AiM> Vol.3 No.1, March 2013

Metabolic Engineering of Thermoanaerobacterium thermosaccharolyticum for Increased n-Butanol Production

DownloadDownload as PDF (Size:522KB) Full-Text HTML PP. 46-51   DOI: 10.4236/aim.2013.31007


Thermoanaerobacterium thermosaccharolyticum shows promise as a host for n-butanol production since it natively has the required genes involved in the n-butanol biosynthetic pathway. Overexpression of the natively occurring bcs operon containing the genes thl, hbd, crt, bcd, etfA, and etfB responsible for the formation of butyryl CoA increased the n-butanol production by 180% compared to the wild type from a n-butanol titer of 1.8 mM to 5.1 mM. The deletion of one of the six alcohol dehydrogenase genes confirmed that it was the primary gene responsible for ethanol and n-butanol production from acetyl CoA and butyryl CoA respectively.


Cite this paper

A. Bhandiwad, A. Guseva and L. Lynd, "Metabolic Engineering of Thermoanaerobacterium thermosaccharolyticum for Increased n-Butanol Production," Advances in Microbiology, Vol. 3 No. 1, 2013, pp. 46-51. doi: 10.4236/aim.2013.31007.


[1] D. T. Jones and D. R. Woods, “Acetone-Butanol Fermentation Revisited,” Microbiology Review, Vol. 50, No. 4, 1986, pp. 484-524.
[2] P. Durre, “Fermentative Butanol Production: Bulk Chemical and Biofuel,” Annals of the New York Academy of Sciences, Vol. 1125, 2008, pp. 353-362.
[3] S. Atsumi, A. F. Cann, M. R. Connor, C. R. Shen, K. M. Smith, M. P. Brynildsen, K. J. Chou, T. Hanai and J. C. Liao, “Metabolic Engineering of Escherichia coli for 1-Butanol Production,” Metabolic Engineering, Vol. 10, No. 6, 2008, pp. 305-311. doi:10.1016/j.ymben.2007.08.003
[4] M. Inui, M. Suda, S. Kimura, K. Yasuda, H. Suzuki, H. Toda, S. Yamamoto, S. Okino, N. Suzuki and H. Yukawa, “Expression of Clostridium Acetobutylicum Butanol Synthetic Genes in Escherichia coli,” Applied and Environmental Microbiology, Vol. 77, No. 6, 2008, pp. 1305-1316. doi:10.1007/s00253-007-1257-5
[5] D. R. Nielsen, E. Leonard, S. H. Yoon, H. C. Tseng, C. Yuan and K. L. Prather, “Engineering Alternative Butanol Production Platforms in Heterologous Bacteria,” Metabolic Engineering, Vol. 11, No. 4-5, 2009, pp. 262-273. doi:10.1016/j.ymben.2009.05.003
[6] B. B. Bond-Watts, R. J. Bellerose and M. C. Y. Chang, “Enzyme Mechanism as a Kinetic Control Element for Designing Synthetic Biofuel Pathways,” Nature Chemical Biology, Vol. 7, No. 4, 2011, pp. 222-227. doi:10.1038/nchembio.537
[7] L. D. Mermelstein, E. T. Papoutsakis, D. J. Petersen and G. N. Bennett, “Metabolic Engineering of Clostridium Acetobutylicum ATCC 824 for Increased Solvent Production by Enhancement of Acetone Formation Enzyme Activities Using a Synthetic Acetone Operon,” Biotechnology and Bioengineering, Vol. 42, No. 9, 1993, pp. 1053-1060. doi:10.1002/bit.260420906
[8] E. T. Papoutsakis, “Engineering Solventogenic Clostridia,” Current Opinion in Biotechnology, Vol. 19, No. 5, 2008, pp. 420-429. doi:10.1016/j.copbio.2008.08.003
[9] T. Lutke-Eversloh and H. Bahl, “Metabolic Engineering of Clostridium Acetobutylicum: Recent Advances to Improve Butanol Production,” Current Opinion in Biotechnology, Vol. 22, No. 5, 2011, pp. 634-647. doi:10.1016/j.copbio.2011.01.011
[10] R. Sillers, A. Chow, B. Tracy and E. T. Papoutsakis, “Metabolic Engineering of the Non-Sporulating, Non-Solventogenic Clostridium Acetobutylicum Strain M5 to Produce Butanol without Acetone Demonstrate the Robustness of the Acid-Formation Pathways and the Importance of the Electron Balance,” Metabolic Engineering, Vol. 10, No. 6, 2008, pp. 321-332. doi:10.1016/j.ymben.2008.07.005
[11] R. Sillers, M. A. Al-Hinai and E. T. Papoutsakis, “Aldehyde-Alcohol Dehydrogenase and/or Thiolase Over-Expression Coupled with CoA Transferase Down Regulation Lead to Higher Alcohol Titers and Selectivity in Clostridium Acetobutylicum Fermentations,” Biotechnology and Bioengineering, Vol. 102, No. 1, 2009, pp. 38-49. doi:10.1002/bit.22058
[12] J. G. Zeikus, “Thermophilic Bacteria—Ecology, Physio logy and Technology,” Enzyme and Microbial Technology, Vol. 1, No. 4, 1979, pp. 243-252. doi:10.1016/0141-0229(79)90043-7
[13] L. S. McClung, “Studies on Anaerobic Bacteria: III. Historical Review and Technique of Culture of Certain Thermophilic Anaerobes,” Journal of Bacteriology, Vol. 29, No. 2, 1935, pp. 173-187.
[14] L. S. McClung, “Studies on Anaerobic Bacteria: IV. Taxonomy of Cultures of a Thermophilic Species Causing ‘Swells’ of Canned Foods,” Journal of Bacteriology, Vol. 29, No. 2, 1935, pp. 189-203.
[15] D. Freierschroder, J. Wiegel and G. Gottschalk, “Butanol Formation by Clostridium-Thermosaccharolyticum at Neutral Ph,” Biotechnology Letters, Vol. 11, No. 11, 1989, pp. 831-836. doi:10.1007/BF01026107
[16] J. N. Saddler and M. K. H. Chan, “Conversion of Pretreated Lignocellulosic Substrates to Ethanol by Clostridium Thermocellum in Monoand Co-Culture with Clostridium Thermosaccharolyticum and Clostridium Thermohydrosulphuricum,” Canadian Journal of Microbiology, Vol. 30, No. 2, 1984, pp. 212-220. doi:10.1139/m84-032
[17] A. L. Demain, M. Newcomb and J. H. Wu, “Cellulase, Clostridia, and Ethanol,” Microbiology and Molecular Biology Reviews, Vol. 69, No. 1, 2005, pp. 124-154. doi:10.1128/MMBR.69.1.124-154.2005
[18] N. O. Sjolander, “Studies on Anaerobic Bacteria: XII. The Fermentation Products of Clostridium Thermosaccharolyticum,” Journal of Bacteriology, Vol. 34, No. 4, 1937, pp. 419-428.
[19] C. L. Hemme, H. Mouttaki, Y. J. Lee, G. Zhang, L. Goodwin, S. Lucas, A. Copeland, A. Lapidus, T. Glavina del Rio, H. Tice, E. Saunders, T. Brettin, J. C. Detter, C. S. Han, S. Pitluck, M. L. Land, L. J. Hauser, N. Kyrpides, N. Mikhailova, Z. He, L. Wu, J. D. Van Nostrand, B. Henrissat, Q. He, P. A. Lawson, R. S. Tanner, L. R. Lynd, J. Wiegel, M. W. Fields, A. P. Arkin, C. W. Schadt, B. S. Stevenson, M. J. McInerney, Y. Yang, H. Dong, D. Xing, N. Ren, A. Wang, R. L. Huhnke, J. R. Mielenz, S. Y. Ding, M. E. Himmel, S. Taghavi, D. van der Lelie, E. M. Rubin and J. Zhou, “Sequencing of Multiple Clostridial Genomes Related to Biomass Conversion and Biofuel Production,” Journal of Bacteriology, Vol. 192, No. 24, 2010, pp. 6494-6496. doi:10.1128/JB.01064-10
[20] S. Yao and M. J. Mikkelsen, “Identification and Over-Expression of a Bifunctional Aldehyde/Alcohol Dehydrogenase Responsible for Ethanol Production in Thermoanaerobacter Mathranii,” Journal of Molecular Microbiology and Biotechnology, Vol. 19, No. 3, 2010, pp. 123-133. doi:10.1159/000321498
[21] Y. Zhang, M. Yu and S. T. Yang, “Effects of ptb KnockOut on Butyric Acid Fermentation by Clostridium Tyrobutyricum,” Biotechnology Progress, Vol. 28, No. 1, 2012, pp. 52-59. doi:10.1002/btpr.730
[22] X. Liu, Y. Zhu and S. T. Yang, “Construction and Characterization of Ack Deleted Mutant of Clostridium Tyrobutyricum for Enhanced Butyric Acid and Hydrogen Production,” Biotechnology Progress, Vol. 22, No. 5, 2006, pp. 1265-1275. doi:10.1021/bp060082g
[23] C. Zhang, H. Yang, F. Yang and Y. Ma, “Current Progress on Butyric Acid Production by Fermentation,” Current Microbiology, Vol. 59, No. 6, 2009, pp. 656-663. doi:10.1007/s00284-009-9491-y
[24] A. J. Shaw, D. A. Hogsett and L. R. Lynd, “Natural Competence in Thermoanaerobacter and Thermoanaerobacterium Species,” Applied and Environmental Microbiology, Vol. 76, No. 14, 2010, pp. 4713-4719. doi:10.1128/AEM.00402-10
[25] A. J. Shaw, S. F. Covalla, D. A. Hogsett and C. D. Herring, “Marker Removal System for Thermoanaerobacterium Saccharolyticum and Development of a Markerless Ethanologen,” Applied and Environmental Microbiology, Vol. 77, No. 7, 2011, pp. 2534-2536. doi:10.1128/AEM.01731-10
[26] D. G. Gibson, L. Young, R. Y. Chuang, J. C. Venter, C. A. Hutchison and H. O. Smith, “Enzymatic Assembly of DNA Molecules Up to Several Hundred Kilobases,” Nature Methods, Vol. 6, No. 5, 2009, pp. 343-U41. doi:10.1038/nmeth.1318
[27] V. Mai, W. W. Lorenz and J. Wiegel, “Transformation of Thermoanaerobacterium sp. Strain JW/SL-YS485 with Plasmid pIKM1 Conferring Kanamycin Resistance,” FEMS Microbiology Letters, Vol. 148, No. 2, 1997, pp. 163-167. doi:10.1111/j.1574-6968.1997.tb10283.x

comments powered by Disqus

Copyright © 2014 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.