Applied Microbiology and Biotechnology

, Volume 88, Issue 1, pp 199–208 | Cite as

Metabolic engineering to improve ethanol production in Thermoanaerobacter mathranii

  • Shuo Yao
  • Marie Just Mikkelsen
Applied Genetics and Molecular Biotechnology


Thermoanaerobacter mathranii can produce ethanol from lignocellulosic biomass at high temperatures, but its biotechnological exploitation will require metabolic engineering to increase its ethanol yield. With a cofactor-dependent ethanol production pathway in T. mathranii, it may become crucial to regenerate cofactor to increase the ethanol yield. Feeding the cells with a more reduced carbon source, such as mannitol, was shown to increase ethanol yield beyond that obtained with glucose and xylose. The ldh gene coding for lactate dehydrogenase was previously deleted from T. mathranii to eliminate an NADH oxidation pathway. To further facilitate NADH regeneration used for ethanol formation, a heterologous gene gldA encoding an NAD+-dependent glycerol dehydrogenase was expressed in T. mathranii. One of the resulting recombinant strains, T. mathranii BG1G1 (Δldh, P xyl GldA), showed increased ethanol yield in the presence of glycerol using xylose as a substrate. With an inactivated lactate pathway and expressed glycerol dehydrogenase activity, the metabolism of the cells was shifted toward the production of ethanol over acetate, hence restoring the redox balance. It was also shown that strain BG1G1 acquired the capability to utilize glycerol as an extra carbon source in the presence of xylose, and utilization of the more reduced substrate glycerol resulted in a higher ethanol yield.


Metabolic engineering Ethanol production Thermoanaerobacter Glycerol dehydrogenase Lactate dehydrogenase 



We thank Slawomir Dabrowski (A&A Biotechnology, Poland) for kindly providing the pYPA vector, Karin Marie Due for taking good care of the continuous reactor, and Klaus Breddam for scientific and linguistic proofreading of the manuscript.

Supplementary material

253_2010_2703_MOESM1_ESM.doc (113 kb)
ESM 1 (DOC 113 kb)


  1. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  2. Brinen LS, Canaves JM, Dai X, Deacon AM, Elsliger MA, Eshaghi S, Floyd R, Godzik A, Grittini C, Grzechnik SK, Guda C, Jaroszewski L, Karlak C, Klock HE, Koesema E, Kovarik JS, Kreusch A, Kuhn P, Lesley SA, McMullan D, McPhillips TM, Miller MA, Miller MD, Morse A, Moy K, Ouyang J, Robb A, Rodrigues K, Selby TL, Spraggon G, Stevens RC, van den Bedem H, Velasquez J, Vincent J, Wang X, West B, Wolf G, Taylor SS, Hodgson KO, Wooley J, Wilson IA (2003) Crystal structure of a zinc-containing glycerol dehydrogenase (TM0423) from Thermotoga maritima at 1.5 A resolution. Proteins 50:371–374CrossRefGoogle Scholar
  3. Bryant MP (1972) Commentary on the Hungate technique for culture of anaerobic bacteria. Am J Clin Nutr 25:1324–1328Google Scholar
  4. Burton RM (1955) Methods in enzymology, vol 1. Academic Press, New York, p 397CrossRefGoogle Scholar
  5. Dien BS, Cotta MA, Jeffries TW (2003) Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 63:258–266CrossRefGoogle Scholar
  6. Georgieva T, Mikkelsen MJ, Ahring BK (2008) Ethanol production from wet-exploded wheat straw hydrolysate by thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 in a continuous immobilized reactor. Appl Biochem Biotechnol 145:99–110CrossRefGoogle Scholar
  7. Germain P, Toukourou E, Donaduzzi L (1986) Ethanol production by anaerobic thermophilic bacteria: regulation of lactate dehydrogenase activity in Clostridium thermohydrosulfuricum. Appl Microbiol Biotech 24:300–305CrossRefGoogle Scholar
  8. Huber R, Langworthy TA, Köning H, Thomm M, Woese CR, Sleytr UB, Stetter KO (1986) Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90 °C. Arch Microbiol 144:324–333CrossRefGoogle Scholar
  9. Hungate RE (1969) A roll tube method for the cultivation of strict anaerobes. Meth Microbiol 3B:117–132CrossRefGoogle Scholar
  10. Ingram LO, Aldrich HC, Borges AC, Causey TB, Martinez A, Morales F, Saleh A, Underwood SA, Yomano LP, York SW, Zaldivar J, Zhou S (1999) Enteric bacterial catalysts for fuel ethanol production. Biotechnol Prog 15:855–866CrossRefGoogle Scholar
  11. Jones DT, Woods DR (1991) Clostrida. Plenum Pres, New YorkGoogle Scholar
  12. Larsen L, Nielsen P, Ahring BK (1997) Thermoanaerobacter mathranii sp. nov., an ethanol-producing, extremely thermophilic anaerobic bacterium from a hot spring in Iceland. Arch Microbiol 168:114–119CrossRefGoogle Scholar
  13. Lynd LR (1989) Production of ethanol from lignocellulosic materials using thermophilic bacteria: critical evaluation of potential and review. Adv Biochem Eng 38:1–52Google Scholar
  14. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:506–577CrossRefGoogle Scholar
  15. Mikkelsen MJ, Ahring BK (2007) Thermoanaerobacter mathranii strain BG1. WO patent application 2007134607Google Scholar
  16. Mikkelsen MJ, Yao S (2010) Increased ethanol production in recombinant bacteria. WO patent application 2010010116Google Scholar
  17. Mitchell WJ (1998) Physiology of carbohydrate to solvent conversion by clostridia. Adv Microb Physiol 39:31–130CrossRefGoogle Scholar
  18. Nielsen J, Villadsen J, Gunnar L (2003) Bioreaction engineering principles. Kluwer Academic/Plenum Publisher, New YorkGoogle Scholar
  19. Pronk JT, Kuijper SM, Toirkens MJ, Winkler R, van Dijken H, de Laat W (2005) Engineering Saccharomyces cerevisiae for xylose utilization. J Biotechnol 118:S86–S87Google Scholar
  20. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  21. Shaw AJ, Jenney FE, Adams MWW, Lynd LR (2008a) End-product pathways in the xylose fermenting bacterium, Thermoanaerobacterium saccharolyticum. Enzyme Microb Technol 42:453–458CrossRefGoogle Scholar
  22. Shaw AJ, Podkaminer KK, Desai SG, Bardsley JS, Rogers SR, Thorne PG, Hogsett DA, Lynd LR (2008b) Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. PNAS 105(37):13769–13774CrossRefGoogle Scholar
  23. Soboh B, Linder D, Hedderich R (2004) A multisubunit membrane-bound [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase in the fermenting bacterium Thermoanaerobacter tengcongensis. Microbiology 150:2451–2463CrossRefGoogle Scholar
  24. Sommer P, Georgieva T, Ahring BK (2004) Potential for using thermophilic anaerobic bacteria for bioethanol production from hemicelluloses. Biochem Soc Trans 32:283–289CrossRefGoogle Scholar
  25. Sonne-Hansen J, Mathrani IM, Ahring BK (1993) Xylanolytic anaerobic thermophiles from Icelandic hot-springs. Appl Microbiol Biotechnol 38:537–541CrossRefGoogle Scholar
  26. Taylor MP, Eley KL, Martin S, Tuffin MI, Burton SG, Cowan DA (2009) Thermophilic ethanologenesis: future prospects for second-generation bioethanol production. Trends Biotechnol 27:398–40CrossRefGoogle Scholar
  27. Tyurin MV, Desai SG, Lynd LR (2004) Electrotransformation of Clostridium thermocellum. Appl Environ Microbiol 70:883–890CrossRefGoogle Scholar
  28. Vasconcelos I, Girbal L, Soucaille P (1994) Regulation of carbon and electron flow in Clostridium acetobutylicum grown in chemostat culture at neutral pH on mixtures of glucose and glycerol. J Bacteriol 176(5):1443–1450Google Scholar
  29. Yanase H, Nozaki K, Okamoto K (2005) Ethanol production from cellulosic materials by genetically engineered Zymomonas mobilis. Biotechnol Lett 27:259–263CrossRefGoogle Scholar
  30. Yao S (2008) Metabolic engineering of ethanol production in Thermoanaerobacter mathranii BG1. PhD Thesis, Risø -Technical University of Denmark, RoskildeGoogle Scholar
  31. Yazdani SS, Gonzalez R (2007) Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr Opin Biotechnol 18:213–219CrossRefGoogle Scholar
  32. Zaldivar J, Nielsen J, Olsson L (2001) Fuel ethanol production from lignocelluloses: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol 56:17–34CrossRefGoogle Scholar
  33. Zeikus JG, Ben-Bassat A, Ng TK, Lamed RJ (1981) Thermophilic ethanol fermentations. In: Hollaender A (ed) Trends in the biology of fermentations for fuels and chemicals. Plenum Press, New York, p 441Google Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.BioScience and Technology Group, BioCentrumTechnical University of DenmarkLyngbyDenmark
  2. 2.BioGasol ApSBallerupDenmark
  3. 3.Biosystems Department, Risø National Laboratory for Sustainable EnergyTechnical University of DenmarkRoskildeDenmark

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