Physiology of native biomass-fermenting organisms
Currently, most ethanol is produced using various strains of yeast. These organisms are very good at producing ethanol but have no native ability to consume lignocellulose. This has made it difficult to develop cost-effective yeast-based processes for lignocellulosic ethanol production. My group takes an alternative approach of starting with organisms that natively ferment lignocellulosic biomass and engineering them for efficient biofuel formation. To do this, we need to understand key aspects of their physiology, including:
- Which substrates they can consume, and how these substrates are used for growth, energy production, and product formation.
- Factors that limit growth and fermentation.
- Genetic adaptation to stresses associated with industrial fermentation
Key publications
Mazzoli, Roberto, Daniel G. Olson, Angela M. Concu, Evert K. Holwerda, and Lee R. Lynd. “In vivo evolution of lactic acid hyper-tolerant Clostridium thermocellum.” New Biotechnology 67, no. June 2021 (2021): Minor revisions. doi:10.1016/j.nbt.2021.12.003.
Cui, Jingxuan, David Stevenson, Travis Korosh, Daniel Amador-Noguez, Daniel G Olson†, Lee R Lynd, Oak Ridge, and Oak Ridge. “Developing a Cell-Free Extract Reaction ( CFER ) system in Clostridium thermocellum to identify metabolic limitations to ethanol production” 8, no. June (2020). doi:10.3389/fenrg.2020.00072.
Eminoğlu, Ayşenur, Sean Jean-Loup Murphy, Marybeth Maloney, Anthony Lanahan, Richard J. Giannone, Robert L. Hettich, Shital A. Tripathi, Ali Osman Beldüz, Lee R. Lynd, and Daniel G. Olson†. “Deletion of the hfsB gene increases ethanol production in Thermoanaerobacterium saccharolyticum and several other thermophilic anaerobic bacteria.” Biotechnology for Biofuels 10, no. 1 (2017): 282. doi:10.1186/s13068-017-0968-9.
Tian, Liang, Skyler J. Perot, David Stevenson, Tyler Jacobson, Anthony A. Lanahan, Daniel Amador-Noguez, Daniel G. Olson†, and Lee R. Lynd. “Metabolome analysis reveals a role for glyceraldehyde 3-phosphate dehydrogenase in the inhibition of C. thermocellum by ethanol.” Biotechnology for Biofuels 10, no. 1 (2017): 276. doi:10.1186/s13068-017-0961-3.
Lo, Jonathan, Daniel G. Olson†, Sean Jean-Loup Murphy, Liang Tian, Shuen Hon, Anthony Lanahan, Adam M. Guss, and Lee R. Lynd. 2017. “Engineering Electron Metabolism to Increase Ethanol Production in Clostridium thermocellum.” Metabolic Engineering, no. October. Elsevier: 1–9. doi:10.1016/j.ymben.2016.10.018.
Metabolic pathway characterization and development
Metabolism can be understood at many levels of aggregation from individual enzymes to the whole organism. An important intermediate level of aggregation is the metabolic pathway. Developing pathways that enable rapid production of desired compounds at high yield and titer requires a detailed understanding of both the individual components and the systems-level behavior that results from the interaction of these components, including:
- Identification of constituent enzymes in a pathway and the stoichiometry of the reactions they mediate
- Characterization of enzyme inhibition and regulation
- Development of screens and selections to improve properties of key enzymes.
- Protein engineering to increase activity, decrease inhibition, or change substrate specificity
Key publications
Cui, Jingxuan, Daniel G. Olson†, and Lee R. Lynd. “Characterization of the Clostridium thermocellum AdhE, NfnAB, Ferredoxin and Pfor proteins for their ability to support high titer ethanol production in Thermoanaerobacterium saccharolyticum.” Metabolic Engineering 51, no. August 2018 (2019): 32–42. doi:10.1016/j.ymben.2018.09.006.
Hon, Shuen, Daniel G. Olson†, Evert K. Holwerda, Anthony A. Lanahan, Sean J.L. Murphy, Marybeth I. Maloney, Tianyong Zheng, Beth Papanek, Adam M. Guss, and Lee R. Lynd. “The Ethanol Pathway from Thermoanaerobacterium saccharolyticum Improves Ethanol Production in Clostridium thermocellum.” Metabolic Engineering 42, no. June (July 2017): 175–84. doi:10.1016/j.ymben.2017.06.011.
Zheng, Tianyong, Daniel G. Olson†, Sean J. Murphy, Xiongjun Shao, Liang Tian, and Lee R. Lynd. “Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum.” Edited by William W. Metcalf. Journal of Bacteriology 199, no. 3 (February 1, 2017): e00542-16. doi:10.1128/JB.00542-16.
Tian, Liang, Beth Papanek, Daniel G. Olson, Thomas Rydzak, Evert K Holwerda, Tianyong Zheng, Jilai Zhou, et al. “Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum.” Biotechnology for Biofuels 9, no. 1 (December 2, 2016): 116. doi:10.1186/s13068-016-0528-8.
Olson, Daniel G., Richard Sparling, and Lee R Lynd. 2015. “Ethanol Production by Engineered Thermophiles.” Current Opinion in Biotechnology 33: 130–141. doi:10.1016/j.copbio.2015.02.006.
Zheng, Tianyong, Daniel G. Olson, Liang Tian, Yannick J. Bomble, Michael E. Himmel, Jonathan Lo, Shuen Hon, a. Joe Shaw, Johannes P. van Dijken, and Lee R. Lynd. 2015. “Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutants of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.” Journal of Bacteriology (May): JB.00232–15. doi:10.1128/JB.00232-15.
Genetic tools for anaerobic thermophilic bacteria
In large-scale industrial fermentations, it can be expensive to add oxygen, and to cool fermenters to mesophilic temperatures (20-45C). Using anaerobic thermophilic bacteria avoids both problems, however many genetic tools that have been originally developed for model organisms such as Saccharomyces cerevisiae and Escherichia coli do not work in these organisms. My group is working to develop several types of genetic tools necessary for domestication of thermophilic anaerobic bacteria, including:
- Ways to get foreign DNA into cells
- Tightly-controlled inducible promoters for reliable temporal control of gene expression
- Plasmid-based gene expression systems
- Chromosomal editing tools
- Ways to control the mutation rate
Key publications
Lanahan, Anthony, Kamila Zakowicz, Liang Tian, Daniel G. Olson†, and Lee R. Lynd. “ A Single Nucleotide Change in the polC DNA Polymerase III in Clostridium thermocellum Is Sufficient To Create a Hypermutator Phenotype .” Applied and Environmental Microbiology 88, no. 1 (2022). doi:10.1128/aem.01531-21.
Walker, Julie E., Anthony A. Lanahan, Tianyong Zheng, Camilo Toruno, Lee R. Lynd, Jeffrey C. Cameron, Daniel G. Olson†, and Carrie A. Eckert. “Development of both type I–B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum.” Metabolic Engineering Communications 10, no. August 2019 (June 2020): e00116. doi:10.1016/j.mec.2019.e00116.
Hon, Shuen, Anthony A Lanahan, Liang Tian, Richard J Giannone, Robert L Hettich, Daniel G Olson†, and Lee R Lynd. 2016. “Development of a Plasmid-Based Expression System in Clostridium thermocellum and Its Use to Screen Heterologous Expression of Bifunctional Alcohol Dehydrogenases (adhEs).” Metabolic Engineering Communications (3): 120–129.
Olson, Daniel G.†, Marybeth Maloney, Anthony A. Lanahan, Shuen Hon, Loren J. Hauser, and Lee R. Lynd. 2015. “Identifying Promoters for Gene Expression in Clostridium thermocellum.” Metabolic Engineering Communications 2: 23–29. doi:10.1016/j.meteno.2015.03.002.