Glycolysis without pyruvate kinase in Clostridium thermocellum

Olson, Daniel G.; Horl, Manuel; Fuhrer, Tobias; Cui, Jingxuan; Zhou, Jilai; Maloney, Marybeth I.; Amador-Noguez, Daniel; Tian, Liang; Sauer, Uwe; Lynd, Lee R.

doi:10.1016/j.ymben.2016.11.011

Title: Glycolysis without pyruvate kinase in Clostridium thermocellum

Journal Article · Thu Dec 01 00:00:00 EST 2016 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2016.11.011· OSTI ID:1333931

Olson, Daniel G. ^[1]; Horl, Manuel ^[2]; Fuhrer, Tobias ^[2]; Cui, Jingxuan ^[1]; Zhou, Jilai ^[1]; Maloney, Marybeth I. ^[1]; Amador-Noguez, Daniel ^[3]; Tian, Liang ^[1]; Sauer, Uwe ^[2]; Lynd, Lee R. ^[1]

Dartmouth College, Hanover, NH (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
ETH Zurich, Zurich (Switzerland)
Univ. of Wisconsin-Madison, Madison, WI (United States)

The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type organism, there are three proposed pathways for conversion of phosphoenolpyruvate (PEP) to pyruvate, which differ in their cofactor usage. One path uses pyruvate phosphate dikinase (PPDK), another pathway uses the combined activities of PEP carboxykinase (PEPCK) and oxaloacetate decarboxylase (ODC). Yet another pathway, the malate shunt, uses the combined activities of PEPCK, malate dehydrogenase and malic enzyme. First we showed that there is no flux through the ODC pathway by enzyme assay. Flux through the remaining two pathways (PPDK and malate shunt) was determined by dynamic ¹³C labeling. In the wild-type strain, the malate shunt accounts for about 33 ± 2% of the flux to pyruvate, with the remainder via the PPDK pathway. Deletion of the ppdk gene resulted in a redirection of all pyruvate flux through the malate shunt. Lastly, this provides the first direct evidence of the in-vivo function of the malate shunt.

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Research Organization:: Dartmouth College, Hanover, NH (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC05-00OR22725; AC02-05CH11231

OSTI ID:: 1333931

Alternate ID(s):: OSTI ID: 1397061

Journal Information:: Metabolic Engineering, Journal Name: Metabolic Engineering; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 43 works

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References (44)

Systems-Level Metabolic Flux Profiling Elucidates a Complete, Bifurcated Tricarboxylic Acid Cycle in Clostridium acetobutylicum Amador-Noguez, D.; Feng, X. -J.; Fan, J. Journal of Bacteriology, Vol. 192, Issue 17 https://doi.org/10.1128/JB.00490-10	journal	July 2010
High Ethanol Titers from Cellulose by Using Metabolically Engineered Thermophilic, Anaerobic Microbes Argyros, D. Aaron; Tripathi, Shital A.; Barrett, Trisha F. Applied and Environmental Microbiology, Vol. 77, Issue 23, p. 8288-8294 https://doi.org/10.1128/AEM.00646-11	journal	September 2011
Purification and Regulatory Properties of the Oxaloacetate Decarboxylase of Acetobacter xylinum Benziman, Moshe; Russo, Anna; Hochman, Sarah Journal of Bacteriology, Vol. 134, Issue 1 https://doi.org/10.1128/JB.134.1.1-9.1978	journal	April 1978
Increase in Ethanol Yield via Elimination of Lactate Production in an Ethanol-Tolerant Mutant of Clostridium thermocellum Biswas, Ranjita; Prabhu, Sandeep; Lynd, Lee R. PLoS ONE, Vol. 9, Issue 2 https://doi.org/10.1371/journal.pone.0086389	journal	February 2014
Ultrahigh Performance Liquid Chromatography−Tandem Mass Spectrometry Method for Fast and Robust Quantification of Anionic and Aromatic Metabolites Buescher, Joerg Martin; Moco, Sofia; Sauer, Uwe Analytical Chemistry, Vol. 82, Issue 11 https://doi.org/10.1021/ac100101d	journal	June 2010
Redirecting carbon flux through exogenous pyruvate kinase to achieve high ethanol yields in Clostridium thermocellum Deng, Yu; Olson, Daniel G.; Zhou, Jilai Metabolic Engineering, Vol. 15, p. 151-158 https://doi.org/10.1016/j.ymben.2012.11.006	journal	January 2013
Characterization of a Membrane-Bound Biotin-Containing Enzyme: Oxaloacetate Decarboxylase from Klebsiella aerogenes Dimroth, Peter European Journal of Biochemistry, Vol. 115, Issue 2 https://doi.org/10.1111/j.1432-1033.1981.tb05245.x	journal	April 1981
High-throughput metabolic flux analysis based on gas chromatography–mass spectrometry derived 13C constraints Fischer, Eliane; Zamboni, Nicola; Sauer, Uwe Analytical Biochemistry, Vol. 325, Issue 2 https://doi.org/10.1016/j.ab.2003.10.036	journal	February 2004
Enzymatic Assembly of Overlapping DNA Fragments Gibson, Daniel G. Methods in Enzymology https://doi.org/10.1016/B978-0-12-385120-8.00015-2	book	May 2011
Genome-scale metabolic model integrated with RNAseq data to identify metabolic states of Clostridium thermocellum Gowen, Christopher M.; Fong, Stephen S. Biotechnology Journal, Vol. 5, Issue 7 https://doi.org/10.1002/biot.201000084	journal	July 2010
Development and evaluation of methods to infer biosynthesis and substrate consumption in cultures of cellulolytic microorganisms Holwerda, Evert K.; Ellis, Lucas D.; Lynd, Lee R. Biotechnology and Bioengineering, Vol. 110, Issue 9 https://doi.org/10.1002/bit.24915	journal	April 2013
Non-stationary ¹³ C-metabolic flux ratio analysis: Non-Stationary ¹³ C-Metabolic Flux Ratio Analysis Hörl, Manuel; Schnidder, Julian; Sauer, Uwe Biotechnology and Bioengineering, Vol. 110, Issue 12 https://doi.org/10.1002/bit.25004	journal	August 2013
Pigeon Liver Malic Enzyme Hsu, Robert Y.; Lardy, Henry A. Journal of Biological Chemistry, Vol. 242, Issue 3 https://doi.org/10.1016/S0021-9258(18)96304-0	journal	February 1967
KEGG as a reference resource for gene and protein annotation Kanehisa, Minoru; Sato, Yoko; Kawashima, Masayuki Nucleic Acids Research, Vol. 44, Issue D1 https://doi.org/10.1093/nar/gkv1070	journal	October 2015
Genetic and Functional Analysis of the Soluble Oxaloacetate Decarboxylase from Corynebacterium glutamicum Klaffl, Simon; Eikmanns, Bernhard J. Journal of Bacteriology, Vol. 192, Issue 10 https://doi.org/10.1128/JB.01678-09	journal	May 2010
Oxaloacetate Decarboxylase fromPseudomonas stutzeri:Purification and Characterization Labrou, N. E.; Clonis, Y. D. Archives of Biochemistry and Biophysics, Vol. 365, Issue 1 https://doi.org/10.1006/abbi.1999.1144	journal	May 1999
Thermostable, ammonium-activated malic enzyme of Clostridium thermocellum Lamed, R.; Zeikus, J. G. Biochimica et Biophysica Acta (BBA) - Enzymology, Vol. 660, Issue 2 https://doi.org/10.1016/0005-2744(81)90167-4	journal	August 1981
Real-time metabolome profiling of the metabolic switch between starvation and growth Link, Hannes; Fuhrer, Tobias; Gerosa, Luca Nature Methods, Vol. 12, Issue 11 https://doi.org/10.1038/nmeth.3584	journal	September 2015
Determination of Metabolic Flux Ratios From 13C-Experiments and Gas Chromatography-Mass Spectrometry Data Nanchen, Annik; Fuhrer, Tobias; Sauer, Uwe https://doi.org/10.1007/978-1-59745-244-1_11	book	January 2007
Properties of oxaloacetate decarboxylase from Veillonella parvula Ng, S. K.; Wong, M.; Hamilton, I. R. Journal of Bacteriology, Vol. 150, Issue 3 https://doi.org/10.1128/JB.150.3.1252-1258.1982	journal	June 1982
Mode of sugar phosphorylation inClostridium thermocellum Nochur, S. V.; Jacobson, G. R.; Roberts, M. F. Applied Biochemistry and Biotechnology, Vol. 33, Issue 1 https://doi.org/10.1007/BF02922182	journal	April 1992
The benefits of being transient: isotope-based metabolic flux analysis at the short time scale Nöh, Katharina; Wiechert, Wolfgang Applied Microbiology and Biotechnology, Vol. 91, Issue 5 https://doi.org/10.1007/s00253-011-3390-4	journal	July 2011
Novel enzymic machinery for the metabolism of oxalacetate, phosphoenolpyruvate, and pyruvate in Pseudomonas citronellolis. O'Brien, R.; Chuang, D. T.; Taylor, B. L. Journal of Biological Chemistry, Vol. 252, Issue 4 https://doi.org/10.1016/S0021-9258(17)40649-1	journal	February 1977
Transformation of Clostridium Thermocellum by Electroporation Olson, Daniel G.; Lynd, Lee R. Cellulases https://doi.org/10.1016/B978-0-12-415931-0.00017-3	book	January 2012
Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum Papanek, Beth; Biswas, Ranjita; Rydzak, Thomas Metabolic Engineering, Vol. 32 https://doi.org/10.1016/j.ymben.2015.09.002	journal	November 2015
The Oxalacetate Decarboxylase of Azotobacter Vinelandii Plaut, G. W. E.; Lardy, Henry A. Journal of Biological Chemistry, Vol. 180, Issue 1 https://doi.org/10.1016/S0021-9258(18)56717-X	journal	August 1949
Transcriptomic analysis of Clostridium thermocellum ATCC 27405 cellulose fermentation Raman, Babu; McKeown, Catherine K; Rodriguez, Miguel BMC Microbiology, Vol. 11, Issue 1, Article No. 134 https://doi.org/10.1186/1471-2180-11-134	journal	January 2011
Biochemical and Genetic Characterization of the Enterococcus faecalis Oxaloacetate Decarboxylase Complex Repizo, Guillermo D.; Blancato, Víctor S.; Mortera, Pablo Applied and Environmental Microbiology, Vol. 79, Issue 9 https://doi.org/10.1128/AEM.03980-12	journal	May 2013
Global Gene Expression Patterns in Clostridium thermocellum as Determined by Microarray Analysis of Chemostat Cultures on Cellulose or Cellobiose Riederer, Allison; Takasuka, Taichi E.; Makino, Shin-ichi Applied and Environmental Microbiology, Vol. 77, Issue 4 https://doi.org/10.1128/AEM.02008-10	journal	December 2010
Collisional fragmentation of central carbon metabolites in LC-MS/MS increases precision of 13C metabolic flux analysis Rühl, Martin; Rupp, Beat; Nöh, Katharina Biotechnology and Bioengineering, Vol. 109, Issue 3 https://doi.org/10.1002/bit.24344	journal	October 2011
Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression Rydzak, Thomas; McQueen, Peter D.; Krokhin, Oleg V. BMC Microbiology, Vol. 12, Issue 1 https://doi.org/10.1186/1471-2180-12-214	journal	January 2012
Expression and characterization of recombinant pyruvate phosphate dikinase from Entamoeba histolytica Saavedra-Lira, Emma; Ramirez-Silva, Leticia; Perez-Montfort, Ruy Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Vol. 1382, Issue 1 https://doi.org/10.1016/S0167-4838(97)00139-8	journal	January 1998
Saccharomyces cerevisiae-Based Molecular Tool Kit for Manipulation of Genes from Gram-Negative Bacteria Shanks, Robert; Caiazza, Nicky; Hinsa, Shannon Applied and Environmental Microbiology, Vol. 72, Issue 7, p. 5027-5036 https://doi.org/10.1128/AEM.00682-06	journal	July 2006
Elucidation of Enzymes in Fermentation Pathways Used by Clostridium thermosuccinogenes Growing on Inulin Sridhar, Jayanth; Eiteman, Mark A.; Wiegel, Juergen W. Applied and Environmental Microbiology, Vol. 66, Issue 1 https://doi.org/10.1128/AEM.66.1.246-251.2000	journal	January 2000
Elucidating central metabolic redox obstacles hindering ethanol production in Clostridium thermocellum Thompson, R. Adam; Layton, Donovan S.; Guss, Adam M. Metabolic Engineering, Vol. 32 https://doi.org/10.1016/j.ymben.2015.10.004	journal	November 2015
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
Phosphoenolpyruvate synthetase and pyruvate, phosphate dikinase of Thermoproteus tenax: key pieces in the puzzle of archaeal carbohydrate metabolism Tjaden, Britta; Plagens, Andre; Dorr, Christine Molecular Microbiology, Vol. 60, Issue 2 https://doi.org/10.1111/j.1365-2958.2006.05098.x	journal	April 2006
Development of pyrF-Based Genetic System for Targeted Gene Deletion in Clostridium thermocellum and Creation of a pta Mutant Tripathi, S. A.; Olson, D. G.; Argyros, D. A. Applied and Environmental Microbiology, Vol. 76, Issue 19, p. 6591-6599 https://doi.org/10.1128/AEM.01484-10	journal	August 2010
Kinetic Mechanism and Metabolic Role of Pyruvate Phosphate Dikinase from Entamoeba histolytica Varela-Gómez, Marcela; Moreno-Sánchez, Rafael; Pardo, Juan Pablo Journal of Biological Chemistry, Vol. 279, Issue 52 https://doi.org/10.1074/jbc.M401697200	journal	December 2004
13C-based metabolic flux analysis Zamboni, Nicola; Fendt, Sarah-Maria; Rühl, Martin Nature Protocols, Vol. 4, Issue 6 https://doi.org/10.1038/nprot.2009.58	journal	May 2009
Hydrogen Formation and Its Regulation in Ruminococcus albus: Involvement of an Electron-Bifurcating [FeFe]-Hydrogenase, of a Non-Electron-Bifurcating [FeFe]-Hydrogenase, and of a Putative Hydrogen-Sensing [FeFe]-Hydrogenase Zheng, Y.; Kahnt, J.; Kwon, I. H. Journal of Bacteriology, Vol. 196, Issue 22 https://doi.org/10.1128/JB.02070-14	journal	August 2014
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
Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485 Zhou, Jilai; Olson, Daniel G.; Lanahan, Anthony A. Biotechnology for Biofuels, Vol. 8, Issue 1 https://doi.org/10.1186/s13068-015-0304-1	journal	September 2015
Quantification and Mass Isotopomer Profiling of α-Keto Acids in Central Carbon Metabolism Zimmermann, Michael; Sauer, Uwe; Zamboni, Nicola Analytical Chemistry, Vol. 86, Issue 6 https://doi.org/10.1021/ac500472c	journal	February 2014

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Related Subjects

09 BIOMASS FUELS
13C flux analysis
pyruvate kinase
malate shunt
malic enzyme
malate dehydrogenase
oxaloacetate decarboxylase

Title: Glycolysis without pyruvate kinase in Clostridium thermocellum

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