Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Conversion of Lignocellulosic Plant Biomass into Industrial Chemicals via Metabolic Engineering of Two Extreme Thermophiles, Caldicellulosiruptor bescii and Pyrococcus furiosus

Authors:

Hailey C. O’Quinn1* Jason Vailionis2, Tania N. N. Tanwee1, Ryan G. Bing3, Kathryne C. Ford3, Ying Zhang2, Dmitry Rodionov4, Robert M. Kelly3, Michael W. W. Adams1

Institutions:

1University of Georgia; 2University of Rhode Island; 3North Carolina State University; 4Sanford Burnham Prebys Medical Discovery Institute

Goals

This project aims to metabolically engineer two extreme thermophiles, Caldicellulosiruptor bescii (Tmax 90°C) and Pyrococcus furiosus (Tmax 103°C), for the renewable production of key industrial chemicals through the conversion of lignocellulosic biomass, with targets including acetone, 2,3-butanediol, 1-propanol, 3-hydroxypropionate, and ethanol. This work includes efforts of carbon and energy optimization through harnessing carbon dioxide (CO2) and dihydrogen (H2) produced from fermentation into desired products and energy, respectively. Select enzymes responsible for degradation of lignocellulose will be expressed in P. furiosus to allow growth on cellulose and xylan. System-wide metabolic and regulatory models for both organisms will be leveraged to optimize biomass degradation and product yield for the target chemicals.

Abstract

Extremely thermophilic organisms present a valuable opportunity to convert lignocellulosic biomass to industrial chemicals, as conversion at high temperatures offers specific advantages, such as reduced contamination risk and temperature-dependent separation of volatile products. P. furiosus is a hyperthermophilic archaeon (Topt 100°C) with a growth range from 70°C to 103°C. The research group seeks to harness this organism’s extreme thermophily and robust genetic system for the production of chemicals of interest. P. furiosus has been previously engineered to produce lactate, ethanol, 3-hydroxypropionate, acetoin and butanol. The P. furiosus alcohol dehydrogenase F (AdhF) was recently identified as the ethanol-forming enzyme, with AdhF overexpression resulting in increased ethanol yields at temperatures up to 95°C (Lipscomb et al. 2023). P. furiosus strains producing 1-propanol were recently developed by constructing a nine-enzyme pathway consisting of both heterologous and native enzymes, although the characterization of these strains is still underway. Additionally, efforts are underway to express hemicellulases and cellulases in P. furiosus, with the goal of enabling the organism to grow directly on xylan and cellulose, as C. bescii does natively.

The other subject of this work, C. bescii, has been metabolically engineered for the production of acetone, ethanol, and other various alcohols. Recent work engineered 2,3-butanediol production in C. bescii when grown on unpretreated biomass (Tanwee et al. 2023). To further the researchers’ understanding of C. bescii and related thermophiles, this team has sequenced the genomes of many species in the genera Caldicellulosiruptor, Thermoclostridium, and Thermoanaerobacter (Bing et al. 2023a; Bing et al. 2023b; Manesh et al. 2024), leading to a reassessment of the taxonomic classification for the genus Caldicellulosiruptor and the order Thermoanerobacterales (Bing et al. 2023c). To better understand the ability of C. bescii to degrade biomass, the presence of microorganisms indigenous to various types of biomass was explored, alongside work to better understand cell-substrate associations during biomass solubilization (Bing et al. 2023d; Laemthong 2023). Work is also ongoing to engineer the cytoplasmic hydrogenase from P. furiosus into C. bescii to provide redox balancing for pathways dependent on the production of nicotinamide adenine dinucleotide phosphate (NADPH). System-wide metabolic and regulatory models of both C. bescii and P. furiosus have been created; these models have been and are currently being harnessed to predict optimization approaches for biomass conversion and product formation (Rodionov et al. 2021; Zhang et al. 2021; Vailionis 2023).

References

Bing, R. G., et al. 2023a. “Complete Genome Sequences of Caldicellulosiruptor acetigenus DSM 7040, Caldicellulosiruptor morganii DSM 8990 (RT8.B8), and Caldicellulosiruptor naganoensis DSM 8991 (NA10),” Microbiology Resource Announcements 12(3). DOI:10.1128/mra.01292-22.

Bing, R. G., et al. 2023b. “Complete Genome Sequences of Two Thermophilic Indigenous Bacteria Isolated from Wheat Straw, Thermoclostridium stercorarium subsp. Strain RKWS1 and Thermoanaerobacter sp. Strain RKWS2,” Microbiology Resource Announcements 12(3). DOI:10.1128/mra.01193-22.

Bing, R. G., et al. 2023c. “Whither the Genus Caldicellulosiruptor and the Order Thermoanaerobacterales: Phylogeny, Taxonomy, Ecology, and Phenotype,” Frontiers in Microbiology 14, L1212538. DOI:10.3389/fmicb.2023.1212538.

Bing, R. G., et al. 2023d. “Fermentative Conversion of Unpretreated Plant Biomass: A Thermophilic Threshold for Indigenous Microbial Growth,” Bioresource Technology 367. DOI:10.1016/j.biortech.2022.128275.

Laemthong, T., et al. 2023. “Role of Cell-Substrate Association During Plant Biomass Solubilization by the Extreme Thermophile Caldicellulosiruptor bescii,” Extremophiles 27(1), 6. DOI:10.1007/s00792-023-01290-7.

Lipscomb, G. L., et al. 2023. “Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95°C Driven by Carbon Monoxide Oxidation,” Applied and Environmental Microbiology 89(6). DOI:10.1128/aem.00012-23.

Manesh, M. J. H., et al. 2024. “Complete Genome Sequence for the Extremely Thermophilic Bacterium Anaerocellum danielii (DSM:8977),” Microbiology. Resource Announcements 13(2). DOI:10.1128/mra.01229-23.

Rodionov, D. A., et al. 2021. “Transcriptional Regulation of Plant Biomass Degradation and Carbohydrate Utilization Genes in the Extreme Thermophile Caldicellulosiruptor bescii,” mSystems 6(3), e01345-20. DOI:10.1128/mSystems.01345-20.

Tanwee, T. N., et al. 2023. “Metabolic engineering of Caldicellulosiruptor bescii for 2,3-butanediol production from unpretreated lignocellulosic biomass and metabolic strategies for improving yields and titers,” Applied and Environmental Microbiology 90(1). DOI:10.1128/aem.01951-23.

Vailionis, J. L., et al. 2023. “Optimizing Strategies for Bio-Based Ethanol Production Using Genome-Scale Metabolic Modeling of the Hyperthermophilic Archaeon, Pyrococcus furiosus,” Applied and Environmental Microbiology 89(6), e0056323. DOI:10.1128/aem.00563-23.

Zhang, K., et al. 2021. “Genome-Scale Metabolic Model of Caldicellulosiruptor bescii Reveals Optimal Metabolic Engineering Strategies for Bio-based Chemical Production,” mSystems 6(3), e0135120. DOI:10.1128/msystems.01351-20.

Funding Information

This material is based upon work supported by the U.S. DOE, Office of Science, BER program, GSP under Award Number DE-SC0022192.