Cell-Free Systems Biology: Characterizing Central Metabolism of Clostridium thermocellum with a Three-Enzyme Cascade Reaction

Jilani, S. Bilal; Alahuhta, Markus; Bomble, Yannick J.; Olson, Daniel G.

doi:10.1021/acssynbio.4c00405

Cell-Free Systems Biology: Characterizing Central Metabolism of Clostridium thermocellum with a Three-Enzyme Cascade Reaction

Journal Article · Thu Oct 10 00:00:00 EDT 2024 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.4c00405· OSTI ID:2477599

Jilani, S. Bilal ^[1]; Alahuhta, Markus ^[2]; ^[2]; ^[1]

Dartmouth College, Hanover, NH (United States)
National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Genetic approaches have been traditionally used to understand microbial metabolism, but this process can be slow in nonmodel organisms due to limited genetic tools. An alternative approach is to study metabolism directly in the cell lysate. This avoids the need for genetic tools and is routinely used to study individual enzymatic reactions but is not generally used to study systems-level properties of metabolism. Here we demonstrate a new approach that we call “cell-free systems biology”, where we use well-characterized enzymes and multienzyme cascades to serve as sources or sinks of intermediate metabolites. This allows us to isolate subnetworks within metabolism and study their systems-level properties. To demonstrate this, we worked with a threeenzyme cascade reaction that converts pyruvate to 2,3-butanediol. Although it has been previously used in cell-free systems, its pH dependence was not well characterized, limiting its utility as a sink for pyruvate. We showed that improved proton accounting allowed better prediction of pH changes and that active pH control allowed 2,3-butanediol titers of up to 2.1 M (189 g/L) from acetoin and 1.6 M (144 g/L) from pyruvate. The improved proton accounting provided a crucial insight that preventing the escape of CO₂ from the system largely eliminated the need for active pH control, dramatically simplifying our experimental setup. We then used this cascade reaction to understand limits to product formation in Clostridium thermocellum, an organism with potential applications for cellulosic biofuel production. We showed that the fate of pyruvate is largely controlled by electron availability and that reactions upstream of pyruvate limit overall product formation.

View Accepted Manuscript (DOE)

Research Organization:: Dartmouth College, Hanover, NH (United States); National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO); USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC36-08GO28308; SC0022175

OSTI ID:: 2477599

Alternate ID(s):: OSTI ID: 2477388

Report Number(s):: NREL/JA--2700-92039

Journal Information:: ACS Synthetic Biology, Journal Name: ACS Synthetic Biology Journal Issue: 11 Vol. 13; ISSN 2161-5063

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

References (45)

A pH-controlled fed-batch process can overcome inhibition by formate in NADH-dependent enzymatic reductions using formate dehydrogenase-catalyzed coenzyme regeneration Neuhauser, Wilfried; Steininger, Monika; Haltrich, Dietmar Biotechnology and Bioengineering, Vol. 60, Issue 3 https://doi.org/10.1002/(sici)1097-0290(19981105)60:3<277::aid-bit2>3.0.co;2-e	journal	November 1998
Cell-free metabolic engineering: Biomanufacturing beyond the cell Dudley, Quentin M.; Karim, Ashty S.; Jewett, Michael C. Biotechnology Journal, Vol. 10, Issue 1 https://doi.org/10.1002/biot.201400330	journal	October 2014
Mimicking theEscherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis Jewett, Michael C.; Swartz, James R. Biotechnology and Bioengineering, Vol. 86, Issue 1 https://doi.org/10.1002/bit.20026	journal	January 2004
Efficient and scalable method for scaling up cell free protein synthesis in batch mode Voloshin, Alexei M.; Swartz, James R. Biotechnology and Bioengineering, Vol. 91, Issue 4 https://doi.org/10.1002/bit.20528	journal	January 2005
Alkoholische Gährung ohne Hefezellen Buchner, Eduard; Rapp, Rudolf Berichte der deutschen chemischen Gesellschaft, Vol. 34, Issue 2 https://doi.org/10.1002/cber.19010340231	journal	May 1901
A Cell‐Free Multi‐enzyme Cascade Reaction for the Synthesis of CDP‐Glycerol Alberto Alcalá‐Orozco, E.; Grote, Valerian; Fiebig, Timm ChemBioChem, Vol. 24, Issue 21 https://doi.org/10.1002/cbic.202300463	journal	September 2023
The background to Eduard Buchner's discovery of cell-free fermentation Kohler, Robert Journal of the History of Biology, Vol. 4, Issue 1 https://doi.org/10.1007/BF00356976	journal	January 1971
Enhanced 2,3-butanediol production by Klebsiella pneumoniae SDM Ma, Cuiqing; Wang, Ailong; Qin, Jiayang Applied Microbiology and Biotechnology, Vol. 82, Issue 1 https://doi.org/10.1007/s00253-008-1732-7	journal	October 2008
A new NAD(H)-dependent meso-2,3-butanediol dehydrogenase from an industrially potential strain Serratia marcescens H30 Zhang, Liaoyuan; Xu, Quanming; Zhan, Senran Applied Microbiology and Biotechnology, Vol. 98, Issue 3 https://doi.org/10.1007/s00253-013-4959-x	journal	May 2013
Microbial production of 2,3-butanediol by a surfactant (serrawettin)-deficient mutant of Serratia marcescens H30 Zhang, Liaoyuan; Sun, Jian’an; Hao, Yingli Journal of Industrial Microbiology & Biotechnology, Vol. 37, Issue 8 https://doi.org/10.1007/s10295-010-0733-6	journal	May 2010
Studies on cell-free metabolism: Ethanol production by a yeast glycolytic system reconstituted from purified enzymes Welch, Peta; Scopes, Robert K. Journal of Biotechnology, Vol. 2, Issue 5 https://doi.org/10.1016/0168-1656(85)90029-X	journal	July 1985
The pH-activity curve of bovine carbonic anhydrase and its relationship to the inhibition of the enzyme by anions Kernohan, J. C. Biochimica et Biophysica Acta (BBA) - Enzymology and Biological Oxidation, Vol. 96, Issue 2 https://doi.org/10.1016/0926-6593(65)90014-7	journal	February 1965
Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription–translation system Caschera, Filippo; Noireaux, Vincent Biochimie, Vol. 99 https://doi.org/10.1016/j.biochi.2013.11.025	journal	April 2014
Closing the carbon balance for fermentation by Clostridium thermocellum (ATCC 27405) Ellis, Lucas D.; Holwerda, Evert K.; Hogsett, David Bioresource Technology, Vol. 103, Issue 1 https://doi.org/10.1016/j.biortech.2011.09.128	journal	January 2012
A model of yeast glycolysis based on a consistent kinetic characterisation of all its enzymes Smallbone, Kieran; Messiha, Hanan L.; Carroll, Kathleen M. FEBS Letters, Vol. 587, Issue 17 https://doi.org/10.1016/j.febslet.2013.06.043	journal	July 2013
Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol Kay, Jennifer E.; Jewett, Michael C. Metabolic Engineering, Vol. 32 https://doi.org/10.1016/j.ymben.2015.09.015	journal	November 2015
Glycolysis without pyruvate kinase in Clostridium thermocellum Olson, Daniel G.; Hörl, Manuel; Fuhrer, Tobias Metabolic Engineering, Vol. 39 https://doi.org/10.1016/j.ymben.2016.11.011	journal	January 2017
Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways Krüger, Antje; Mueller, Alexander P.; Rybnicky, Grant A. Metabolic Engineering, Vol. 62 https://doi.org/10.1016/j.ymben.2020.06.004	journal	November 2020
A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis Schroeder, Wheaton L.; Kuil, Teun; van Maris, Antonius J. A. Metabolic Engineering, Vol. 77 https://doi.org/10.1016/j.ymben.2023.04.003	journal	May 2023
Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis Dudley, Quentin M.; Anderson, Kim C.; Jewett, Michael C. ACS Synthetic Biology, Vol. 5, Issue 12 https://doi.org/10.1021/acssynbio.6b00154	journal	July 2016
Purification and properties of Salmonella typhimurium acetolactate synthase isozyme II from Escherichia coli HB101/pDU9 Schloss, John V.; Van Dyk, Drew E.; Vasta, Julia F. Biochemistry, Vol. 24, Issue 18 https://doi.org/10.1021/bi00339a034	journal	August 1985
Transformation of Biomass into Commodity Chemicals Using Enzymes or Cells Straathof, Adrie J. J. Chemical Reviews, Vol. 114, Issue 3 https://doi.org/10.1021/cr400309c	journal	August 2013
Effects of carbon dioxide and climate change on ocean acidification and carbonate mineral saturation Cao, Long; Caldeira, Ken; Jain, Atul K. Geophysical Research Letters, Vol. 34, Issue 5 https://doi.org/10.1029/2006GL028605	journal	March 2007
An integrated cell-free metabolic platform for protein production and synthetic biology Jewett, Michael C.; Calhoun, Kara A.; Voloshin, Alexei Molecular Systems Biology, Vol. 4, Issue 1, Article No. 220 https://doi.org/10.1038/msb.2008.57	journal	October 2008
Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis Bujara, Matthias; Schümperli, Michael; Pellaux, René Nature Chemical Biology, Vol. 7, Issue 5 https://doi.org/10.1038/nchembio.541	journal	March 2011
Forward design of a complex enzyme cascade reaction Hold, Christoph; Billerbeck, Sonja; Panke, Sven Nature Communications, Vol. 7, Issue 1 https://doi.org/10.1038/ncomms12971	journal	September 2016
A synthetic biochemistry molecular purge valve module that maintains redox balance Opgenorth, Paul H.; Korman, Tyler P.; Bowie, James U. Nature Communications, Vol. 5, Issue 1 https://doi.org/10.1038/ncomms5113	journal	June 2014
Isobutanol production freed from biological limits using synthetic biochemistry Sherkhanov, Saken; Korman, Tyler P.; Chan, Sum Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-18124-1	journal	August 2020
Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale Liew, Fungmin Eric; Nogle, Robert; Abdalla, Tanus Nature Biotechnology https://doi.org/10.1038/s41587-021-01195-w	journal	February 2022
Toward low-cost biological and hybrid biological/catalytic conversion of cellulosic biomass to fuels Lynd, Lee R.; Beckham, Gregg T.; Guss, Adam M. Energy & Environmental Science, Vol. 15, Issue 3 https://doi.org/10.1039/D1EE02540F	journal	January 2022
Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Testing biochemistry: Do we understand yeast glycolysis? Teusink, Bas; Passarge, Jutta; Reijenga, Corinne A. European Journal of Biochemistry, Vol. 267, Issue 17 https://doi.org/10.1046/j.1432-1327.2000.01527.x	journal	September 2000
BiGG Models: A platform for integrating, standardizing and sharing genome-scale models King, Zachary A.; Lu, Justin; Dräger, Andreas Nucleic Acids Research, Vol. 44, Issue D1 https://doi.org/10.1093/nar/gkv1049	journal	October 2015
The MetaCyc database of metabolic pathways and enzymes - a 2019 update Caspi, Ron; Billington, Richard; Keseler, Ingrid M. Nucleic Acids Research, Vol. 48, Issue D1 https://doi.org/10.1093/nar/gkz862	journal	October 2019
Enhancing control of cell-free metabolism through pH modulation Karim, Ashty S.; Rasor, Blake J.; Jewett, Michael C. Synthetic Biology, Vol. 5, Issue 1 https://doi.org/10.1093/synbio/ysz027	journal	November 2019
A model of carbon dioxide dissolution and mineral carbonation kinetics Mitchell, Mark J.; Jensen, Oliver E.; Cliffe, K. Andrew Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences, Vol. 466, Issue 2117 https://doi.org/10.1098/rspa.2009.0349	journal	September 2009
Atypical Glycolysis in Clostridium thermocellum Zhou, Jilai; Olson, Daniel G.; Argyros, D. Aaron Applied and Environmental Microbiology, Vol. 79, Issue 9, p. 3000-3008 https://doi.org/10.1128/AEM.04037-12	journal	February 2013
Increasing the Thermodynamic Driving Force of the Phosphofructokinase Reaction in Clostridium thermocellum Hon, Shuen; Jacobson, Tyler; Stevenson, David M. Applied and Environmental Microbiology, Vol. 88, Issue 22 https://doi.org/10.1128/aem.01258-22	journal	November 2022
Functional Analysis of H ⁺ -Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum Kuil, Teun; Hon, Shuen; Yayo, Johannes Applied and Environmental Microbiology, Vol. 88, Issue 4 https://doi.org/10.1128/aem.01857-21	journal	February 2022
In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using ¹³ C and ² H Tracers Jacobson, Tyler B.; Korosh, Travis K.; Stevenson, David M. mSystems, Vol. 5, Issue 2 https://doi.org/10.1128/mSystems.00736-19	journal	March 2020
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
Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum Tian, Liang; Papanek, Beth; Olson, Daniel G. Biotechnology for Biofuels, Vol. 9, Issue 1 https://doi.org/10.1186/s13068-016-0528-8	journal	June 2016
Enhanced production of 2,3-butanediol by engineered Saccharomyces cerevisiae through fine-tuning of pyruvate decarboxylase and NADH oxidase activities Kim, Jin-Woo; Kim, Jungyeon; Seo, Seung-Oh Biotechnology for Biofuels, Vol. 9, Issue 1 https://doi.org/10.1186/s13068-016-0677-9	journal	December 2016
Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production Hon, Shuen; Holwerda, Evert K.; Worthen, Robert S. Biotechnology for Biofuels, Vol. 11, Issue 1 https://doi.org/10.1186/s13068-018-1245-2	journal	September 2018
Method for the simultaneous assay of diacetyl and acetoin in the presence of α-acetolactate: Application in determining the kinetic parameters for the decomposition of α-acetolactate Kobayashi, Ken; Kusaka, Kazutaka; Takahashi, Tosirou Journal of Bioscience and Bioengineering, Vol. 99, Issue 5 https://doi.org/10.1263/jbb.99.502	journal	May 2005
Developing a Cell-Free Extract Reaction (CFER) System in Clostridium thermocellum to Identify Metabolic Limitations to Ethanol Production Cui, Jingxuan; Stevenson, David; Korosh, Travis Frontiers in Energy Research, Vol. 8 https://doi.org/10.3389/fenrg.2020.00072	journal	June 2020

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Cell-Free Systems Biology: Characterizing Central Metabolism of Clostridium thermocellum with a Three-Enzyme Cascade Reaction

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