Applied Microbiology and Biotechnology

, Volume 92, Issue 5, pp 985–996 | Cite as

Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing Corynebacterium glutamicum

  • Vipin Gopinath
  • Tobias M. Meiswinkel
  • Volker F. WendischEmail author
  • K. Madhavan NampoothiriEmail author
Applied genetics and molecular biotechnology


Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the l-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more l-glutamate and l-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM l-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM l-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.


Corynebacterium glutamicum Amino acid production Lignocellulosic hydrolysates Pentose utilization Renewables Metabolic engineering 



Work in the laboratories of the corresponding authors was supported in part by grants from the Department of Biotechnology (DBT), New Delhi, India, and from the International Bureau (IB) of the Federal Ministry of Education and Research (BMBF), Germany (IND 07/030) under the Indo-German bilateral program.

Supplementary material

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  1. Aristidou A, Penttila M (2000) Metabolic engineering applications to renewable resource utilization. Curr Opin Biotechnol 11(2):187–198CrossRefGoogle Scholar
  2. Arndt A, Auchter M, Ishige T, Wendisch VF, Eikmanns BJ (2008) Ethanol catabolism in Corynebacterium glutamicum. J Mol Microbiol Biotechnol 15(4):222–233CrossRefGoogle Scholar
  3. Arndt A, Eikmanns BJ (2008) Regulation of carbon metabolism in Corynebacterium glutamicum. In: Burkovski A (ed) Corynebacteria: genomics and molecular biology. Caister Academic, Wymondham, pp 155–182Google Scholar
  4. Barrett E, Stanton C, Zelder O, Fitzgerald G, Ross RP (2004) Heterologous expression of lactose- and galactose-utilizing pathways from lactic acid bacteria in Corynebacterium glutamicum for production of lysine in whey. Appl Environ Microbiol 70(5):2861–2866CrossRefGoogle Scholar
  5. Becker J, Boles E (2003) A modified Saccharomyces cerevisiae strain that consumes L-arabinose and produces ethanol. Appl Environ Microbiol 69(7):4144–4150CrossRefGoogle Scholar
  6. Blombach B, Riester T, Wieschalka S, Ziert C, Youn JW, Wendisch VF, Eikmanns BJ (2011) Corynebacterium glutamicum tailored for efficient isobutanol production. Appl Environ Microbiol 77(10):3300–3310CrossRefGoogle Scholar
  7. Brabetz W, Liebl W, Schleifer KH (1991) Studies on the utilization of lactose by Corynebacterium glutamicum, bearing the lactose operon of Escherichia coli. Arch Microbiol 155(6):607–612CrossRefGoogle Scholar
  8. Buschke N, Schroder H, Wittmann C (2011) Metabolic engineering of Corynebacterium glutamicum for production of 1,5-diaminopentane from hemicellulose. Biotechnol J 6(3):306–317CrossRefGoogle Scholar
  9. Cadenas RF, Gil JA, Martin JF (1992) Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans. Appl Microbiol Biotechnol 38(3):362–369CrossRefGoogle Scholar
  10. Cerdeno-Tarraga AM, Efstratiou A, Dover LG, Holden MT, Pallen M, Bentley SD, Besra GS, Churcher C, James KD, De Zoysa A, Chillingworth T, Cronin A, Dowd L, Feltwell T, Hamlin N, Holroyd S, Jagels K, Moule S, Quail MA, Rabbinowitsch E, Rutherford KM, Thomson NR, Unwin L, Whitehead S, Barrell BG, Parkhill J (2003) The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129. Nucleic Acids Res 31(22):6516–6523CrossRefGoogle Scholar
  11. Dominguez H, Cocaign-Bousquet M, Lindley ND (1997) Simultaneous consumption of glucose and fructose from sugar mixtures during batch growth of Corynebacterium glutamicum. Appl Microbiol Biot 47(5):600–603CrossRefGoogle Scholar
  12. Dominguez H, Rollin C, Guyonvarch A, Guerquin-Kern JL, Cocaign-Bousquet M, Lindley ND (1998) Carbon-flux distribution in the central metabolic pathways of Corynebacterium glutamicum during growth on fructose. Eur J Biochem 254(1):96–102CrossRefGoogle Scholar
  13. Eggeling L, Bott M (2005) Handbook of Corynebacterium glutamicum. CRC, USAGoogle Scholar
  14. Eggeling L, Reyes O (2005) Experiments. In: Eggeling L, Bott M (eds) Handbook of Corynebacterium glutamicum. CRC Press, USA, pp 3535–3566Google Scholar
  15. Eikmanns BJ (2005) Central metabolism: tricarboxylic acid cycle and anaplerotic reactions. In: Eggeling L, Bott M (eds) Handbook on Corynebacterium glutamicum. CRC Press, USA, pp 241–276Google Scholar
  16. Eikmanns BJ, Thum-Schmitz N, Eggeling L, Lüdtke KU, Sahm H (1994) Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. Microbiology 140(Pt 8):1817–1828CrossRefGoogle Scholar
  17. Engels V, Georgi T, Wendisch VF (2008) ScrB (Cg2927) is a sucrose-6-phosphate hydrolase essential for sucrose utilization by Corynebacterium glutamicum. FEMS Microbiol Lett 289(1):80–89CrossRefGoogle Scholar
  18. Frunzke J, Engels V, Hasenbein S, Gatgens C, Bott M (2008) Co-ordinated regulation of gluconate catabolism and glucose uptake in Corynebacterium glutamicum by two functionally equivalent transcriptional regulators, GntR1 and GntR2. Mol Microbiol 67(2):305–322CrossRefGoogle Scholar
  19. Georgi T, Rittmann D, Wendisch VF (2005) Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose: roles of malic enzyme and fructose-1,6-bisphosphatase. Metab Eng 7(4):291–301CrossRefGoogle Scholar
  20. Gerstmeir R, Wendisch VF, Schnicke S, Ruan H, Farwick M, Reinscheid D, Eikmanns BJ (2003) Acetate metabolism and its regulation in Corynebacterium glutamicum. J Biotechnol 104(1–3):99–122CrossRefGoogle Scholar
  21. Hahn-Hagerdal B, Karhumaa K, Fonseca C, Spencer-Martins I, Gorwa-Grauslund MF (2007a) Towards industrial pentose-fermenting yeast strains. Appl Microbiol Biotechnol 74(5):937–953CrossRefGoogle Scholar
  22. Hahn-Hagerdal B, Karhumaa K, Jeppsson M, Gorwa-Grauslund MF (2007b) Metabolic engineering for pentose utilization in Saccharomyces cerevisiae. Adv Biochem Eng Biotechnol 108:147–177Google Scholar
  23. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166(4):557–580CrossRefGoogle Scholar
  24. Heer D, Sauer U (2008) Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain. Microb Biotechnol 1(6):497–506CrossRefGoogle Scholar
  25. Jeffries TW, Jin YS (2000) Ethanol and thermotolerance in the bioconversion of xylose by yeasts. Adv Appl Microbiol 47:221–268CrossRefGoogle Scholar
  26. Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, Dusch N, Eggeling L, Eikmanns BJ, Gaigalat L, Goesmann A, Hartmann M, Huthmacher K, Kramer R, Linke B, McHardy AC, Meyer F, Mockel B, Pfefferle W, Puhler A, Rey DA, Ruckert C, Rupp O, Sahm H, Wendisch VF, Wiegrabe I, Tauch A (2003) The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. J Biotechnol 104(1–3):5–25CrossRefGoogle Scholar
  27. Karhumaa K, Wiedemann B, Hahn-Hagerdal B, Boles E, Gorwa-Grauslund MF (2006) Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains. Microb Cell Fact 5:18CrossRefGoogle Scholar
  28. Kato O, Youn JW, Stansen KC, Matsui D, Oikawa T, Wendisch VF (2010) Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate. BMC Microbiol 10:321CrossRefGoogle Scholar
  29. Kawaguchi H, Sasaki M, Vertes AA, Inui M, Yukawa H (2008) Engineering of an L-arabinose metabolic pathway in Corynebacterium glutamicum. Appl Microbiol Biotechnol 77(5):1053–1062CrossRefGoogle Scholar
  30. Kawaguchi H, Sasaki M, Vertes AA, Inui M, Yukawa H (2009) Identification and functional analysis of the gene cluster for L-arabinose utilization in Corynebacterium glutamicum. Appl Environ Microbiol 75(11):3419–3429CrossRefGoogle Scholar
  31. Kawaguchi H, Vertes AA, Okino S, Inui M, Yukawa H (2006) Engineering of a xylose metabolic pathway in Corynebacterium glutamicum. Appl Environ Microbiol 72(5):3418–3428CrossRefGoogle Scholar
  32. Kiefer P, Heinzle E, Wittmann C (2002) Influence of glucose, fructose and sucrose as carbon sources on kinetics and stoichiometry of lysine production by Corynebacterium glutamicum. J Industrial Microbiol Biotechnol 28:338–343CrossRefGoogle Scholar
  33. Kinoshita S, Udaka S, Shimono M (1957) Studies on the amino acid fermentation. Production of L-glutamic acid by various microorganisms. J Gen Appl Microbiol 3:193–205CrossRefGoogle Scholar
  34. Klinke HB, Thomsen AB, Ahring BK (2004) Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 66(1):10–26CrossRefGoogle Scholar
  35. Koenig K, Andreesen JR (1990) Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition. J Bacteriol 172(10):5999–6009Google Scholar
  36. Koopman F, Wierckx N, de Winde JH, Ruijssenaars HJ (2010) Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degradation pathways of Cupriavidus basilensis HMF14. Proc Natl Acad Sci USA 107(11):4919–4924CrossRefGoogle Scholar
  37. Kotrba P, Inui M, Yukawa H (2003) A single V317A or V317M substitution in enzyme II of a newly identified beta-glucoside phosphotransferase and utilization system of Corynebacterium glutamicum R extends its specificity towards cellobiose. Microbiology 149(Pt 6):1569–1580CrossRefGoogle Scholar
  38. Krämer R, Lambert C, Hoischen C, Ebbighausen H (1990) Uptake of glutamate in Corynebacterium glutamicum. 1. Kinetic properties and regulation by internal pH and potassium. Eur J Biochem 194(3):929–935CrossRefGoogle Scholar
  39. Krause FS, Blombach B, Eikmanns BJ (2010) Metabolic engineering of Corynebacterium glutamicum for 2-ketoisovalerate production. Appl Environ Microbiol 76(24):8053–8061CrossRefGoogle Scholar
  40. Krings E, Krumbach K, Bathe B, Kelle R, Wendisch VF, Sahm H, Eggeling L (2006) Characterization of myo-inositol utilization by Corynebacterium glutamicum: the stimulon, identification of transporters, and influence on L-lysine formation. J Bacteriol 188(23):8054–8061CrossRefGoogle Scholar
  41. Kronemeyer W, Peekhaus N, Kramer R, Sahm H, Eggeling L (1995) Structure of the gluABCD cluster encoding the glutamate uptake system of Corynebacterium glutamicum. J Bacteriol 177(5):1152–1158Google Scholar
  42. Lee HW, Pan JG, Lebeault JM (1998) Enhanced L-lysine production in threonine-limited continuous culture of Corynebacterium glutamicum by using gluconate as a secondary carbon source with glucose. Appl Microbiol Biotechnol 49(1):9–15CrossRefGoogle Scholar
  43. Li Y, Park JY, Shiroma R, Tokuyasu K (2011) Bioethanol production from rice straw by a sequential use of Saccharomyces cerevisiae and Pichia stipitis with heat inactivation of Saccharomyces cerevisiae cells prior to xylose fermentation. J Biosci Bioeng 111(6):682–686CrossRefGoogle Scholar
  44. Lin ECC (1996) Dissimilatory pathways for sugars, polyols and carboxylates. In: Neidhardt FC, Curtiss R III, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella: cellular and molecular biology, 2nd edn. ASM, Washington, pp 307–342Google Scholar
  45. Lindner SN, Knebel S, Pallerla SR, Schoberth SM, Wendisch VF (2010) Cg2091 encodes a polyphosphate/ATP-dependent glucokinase of Corynebacterium glutamicum. Appl Microbiol Biotechnol 87(2):703–713CrossRefGoogle Scholar
  46. Lindner SN, Seibold GM, Henrich A, Kramer R, Wendisch VF (2011) Phosphotransferase system-independent glucose utilization in Corynebacterium glutamicum by inositol permeases and glucokinases. Appl Environ Microbiol 77(11):3571–3581CrossRefGoogle Scholar
  47. Mimitsuka T, Sawai H, Hatsu M, Yamada K (2007) Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation. Biosci Biotechnol Biochem 71(9):2130–2135CrossRefGoogle Scholar
  48. Moon MW, Kim HJ, Oh TK, Shin CS, Lee JS, Kim SJ, Lee JK (2005) Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicum ATCC 13032. FEMS Microbiol Lett 244(2):259–266CrossRefGoogle Scholar
  49. Nishio Y, Nakamura Y, Kawarabayasi Y, Usuda Y, Kimura E, Sugimoto S, Matsui K, Yamagishi A, Kikuchi H, Ikeo K, Gojobori T (2003) Comparative complete genome sequence analysis of the amino acid replacements responsible for the thermostability of Corynebacterium efficiens. Genome Res 13(7):1572–1579CrossRefGoogle Scholar
  50. Palmqvist E, Grage H, Meinander NQ, Hahn-Hagerdal B (1999) Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts. Biotechnol Bioeng 63(1):46–55CrossRefGoogle Scholar
  51. Peters-Wendisch PG, Schiel B, Wendisch VF, Katsoulidis E, Mockel B, Sahm H, Eikmanns BJ (2001) Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. J Mol Microbiol Biotechnol 3(2):295–300Google Scholar
  52. Polen T, Kramer M, Bongaerts J, Wubbolts M, Wendisch VF (2005) The global gene expression response of Escherichia coli to L-phenylalanine. J Biotechnol 115(3):221–237CrossRefGoogle Scholar
  53. Radmacher E, Stansen KC, Besra GS, Alderwick LJ, Maughan WN, Hollweg G, Sahm H, Wendisch VF, Eggeling L (2005) Ethambutol, a cell wall inhibitor of Mycobacterium tuberculosis, elicits L-glutamate efflux of Corynebacterium glutamicum. Microbiology 151(Pt 5):1359–1368CrossRefGoogle Scholar
  54. Rittmann D, Lindner SN, Wendisch VF (2008) Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum. Appl Environ Microbiol 74(20):6216–6222CrossRefGoogle Scholar
  55. Sakai S, Tsuchida Y, 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(7):2349–2353CrossRefGoogle Scholar
  56. Sasaki M, Jojima T, Inui M, Yukawa H (2008) Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions. Appl Microbiol Biotechnol 81(4):691–699CrossRefGoogle Scholar
  57. Sasaki M, Jojima T, Kawaguchi H, Inui M, Yukawa H (2009) Engineering of pentose transport in Corynebacterium glutamicum to improve simultaneous utilization of mixed sugars. Appl Microbiol Biotechnol 85(1):105–115CrossRefGoogle Scholar
  58. Schneider J, Niermann K, Wendisch VF (2011) Production of the amino acids l-glutamate, l-lysine, l-ornithine and l-arginine from arabinose by recombinant Corynebacterium glutamicum. J Biotechnol 154(2–3):191–198CrossRefGoogle Scholar
  59. Schneider J, Wendisch VF (2010) Putrescine production by engineered Corynebacterium glutamicum. Appl Microbiol Biotechnol 88(4):859–868CrossRefGoogle Scholar
  60. Seibold G, Auchter M, Berens S, Kalinowski J, Eikmanns BJ (2006) Utilization of soluble starch by a recombinant Corynebacterium glutamicum strain: growth and lysine production. J Biotechnol 124(2):381–391CrossRefGoogle Scholar
  61. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D (2008) Determination of sugars, byproducts, and degradation products in liquid fraction process samples. NREL Report No TP-510-42623 (January 2008)Google Scholar
  62. Stansen C, Uy D, Delaunay S, Eggeling L, Goergen JL, Wendisch VF (2005) Characterization of a Corynebacterium glutamicum lactate utilization operon induced during temperature-triggered glutamate production. Appl Environ Microbiol 71(10):5920–5928CrossRefGoogle Scholar
  63. Tateno T, Fukuda H, Kondo A (2007) Production of L-lysine from starch by Corynebacterium glutamicum displaying alpha-amylase on its cell surface. Appl Microbiol Biotechnol 74(6):1213–1220CrossRefGoogle Scholar
  64. Tauch A, Kaiser O, Hain T, Goesmann A, Weisshaar B, Albersmeier A, Bekel T, Bischoff N, Brune I, Chakraborty T, Kalinowski J, Meyer F, Rupp O, Schneiker S, Viehoever P, Puhler A (2005) Complete genome sequence and analysis of the multiresistant nosocomial pathogen Corynebacterium jeikeium K411, a lipid-requiring bacterium of the human skin flora. J Bacteriol 187(13):4671–4682CrossRefGoogle Scholar
  65. van Maris AJ, Abbott DA, Bellissimi E, van den Brink J, Kuyper M, Luttik MA, Wisselink HW, Scheffers WA, van Dijken JP, Pronk JT (2006) Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Antonie Van Leeuwenhoek 90(4):391–418CrossRefGoogle Scholar
  66. Wendisch VF (2006) Genetic regulation of Corynebacterium glutamicum metabolism. J Microbiol Biotechnol 16(7):999–1009Google Scholar
  67. Wendisch VF, Bott M, Eikmanns BJ (2006) Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids. Curr Opin Microbiol 9(3):268–274CrossRefGoogle Scholar
  68. Wendisch VF, de Graaf AA, Sahm H, Eikmanns BJ (2000) Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose. J Bacteriol 182(11):3088–3096CrossRefGoogle Scholar
  69. Wyman CE (1999) Production of low cost sugars from biomass: progress, opportunities, and challenges. In: Overend RP, Chornet E (eds) Biomass: a growth opportunity in green energy and value added products, vol 1. Pergamon Press, United Kingdom, pp 867–872Google Scholar
  70. Youn JW, Jolkver E, Kramer R, Marin K, Wendisch VF (2008) Identification and characterization of the dicarboxylate uptake system DccT in Corynebacterium glutamicum. J Bacteriol 190(19):6458–6466CrossRefGoogle Scholar
  71. Youn JW, Jolkver E, Kramer R, Marin K, Wendisch VF (2009) Characterization of the dicarboxylate transporter DctA in Corynebacterium glutamicum. J Bacteriol 191(17):5480–5488CrossRefGoogle Scholar
  72. Zaldivar J, Ingram LO (1999) Effect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01. Biotechnol Bioeng 66(4):203–210CrossRefGoogle Scholar
  73. Zaldivar J, Martinez A, Ingram LO (1999) Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli. Biotechnol Bioeng 65(1):24–33CrossRefGoogle Scholar
  74. Zaldivar J, Martinez A, Ingram LO (2000) Effect of alcohol compounds found in hemicellulose hydrolysate on the growth and fermentation of ethanologenic Escherichia coli. Biotechnol Bioeng 68(5):524–530CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Biotechnology DivisionNational Institute for Interdisciplinary Science and Technology (NIIST), CSIRTrivandrumIndia
  2. 2.Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTecBielefeld UniversityBielefeldGermany

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