Genetic Engineering of Corynebacteria

Masato Ikeda; Seiki Takeno

doi:10.1128/9781555816827.ch16

Chapter 16 : Genetic Engineering of Corynebacteria

Authors: Masato Ikeda, Seiki Takeno

Content Type: Reference

Publication Year: 2010

Category: Applied and Industrial Microbiology

Book DOI: 10.1128/9781555816827

Chapter DOI: 10.1128/9781555816827.ch16

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter

Preview this chapter:

Genetic Engineering of Corynebacteria, Page 1 of 2

< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555816827/9781555815127_Chap16-1.gif /docserver/preview/fulltext/10.1128/9781555816827/9781555815127_Chap16-2.gif

Abstract:

This chapter describes the technology and provides strategies for molecular strain improvement using current genetic engineering tools, global analysis techniques, and genome-based engineering approaches, with particular emphasis on the industrially important Corynebacterium glutamicum. The transposon (Tn) mutagenesis experiments described in this chapter were performed using special Tn delivery vectors containing insertion sequences (ISs). As a tool for the mutagenesis of C. glutamicum ATCC 13032, Tn vector pAT6100 has been constructed. In C. glutamicum, comparative genomic analysis between two C. glutamicum strains, R and ATCC 13032, revealed that 11 strain-specific islands are scattered in the genomes. Such strain-specific islands are thought to be composed of dispensable genes acquired by horizontal gene transfer. Determining the whole genome sequence of C. glutamicum is aimed at gaining sufficient information to manipulate the metabolism or physiology on a global scale, eventually integrating the information for development of more efficient production strains. This chapter discusses strain improvement strategies, and genome-based strain reconstruction. Exhaustive studies have been directed to metabolic engineering of C. glutamicum for lysine production, resulting in a large body of literature. Comprehensive screening of secretion signal sequences has been conducted in C. glutamicum R by using bioinformatic analysis and a high-throughput secretion assay, which identified a total of 108 candidate signal sequences that could secrete heterologous α-amylase in the organism. The two examples of lysine and arg+cit production described in this chapter will be a paradigm for future strain development in the fermentation industry.

Citation: Ikeda M, Takeno S. 2010. Genetic Engineering of Corynebacteria, p 225-237. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch16

Key Concept Ranking

Basic Amino Acids: 0.7577955
Genetic Elements: 0.5128887
Amino Acids: 0.47721872
Bacterial Proteins: 0.4746503
Chemicals: 0.45239395
Horizontal Gene Transfer: 0.42106524

0.7577955

Highlighted Text: Show | Hide

Full text loading...

Figures

Genetic Engineering of Corynebacteria

[com.pub2web.rdf.cci.facet.ContentItem[id=http://asm.metastore.ingenta.com/content/author/10.1128/9781555816827.ch16-1,webId=/content/author/10.1128/9781555816827.ch16-1,properties={foaf_givenname=Masato, foaf_name=Masato Ikeda, foaf_surname=Ikeda}], com.pub2web.rdf.cci.facet.ContentItem[id=http://asm.metastore.ingenta.com/content/author/10.1128/9781555816827.ch16-2,webId=/content/author/10.1128/9781555816827.ch16-2,properties={foaf_givenname=Seiki, foaf_name=Seiki Takeno, foaf_surname=Takeno}]]

/content/10.1128/9781555816827.ch16.ch16fig01

FIGURE 1

Cre/loxP-mediated deletion of the C. glutamicum genome. Boxes A and B are short segments of the C. glutamicum genome. These PCR-amplified segments are integrated into the genome by homologous recombination using two separate vectors. After integration, the Cre-containing plasmid is introduced into the recombinant cell in order to excise the target region. Kmr, kanamycin resistance gene; Spr, spectinomycin resistance gene.

10.1128/9781555816827/f0227-01_thmb.gif

10.1128/9781555816827/f0227-01.gif

FIGURE 1

/content/10.1128/9781555816827.ch16.ch16fig02

FIGURE 2

Random segment deletion of the C. glutamicum genome. Two IS31831-based transposon vectors are serially introduced into the C. glutamicum cell, randomly integrating two loxP sites into the genome, followed by Cre-mediated recombination. Cmr, chloramphenicol resistance gene; L-IR, left inverted repeat; R-IR, right inverted repeat.

10.1128/9781555816827/f0227-02_thmb.gif

10.1128/9781555816827/f0227-02.gif

FIGURE 2

/content/10.1128/9781555816827.ch16.ch16fig03

FIGURE 3

Methodology to create a minimally mutated strain. Useful mutations relevant to amino acid production are indicated (stars), together with unnecessary mutations (×).

10.1128/9781555816827/f0231-01_thmb.gif

10.1128/9781555816827/f0231-01.gif

FIGURE 3

Methodology to create a minimally mutated strain. Useful mutations relevant to amino acid production are indicated (stars), together with unnecessary mutations (×).

/content/10.1128/9781555816827.ch16.ch16fig04

FIGURE 4

Fermentation kinetics of the newly developed strain RBid at 38°C in 5-liter jar fermentor cultivation. For comparison, the profiles of the best classical producer, A-27, which was cultured under its optimal 30°C conditions, are shown as controls.

, arg+cit of strain RBid; •, growth of strain RBid;

, arg+cit of strain A-27;

, growth of strain A-27.

10.1128/9781555816827/f0232-01_thmb.gif

10.1128/9781555816827/f0232-01.gif

FIGURE 4

/content/10.1128/9781555816827.ch16.ch16fig05

FIGURE 5

Schematic diagram of the creation of new strain RBid. Useful mutations identified in classical producers I-30, α-27, and D-77 are indicated (stars), together with unnecessary mutations (×).

10.1128/9781555816827/f0232-02_thmb.gif

10.1128/9781555816827/f0232-02.gif

FIGURE 5

Schematic diagram of the creation of new strain RBid. Useful mutations identified in classical producers I-30, α-27, and D-77 are indicated (stars), together with unnecessary mutations (×).

References

/content/book/10.1128/9781555816827.ch16

1. Ankri, S.,, I. Serebrijski,, O. Reyes, and, G. Leblon. 1996. Mutations in the Corynebacterium glutamicum proline biosynthetic pathway: a natural bypass of the proA step. J. Bacteriol. 178:4412–4419.

2. Ara, K.,, K. Ozaki,, K. Nakamura,, K. Yamane,, J. Sekiguchi, and, N. Ogasawara. 2007. Bacillus minimum genome factory: effective utilization of microbial genome information. Biotechnol. Appl. Biochem. 46:169–178.

3. Barreiro, C.,, E. González-Lavado,, S. Brand,, A. Tauch, and, J. F. Martín. 2005. Heat shock proteome analysis of wild-type Corynebacterium glutamicum ATCC 13032 and a spontaneous mutant lacking GroEL1, a dispensable chaperone. J. Bacteriol. 187:884–889.

4. Barrett, E.,, C. Stanton,, O. Zelder,, G. Fitzgerald, and, R. P. Ross. 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. 70:2861–2866.

5. Barriuso-Iglesias, M.,, D. Schluesener,, C. Barreiro,, A. Poetsch, and, J. F. Martín. 2008. Response of the cytoplas-mic and membrane proteome of Corynebacterium glutamicum ATCC 13032 to pH changes. BMC Microbiol. 8:225.

6. Becker, J.,, C. Klopprogge,, A. Herold,, O. Zelder,, C. J. Bolten, and, C. Wittmann. 2007. Metabolic flux engineering of l-lysine production in Corynebacterium glutamicum—over expression and modification of G6P dehydrogenase. J. Biotechnol. 132:99–109.

7. Becker, J.,, C. Klopprogge,, O. Zelder,, E. Heinzle, and, C. Wittmann. 2005. Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl. Environ. Microbiol. 71:8587–8596.

8. Beckers, G.,, J. Strösser,, U. Hildebrandt,, J. Kalinowski,, M. Farwick,, R. Krämer, and, A. Burkovski. 2005. Regulation of AmtR-controlled gene expression in Corynebacterium glutamicum: mechanism and characterization of the AmtR regulon. Mol. Microbiol. 58:580–595.

9. Bendt, A. K.,, A. Burkovski,, S. Schaffer,, M. Bott,, M. Farwick, and, T. Hermann. 2003. Towards a phospho-proteome map of Corynebacterium glutamicum. Proteomics 3:1637–1646.

10. Billman-Jacobe, H.,, L. Wang,, A. Kortt,, D. Stewart, and, A. Radford. 1995. Expression and secretion of heterologous proteases by Corynebacterium glutamicum. Appl. Environ. Microbiol. 61:1610–1613.

11. Blombach, B.,, M. E. Schreiner,, M. Moch,, M. Oldiges, and, B. J. Eikmanns. 2007. Effect of pyruvate dehydro-genase complex deficiency on l-lysine production with Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 76:615–623.

12. Bonamy, C.,, J. Labarre,, L. Cazaubon,, C. Jacob,, F. L. Bohec,, O. Reyes, and, G. Leblon. 2003. The mobile element IS1207 of Brevibacterium lactofermentum ATCC 21086: isolation and use in the construction of Tn5531, a versatile transposon for insertional mutagenesis of Coryne-bacterium glutamicum. J. Biotechnol. 104:301–309.

13. Bott, M., and, A. Niebisch. 2003. The respiratory chain of Corynebacterium glutamicum. J. Biotechnol. 104:129–153.

14. Brockmann-Gretza, O., and, J. Kalinowski. 2006. Global gene expression during stringent response in Corynebacterium glutamicum in presence and absence of the rel gene encoding (p)ppGpp synthase. BMC Genomics 7:230.

15. Brune, I.,, N. Jochmann,, K. Brinkrolf,, A. T. Hüser,, R. Gerstmeir,, B. J. Eikmanns,, J. Kalinowski,, A. Püh-ler, and, A. Tauch. 2007. The IclR-type transcriptional repressor LtbR regulates the expression of leucine and tryptophan biosynthesis genes in the amino acid producer Corynebacterium glutamicum. J. Bacteriol. 189:2720–2733.

16. Burkovski, A. 2008. Corynebacteria: Genomics and Molecular Biology. Caister Academic Press, Norfolk, United Kingdom.

17. Burkovski, A., and, R. Krämer. 2002. Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications. Appl. Microbiol. Biotechnol. 58:265–274.

18. Caspers, M., and, R. Freudl. 2008. Corynebacterium glu-tamicum possesses two secA homologous genes that are essential for viability. Arch. Microbiol. 189:605–610.

19. Cerdeño-Tárraga, A. M.,, A. Efstratiou,, L. G. Dover,, M. T. Holden,, M. Pallen,, S. D. Bentley,, G. S. Besra,, C. Churcher,, K. D. James,, A. De Zoysa,, T. Chilling-worth,, A. Cronin,, L. Dowd,, T. Feltwell,, N. Hamlin,, S. Holroyd,, K. Jagels,, S. Moule,, M. A. Quail,, E. Rabbin-owitsch,, K. M. Rutherford,, N. R. Thomson,, L. Unwin,, S. Whitehead,, B. G. Barrell, and, J. Parkhill. 2003. The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129. Nucleic Acids Res. 31:6516–6523.

20. Cremer, J.,, L. Eggeling, and, H. Sahm. 1991. Control of the lysine biosynthesis sequence in Corynebacterium glutamicum as analyzed by overexpression of the individual corresponding genes. Appl. Environ. Microbiol. 57:1746–1752.

21. Date, M.,, H. Itaya,, H. Matsui, and, Y. Kikuchi. 2006. Secretion of human epidermal growth factor by Coryne-bacterium glutamicum. Lett. Appl. Microbiol. 42:66–70.

22. Diesveld, R.,, N. Tietze,, O. Fürst,, A. Reth,, B. Bathe,, H. Sahm, and, L. Eggeling. 2008. Activity of exporters of Escherichia coli in Corynebacterium glutamicum, and their use to increase l-threonine production. J. Mol. Microbiol. Biotechnol. 16:198–207.

23. Eggeling, L., and, M. Bott. 2005. Handbook of Corynebacterium glutamicum. CRC Press, Inc., Boca Raton, FL.

24. Engels, V.,, S. N. Lindner, and, V. F. Wendisch. 2008. The global repressor SugR controls expression of genes of glycolysis and of the l-lactate dehydrogenase LdhA in Corynebacterium glutamicum. J. Bacteriol. 190:8033–8044.

25. Gunji, Y., and, H. Yasueda. 2006. Enhancement of l-lysine production in methylotroph Methylophilus methylotrophus by introducing a mutant LysE exporter. J. Biotechnol. 127:1–13.

26. Hänssler, E.,, T. Müller,, N. Jessberger,, A. Völzke,, J. Plassmeier,, J. Kalinowski,, R. Krämer, and, A. Burkovski. 2007. FarR, a putative regulator of amino acid metabolism in Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 76:625–632.

27. Hayashi, M.,, H. Mizoguchi,, J. Ohnishi,, S. Mitsuhashi,, Y. Yonetani,, S. Hashimoto, and, M. Ikeda. 2006. A leuC mutation leading to increased l-lysine production and rel-independent global expression changes in Cory-nebacterium glutamicum. Appl. Microbiol. Biotechnol. 72:783–789.

28. Hayashi, M.,, H. Mizoguchi,, N. Shiraishi,, M. Obayashi,, S. Nakagawa,, J. Imai,, S. Watanabe,, T. Ota, and, M. Ikeda. 2002. Transcriptome analysis of acetate metabolism in Corynebacterium glutamicum using a newly developed metabolic array. Biosci. Biotechnol. Biochem. 66:1337–1344.

29. Hayashi, M.,, J. Ohnishi,, S. Mitsuhashi,, Y. Yonetani,, S. Hashimoto, and, M. Ikeda. 2006. Transcriptome analysis reveals global expression changes in an industrial l-lysine producer of Corynebacterium glutamicum. Biosci. Biotechnol. Biochem. 70:546–550.

30. Hermann, T.,, W. Pfefferle,, C. Baumann,, E. Busker,, S. Schaffer,, M. Bott,, H. Sahm,, N. Dusch,, J. Kalinowski,, A. Pühler,, A. K. Bendt,, R. Krämer, and, A. Burkovski. 2001. Proteome analysis of Corynebacterium glutamicum. Electrophoresis 22:1712–1723.

31. Hüser, A. T.,, C. Chassagnole,, N. D. Lindley,, M. Merkamm,, A. Guyonvarch,, V. Elisáková,, M. Pátek,, J. Kalinowski,, I. Brune,, A. Pühler, and, A. Tauch. 2005. Rational design of a Corynebacterium glutamicum pantothenate production strain and its characterization by metabolic flux analysis and genome-wide transcriptional profiling. Appl. Environ. Microbiol. 71:3255–3268.

32. Hwang, B. J.,, S. D. Park,, Y. Kim,, P. Kim, and, H. S. Lee. 2007. Biochemical analysis on the parallel pathways of methionine biosynthesis in Corynebacterium glutamicum. J. Microbiol. Biotechnol. 17:1010–1017.

33. Ikeda, M. 2003. Amino acid production processes, p. 1–35. In R. Faurie and, J. Thommel (ed.), Advances in Biochemical Engineering/Biotechnology, vol. 79. Microbial Production of l-Amino Acids. Springer-Verlag, Berlin, Germany.

34. Ikeda, M.,, S. Mitsuhashi,, K. Tanaka, and, M. Hayashi. 2009. Reengineering of a Corynebacterium glutamicum l-arginine and l-citrulline producer. Appl. Environ. Microbiol. 75:1635–1641.

35. Ikeda, M., and, S. Nakagawa. 2003. The Corynebacterium glutamicum genome: features and impacts on biotechno-logical process. Appl. Microbiol. Biotechnol. 62:99–109.

36. Ikeda, M.,, J. Ohnishi,, M. Hayashi, and, S. Mitsuhashi. 2006. A genome-based approach to create a minimally mutated Corynebacterium glutamicum strain for efficient l-lysine production. J. Ind. Microbiol. Biotechnol. 33:610–615.

37. Ikeda, M.,, J. Ohnishi, and, S. Mitsuhashi. 2005. Genome breeding of an amino acid-producing Corynebacterium glu-tamicum mutant, p. 179–189. In J. L. S. Barredo (ed.), Microbial Processes and Products. Humana Press, Totowa, NJ.

38. Inui, M.,, S. Murakami,, S. Okino,, H. Kawaguchi,, A. A. Vertes, and, H. Yukawa. 2004. Metabolic analysis of Corynebacterium glutamicum during lactate and succinate production under oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol. 7:182–196.

39. Inui, M.,, Y. Tsuge,, N. Suzuki,, A. A. Vertès, and, H. Yukawa. 2005. Isolation and characterization of a native composite transposon, Tn14751, carrying 17.4 kilobases of Corynebacterium glutamicum chromosomal DNA. Appl. Environ. Microbiol. 71:407–416.

40. Jo, S. J.,, M. Maeda,, T. Ooi, and, S. Taguchi. 2006. Production system for biodegradable polyester polyhydroxybutyr-ate by Corynebacterium glutamicum. J. Biosci. Bioeng. 102:233–236.

41. Jo, S. J.,, K. Matsumoto,, C. R. Leong,, T. Ooi, and, S. Taguchi. 2007. Improvement of poly(3-hydroxybutyrate) [P(3HB)] production in Corynebacterium glutamicum by codon optimization, point mutation and gene dosage of P(3HB) biosynthetic genes. J. Biosci. Bioeng. 104:457–463.

42. Kabus, A.,, T. Georgi,, V. F. Wendisch, and, M. Bott. 2007. Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation. Appl. Microbiol. Biotechnol. 75:47–53.

43. Kabus, A.,, A. Niebisch, and, M. Bott. 2007. Role of cytochrome bd oxidase from Corynebacterium glutamicum in growth and lysine production. Appl. Environ. Microbiol. 73:861–868.

44. Kalinowski, J.,, B. Bathe,, D. Bartels,, N. Bischoff,, M. Bott,, A. Burkovski,, N. Dusch,, L. Eggeling,, B. J. Eikmanns,, L. Gaigalat,, A. Goesmann,, M. Hart-mann,, K. Huthmacher,, R. Krämer,, B. Linke,, A. C. McHardy,, F. Meyer,, B. Möckel,, W. Pfefferle,, A. Pühler,, D. A. Rey,, C. Rückert,, O. Rupp,, H. Sahm,, V. F. Wendisch,, I. Wiegräbe, and, A. Tauch. 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.

45. Kase, H., and, K. Nakayama. 1974. Mechanism of l-threonine and l-lysine production by analog-resistant mutants of Corynebacterium glutamicum. Agr. Biol. Chem. 38:993–1000.

46. Katsumata, R.,, A. Ozaki,, T. Oka, and, A. Furuya. 1984. Protoplast transformation of glutamate-producing bacteria with plasmid DNA. J. Bacteriol. 159:306–311.

47. Kawaguchi, H.,, M. Sasaki,, A. A Vertès,, M. Inui, and, H. Yukawa. 2008. Engineering of an l-arabinose metabolic pathway in Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 77:1053–1062.

48. Kawaguchi, H.,, A. A Vertès,, S. Okino,, M. Inui, and, H. Yukawa. 2006. Engineering of a xylose metabolic pathway in Corynebacterium glutamicum. Appl. Environ. Microbiol. 72:3418–3428.

49. Kennerknecht, N.,, H. Sahm,, M. R. Yen,, M. Patek,, M. H. Saier, Jr., and, L. Eggeling. 2002. Export of l-isoleucine from Corynebacterium glutamicum: a two-gene-encoded member of a new translocator family. J. Bacteriol. 184:3947–3956.

50. Kikuchi, Y.,, M. Date,, K. Yokoyama,, Y. Umezawa, and, H. Matsui. 2003. Secretion of active-form Streptoverti-cillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-like protease from Streptomyces al-bogriseolus. Appl. Environ. Microbiol. 69:358–366.

51. Kikuchi, Y.,, H. Itaya,, M. Date,, K. Matsui, and, L. F. Wu. 2008. Production of Chryseobacterium proteolyticum protein-glutaminase using the twin-arginine translocation pathway in Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 78:67–74.

52. Kikuchi, Y.,, H. Itaya,, M. Date,, K. Matsui, and, L. F. Wu. 2009. TatABC overexpression improves Corynebacterium glutamicum Tat-dependent protein secretion. Appl. Environ. Microbiol. 75:603–607.

53. Kim, H. J.,, T. H. Kim,, Y. Kim, and, H. S. Lee. 2004. Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum. J. Bacteriol. 186:3453–3460.

54. Kim, T. H.,, H. J. Kim,, J. S. Park,, Y. Kim,, P. Kim, and, H. S. Lee. 2005. Functional analysis of sigH expression in Corynebacterium glutamicum. Biochem. Biophys. Res. Commun. 331:1542–1547.

55. Kim, T. H.,, J. S. Park,, H. J. Kim,, Y. Kim,, P. Kim, and, H. S. Lee. 2005. The whcE gene of Corynebacterium glutamicum is important for survival following heat and oxidative stress. Biochem. Biophys. Res. Commun. 337:757–764.

56. Kjeldsen, K. R., and, J. Nielsen. 2009. In silico genome-scale reconstruction and validation of the Corynebacterium glutamicum metabolic network. Biotechnol. Bioeng. 102:583–597.

57. Koch, D. J.,, C. Rückert,, A. Albersmeier,, A. T. Hüser,, A. Tauch,, A. Pühler, and, J. Kalinowski. 2005. The transcriptional regulator SsuR activates expression of the Corynebacterium glutamicum sulphonate utilization genes in the absence of sulphate. Mol. Microbiol. 58:480–494.

58. Koffas, M. A. G.,, G. Y. Jung, and, G. Stephanopoulos. 2003. Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overex-pression. Metab. Eng. 5:32–41.

59. Kolisnychenko, V.,, G. Plunkett III,, C. D. Herring,, T. Fehér,, J. Pósfai,, F. R. Blattner, and, G. Pósfai. 2002. Engineering a reduced Escherichia coli genome. Genome Res. 12:640–647.

60. Krömer, J. O.,, C. J. Bolten,, E. Heinzle,, H. Schröder, and, C. Wittmann. 2008. Physiological response of Cory-nebacterium glutamicum to oxidative stress induced by deletion of the transcriptional repressor McbR. Microbiology 154:3917–3930.

61. Krömer, J. O.,, O. Sorgenfrei,, K. Klopprogge,, E. Heinzle, and, C. Wittmann. 2004. In-depth profiling of lysine-pro-ducing Corynebacterium glutamicum by combined analysis of the transcriptome, metabolome, and fluxome. J. Bacteriol. 186:1769–1784.

62. Krömer, J. O.,, C. Wittmann,, H. Schröder, and, E. Heinzle. 2006. Metabolic pathway analysis for rational design of l-methionine production by Eschrichia coli and Corynebacterium glutamicum. Metab. Eng. 8:353–369.

63. Lange, C.,, D. Rittmann,, V. F. Wendisch,, M. Bott, and, H. Sahm. 2003. Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of l-valine. Appl. Environ. Microbiol. 69:2521–2532.

64. Lee, H. S., and, B. J. Hwang. 2003. Methionine biosynthesis and its regulation in Corynebacterium glutamicum: parallel pathways of transsulfuration and direct sulfhydry-lation. Appl. Microbiol. Biotechnol. 62:459–467.

65. Lee, J. H.,, B. H. Sung,, M. S. Kim,, F. R. Blattner,, B. H. Yoon,, J. H. Kim, and, S. C. Kim. 2009. Metabolic engineering of a reduced-genome strain of Escherichia coli for l-threonine production. Microb. Cell Fact. 8:2–13.

66. Li, L.,, M. Wada, and, A. Yokota. 2007. A comparative proteomic approach to understand the adaptations of an H1-ATPase-defective mutant of Corynebacterium glutamicum ATCC14067 to energy deficiencies. Proteomics 7:3348–3357.

67. Li, L.,, M. Wada, and, A. Yokota. 2007. Cytoplasmic proteome reference map for a glutamic acid-producing Corynebacterium glutamicum ATCC 14067. Proteomics 7:4317–4322.

68. Liebl, W.,, A. J. Sinskey, and, K. H. Schleifer, 1992. Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum. J. Bacteriol. 174:1854–1861.

69. Loos, A.,, C. Glanemann,, L. B. Willis,, X. M. O’Brien,, P. A. Lessard,, R. Gerstmeir,, S. Guillouet, and, A. J. Sinskey. 2001. Development and validation of Coryne-bacterium DNA microarrays. Appl. Environ. Microbiol. 67:2310–2318.

70. Magnus, J. B.,, D. Hollwedel,, M. Oldiges, and, R. Takors. 2006. Monitoring and modeling of the reaction dynamics in the valine/leucine synthesis pathway in Corynebacterium glutamicum. Biotechnol. Prog. 22:1071–1083.

71. Mampel, J.,, H. Schröder,, S. Haefner, and, U. Sauer. 2005. Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 68:228–236.

72. Marx, A.,, S. Hans,, B. Mockel,, B. Bathe, and, A. A. de Graaf. 2003. Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum. J. Biotechnol. 104:185–197.

73. Mimitsuka, T.,, H. Sawai,, M. Hatsu, and, K. Yamada. 2007. Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation. Biosci. Biotechnol. Biochem. 71:2130–2135.

74. Mitsuhashi, S.,, M. Hayashi,, J. Ohnishi, and, M. Ikeda. 2006. Disruption of malate:quinone oxidoreductase in creases l-lysine production by Corynebacterium glutamicum. Biosci. Biotechnol. Biochem. 70:2803–2806.

75. Mizoguchi, H.,, H. Mori, and, T. Fujio. 2007. Escherichia coli minimum genome factory. Biotechnol. Appl. Biochem. 46:157–167.

76. Möker, N.,, J. Krämer,, G. Unden,, R. Krämer, and, S. Morbach. 2007. In vitro analysis of the two-component system MtrB-MtrA from Corynebacterium glutamicum. J Bacteriol. 189:3645–3649.

77. Mormann, S.,, A. Lömker,, C. Rückert,, L. Gaigalat,, A. Tauch,, A. Pühler, and, J. Kalinowski. 2006. Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway. BMC Genomics 7:205–224.

78. Muffler, A.,, S. Bettermann,, M. Haushalter,, A. Hörlein,, U. Neveling,, M. Schramm, and, O. Sorgenfrei. 2002. Genome-wide transcription profiling of Corynebacterium glutamicum after heat shock and during growth on acetate and glucose. J. Biotechnol. 98:255–268.

79. Nakamura, J.,, S. Hirano,, H. Ito, and, M. Wachi. 2007. Mutations of the Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel homolog, induce l-glutamic acid production. Appl. Environ. Microbiol. 73:4491–4498.

80. Netzer, R.,, M. Krause,, D. Rittmann,, P. G. Peters-Wendisch,, L. Eggeling,, V. F. Wendisch, and, H. Sahm. 2004. Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis. Arch. Microbiol. 182:354–363.

81. Nishimura, T.,, A. A. Vertès,, Y. Shinoda,, M. Inui, and, H. Yukawa. 2007. Anaerobic growth of Corynebacterium glutamicum using nitrate as a terminal electron acceptor. Appl. Microbiol. Biotechnol. 75:889–897.

82. Nishio, Y.,, Y. Nakamura,, Y. Kawarabayasi,, Y. Usuda,, E. Kimura,, S. Sugimoto,, K. Matsui,, A. Yamagishi,, H. Kikuchi,, K. Ikeo, and, T. Gojobori. 2003. Comparative complete genome sequence analysis of the amino acid replacements responsible for the thermostability of Cory-nebacterium efficiens. Genome Res. 13:1572–1579.

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

84. Ohnishi, J., and, M. Ikeda. 2006. Comparisons of potentials for l-lysine production among different Coryne-bacterium glutamicum strains. Biosci. Biotechnol. Biochem. 70:1017–1020.

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

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

87. Park, S. D.,, J. Y. Lee,, S. Y. Sim,, Y. Kim, and, H. S. Lee. 2007. Characteristics of methionine production by an en gineered Corynebacterium glutamicum strain. Metab. Eng. 9:327–336.

88. Peter, H.,, A. Burkovski, and, R. Krämer. 1996. Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine. J. Bacteriol. 178:5229–5234.

89. Peter, H.,, B. Weil,, A. Burkovski,, R. Krämer, and, S. Morbach. 1998. Corynebacterium glutamicum is equipped with four secondary carriers for compatible solutes: identification, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine betaine carrier, EctP. J. Bacteriol. 180:6005–6012.

90. Petersen, S.,, C. Mack,, A. A. de Graaf,, C. Riedel,, B. J. Eikmanns, and, H. Sahm. 2001. Metabolic consequences of altered phosphoenolpyruvate carboxykinase activity in Corynebacterium glutamicum reveal anaplerotic mechanisms in vivo. Metab. Eng. 3:344–361.

91. Peters-Wendisch, P. G.,, B. Schiel,, V. F. Wendisch,, E. Katsoulidis,, B. Möckel,, H. Sahm, and, B. J. Eikmanns. 2001. Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glu-tamicum. J. Mol. Microbiol. Biotechnol. 3:295–300.

92. Peters-Wendisch, P.,, M. Stolz,, H. Etterich,, N. Kennerknecht,, H. Sahm, and, L. Eggeling. 2005. Metabolic engineering of Corynebacterium glutamicum for l-serine production. Appl. Environ. Microbiol. 71:7139–7144.

93. Pfefferle, W.,, B. Möckel,, B. Bathe, and, A. Marx. 2003. Biotechnological manufacture of lysine. Adv. Biochem. Eng. Biotechnol. 79:59–112.

94. Rey, D. A.,, S. S. Nentwich,, D. J. Koch,, C. Rückert,, A. Pühler,, A. Tauch, and, J. Kalinowski. 2005. The McbR repressor modulated by the effector substance S-adeno-sylhomocysteine controls directly the transcription of a regulon involved in sulphur metabolism of Corynebacterium glutamicum ATCC 13032. Mol. Microbiol. 56:871–887.

95. Rey, D. A.,, A. Pühler, and, J. Kalinowski. 2003. The putative transcriptional repressor McbR, member of the TetR-family, is involved in the regulation of the metabolic network directing the synthesis of sulfur containing amino acids in Corynebacterium glutamicum. J. Biotechnol. 103:51–65.

96. Riedel, C.,, D. Rittmann,, P. Dangel,, B. Möckel,, H. Sahm, and, B. J. Eikmanns. 2001. Characterization, expression, and inactivation of the phosphoenolpyruvate carboxykinase gene from Corynebacterium glutamicum and significance of the enzyme for growth and amino acid production. J. Mol. Microbiol. Biotechnol. 3:573–583.

97. Rittmann, D.,, S. N. Lindner, and, V. F. Wendisch. 2008. Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum. Appl. Environ. Microbiol. 74:6216–6222.

98. Salim, K.,, V. Haedens,, J. Content,, G. Leblon, and, K. Huygen. 1997. Heterologous expression of the My-cobacterium tuberculosis gene encoding antigen 85A in Corynebacterium glutamicum. Appl. Environ. Microbiol. 63:4392–4400.

99. Sano, K., and, I. Shiio. 1971. Microbial production of l-lysine. I V. Selection of lysine-producing mutants from Brevibacterium flavum by detecting threonine sensitivity or halo-forming method. J. Gen. Appl. Microbiol. 17:97–113.

100. Schaaf, S., and, M. Bott. 2007. Target genes and DNA-binding sites of the response regulator PhoR from Cory-nebacterium glutamicum. J. Bacteriol. 189:5002–5011.

101. Schaffer, S.,, B. Weil,, V. D. Nguyen,, G. Dongmann,, K. Günther,, M. Nickolaus,, T. Hermann, and, M. Bott. 2001. A high-resolution reference map for cytoplasmic and membrane-associated proteins of Corynebacterium glutamicum. Electrophoresis 22:4404–4422.

102. Schluesener, D.,, F. Fischer,, J. Kruip,, M. Rögner, and, A. Poetsch. 2005. Mapping the membrane proteome of Corynebacterium glutamicum. Proteomics 5:1317–1330.

103. Schluesener, D.,, M. Rögner, and, A. Poetsch. 2007. Evaluation of two proteomics technologies used to screen the membrane proteomes of wild-type Corynebacterium glutamicum and an l-lysine-producing strain. Anal. Bio-anal. Chem. 389:1055–1064.

104. Seibold, G.,, M. Auchter,, S. Berens,, J. Kalinowski, and, B. J. Eikmanns. 2006. Utilization of soluble starch by a recombinant Corynebacterium glutamicum strain: growth and lysine production. J. Biotechnol. 124:381–391.

105. Shiio, I., and, R. Miyajima. 1969. Concerted inhibition and its reversal by end products of aspartate kinase in Brevibacterium flavum. J. Biochem. 65:849–859.

106. Shiio, I.,, Y. Toride, and, S. Sugimoto. 1984. Production of lysine by pyruvate dehydrogenase mutants of Brevibacterium flavum. Agric. Biol. Chem. 48:3091–3098.

107. Silberbach, M.,, M. Schäfer,, A. T Hüser,, J. Kalinowski,, A. Pühler,, R. Krämer, and, A. Burkovski. 2005. Adaptation of Corynebacterium glutamicum to ammonium limitation: a global analysis using transcriptome and proteome techniques. Appl. Environ. Microbiol. 71:2391–2402.

108. Simic, P.,, H. Sahm, and, L. Eggeling. 2001. l-Threonine export: use of peptides to identify a new translocator from Corynebacterium glutamicum. J. Bacteriol. 183:5317–5324.

109. Sindelar, G., and, V. F. Wendisch. 2007. Improving lysine production by Corynebacterium glutamicum through DNA microarray-based identification of novel target genes. Appl. Microbiol. Biotechnol. 76:677–689.

110. Steger, R.,, M. Weinand,, R. Krämer, and, S. Morbach. 2004. LcoP, an osmoregulated betaine/ectoine uptake system from Corynebacterium glutamicum. FEBS Lett. 573:155–160.

111. Stolz, M.,, P. Peters-Wendisch,, H. Etterich,, T. Gerharz,, R. Faurie,, H. Sahm,, H. Fersterra, and, L. Eggeling. 2007. Reduced folate supply as a key to enhanced l-serine production by Corynebacterium glutamicum. Appl. Environ. Microbiol. 73:750–755.

112. Strelkov, S.,, M. von Elstermann, and, D. Schomburg. 2004. Comprehensive analysis of metabolites in Co-rynebacterium glutamicum by gas chromatography/mass spectrometry. Biol. Chem. 385:853–861.

113. Suzuki, N.,, H. Nonaka,, Y, Tsuge,, M. Inui, and, H. Yukawa. 2005. New multiple-deletion method for the Corynebacterium glutamicum genome, using a mutant lox sequence. Appl. Environ. Microbiol. 71:8472–8480.

114. Suzuki, N.,, H. Nonaka,, Y. Tsuge,, S. Okayama,, M. Inui, and, H. Yukawa. 2005. Multiple large segment deletion method for Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 69:151–161.

115. Suzuki, N.,, N. Okai,, H. Nonaka,, Y. Tsuge,, M. Inui, and, H. Yukawa. 2006. High-throughput transposon mu-tagenesis of Corynebacterium glutamicum and construction of a single-gene disruptant mutant library. Appl. Environ. Microbiol. 72:3750–3755.

116. Suzuki, N.,, S. Okayama,, H. Nonaka,, Y. Tsuge,, M. Inui, and, H. Yukawa. 2005. Large-scale engineering of the Corynebacterium glutamicum genome. Appl. Environ. Microbiol. 71:3369–3372.

117. Suzuki, N.,, Y. Tsuge,, M. Inui, and, H. Yukawa. 2005. CreloxP-mediated deletion system for large genome rearrangements in Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 67:225–233.

118. Takeno, S.,, M. Nakamura,, R. Fukai,, J. Ohnishi, and, M. Ikeda. 2008. The Cgl1281-encoding putative transporter of the cation diffusion facilitator family is responsible for alkali-tolerance in Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 190:531–538.

119. Takeno, S.,, J. Ohnishi,, T. Komatsu,, T. Masaki,, K. Sen, and, M. Ikeda. 2007. Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 75:1173–1182.

120. Tateno, T.,, H. Fukuda, and, A. Kondo. 2007. Production of l-lysine from starch by Corynebacterium glu-tamicum displaying α-amylase on its cell surface. Appl. Microbiol. Biotechnol. 74:1213–1220.

121. Tauch, A.,, S. Götker,, A. Pühler,, J. Kalinowski, and, G. Thierbach. 2002. The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tet-racycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100. Plasmid 48:117–129.

122. Tauch, A.,, O. Kaiser,, T. Hain,, A. Goesmann,, B. Weis-shaar,, A. Albersmeier,, T. Bekel,, N. Bischoff,, I. Brune,, T. Chakraborty,, J. Kalinowski,, F. Meyer,, O. Rupp,, S. Schneiker,, P. Viehoever, and, A. Pühler. 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. 187:4671–4682.

123. Trötschel, C.,, D. Deutenberg,, B. Bathe,, A. Burkovski, and, R. Krämer. 2005. Characterization of methionine export in Corynebacterium glutamicum. J. Bacteriol. 187:3786–3794.

124. Tsuge, Y.,, K. Ninomiya,, N. Suzuki,, M. Inui, and, H. Yukawa. 2005. A new insertion sequence, IS14999, from Corynebacterium glutamicum. Microbiology 151:501–508.

125. Tsuge, Y.,, N. Suzuki,, M. Inui, and, H. Yukawa. 2007. Random segment deletion based on IS31831 and Cre/loxP excision system in Corynebacterium glutamicum. Appl. Mi-crobiol. Biotechnol. 74:1333–1341.

126. Tsuge, Y.,, N. Suzuki,, K. Ninomiya,, M. Inui, and, H. Yukawa. 2007. Isolation of a new insertion sequence, IS13655, and its application to Corynebacterium glutamicum genome mutagenesis. Biosci. Biotechnol. Biochem. 71:1683–1690.

127. Vertès, A. A.,, Y. Asai,, M. Inui,, M. Kobayashi,, Y. Ku-rusu, and, H. Yukawa. 1994. Transposon mutagenesis of coryneform bacteria. Mol. Gen. Genet. 245:397–405.

128. Vrljié, M.,, H. Sahm, and, L. Eggeling. 1996. A new type of transporter with a new type of cellular function: l-lysine export from Corynebacterium glutamicum. Mol. Microbiol. 22:815–826.

129. Watanabe, K.,, Y. Tsuchida,, N. Okibe,, H. Teramoto,, N. Suzuki,, M. Inui, and, H. Yukawa. 2009. Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences. Microbiology 155:741–750.

130. Wendisch, V. F. 2003. Genome-wide expression analysis in Corynebacterium glutamicum using DNA microarrays. J. Biotechnol. 104:273–285.

131. Wendisch, V. F.,, M. Bott.,, J. Kalinowski,, M. Oldiges, and, W. Wiechert. 2006. Emerging Corynebacterium glu-tamicum systems biology. J. Biotechnol. 124:74–92.

132. Wennerhold, J., and, M. Bott. 2006. The DtxR regulon of Corynebacterium glutamicum. J. Bacteriol. 188:2907–2918.

133. Wittmann, C., and, E. Heinzle. 2002. Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria. Appl. Environ. Microbiol. 68:5843–5859.

134. Yukawa, H.,, C. A. Omumasaba,, H. Nonaka,, P. Kós,, N. Okai,, N. Suzuki,, M. Suda,, Y. Tsuge,, J. Watanabe,, Y. Ikeda,, A. A Vertès, and, M. Inui. 2007. Comparative analysis of the Corynebacterium glutamicum group and complete genome sequence of strain R. Microbiology 153:1042–1058.