Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Production of l-Lysine from starch by Corynebacterium glutamicum displaying α-amylase on its cell surface


We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Bayan N, Houssin C, Chami M, Leblon G (2003) Mycomembrane and S-layer: two important structures of Corynebacterium glutamicum cell envelope with promising biotechnology applications. J Biotechnol 104:55–67

  2. Costa-Riu N, Burkovski A, Krämer R, Benz R (2003) PorA represents the major cell wall channel of the Gram-positive bacterium Corynebacterium glutamicum. J Bacteriol 185:4779–4786

  3. Dubois M, Gilles KA, Hamilton JK, Reberse PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356

  4. Hansmeier N, Bartels FW, Ros R, Anselmetti D, Tauch A, Puhler A, Kalinowski J (2004) Classification of hyper-variable Corynebacterium glutamicum surface-layer proteins by sequence analyses and atomic force microscopy. J Biotechnol 112:177–193

  5. Hermann T (2003) Industrial production of amino acids by coryneform bacteria. J Biotechnol 104:155–172

  6. Ikeda M, Nakagawa S (2003) The Corynebacteirum glutamicum genome: features and impacts on biotechnological processes. Appl Microbiol Biotechnol 62:99–109

  7. Ikeda M, Ohnishi J, Hayashi M, Mitsuhashi S (2006) A genome-based approach to create a minimally mutated Corynebacteirum glutamicum strain for efficient l-lysine production. J Ind Microbiol Biotech 33:610–615

  8. Inui M, Murakami S, Okino S, Kawaguchi H, Vertes AA, Yukawa H (2004a) Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J Mol Microbiol Biotechnol 7:182–196

  9. Inui M, Kawaguchi H, Murakami S, Vertes AA, Yukawa H (2004b) Metabolic engineering of Corynebacterium glutamicum for fuel production under oxygen-deprivation conditions. J Mol Microbiol Biotechnol 8:243–254

  10. Kacem R, Sousa-D’Auria CD, Tropis M, Chami M, Gounon P, Leblon G, Houssin C, Daffé M (2004) Importance of mycoloyltransferases on the physiology of Corynebacterium glutamicum. Microbiology 150:73–84

  11. 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, Krämer R, Linke B, McHardy AC, Meyer F, Möckel B, Pfefferle W, Pühler A, Rey DA, Rückert C, Rupp O, Sahm H, Wendisch VF, Wiegräbe 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 104:5–25

  12. Katahira S, Fujita Y, Mizuike A, Fukuda H, Kondo A (2004) Construction of a xylan-fermenting yeast strain through codisplay of xylanolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells. Appl Environ Microbiol 70:5407–5414

  13. Koffas M, Stephanopoulos G (2005) Strain improvement by metabolic engineering: lysine production as a case study for systems biology. Curr Opin Biotechnol 16:361–366

  14. Leuchtenberger W, Klaus H, Drauz K (2005) Biotechnological production of amino acids and derivatives: current status and prospects. Appl Microbiol Biotechnol 69:1–8

  15. Murai T, Ueda M, Shibasaki Y, Kamasawa N, Osumi M, Imanaka T, Tanaka A (1999) Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylotic enzymes on the cell surface. Appl Microbiol Biotechnol 51:65–70

  16. Narita J, Nakahara S, Fukuda H, Kondo A (2004) Efficient production of l-(+)-lactic acid from raw starch by Streptococcus bovis 148. J Biosci Bioeng 97:423–425

  17. Narita J, Okano K, Tateno T, Tanino T, Sewaki T, Sung MH, Fukuda H, Kondo A (2005) Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion. Appl Microbiol Biotechnol 20:1–9

  18. Narita J, Okano K, Kitao T, Ishida S, Sewaki T, Sung MH, Fukuda H, Kondo A (2006) Display of α-amylase on the surface of Lactobacillus casei cells by use of the pgsA anchor protein, and production of lactic acid from starch. Appl Environ Microbiol 72:269–275

  19. Ohnishi J, Mitsuhashi S, Hayashi M, Ando S, Yokoi H, Ochiai K, Ikeda M (2002) A novel methodology employing Corynebacterium glutamicum genome information to generate a new l-lysine-producing mutant. Appl Microbiol Biotechnol 58:217–223

  20. Ohnishi J, Hayashi M, Mitsuhashi S, Ikeda M (2003) Efficient 40°C fermentation of l-lysine by a new Corynebacterium glutamicum mutant developed by genome breeding. Appl Microbiol Biotechnol 62:69–75

  21. Ohnishi J, Katahira R, Mitsuhashi S, Kakita S, Ikeda M (2005) A novel gnd mutation leading to increased l-lysine production in Corynebacterium glutamicum. FEMS Microbial Lett 242:265–274

  22. Okino S, Inui M, Yukawa H (2005) Production of organic acids by Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 68:475–480

  23. Ozaki A, Katsumata R, Oka T, Furuya A (1985) Cloning of the genes concerned in phenylalanine biosynthesis in Corynebacterium glutamicum and its application to breeding of a phenylalanine producing strain. Agric Biol Chem 49:2925–2930

  24. Puech V, Chami M, Lemassu A, Lanéelle MA, Schiffler B, Gounon P, Bayan N, Benz R, Daffé M (2001) Structure of the cell envelope of corynebacteria: importance of the noncovalently bound lipids in the formation of the cell wall permeability barrier and fracture plane. Microbiology 147:1365–1382

  25. Samuelson P, Gunneriusson E, Nygren PA, Ståhl S (2002) Display of proteins on bacteria. J Biotechnol 96:129–154

  26. Satoh E, Niimura Y, Uchimura T, Kozaki M, Komagata K (1993) Molecular cloning and expression of two α-amylase genes from Streptococcus bovis 148 in Escherichia coli. Appl Environ Microbiol 63:4941–4944

  27. Shigechi H, Koh J, Fujita Y, Matsumoto T, Bito Y, Ueda M, Satoh E, Fukuda H, Kondo A (2004) Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and α-amylase. Appl Environ Microbiol 70:5037–5040

  28. 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 124:381–391

Download references


We would like to express our gratitude to Dr. Masahiro Hatsu and Mr. Takashi Mimitsuka of New Frontiers Research Laboratories, Toray Industries for providing the plasmid pTM44 and for advices on the experimental procedures, and to Dr. Moon-Hee Sung and Dr. Tomomitsu Sewaki of Bioleaders Japan Corporation for providing the PgsA gene.

Author information

Correspondence to Akihiko Kondo.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tateno, T., Fukuda, H. & Kondo, A. Production of l-Lysine from starch by Corynebacterium glutamicum displaying α-amylase on its cell surface. Appl Microbiol Biotechnol 74, 1213–1220 (2007).

Download citation


  • Fermentation
  • Sole Carbon Source
  • Soluble Starch
  • Corynebacterium Glutamicum
  • Cell Surface Display