Conversion of Lignocellulosic Plant Biomass into Industrial Chemicals via Metabolic Engineering of Two Extreme Thermophiles, Caldicellulosiruptor bescii and Pyrococcus furiosus
Ryan G. Bing1* (email@example.com), Tania N. N. Tanwee2, Hailey O’Quinn2, Jason Vailionis3, Gina L. Lipscomb2, James Crosby1, Tunyaboon Laemthong1, Ke Zhang3, Ying Zhang3, Dmitry Rodionov4, Robert M. Kelly1, and Michael W. W. Adams2
1North Carolina State University; 2University of Georgia; 3University of Rhode Island; and 4Sanford Burnham Prebys Medical Discovery Institute
This project aims to metabolically engineer two extreme thermophiles, Caldicellulosiruptor bescii (Tmax 90°C) and Pyrococcus furiosus (Tmax 103°C), to convert lignocellulosic plant biomass into industrial chemicals including acetone, 2,3-butanediol, 1-propanol, 3-hydroxypropionate, and ethanol. This work includes efforts to reincorporate CO2, which is generated by fermentation, into desired products powered by energy recovered from H2, which is also produced during fermentation. Some of the native enzymes of C. bescii that degrade lignocellulose will be expressed in P. furiosus to allow growth on cellulose and xylan. Additionally, system-wide metabolic and regulatory models for both organisms will be leveraged to optimize conversion, yield, and selectivity of plant biomass to industrial chemicals.
Conversion of lignocellulosic plant biomass into industrial chemicals has potential to provide renewable, sustainable sources of non-electrifiable fuels and chemicals. Enzymatic degradation and conversion of lignocellulose into desirable chemicals offers possible energy and monetary savings compared to chemical or mechanical methods. Consolidated bioprocessing aims to combine biological deconstruction and conversion of plant biomass into a single step to further increase these savings. Some extreme thermophiles, like C. bescii, excel at deconstruction of plant biomass which is in part aided by the high temperatures (Straub et al. 2018; Bing et al. 2021). Labs have shown how extremely thermophilic fermentation temperatures (>70°C) offer additional specific advantages including reduced contamination risk and opportunities for novel product separations (Bing et al. 2022, 2023). Previously, C. bescii was metabolically engineered to produce ethanol, acetone, and various alcohols, but not yet at industrially relevant titers (Williams-Rhaesa et al. 2018; Straub et al. 2020; Rubinstein et al. 2020). As such, the project is currently working to improve selectivity, yield, and titers of these products in C. bescii, as well as looking at additional target products, such as 2,3-butanediol and 3-hydroxypropionate.
Additional work involving the hyperthermophilic archaeon, P. furiosus, is also underway with the aim to leverage its high thermophily, efficient and established genetic system, as well as unique CO2 fixing and energy conservation enzymes (Hawkins et al. 2015; Keller et al. 2015, 2017). This work not only includes similar efforts to produce industrial chemicals (3-hydroxypropionate, 1-propanol, and ethanol), but also to leverage knowledge of C. bescii to engineer P. furiosus with (hemi)cellulases from C. bescii to enable growth on cellulose and xylan. Likewise, researchers are using enzymes from P. furiosus in C. bescii to improve production of target chemicals. Research is ongoing to engineer an NADPH regenerating soluble hydrogenase I from P. furiosus into C. bescii to increase redox factors for different target chemicals, such as 3-hydroxypropionate. Throughout all this work, system-wide metabolic and regulatory models for the organisms were created to evaluate, guide, assist, and optimize the production of target chemicals. The C. bescii models that were created previously continue to be updated, and P. furiosus models were created more recently as part of this project (Rodionov et al. 2021; Zhang et al. 2021).
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This material is based upon work supported by the U.S. Department of Energy, Office of Science, Biological and Environmental Research (BER) Program, Genomic Science program under Award Number DE-SC0022192.