Sequencing of multiple clostridial genomes related to biomass conversion and biofuel production.

Abstract

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.

Full text

Page 1

Published Ahead of Print 1 October 2010.
2010, 192(24):6494. DOI: 10.1128/JB.01064-10. J. Bacteriol.
Rubin and Jizhong Zhou
Himmel, Safiyh Taghavi, Daniël van der Lelie, Edward M.
Huhnke, Jonathan R. Mielenz, Shi-You Ding, Michael E.
Dong, Defeng Xing, Nanqi Ren, Aijie Wang, Raymond L.
Stevenson, Michael J. McInerney, Yunfeng Yang, Hailiang
Fields, Adam P. Arkin, Christopher W. Schadt, Bradley S.
Ralph S. Tanner, Lee R. Lynd, Juergen Wiegel, Matthew W.
Nostrand, Bernard Henrissat, Qiang He, Paul A. Lawson,
Van Kyrpides, Natalia Mikhailova, Zhili He, Liyou Wu, Joy D.
Han, Sam Pitluck, Miriam L. Land, Loren J. Hauser, Nikos
Elizabeth Saunders, Thomas Brettin, John C. Detter, Cliff S.
Copeland, Alla Lapidus, Tijana Glavina del Rio, Hope Tice,
Gengxin Zhang, Lynne Goodwin, Susan Lucas, Alex
Christopher L. Hemme, Housna Mouttaki, Yong-Jin Lee,
and Biofuel Production
Genomes Related to Biomass Conversion
Sequencing of Multiple Clostridial
http://jb.asm.org/content/192/24/6494
Updated information and services can be found at:
These include:
REFERENCES
http://jb.asm.org/content/192/24/6494#ref-list-1at:
This article cites 20 articles, 14 of which can be accessed free
CONTENT ALERTS
more»articles cite this article),
Receive: RSS Feeds, eTOCs, free email alerts (when new
http://jb.asm.org/site/misc/reprints.xhtml
http://journals.asm.org/site/subscriptions/
Information about commercial reprint orders:
To subscribe to to another ASM Journal go to:
on March 9, 2012 by UNIV OF OKLA LIBRARIES
http://jb.asm.org/
Downloaded from

Page 2

JOURNAL OF BACTERIOLOGY, Dec. 2010, p. 6494–6496
0021-9193/10/$12.00 doi:10.1128/JB.01064-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 192, No. 24
Sequencing of Multiple Clostridial Genomes Related to Biomass
Conversion and Biofuel Production?
Christopher L. Hemme,1,2Housna Mouttaki,1,2,3Yong-Jin Lee,1,2Gengxin Zhang,14Lynne Goodwin,5
Susan Lucas,4Alex Copeland,4Alla Lapidus,4Tijana Glavina del Rio,4Hope Tice,4
Elizabeth Saunders,5Thomas Brettin,5,6John C. Detter,5Cliff S. Han,5Sam Pitluck,4
Miriam L. Land,6Loren J. Hauser,6Nikos Kyrpides,4Natalia Mikhailova,4Zhili He,1,2
Liyou Wu,1,2Joy D. Van Nostrand,1,2Bernard Henrissat,7Qiang He,8
Paul A. Lawson,1Ralph S. Tanner,1Lee R. Lynd,9Juergen Wiegel,10
Matthew W. Fields,11Adam P. Arkin,12Christopher W. Schadt,13
Bradley S. Stevenson,1Michael J. McInerney,1Yunfeng Yang,13
Hailiang Dong,14Defeng Xing,15Nanqi Ren,15Aijie Wang,15
Raymond L. Huhnke,16Jonathan R. Mielenz,17Shi-You Ding,18
Michael E. Himmel,18Safiyh Taghavi,19Danie ¨l van der Lelie,19
Edward M. Rubin,4and Jizhong Zhou1,2*
Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma1; Institute for Environmental Genomics,
University of Oklahoma, Norman, Oklahoma2; German Research Center for Environment and Health, Neuherberg,
Germany3; Joint Genome Institute Production Genomics Facility, Walnut Creek, California4; Joint Genome Institute,
Los Alamos National Laboratory, Los Alamos, New Mexico5; Computational Biology and Bioinformatics Group,
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee6; CNRS, Universite ´s Aix-Marseille,
13628 Aix-en-Provence, France7; Department of Civil and Environmental Engineering, University of
Tennessee—Knoxville, Knoxville, Tennessee8; Department of Biological Sciences, Dartmouth College,
Hanover, New Hampshire9; Department of Microbiology, University of Georgia, Athens, Georgia10;
Department of Microbiology, Montana State University, Bozeman, Montana11; University of
California—Berkeley, Berkeley, California12; Molecular Microbial Ecology Group, Biosciences Division,
Oak Ridge National Laboratory, Oak Ridge, Tennessee13; Miami University, Oxford, Ohio14;
State Key Lab of Urban Water Resource and Environment, Harbin 160090, People’s Republic of
China15; Oklahoma State University, Stillwater, Oklahoma16; Bioconversion and Science
Technology Group, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge,
Tennessee17; National Renewable Energy Laboratory, Golden, Colorado18; and
Brookhaven National Laboratory, Upton, New York19
Received 7 September 2010/Accepted 20 September 2010
Modern methods to develop microbe-based biomass conversion processes require a system-level under-
standing of the microbes involved. Clostridium species have long been recognized as ideal candidates for
processes involving biomass conversion and production of various biofuels and other industrial products.
To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have
sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall
within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacter-
aceae. Species were chosen based on representation in the experimental literature as model organisms,
ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment
hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced
strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide
a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial
systems biology.
Clostridial genomes were sequenced using a combination of
Sanger (3? coverage, 8 kb, pMCL200), 454 (20? coverage),
and Solexa methods. Standardsequencingprotocolsarelistedon
the Joint Genome Institute (JGI) website (http://www.jgi.doe.gov
/sequencing/protocols/prots_production.html). Sanger and 454
reads were assembled as previously described (5). Automatic
annotations were conducted for all draft genomes using the
JGI-Oak Ridge National Laboratory (ORNL) annotation
pipeline, and all draft genomes and annotations were loaded
into the JGI Integrated Microbial Resource (IMG) for analysis
(11). Due to difficulties in assembling and finishing low-GC,
high-repeat genomes, many of the genomes targeted for
sequencing could not be finished and are presented as a high-
* Corresponding author. Mailing address: Stephenson Research and
Technology Center, University of Oklahoma, Norman, OK 73019.
Phone: (405) 325-6073. Fax: (405) 325-7552. E-mail: jzhou@ou.edu.
?Published ahead of print on 1 October 2010.
6494
on March 9, 2012 by UNIV OF OKLA LIBRARIES
http://jb.asm.org/
Downloaded from

Page 3

quality permanent draft. Sequences available at the time of this
analysis are listed in Table 1 and are categorized based on the
latest Bergey’s taxonomy (9).
The biomass-converting clostridium sequencing project was initiated
by the Clostridium Sequencing Consortium (see author list) through
the Joint Genome Institute. The genome sequencing work was per-
formed under the auspices of the U.S. Department of Energy’s Office
of Science, Biological and Environmental Research Program, and by
the University of California, Lawrence Berkeley National Laboratory,
under contract no. DE-AC02-05CH11231, Lawrence Livermore Na-
tional Laboratory, under contract no. DE-AC52-07NA27344, Los
Alamos National Laboratory,
06NA25396, and Oak Ridge National Laboratory, under contract no.
DE-AC05-00OR22725. This work was supported by NSF-EPSC.R
grant 105118300.
under contractno.DE-AC02-
REFERENCES
1. Cook, G. M., F. A. Rainey, B. K. C. Patel, and H. W. Morgan. 1996. Char-
acterization of a new obligately anaerobic thermophile, Thermoanaer-
obacter wiegelii sp. nov. Int. J. Syst. Bacteriol. 46:123–127.
2. Freier, D., C. P. Mothershed, and J. Wiegel. 1988. Characterization of Clos-
tridium thermocellum JW20. Appl. Environ. Microbiol. 54:204–211.
3. Huhnke, R. L., R. S. Lewis, and R. S. Tanner. 27 April 2010. Isolation and
characterization of novel clostridial species. U.S. patent 7,704,723.
4. Kozianowski, G., F. Canganella, F. A. Rainey, H. Hippe, and G. Antranikian.
1997. Purification and characterization of thermostable pectate-lyases from a
newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov.
Extremophiles 1:171–182.
5. Land, M., R. Pukall, B. Abt, M. Go ¨ker, M. Rohde, T. G. D. Rio, H. Tice, A.
Copeland, J.-F. Cheng, S. Lucas, F. Chen, M. Nolan, D. Bruce, L. Goodwin,
S. Pitluck, N. Ivanova, K. Mavromatis, G. Ovchinnikova, A. Pati, A. Chen, K.
Palaniappan, L. Hauser, Y.-J. Chang, C. C. Jefferies, E. Saunders, T. Bret-
tin, J. C. Detter, C. Han, P. Chain, J. Bristow, J. A. Eisen, V. Markowitz, P.
Hugenholtz, N. C. Kyrpides, H.-P. Klenk, and A. Lapidus. 2009. Complete
genome sequence of Beutenbergia cavernae type strain (HKI 0122T). Stand.
Genomic Sci. doi:10.4056/sigs.1162.
6. Larsen, L., P. Nielsen, and B. K. Ahring. 1997. Thermoanaerobacter math-
ranii sp. nov., an ethanol-producing, extremely thermophilic anaerobic bac-
terium from a hot spring in Iceland. Arch. Microbiol. 168:114–119.
7. Lee, Y.-E., M. K. Jain, C. Lee, S. E. Lowe, and J. G. Zeikus. 1993. Taxonomic
distinction of saccharolytic thermophilic anaerobes: description of Thermo-
anaerobacterium xylanolyticum gen. nov., sp. nov., and Thermoanaerobacte-
rium saccharolyticum gen. nov., sp. nov.; reclassification of Thermoanaero-
bium brockii, Clostridiumthermosulfurogenes,
thermohydrosulfiricum ElO0-69 as Thermoanaerobacter brockii comb. nov.,
Thermoanaerobacterium thermosulfurigenes comb. nov., and Thermoanaer-
obacter thermohydrosulfuricus comb. nov., respectively; and transfer of Clos-
tridium thermohydrosulfuricum 39E to Thermoanaerobacter ethanolicus. Int. J.
Syst. Microbiol. 43:41–51.
8. Liou, J. S.-C., D. L. Balkwill, G. R. Drake, and R. S. Tanner. 2005. Clostrid-
ium carboxidivorans sp. nov., a solvent-producing clostridium isolated from
an agricultural settling lagoon, and reclassification of the acetogen Clostrid-
ium scatologenes strain SL1 as Clostridium drakei sp. nov. Int. J. Syst. Evol.
Microbiol. 55:2085–2091.
9. Ludwig, W., K. H. Schleifer, and W. B. Whitman. 2008. Revised roadmap to
the phylum Firmicutes, 2nd ed., vol. 3. Springer, New York, NY.
10. Madden, R. H., M. J. Bryder, and N. J. Poole. 1982. Isolation and charac-
terization of an anaerobic, cellulolytic bacterium, Clostridium papyrosolvens
sp. nov. Int. J. Syst. Bacteriol. 32:87–91.
11. Markowitz, V. M., I. M. A. Chen, K. Palaniappan, K. Chu, E. Szeto, Y.
Grechkin, A. Ratner, I. Anderson, A. Lykidis, K. Mavromatis, N. N. Ivanova,
and N. C. Kyrpides. 2010. The integrated microbial genomes system: an
and Clostridium
TABLE 1. Clostridium genome sequencing projects related to biomass conversion and biofuels production
Organism name Reference(s)
Genome analysis
Accession no.c
Seq.
statusa
Size (Mb)
No. of
contigs
% GC CDSb
Clostridium sensu stricto
Clostridium cellulovorans 743B
Clostridium carboxidivorans P7
Clostridium ragsdalei P11
17
3, 8
3
F
PD
P
5.2
5.5
1 31.2
29.7
4,256
5,462
CP002160
ACVI0000000050
Ruminococcaceae
Acetivibrio cellulolyticus CD2
Clostridium cellulolyticum H10
Clostridium papyrosolvens DSM 2782
Clostridium thermocellum JW20 DSM 4150
Clostridium thermocellum LQRI DSM 2360
14
15
10
2
12
D
F
PD
PD
PD
6.1
4.1
4.8
3.8
3.5
19335.5
37.0
36.9
39.0
39.1
5,146
3,390
4,425
3,077
2,914
AEDB00000000
CP001348
ACXX00000000
ABVG00000000
ACVX00000000
1
31
26
23
Thermoanaerobacteraceae
Thermoanaerobacter wiegelii Rt8.B1
Thermoanaerobacter brockii sp. finnii Ako-1
Thermoanaerobacter sp. CCSD1
Thermoanaerobacter sp. X513
Thermoanaerobacter sp. X561
Thermoanaerobacter pseudethanolicus 39E
Thermoanaerobacter sp. X514
Thermoanaerobacter italicus Ab9
Thermoanaerobacter mathranii subsp. mathranii A3
DSM 11426
Thermoanaerobacter ethanolicus JW200
Thermoanaerobacter siderophilus L-64
Thermoanaerobacterium thermosaccharolyticum
DSM 571
Thermoanaerobacterium xylanolyticum LX11
1
7, 21
22
16
16
13
16
4
6
D
PD
PD
F
PD
F
F
F
F
2.7
2.2
2.2
2.3
2.4
2.4
2.5
2.4
2.3
169
85
18
34.2
34.3
34.3
34.5
34.5
34.0
34.0
34.1
34.3
2,906
2,279
2,146
2,330
2,332
2,243
2,349
2,271
2,161
Pending
ACQZ00000000
ACXY00000000
ACPF00000000
ACXP00000000
CP000924
CP000923
CP001936
CP002032
1
8
1
1
1
1
19
18
7
D
P
F
2.2 37634.02423 ACXY00000000
2.71 34.12,161 CP002171
7D 2.5 91 34.82,509 Pending
Miscellaneous
Clostridium saccharolyticum DSM 2544
Ethanoligenens harbinense YUAN-3
7
20
F
D
4.6
3.0
1
3
45.0
55.5
4,160
2,787
CP002109
ADJQ00000000
aSequencing status: F, finished; PD, permanent high-quality draft; D, draft (ongoing); P, sequence pending.
bNo. of annotated protein coding sequences.
cGenBank accession number.
VOL. 192, 2010GENOME ANNOUNCEMENTS6495
on March 9, 2012 by UNIV OF OKLA LIBRARIES
http://jb.asm.org/
Downloaded from

Page 4

expanding comparative analysis resource. Nucleic Acids Res. 38:D382–
D390.
12. Ng, T. K., and J. G. Zeikus. 1981. Comparison of extracellular cellulase
activities of Clostridium thermocellum LQRI and Trichoderma reesei
QM9414. Appl. Environ. Microbiol. 42:231–240.
13. Onyenwoke, R. U., V. V. Kevbrin, A. M. Lysenko, and J. Wiegel. 2007.
Thermoanaerobacter pseudethanolicus sp. nov., a thermophilic heterotrophic
anaerobe from Yellowstone National Park. Int. J. Syst. Evol. Microbiol.
57:2191–2193.
14. Patel, G. B., A. W. Khan, B. J. Agnew, and J. R. Colvin. 1980. Isolation and
characterization of an anaerobic, cellulolytic microorganism, Acetivibrio cel-
lulolyticus gen. nov., sp. nov. Int. J. Syst. Bacteriol. 30:179–185.
15. Petitdemange, E., F. Caillet, J. Giallo, and C. Gaudin. 1984. Clostridium
cellulolyticum sp. nov., a cellulolytic, mesophilic species from decayed grass.
Int. J. Syst. Bacteriol. 34:155–159.
16. Roh, Y., S. V. Liu, G. Li, H. Huang, T. J. Phelps, and J. Zhou. 2002. Isolation
and characterization of metal-reducing Thermoanaerobacter strains from
deep subsurface environments of the Piceance Basin, Colorado. Appl. En-
viron. Microbiol. 68:6013–6020.
17. Sleat, R., R. A. Mah, and R. Robinson. 1984. Isolation and characterization
of an anaerobic, cellulolytic bacterium, Clostridium cellulovorans sp. nov.
Appl. Environ. Microbiol. 48:88–93.
18. Slobodkin, A. I., T. P. Tourova, B. B. Kuznetsov, N. A. Kostrikina, N. A.
Chernyh, and E. A. Bonch-Osmolovskaya. 1999. Thermoanaerobacter sid-
erophilus sp. nov., a novel dissimilatory Fe(III)-reducing, anaerobic, thermo-
philic bacterium. Int. J. Syst. Bacteriol. 49:1471–1478.
19. Wiegel, J., and L. G. Ljungdahl. 1981. Thermoanaerobacter ethanolicus gen.
nov., spec. nov., a new, extreme thermophilic, anaerobic bacterium. Arch.
Microbiol. 128:343–348.
20. Xing, D., N. Ren, Q. Li, M. Lin, A. Wang, and L. Zhao. 2006. Ethanoligenens
harbinense gen. nov., sp. nov., isolated from molasses wastewater. Int. J. Syst.
Evol. Microbiol. 56:755–760.
21. Zeikus, J. G., P. W. Hegge, and M. A. Anderson. 1979. Thermoanaerobium
brockii gen. nov. and sp. nov., a new chemoorganotrophic, caldoactive, an-
aerobic bacterium. Arch. Microbiol. 122:41–48.
22. Zhang, G., H. Dong, Z. Xu, D. Zhao, and C. Zhang. 2005. Microbial diversity
in ultra-high-pressure rocks and fluids from the Chinese Continental Scien-
tific Drilling Project in China. Appl. Environ. Microbiol. 71:3213–3227.
6496GENOME ANNOUNCEMENTS J. BACTERIOL.
on March 9, 2012 by UNIV OF OKLA LIBRARIES
http://jb.asm.org/
Downloaded from