Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii

Chung, Daehwan; Cha, Minseok; Snyder, Elise N.; Elkins, James G.; Guss, Adam M.; Westpheling, Janet

doi:10.1186/s13068-015-0346-4

Title: Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii

Journal Article · Tue Oct 06 00:00:00 EDT 2015 · Biotechnology for Biofuels

DOI:https://doi.org/10.1186/s13068-015-0346-4· OSTI ID:1618875

Chung, Daehwan; Cha, Minseok; Snyder, Elise N.; Elkins, James G.; Guss, Adam M.; Westpheling, Janet

In this paper, we report that the C. bescii genome does not encode an acetaldehyde/alcohol dehydrogenase or an acetaldehyde dehydrogenase and no ethanol production is detected in this strain. The recent introduction of an NADH-dependent AdhE from C. thermocellum (Fig. 1a) in an ldh mutant of this strain resulted in production of ethanol from un-pretreated switchgrass, but the thermolability of the C. thermocellum AdhE at the optimum growth temperature of C. bescii (78 °C) meant that ethanol was not produced above 65 °C. The adhB and adhE genes from Thermoanaerobacter pseudethanolicus 39E, an anaerobic thermophile that produces ethanol as a major fermentation product at 70 °C, were cloned and expressed in an ldh deletion mutant of C. bescii. The engineered strains produced ethanol at 75 °C, near the ethanol boiling point. The AdhB expressing strain produced ethanol (1.4 mM on Avicel, 0.4 mM on switchgrass) as well as acetate (13.0 mM on Avicel, 15.7 mM on switchgrass). The AdhE expressing strain produced more ethanol (2.3 mM on Avicel, 1.6 mM on switchgrass) and reduced levels of acetate (12.3 mM on Avicel, 15.1 mM on switchgrass). These engineered strains produce cellulosic ethanol at the highest temperature of any microorganism to date. In addition, the addition of 40 mM MOPS to the growth medium increased the maximal growth yield of C. bescii by approximately twofold. In conclusion, the utilization of thermostable enzymes will be critical to achieving high temperature CBP in bacteria such as C. bescii. The ability to produce ethanol at 75 °C, near its boiling point, raises the possibility that process optimization could allow in situ product removal of this end product to mitigate ethanol toxicity.

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Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)

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

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1618875

Alternate ID(s):: OSTI ID: 1260585; OSTI ID: 1324147

Journal Information:: Biotechnology for Biofuels, Journal Name: Biotechnology for Biofuels Vol. 8 Journal Issue: 1; ISSN 1754-6834

Publisher:: Springer Science + Business MediaCopyright Statement

Country of Publication:: Netherlands

Language:: English

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Cited by: 32 works

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References (44)

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
Classification of 'Anaerocellum thermophilum' strain DSM 6725 as Caldicellulosiruptor bescii sp. nov. Yang, S. -J.; Kataeva, I.; Wiegel, J. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, Vol. 60, Issue 9 https://doi.org/10.1099/ijs.0.017731-0	journal	October 2009
Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2° Adh) as a bifunctional alcohol dehydrogenase-acetyl-CoA reductive thioesterase Burdette, D.; Zeikus, J. G. Biochemical Journal, Vol. 302, Issue 1 https://doi.org/10.1042/bj3020163	journal	August 1994
Extreme thermophiles: moving beyond single-enzyme biocatalysis Frock, Andrew D.; Kelly, Robert M. Current Opinion in Chemical Engineering, Vol. 1, Issue 4 https://doi.org/10.1016/j.coche.2012.07.003	journal	November 2012
Extremely thermophilic microorganisms for biomass conversion status and prospects Blumer-Schuette, Sara E.; Kataeva, Irina; Westpheling, Janet Current Opinion in Biotechnology, Vol. 19, Issue 3, p. 210-217 https://doi.org/10.1016/j.copbio.2008.04.007	journal	June 2008
Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production Himmel, M. E.; Ding, S.-Y.; Johnson, D. K. Science, Vol. 315, Issue 5813, p. 804-807 https://doi.org/10.1126/science.1137016	journal	February 2007
Biofuels and sustainability: Biofuels and sustainability Solomon, Barry D. Annals of the New York Academy of Sciences, Vol. 1185, Issue 1 https://doi.org/10.1111/j.1749-6632.2009.05279.x	journal	January 2010
Thermophilic lignocellulose deconstruction Blumer-Schuette, Sara E.; Brown, Steven D.; Sander, Kyle B. FEMS Microbiology Reviews, Vol. 38, Issue 3 https://doi.org/10.1111/1574-6976.12044	journal	May 2014
How biotech can transform biofuels Lynd, Lee R.; Laser, Mark S.; Bransby, David Nature Biotechnology, Vol. 26, Issue 2, p. 169-172 https://doi.org/10.1038/nbt0208-169	journal	February 2008
Recent progress in consolidated bioprocessing Olson, Daniel G.; McBride, John E.; Joe Shaw, A. Current Opinion in Biotechnology, Vol. 23, Issue 3, p. 396-405 https://doi.org/10.1016/j.copbio.2011.11.026	journal	June 2012
Ethanol production by engineered thermophiles Olson, Daniel G.; Sparling, Richard; Lynd, Lee R. Current Opinion in Biotechnology, Vol. 33 https://doi.org/10.1016/j.copbio.2015.02.006	journal	June 2015
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
Genome Sequence of the Anaerobic, Thermophilic, and Cellulolytic Bacterium "Anaerocellum thermophilum" DSM 6725 Kataeva, I. A.; Yang, S. -J.; Dam, P. Journal of Bacteriology, Vol. 191, Issue 11 https://doi.org/10.1128/JB.00256-09	journal	April 2009
Fed-batch mode in shake flasks by slow-release technique Jeude, M.; Dittrich, B.; Niederschulte, H. Biotechnology and Bioengineering, Vol. 95, Issue 3 https://doi.org/10.1002/bit.21012	journal	January 2006
Thermophilic ethanologenesis: future prospects for second-generation bioethanol production Taylor, Mark P.; Eley, Kirsten L.; Martin, Steve Trends in Biotechnology, Vol. 27, Issue 7, p. 398-405 https://doi.org/10.1016/j.tibtech.2009.03.006	journal	July 2009
Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Zheng, Tianyong; Olson, Daniel G.; Tian, Liang Journal of Bacteriology, Vol. 197, Issue 15 https://doi.org/10.1128/JB.00232-15	journal	May 2015
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
Thermoanaerobacter spp. control ethanol pathway via transcriptional regulation and versatility of key enzymes Pei, Jianjun; Zhou, Qing; Jiang, Yu Metabolic Engineering, Vol. 12, Issue 5 https://doi.org/10.1016/j.ymben.2010.06.001	journal	September 2010
Degradation of high loads of crystalline cellulose and of unpretreated plant biomass by the thermophilic bacterium Caldicellulosiruptor bescii Basen, Mirko; Rhaesa, Amanda M.; Kataeva, Irina Bioresource Technology, Vol. 152 https://doi.org/10.1016/j.biortech.2013.11.024	journal	January 2014
Microbial Cellulose Utilization: Fundamentals and Biotechnology Lynd, L. R.; Weimer, P. J.; van Zyl, W. H. Microbiology and Molecular Biology Reviews, Vol. 66, Issue 3, p. 506-577 https://doi.org/10.1128/MMBR.66.3.506-577.2002	journal	September 2002
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
Isolation and bioinformatic analysis of a novel transposable element, ISCbe4, from the hyperthermophilic bacterium, Caldicellulosiruptor bescii Cha, Minseok; Wang, Hao; Chung, Daehwan Journal of Industrial Microbiology & Biotechnology, Vol. 40, Issue 12 https://doi.org/10.1007/s10295-013-1345-8	journal	October 2013
Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii Chung, D.; Cha, M.; Guss, A. M. Proceedings of the National Academy of Sciences, Vol. 111, Issue 24, p. 8931-8936 https://doi.org/10.1073/pnas.1402210111	journal	June 2014
Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum Biswas, Ranjita; Zheng, Tianyong; Olson, Daniel G. Biotechnology for Biofuels, Vol. 8, Issue 1 https://doi.org/10.1186/s13068-015-0204-4	journal	January 2015
Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes Bhalla, Aditya; Bansal, Namita; Kumar, Sudhir Bioresource Technology, Vol. 128 https://doi.org/10.1016/j.biortech.2012.10.145	journal	January 2013
Elimination of formate production in Clostridium thermocellum Rydzak, Thomas; Lynd, Lee R.; Guss, Adam M. Journal of Industrial Microbiology & Biotechnology, Vol. 42, Issue 9 https://doi.org/10.1007/s10295-015-1644-3	journal	July 2015
NADP+ Reduction with Reduced Ferredoxin and NADP+ Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex in Clostridium kluyveri Wang, S.; Huang, H.; Moll, J. Journal of Bacteriology, Vol. 192, Issue 19 https://doi.org/10.1128/JB.00612-10	journal	July 2010
Construction of a Stable Replicating Shuttle Vector for Caldicellulosiruptor Species: Use for Extending Genetic Methodologies to Other Members of This Genus Chung, Daehwan; Cha, Minseok; Farkas, Joel PLoS ONE, Vol. 8, Issue 5, Article No. e62881 https://doi.org/10.1371/journal.pone.0062881	journal	May 2013
Cloning and expression of the gene encoding the Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase and biochemical characterization of the enzyme Burdette, Douglas S.; Vieille, Claire; Zeikus, J. Gregory Biochemical Journal, Vol. 316, Issue 1 https://doi.org/10.1042/bj3160115	journal	May 1996
Taxonomic Distinction of Saccharolytic Thermophilic Anaerobes: Description of Thermoanaerobacterium xylanolyticum gen. nov., sp. nov., and Thermoanaerobacterium saccharolyticum gen. nov., sp. nov.; Reclassification of Thermoanaerobium brockii, Clostridium thermosulfurogenes, and Clostridium thermohydrosulfuricum E100-69 as Thermoanaerobacter brockii comb. nov., Thermoanaerobacterium thermosulfurigenes comb. nov., and Thermoanaerobacter thermohydrosulfuricus comb. nov., Respectively; and Transfer of Clostridium thermohydrosulfuricum 39E to Thermoanaerobacter ethanolicus Lee, Y. -E.; Jain, M. K.; Lee, C. International Journal of Systematic Bacteriology, Vol. 43, Issue 1 https://doi.org/10.1099/00207713-43-1-41	journal	January 1993
Homologous Expression of the Caldicellulosiruptor bescii CelA Reveals that the Extracellular Protein Is Glycosylated Chung, Daehwan; Young, Jenna; Bomble, Yannick J. PLOS ONE, Vol. 10, Issue 3 https://doi.org/10.1371/journal.pone.0119508	journal	March 2015
Salt Accumulation Resulting from Base Added for pH Control, and Not Ethanol, Limits Growth of Thermoanaerobacterium thermosaccharolyticum HG-8 at Elevated Feed Xylose Concentrations in Continuous Culture Lynd, L. R.; Baskaran, S.; Casten, S. Biotechnology Progress, Vol. 17, Issue 1 https://doi.org/10.1021/bp000158n	journal	February 2001
The Iron-Hydrogenase of Thermotoga maritima Utilizes Ferredoxin and NADH Synergistically: a New Perspective on Anaerobic Hydrogen Production Schut, G. J.; Adams, M. W. W. Journal of Bacteriology, Vol. 191, Issue 13 https://doi.org/10.1128/JB.01582-08	journal	May 2009
Substrate and product inhibition of hydrogen production by the extreme thermophile,Caldicellulosiruptor saccharolyticus van Niel, Ed W. J.; Claassen, Pieternel A. M.; Stams, Alfons J. M. Biotechnology and Bioengineering, Vol. 81, Issue 3 https://doi.org/10.1002/bit.10463	journal	December 2002
Controlling pH in shake flasks using polymer-based controlled-release discs with pre-determined release kinetics Scheidle, Marco; Dittrich, Barbara; Klinger, Johannes BMC Biotechnology, Vol. 11, Issue 1 https://doi.org/10.1186/1472-6750-11-25	journal	January 2011
Thermoanaerobacter pseudethanolicus sp. nov., a thermophilic heterotrophic anaerobe from Yellowstone National Park Onyenwoke, R. U.; Kevbrin, V. V.; Lysenko, Anatolly. M. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, Vol. 57, Issue 10 https://doi.org/10.1099/ijs.0.65051-0	journal	October 2007
Review: Minibioreactors Kumar, Sathish; Wittmann, Christoph; Heinzle, Elmar Biotechnology Letters, Vol. 26, Issue 1 https://doi.org/10.1023/B:BILE.0000009469.69116.03	journal	January 2004
Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum Shao, Xiongjun; Raman, Babu; Zhu, Mingjun Applied Microbiology and Biotechnology, Vol. 92, Issue 3 https://doi.org/10.1007/s00253-011-3492-z	journal	August 2011
Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement Chung, Daehwan; Farkas, Joel; Westpheling, Janet Biotechnology for Biofuels, Vol. 6, Issue 1 https://doi.org/10.1186/1754-6834-6-82	journal	January 2013
Linking genome content to biofuel production yields: a meta-analysis of major catabolic pathways among select H2 and ethanol-producing bacteria Carere, Carlo R.; Rydzak, Thomas; Verbeke, Tobin J. BMC Microbiology, Vol. 12, Issue 1 https://doi.org/10.1186/1471-2180-12-295	journal	January 2012
Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii Farkas, Joel; Chung, Daehwan; Cha, Minseok Journal of Industrial Microbiology & Biotechnology, Vol. 40, Issue 1, p. 41-49 https://doi.org/10.1007/s10295-012-1202-1	journal	November 2012
Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum Brown, S. D.; Guss, A. M.; Karpinets, T. V. Proceedings of the National Academy of Sciences, Vol. 108, Issue 33 https://doi.org/10.1073/pnas.1102444108	journal	August 2011
Metabolic engineering of Caldicellulosiruptor bescii yields increased hydrogen production from lignocellulosic biomass Cha, Minseok; Chung, Daehwan; Elkins, James G. Biotechnology for Biofuels, Vol. 6, Issue 1 https://doi.org/10.1186/1754-6834-6-85	journal	January 2013
Controlling pH in shake flasks using polymer-based controlled-release discs with pre-determined release kinetics Scheidle, Marco; Dittrich, Barbara; Klinger, Johannes RWTH Aachen University https://doi.org/10.18154/rwth-conv-060077	text	January 2011

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59 BASIC BIOLOGICAL SCIENCES
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Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii [Supplemental Data] Chung, Daehwan; Cha, Minseok; Snyder, Elise https://doi.org/10.6084/m9.figshare.c.3617006 The current record is supplemented by this dataset	dataset	October 2015
MOESM1 of Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii Chung, Daehwan; Cha, Minseok; Snyder, Elise https://doi.org/10.6084/m9.figshare.c.3617006_d1.v1 The current record is supplemented by this figure	figure	October 2015
Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii [Supplemental Data] Chung, Daehwan; Cha, Minseok; Snyder, Elise https://doi.org/10.6084/m9.figshare.c.3617006.v1 The current record is supplemented by this dataset	dataset	October 2015
MOESM1 of Cellulosic ethanol production via consolidated bioprocessing at 75Â Â°C by engineered Caldicellulosiruptor bescii Chung, Daehwan; Cha, Minseok; Snyder, Elise https://doi.org/10.6084/m9.figshare.c.3617006_d1 The current record is supplemented by this text	text	January 2015