	journal	September 2012
Deletion of acetyl-CoA synthetases I and II increases production of 3-hydroxypropionate by the metabolically-engineered hyperthermophile Pyrococcus furiosus Thorgersen, Michael P.; Lipscomb, Gina L.; Schut, Gerrit J. Metabolic Engineering, Vol. 22 https://doi.org/10.1016/j.ymben.2013.12.006	journal	March 2014
A hybrid synthetic pathway for butanol production by a hyperthermophilic microbe Keller, Matthew W.; Lipscomb, Gina L.; Loder, Andrew J. Metabolic Engineering, Vol. 27 https://doi.org/10.1016/j.ymben.2014.11.004	journal	January 2015
Temperature-dependent acetoin production by Pyrococcus furiosus is catalyzed by a biosynthetic acetolactate synthase and its deletion improves ethanol production Nguyen, Diep M. N.; Lipscomb, Gina L.; Schut, Gerrit J. Metabolic Engineering, Vol. 34 https://doi.org/10.1016/j.ymben.2015.12.006	journal	March 2016
Reaction kinetic analysis of the 3-hydroxypropionate/4-hydroxybutyrate CO2 fixation cycle in extremely thermoacidophilic archaea Loder, Andrew J.; Han, Yejun; Hawkins, Aaron B. Metabolic Engineering, Vol. 38 https://doi.org/10.1016/j.ymben.2016.10.009	journal	November 2016
Identification of Core Regulatory Genes and Metabolic Pathways for the n-Propanol Synthesis in Saccharomyces cerevisiae Wang, Ya-Ping; Sun, Zhong-guan; Wei, Xiao-Qing Journal of Agricultural and Food Chemistry, Vol. 69, Issue 5 https://doi.org/10.1021/acs.jafc.0c06810	journal	January 2021
Formate-driven growth coupled with H2 production Kim, Yun Jae; Lee, Hyun Sook; Kim, Eun Sook Nature, Vol. 467, Issue 7313 https://doi.org/10.1038/nature09375	journal	September 2010
Direct propanol synthesis from CO ₂ , C ₂ H ₄ , and H ₂ over Cs–Au/TiO ₂ rutile: effect of promoter loading, temperature and feed composition Ahlers, S. J.; Pohl, M. -M.; Holena, M. Catalysis Science & Technology, Vol. 6, Issue 7 https://doi.org/10.1039/C5CY01425E	journal	January 2016
Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide Keller, M. W.; Schut, G. J.; Lipscomb, G. L. Proceedings of the National Academy of Sciences, Vol. 110, Issue 15, p. 5840-5845 https://doi.org/10.1073/pnas.1222607110	journal	March 2013
Energy conservation by oxidation of formate to carbon dioxide and hydrogen via a sodium ion current in a hyperthermophilic archaeon Lim, J. K.; Mayer, F.; Kang, S. G. Proceedings of the National Academy of Sciences, Vol. 111, Issue 31 https://doi.org/10.1073/pnas.1407056111	journal	July 2014
Single gene insertion drives bioalcohol production by a thermophilic archaeon Basen, Mirko; Schut, Gerrit J.; Nguyen, Diep M. Proceedings of the National Academy of Sciences, Vol. 111, Issue 49 https://doi.org/10.1073/pnas.1413789111	journal	November 2014
Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase Ma, K.; Hutchins, A.; Sung, S. -J. S. Proceedings of the National Academy of Sciences, Vol. 94, Issue 18 https://doi.org/10.1073/pnas.94.18.9608	journal	September 1997
Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex Lipscomb, Gina L.; Schut, Gerrit J.; Thorgersen, Michael P. Journal of Biological Chemistry, Vol. 289, Issue 5 https://doi.org/10.1074/jbc.M113.530725	journal	December 2013
Proteome Analyses of Hydrogen-producing Hyperthermophilic Archaeon Thermococcus onnurineus NA1 in Different One-carbon Substrate Culture Conditions Moon, Yoon-Jung; Kwon, Joseph; Yun, Sung-Ho Molecular & Cellular Proteomics, Vol. 11, Issue 6 https://doi.org/10.1074/mcp.M111.015420	journal	June 2012
Biotechnology of extremely thermophilic archaea Straub, Christopher T.; Counts, James A.; Nguyen, Diep M. N. FEMS Microbiology Reviews, Vol. 42, Issue 5 https://doi.org/10.1093/femsre/fuy012	journal	June 2018
Parallel evolution of transcriptome architecture during genome reorganization Yoon, S. H.; Reiss, D. J.; Bare, J. C. Genome Research, Vol. 21, Issue 11, p. 1892-1904 https://doi.org/10.1101/gr.122218.111	journal	July 2011
A 3-Hydroxypropionate/4-Hydroxybutyrate Autotrophic Carbon Dioxide Assimilation Pathway in Archaea Berg, Ivan A.; Kockelkorn, Daniel; Buckel, Wolfgang Science, Vol. 318, Issue 5857, p. 1782-1786 https://doi.org/10.1126/science.1149976	journal	December 2007
Mannosylglycerate and Di- myo -Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress Esteves, Ana M.; Chandrayan, Sanjeev K.; McTernan, Patrick M. Applied and Environmental Microbiology, Vol. 80, Issue 14 https://doi.org/10.1128/AEM.00559-14	journal	July 2014
Natural Competence in the Hyperthermophilic Archaeon Pyrococcus furiosus Facilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases Lipscomb, Gina L.; Stirrett, Karen; Schut, Gerrit J. Applied and Environmental Microbiology, Vol. 77, Issue 7, p. 2232-2238 https://doi.org/10.1128/AEM.02624-10	journal	February 2011
Isolation and Characterization of Metal-Reducing Thermoanaerobacter Strains from Deep Subsurface Environments of the Piceance Basin, Colorado Roh, Y.; Liu, S. V.; Li, G. Applied and Environmental Microbiology, Vol. 68, Issue 12 https://doi.org/10.1128/AEM.68.12.6013-6020.2002	journal	December 2002
Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production Ward, Donald E.; Kengen, Servé W. M.; van der Oost, John Journal of Bacteriology, Vol. 182, Issue 9 https://doi.org/10.1128/JB.182.9.2559-2566.2000	journal	May 2000
Whole-Genome DNA Microarray Analysis of a Hyperthermophile and an Archaeon: Pyrococcus furiosus Grown on Carbohydrates or Peptides Schut, G. J.; Brehm, S. D.; Datta, S. Journal of Bacteriology, Vol. 185, Issue 13, p. 3935-3947 https://doi.org/10.1128/JB.185.13.3935-3947.2003	journal	July 2003
Cold Shock of a Hyperthermophilic Archaeon: Pyrococcus furiosus Exhibits Multiple Responses to a Suboptimal Growth Temperature with a Key Role for Membrane-Bound Glycoproteins Weinberg, M. V.; Schut, G. J.; Brehm, S. Journal of Bacteriology, Vol. 187, Issue 1, p. 336-348 https://doi.org/10.1128/JB.187.1.336-348.2005	journal	January 2005
Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95°C Driven by Carbon Monoxide Oxidation Lipscomb, Gina L.; Crowley, Alexander T.; Nguyen, Diep M. N. Applied and Environmental Microbiology, Vol. 89, Issue 6 https://doi.org/10.1128/aem.00012-23	journal	June 2023
Optimizing Strategies for Bio-Based Ethanol Production Using Genome-Scale Metabolic Modeling of the Hyperthermophilic Archaeon, Pyrococcus furiosus Vailionis, Jason L.; Zhao, Weishu; Zhang, Ke Applied and Environmental Microbiology, Vol. 89, Issue 6 https://doi.org/10.1128/aem.00563-23	journal	June 2023
Metabolic engineering of Caldicellulosiruptor bescii for 2,3-butanediol production from unpretreated lignocellulosic biomass and metabolic strategies for improving yields and titers Tanwee, Tania N. N.; Lipscomb, Gina L.; Vailionis, Jason L. Applied and Environmental Microbiology, Vol. 90, Issue 1 https://doi.org/10.1128/aem.01951-23	journal	December 2023
Growth Physiology of the Hyperthermophilic Archaeon Thermococcus litoralis: Development of a Sulfur-Free Defined Medium, Characterization of an Exopolysaccharide, and Evidence of Biofilm Formation. Rinker, K. D.; Kelly, R. M. Applied and environmental microbiology, Vol. 62, Issue 12 https://doi.org/10.1128/aem.62.12.4478-4485.1996	journal	January 1996
Engineering a Hyperthermophilic Archaeon for Temperature-Dependent Product Formation Basen, M.; Sun, J.; Adams, M. W. W. mBio, Vol. 3, Issue 2, Article No. e00053-12 https://doi.org/10.1128/mBio.00053-12	journal	April 2012
Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic Archaeon Pyrococcus furiosus Scott, Joseph W.; Poole, Farris L.; Adams, Michael W. W. Archaea, Vol. 2014 https://doi.org/10.1155/2014/176863	journal	January 2014
COBRApy: COnstraints-Based Reconstruction and Analysis for Python Ebrahim, Ali; Lerman, Joshua A.; Palsson, Bernhard O. BMC Systems Biology, Vol. 7, Issue 1 https://doi.org/10.1186/1752-0509-7-74	journal	January 2013
Metabolic pathway engineering for production of 1,2-propanediol and 1-propanol by Corynebacterium glutamicum Siebert, Daniel; Wendisch, Volker F. Biotechnology for Biofuels, Vol. 8, Issue 1 https://doi.org/10.1186/s13068-015-0269-0	journal	June 2015
A seamless and iterative DNA assembly method named PS-Brick and its assisted metabolic engineering for threonine and 1-propanol production Liu, Shuwen; Xiao, Haihan; Zhang, Fangfang Biotechnology for Biofuels, Vol. 12, Issue 1 https://doi.org/10.1186/s13068-019-1520-x	journal	July 2019
Alcohol dehydrogenases AdhE and AdhB with broad substrate ranges are important enzymes for organic acid reduction in Thermoanaerobacter sp. strain X514 Hitschler, Lisa; Nissen, Laura Sofie; Kuntz, Michelle Biotechnology for Biofuels, Vol. 14, Issue 1 https://doi.org/10.1186/s13068-021-02038-1	journal	September 2021
PSAMM: A Portable System for the Analysis of Metabolic Models Steffensen, Jon Lund; Dufault-Thompson, Keith; Zhang, Ying PLOS Computational Biology, Vol. 12, Issue 2 https://doi.org/10.1371/journal.pcbi.1004732	journal	February 2016
optGpSampler: An Improved Tool for Uniformly Sampling the Solution-Space of Genome-Scale Metabolic Networks Megchelenbrink, Wout; Huynen, Martijn; Marchiori, Elena PLoS ONE, Vol. 9, Issue 2 https://doi.org/10.1371/journal.pone.0086587	journal	February 2014
Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals Zeldes, Benjamin M.; Keller, Matthew W.; Loder, Andrew J. Frontiers in Microbiology, Vol. 6 https://doi.org/10.3389/fmicb.2015.01209	journal	November 2015
Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus Schut, Gerrit J.; Lipscomb, Gina L.; Nguyen, Diep M. N. Frontiers in Microbiology, Vol. 7 https://doi.org/10.3389/fmicb.2016.00029	journal	January 2016

Similar Records

Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO₂-based 3-hydroxypropionate production

Journal Article · Thu Jun 11 00:00:00 EDT 2015 · Biotechnology and Bioengineering · OSTI ID:1342894

Engineering a Hyperthermophilic Archaeon for Temperature-Dependent Product Formation

Journal Article · Thu Feb 23 23:00:00 EST 2012 · mBio (Online) · OSTI ID:1211477

Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95°C Driven by Carbon Monoxide Oxidation

Journal Article · Wed Jun 28 00:00:00 EDT 2023 · Applied and Environmental Microbiology · OSTI ID:2422078

Engineering the hyperthermophilic archaeon Pyrococcus furiosus for 1-propanol production

Citation Formats

References (49)

Similar Records

Related Subjects