Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously

Xiong, Wei; Reyes, Luis H.; Michener, William E.; Maness, Pin -Ching; Chou, Katherine J.

doi:10.1002/bit.26590

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously

Journal Article · Tue Apr 10 00:00:00 EDT 2018 · Biotechnology and Bioengineering

DOI:https://doi.org/10.1002/bit.26590· OSTI ID:1430817

Xiong, Wei ^[1]; Reyes, Luis H. ^[2]; Michener, William E. ^[1]; Maness, Pin -Ching ^[1]; ^[1]

National Renewable Energy Lab. (NREL), Golden, CO (United States)
National Renewable Energy Lab. (NREL), Golden, CO (United States); Univ. de los Andes, Bogota (Columbia)

Here, cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H₂ and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.

View Accepted Manuscript (DOE)

Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE; USDOE Office of Energy Efficiency and Renewable Energy (EERE), Hydrogen and Fuel Cell Technologies Program (EE-3F); USDOE Office of Energy Efficiency and Renewable Energy (EERE), NREL Laboratory Directed Research and Development (LDRD)

Grant/Contract Number:: AC36-08GO28308

OSTI ID:: 1430817

Alternate ID(s):: OSTI ID: 1432725

Report Number(s):: NREL/JA--2700-71150

Journal Information:: Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Journal Issue: 7 Vol. 115; ISSN 0006-3592

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

References (40)

Allocation of ATP to synthesis of cells and hydrolytic enzymes in cellulolytic fermentative microorganisms: Bioenergetics, kinetics, and bioprocessing van Walsum, G. Peter; Lynd, Lee R. Biotechnology and Bioengineering, Vol. 58, Issue 2-3 https://doi.org/10.1002/(SICI)1097-0290(19980420)58:2/3<316::AID-BIT31>3.0.CO;2-7	journal	April 1998
Continuous hydrogen production during fermentation of α-cellulose by the thermophillic bacterium Clostridium thermocellum Magnusson, Lauren; Cicek, Nazim; Sparling, Richard Biotechnology and Bioengineering, Vol. 102, Issue 3 https://doi.org/10.1002/bit.22092	journal	February 2009
Engineering the N -terminal end of CelA results in improved performance and growth of Caldicellulosiruptor bescii on crystalline cellulose : Engineering the Kim, Sun-Ki; Chung, Daehwan; Himmel, Michael E. Biotechnology and Bioengineering, Vol. 114, Issue 5 https://doi.org/10.1002/bit.26242	journal	March 2017
Consolidated Bioprocessing for Bioethanol Production Using Saccharomyces cerevisiae van Zyl, Willem H.; Lynd, Lee R.; den Haan, Riaan Biofuels https://doi.org/10.1007/10_2007_061	book	January 2007
The cellulosome and cellulose degradation by anaerobic bacteria Schwarz, W. H. Applied Microbiology and Biotechnology, Vol. 56, Issue 5-6 https://doi.org/10.1007/s002530100710	journal	September 2001
Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405 Carere, Carlo R.; Kalia, Vipin; Sparling, Richard Indian Journal of Microbiology, Vol. 48, Issue 2 https://doi.org/10.1007/s12088-008-0036-z	journal	June 2008
Carbon catabolite repression in bacteria: choice of the carbon source and autoregulatory limitation of sugar utilization Brückner, R. FEMS Microbiology Letters, Vol. 209, Issue 2 https://doi.org/10.1016/S0378-1097(02)00559-1	journal	April 2002
Engineered Escherichia coli capable of co-utilization of cellobiose and xylose Vinuselvi, Parisutham; Lee, Sung Kuk Enzyme and Microbial Technology, Vol. 50, Issue 1 https://doi.org/10.1016/j.enzmictec.2011.10.001	journal	January 2012
Biohydrogen as a renewable energy resource—Prospects and potentials Meherkotay, S.; Das, D. International Journal of Hydrogen Energy, Vol. 33, Issue 1 https://doi.org/10.1016/j.ijhydene.2007.07.031	journal	January 2008
Hydrogen production from cellulose in a two-stage process combining fermentation and electrohydrogenesis Lalaurette, Elodie; Thammannagowda, Shivegowda; Mohagheghi, Ali International Journal of Hydrogen Energy, Vol. 34, Issue 15 https://doi.org/10.1016/j.ijhydene.2009.05.112	journal	August 2009
Biocommodity Engineering Lynd, L. R.; Wyman, C. E.; Gerngross, T. U. Biotechnology Progress, Vol. 15, Issue 5 https://doi.org/10.1021/bp990109e	journal	October 1999
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
Carbon catabolite repression in bacteria: many ways to make the most out of nutrients Görke, Boris; Stülke, Jörg Nature Reviews Microbiology, Vol. 6, Issue 8, p. 613-624 https://doi.org/10.1038/nrmicro1932	journal	August 2008
Cellulosomes: plant-cell-wall-degrading enzyme complexes Doi, Roy H.; Kosugi, Akihiko Nature Reviews Microbiology, Vol. 2, Issue 7 https://doi.org/10.1038/nrmicro925	journal	July 2004
Crotonylation of key metabolic enzymes regulates carbon catabolite repression in Streptomyces roseosporus Sun, Chen-Fan; Xu, Wei-Feng; Zhao, Qing-Wei Communications Biology, Vol. 3, Issue 1 https://doi.org/10.1038/s42003-020-0924-2	journal	April 2020
Engineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic biofuel production Liu, Zhuo; Ho, Shih-Hsin; Sasaki, Kengo Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep24550	journal	April 2016
DMR (deacetylation and mechanical refining) processing of corn stover achieves high monomeric sugar concentrations (230 g L ⁻¹ ) during enzymatic hydrolysis and high ethanol concentrations (>10% v/v) during fermentation without hydrolysate purification or concentration Chen, Xiaowen; Kuhn, Erik; Jennings, Edward W. Energy & Environmental Science, Vol. 9, Issue 4 https://doi.org/10.1039/C5EE03718B	journal	January 2016
Cellulose utilization by Clostridium thermocellum: Bioenergetics and hydrolysis product assimilation Zhang, Y. -H. P.; Lynd, L. R. Proceedings of the National Academy of Sciences, Vol. 102, Issue 20 https://doi.org/10.1073/pnas.0408734102	journal	May 2005
Deletion of the Cel48S cellulase from Clostridium thermocellum Olson, Daniel G.; Tripathi, Shital A.; Giannone, Richard J. Proceedings of the National Academy of Sciences, Vol. 107, Issue 41, p. 17727-17732 https://doi.org/10.1073/pnas.1003584107	journal	September 2010
Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation Ha, S. -J.; Galazka, J. M.; Rin Kim, S. Proceedings of the National Academy of Sciences, Vol. 108, Issue 2 https://doi.org/10.1073/pnas.1010456108	journal	December 2010
Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factors Nataf, Y.; Bahari, L.; Kahel-Raifer, H. Proceedings of the National Academy of Sciences, Vol. 107, Issue 43 https://doi.org/10.1073/pnas.1012175107	journal	October 2010
Induction of lignocellulose-degrading enzymes in Neurospora crassa by cellodextrins Znameroski, E. A.; Coradetti, S. T.; Roche, C. M. Proceedings of the National Academy of Sciences, Vol. 109, Issue 16 https://doi.org/10.1073/pnas.1118440109	journal	April 2012
CO ₂ -fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum Xiong, Wei; Lin, Paul P.; Magnusson, Lauren Proceedings of the National Academy of Sciences, Vol. 113, Issue 46 https://doi.org/10.1073/pnas.1605482113	journal	October 2016
Carbon catabolite repression in bacteria: choice of the carbon source and autoregulatory limitation of sugar utilization BrÃ¼ckner, Reinhold; Titgemeyer, Fritz FEMS Microbiology Letters, Vol. 209, Issue 2 https://doi.org/10.1111/j.1574-6968.2002.tb11123.x	journal	April 2002
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
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
Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose Higashide, Wendy; Li, Yongchao; Yang, Yunfeng Applied and Environmental Microbiology, Vol. 77, Issue 8 https://doi.org/10.1128/AEM.02454-10	journal	March 2011
Cellodextrin and Laminaribiose ABC Transporters in Clostridium thermocellum Nataf, Y.; Yaron, S.; Stahl, F. Journal of Bacteriology, Vol. 191, Issue 1 https://doi.org/10.1128/JB.01190-08	journal	October 2008
Complete Genome Sequences for the Anaerobic, Extremely Thermophilic Plant Biomass-Degrading Bacteria Caldicellulosiruptor hydrothermalis, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor kronotskyensis, Caldicellulosiruptor owensensis, and Caldicellulosiruptor lactoaceticus Blumer-Schuette, S. E.; Ozdemir, I.; Mistry, D. Journal of Bacteriology, Vol. 193, Issue 6, p. 1483-1484 https://doi.org/10.1128/JB.01515-10	journal	January 2011
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
Cellulase, Clostridia, and Ethanol Demain, A. L.; Newcomb, M.; Wu, J. H. D. Microbiology and Molecular Biology Reviews, Vol. 69, Issue 1, p. 124-154 https://doi.org/10.1128/MMBR.69.1.124-154.2005	journal	March 2005
Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose Higashide, Wendy; Li, Yongchao; Yang, Yunfeng Applied and Environmental Microbiology, Vol. 78, Issue 19 https://doi.org/10.1128/aem.02335-12	journal	September 2012
Relationship of cellulosomal and noncellulosomal xylanases of Clostridium thermocellum to cellulose-degrading enzymes. Morag, E.; Bayer, E. A.; Lamed, R. Journal of Bacteriology, Vol. 172, Issue 10 https://doi.org/10.1128/jb.172.10.6098-6105.1990	journal	October 1990
Microbial Cellulose Utilization: Fundamentals and Biotechnology Lynd, L. R.; Weimer, P. J.; van Zyl, W. H. Microbiology and Molecular Biology Reviews, Vol. 66, Issue 4, p. 739-739 https://doi.org/10.1128/mmbr.66.4.739.2002	journal	December 2002
The Cellulosomes: Multienzyme Machines for Degradation of Plant Cell Wall Polysaccharides Bayer, Edward A.; Belaich, Jean-Pierre; Shoham, Yuval Annual Review of Microbiology, Vol. 58, Issue 1 https://doi.org/10.1146/annurev.micro.57.030502.091022	journal	October 2004
Carbon catabolite repression in Thermoanaerobacterium saccharolyticum Tsakraklides, Vasiliki; Shaw, A.; Miller, Bethany B. Biotechnology for Biofuels, Vol. 5, Issue 1 https://doi.org/10.1186/1754-6834-5-85	journal	January 2012
Transcriptomic and proteomic analyses of core metabolism in Clostridium termitidis CT1112 during growth on α-cellulose, xylan, cellobiose and xylose Munir, Riffat I.; Spicer, Victor; Krokhin, Oleg V. BMC Microbiology, Vol. 16, Issue 1 https://doi.org/10.1186/s12866-016-0711-x	journal	May 2016
Impact of Pretreated Switchgrass and Biomass Carbohydrates on Clostridium thermocellum ATCC 27405 Cellulosome Composition: A Quantitative Proteomic Analysis Raman, Babu; Pan, Chongle; Hurst, Gregory B. PLoS ONE, Vol. 4, Issue 4, Article No. e5271 https://doi.org/10.1371/journal.pone.0005271	journal	April 2009
Dilute-Sulfuric Acid Pretreatment of Corn Stover in Pilot-Scale Reactor: Investigation of Yields, Kinetics, and Enzymatic Digestibilities of Solids Schell, Daniel J.; Farmer, Jody; Newman, Millie Applied Biochemistry and Biotechnology, Vol. 105, Issue 1-3, p. 69-86 https://doi.org/10.1385/ABAB:105:1-3:69	journal	January 2003
Enzymatic hydrolysis of cellulosic biomass Yang, Bin; Dai, Ziyu; Ding, Shi-You Biofuels, Vol. 2, Issue 4 https://doi.org/10.4155/bfs.11.116	journal	July 2011

Cited By (3)

Isotope-Assisted Metabolite Analysis Sheds Light on Central Carbon Metabolism of a Model Cellulolytic Bacterium Clostridium thermocellum Xiong, Wei; Lo, Jonathan; Chou, Katherine J. Frontiers in Microbiology, Vol. 9 https://doi.org/10.3389/fmicb.2018.01947	journal	August 2018
Clostridial whole cell and enzyme systems for hydrogen production: current state and perspectives Latifi, Amel; Avilan, Luisana; Brugna, Myriam Applied Microbiology and Biotechnology, Vol. 103, Issue 2 https://doi.org/10.1007/s00253-018-9514-3	journal	November 2018
Consolidated bioprocessing for butanol production of cellulolytic Clostridia: development and optimization Wen, Zhiqiang; Li, Qi; Liu, Jinle Microbial Biotechnology, Vol. 13, Issue 2 https://doi.org/10.1111/1751-7915.13478	journal	August 2019

Similar Records

Engineered xylose utilization enhances bio-products productivity in the cyanobacterium Synechocystis sp. PCC 6803

Journal Article · Fri Jun 12 00:00:00 EDT 2015 · Metabolic Engineering · OSTI ID:1225916

Simultaneous bioconversion of cellulose and hemicellulose to ethanol

Journal Article · Wed Dec 30 23:00:00 EST 1998 · CRC Critical Reviews in Biotechnology · OSTI ID:318614

Ethanol production by thermophilic bacteria: fermentation of cellulosic substrates by cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum

Journal Article · Mon Jun 01 00:00:00 EDT 1981 · Appl. Environ. Microbiol.; (United States) · OSTI ID:6298673

Related Subjects

09 BIOMASS FUELS
Clostridium thermocellum
biohydrogen
consolidated bioprocessing (CBP)
lignocellulose
thermophile
xylose

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously

Citation Formats

References (40)

Cited By (3)

Similar Records

Related Subjects