Applied Microbiology and Biotechnology

, Volume 90, Issue 3, pp 895–901 | Cite as

Glutamate production from β-glucan using endoglucanase-secreting Corynebacterium glutamicum

  • Takeyuki Tsuchidate
  • Toshihiro Tateno
  • Naoko Okai
  • Tsutomu Tanaka
  • Chiaki Ogino
  • Akihiko Kondo
Biotechnological Products and Process Engineering


We demonstrate glutamate production from β-glucan using endoglucanase (EG)-expressing Corynebacterium glutamicum. The signal sequence torA derived from Escherichia coli K12, which belongs to the Tat pathway, was suitable for secreting EG of Clostridium thermocellum using C. glutamicum as a host. Using the torA signal sequence, endoglucanase from Clostridium cellulovorans 743B was successfully expressed, and the secreted EG produced 123 mg of reducing sugar from 5 g of β-glucan at 30 °C for 72 h, which is the optimal condition for C. glutamicum growth. Subsequently, glutamate fermentation from β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae. Using EG-secreting C. glutamicum, 178 mg/l of glutamate was produced from 15 g of β-glucan. This is the first report of glutamate fermentation from β-glucan using endoglucanase-secreting C. glutamicum.


Corynebacterium glutamicum Endoglucanase Signal sequence Glutamate fermentation β-Glucan 


  1. Adachi T, Ito J, Kawata K, Kaya M, Ishida H, Sahara H, Hata Y, Ogino C, Fukuda H, Kondo A (2008) Construction of an Aspergillus oryzae cell-surface display system using a putative GPI-anchored protein. Appl Microbiol Biotechnol 81:711–719CrossRefGoogle Scholar
  2. Adham SA, Honrubia P, Díaz M, Fernández-Abalos JM, Santamaría RI, Gil JA (2001) Expression of the genes coding for the xylanase Xys1 and the cellulase Cel1 from the straw-decomposing Streptomyces halstedii JM8 cloned into the amino-acid producer Brevibacterium lactofermentum ATCC13869. Arch Microbiol 177:91–97CrossRefGoogle Scholar
  3. Berks BC, Sargent F, Palmer T (2000) The Tat protein export pathway. Mol Microbiol 35:260–274CrossRefGoogle Scholar
  4. Bourke SL, Kohn J (2003) Polymers derived from the amino acid L-tyrosine: polycarbonates, polyarylates copolymers with poly(ethylene glycol). Adv Drug Deliv Rev 25:447–466CrossRefGoogle Scholar
  5. Date M, Yokoyama K, Umezawa Y, Matsui H, Kikuchi Y (2003) Production of native-type Streptoverticillium mobaraense transglutaminase in Corynebacterium glutamicum. Appl Environ Microbiol 69:3011–3014CrossRefGoogle Scholar
  6. Fujita Y, Takahashi S, Ueda M, Tanaka A, Okada H, Morikawa Y, Kawaguchi T, Arai M, Fukuda H, Kondo A (2002) Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes. Appl Environ Microbiol 68:5136–5141CrossRefGoogle Scholar
  7. Fujita Y, Ito J, Ueda M, Fukuda H, Kondo A (2004) Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. Appl Environ Microbiol 70:1207–1212CrossRefGoogle Scholar
  8. Fukuoka T, Uyama H, Kobayashi S (2004) Polymerization of polyfunctional macromolecules: synthesis of a new class of high molecular weight poly(amino acid)s by oxidative coupling of phenol-containing precursor polymers. Biomacromolecules 5:977–983CrossRefGoogle Scholar
  9. George MG (2001) Bergey’s manual of systematic bacteriology, 2nd edn. Springer, New YorkGoogle Scholar
  10. Hermann T (2003) Industrial production of amino acids by coryneform bacteria. J Biotechnol 104:155–172CrossRefGoogle Scholar
  11. Inui M, Murakami S, Okino S, Kawaguchi H, Vertès AA, Yukawa H (2004) Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J Mol Microbiol Biotechnol 7:182–196CrossRefGoogle Scholar
  12. Katsumata R, Ozaki A, Oka T, Furuya A (1984) Protoplast transformation of glutamate-producing bacteria with plasmid DNA. J Bacteriol 159:306–311Google Scholar
  13. Kikuchi Y, Date M, Itaya H, Matsui K, Wu LF (2006) Functional analysis of the twin-arginine translocation pathway in Corynebacterium glutamicum ATCC 13869. Appl Environ Microbiol 72:7183–7192CrossRefGoogle Scholar
  14. Kikuchi Y, Itaya H, Date M, Matsui K, Wu LF (2009) TatABC overexpression improves Corynebacterium glutamicum Tat-dependent protein secretion. Appl Environ Microbiol 75:603–607CrossRefGoogle Scholar
  15. Leuchtenberger W, Huthmacher K, Drauz K (2005) Biotechnological production of amino acids and derivatives: current status and prospects. Appl Microbiol Biotechnol 69:1–8CrossRefGoogle Scholar
  16. Mimitsuka T, Sawai H, Hatsu M, Yamada K (2007) Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation. Biosci Biotechnol Biochem 71:2130–2135Google Scholar
  17. Okano K, Zhang Q, Yoshida S, Tanaka T, Ogino C, Fukuda H, Kondo A (2010) D-lactic acid production from cellooligosaccharides and β-glucan using genetically modified L-lactate dehydrogenase gene-deficient and endoglucanase-secreting Lactobacillus plantarum. Appl Microbiol Biotechnol 85:643–650CrossRefGoogle Scholar
  18. Okino S, Noburyu R, Suda M, Jojima T, Inui M, Yukawa H (2008) An efficient succinic acid production process in a metabolically engineered Corynebacterium glutamicum strain. Appl Microbiol Biotechnol 81:459–464CrossRefGoogle Scholar
  19. Paradis FW, Warren RA, Kilburn DG, Miller RC (1987) The expression of Cellulomonas fimi cellulase genes in Brevibacterium lactofermentum. Gene 61:199–206CrossRefGoogle Scholar
  20. Peyret JL, Bayan N, Joliff G, Gulik-Krzywicki T, Mathieu L, Schechter E, Leblon G (1993) Characterization of the cspB gene encoding PS2, an ordered surface-layer protein in Corynebacterium glutamicum. Mol Microbiol 9:97–109CrossRefGoogle Scholar
  21. Sakai S, Tsuchida Y, Nakamoto H, Okino S, Ichihashi O, Kawaguchi H, Watanabe T, Inui M, Yukawa H (2007) Effect of lignocellulose-derived inhibitors on growth of and ethanol production by growth-arrested Corynebacterium glutamicum R. Appl Environ Microbiol 73:2349–2353CrossRefGoogle Scholar
  22. Settles AM, Martienssen R (1998) Old and new pathways of protein export in chloroplasts and bacteria. Trends Cell Biol 8:494–501CrossRefGoogle Scholar
  23. Somogyi M (1952) Notes on sugar determination. J Biol Chem 195:19–23Google Scholar
  24. Tateno T, Fukuda H, Kondo A (2007) Direct production of L-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis alpha-amylase using cspB promoter and signal sequence. Appl Microbiol Biotechnol 77:533–541CrossRefGoogle Scholar
  25. Tateno T, Okada Y, Tsuchidate T, Tanaka T, Fukuda H, Kondo A (2009) Direct production of cadaverine from soluble starch using Corynebacterium glutamicum coexpressing α-amylase and lysine decarboxylase. Appl Microbiol Biotechnol 82:115–121CrossRefGoogle Scholar
  26. US Department of Energy (2004) Top value added chemicals from biomass, volume I—results of screening for potential candidates from sugars and synthesis gas. T. Werpy and G. Petersen, the Pacific Northwest National Laboratory (PNNL)Google Scholar
  27. Yao W, Deng X, Zhong H, Liu M, Zheng P, Sun Z, Zhang Y (2009) Double deletion of dtsR1 and pyc induce efficient L-glutamate overproduction in Corynebacterium glutamicum. J Ind Microbiol Biotechnol 36:911–921CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Takeyuki Tsuchidate
    • 1
  • Toshihiro Tateno
    • 2
  • Naoko Okai
    • 3
  • Tsutomu Tanaka
    • 3
  • Chiaki Ogino
    • 1
  • Akihiko Kondo
    • 1
  1. 1.Department of Chemical Science and Engineering, Graduate School of EngineeringKobe UniversityKobeJapan
  2. 2.Department of Molecular Science and Material Engineering, Graduate School of Science and TechnologyKobe UniversityKobeJapan
  3. 3.Organization of Advanced Science and TechnologyKobe UniversityKobeJapan

Personalised recommendations