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Factors enhancing l-valine production by the growth-limited l-isoleucine auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB ilvNM13 (pECKAilvBNC)

  • Original Paper
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Journal of Industrial Microbiology & Biotechnology

Abstract

Cell growth limitation is known to be an important condition that enhances l-valine synthesis in Corynebacterium glutamicum recombinant strains with l-isoleucine auxotrophy. To identify whether it is the limited availability of l-isoleucine itself or the l-isoleucine limitation-induced rel-dependent ppGpp-mediated stringent response that is essential for the enhancement of l-valine synthesis in growth-limited C. glutamicum cells, we deleted the rel gene, thereby constructing a relaxed (rel − ) C. glutamicum ΔilvA ΔpanB Δrel ilvNM13 (pECKAilvBNC) strain. Variations in enzyme activity and l-valine synthesis in rel + and rel − strains under conditions of l-isoleucine excess and limitation were investigated. A sharp increase in acetohydroxy acid synthase (AHAS) activity, a slight increase in acetohydroxyacid isomeroreductase (AHAIR) activity, and a dramatic increase in l-valine synthesis were observed in both rel + and rel − cells exposed to l-isoleucine limitation. Although the positive effect of induction of the stringent response on AHAS and AHAIR upregulation in cells was not confirmed, we found the stringent response to be beneficial for maintaining increased AHAS, dihydroxyacid dehydratase, and transaminase B activity and l-valine synthesis in cells during the stationary growth phase.

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References

  1. Arfin SM, Umbarger HE (1969) Purification and properties of the actehydroxy acid isomeroreductase of Salmonella typhimurium. J Biol Chem 244:1118–1127

    CAS  PubMed  Google Scholar 

  2. Blombach B, Schreiner ME, Holátko J, Bartek T, Oldiges M, Eikmanns BJ (2007) l-Valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum. Appl Environ Microbiol 73:2079–2084. doi:10.1007/s00253-008-1444-z

    Article  CAS  PubMed  Google Scholar 

  3. Blombach B, Schreiner ME, Bartek T, Oldiges M, Eikmanns BJ (2008) Corynebacterium glutamicum tailored for high-yield l-valine production. Appl Microbiol Biotechnol 79:471–479. doi:10.1128/AEM.02826-06

    Article  CAS  PubMed  Google Scholar 

  4. Brockmann-Gretza O, Kalinowski J (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. doi:10.1186/1471-2164-7-230

    Article  PubMed  Google Scholar 

  5. Cabiscol E, Tamarit J, Ros J (2000) Oxidative stress in bacteria and protein damage by reactive oxygen species. Int Microbiol 3:3–8

    CAS  PubMed  Google Scholar 

  6. Cioffi EA, Shaw KJ, Bailey WF, Berg CM (1980) Improved synthesis of the sodium salt of dl-α, β-dihydroxyisovaleric acid. Anal Biochem 104:485–488

    Article  CAS  PubMed  Google Scholar 

  7. Cordes C, Mockel B, Eggeling L, Sahm H (1992) Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum. Gene 112:113–116

    Article  CAS  PubMed  Google Scholar 

  8. Eggeling L (2001) Amino acids. In: Ratledge C, Kristiansen B (eds) Basic biotechnology. Cambridge University Press, London, pp 281–303

    Google Scholar 

  9. Eggeling L, Bott M (2005) Handbook of Corynebacterium glutamicum. CRC Press, Boca Raton

    Google Scholar 

  10. Eggeling I, Cordes C, Eggeling L, Sahm H (1987) Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to l-isoleucine. Appl Microbiol Biotechnol 25:346–351

    Article  CAS  Google Scholar 

  11. Eggeling L, Oberle S, Sahm H (1998) Improved l-lysine yield with Corynebacterium glutamicum: use of dapA resulting in increased flux combined with growth limitation. Appl Microbiol Biotechnol 49:24–30

    Article  CAS  PubMed  Google Scholar 

  12. Elišáková V, Pátek M, Holátko J, Nešvera J, Leyval D, Goergen JL, Delaunay S (2005) Feedback resistant acetoxydroxy acid synthase increases valine production by Corynebacterium glutamicum. Appl Environ Microbiol 71:207–213. doi:10.1128/AEM.71.1.207-213.2005

    Article  PubMed  Google Scholar 

  13. Eymann C, Homuth G, Scharf C, Hecker M (2002) Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis. J Bacteriol 184:2500–2520. doi:10.1128/JB.184.9.2500-2520.2002

    Article  CAS  PubMed  Google Scholar 

  14. Flint DH, Emptage MH, Finnegan MG, Fu W, Johnson MK (1993) The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase. J Biol Chem 268:14732–14742

    CAS  PubMed  Google Scholar 

  15. Hayashi M, Mizoguchi H, Ohnishi J, Mitsubashi H, Yonetani Y, Hashimoto S, Ikeda M (2006) The leuC mutation leading to increased l-lysine production and rel-independent global expression changes in Corynebacterium glutamicum. Appl Microbiol Biotechnol 72:783–789. doi:10.1007/s00253-006-0539-7

    Article  CAS  PubMed  Google Scholar 

  16. Hermann T, Kramer R (1996) Mechanism and regulation of isoleucine excretion in Corynebacterium glutamicum. Appl Environ Microbiol 62:3238–3244

    CAS  PubMed  Google Scholar 

  17. Holátko J, Elišáková V, Prouza M, Sobotka M, Nešvera J, Pátek M (2009) Metabolic engineering of the l-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation. J Biotechnol 139:203–210. doi:10.1016/j.jbiotec.2008.12.005

    Article  PubMed  Google Scholar 

  18. Horton RM (1995) PCR-mediated recombination and mutagenesis. SOEing together tailor-made genes. Mol Biotechnol 3:93–99

    Article  CAS  PubMed  Google Scholar 

  19. Jäger W, Schäfer A, Pühler A, Labes G, Wohlleben W (1992) Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans. J Bacteriol 174:5462–5465

    PubMed  Google Scholar 

  20. Keilhauer C, Eggeling L, Sahm H (1993) Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175:5595–5603

    CAS  PubMed  Google Scholar 

  21. Kim TH, Kim HJ, Park JS, Kim Y, Kim P, Lee HS (2005) Functional analysis of SigH expression in Corynebacterium glutamicum. Biochem Biophys Res Commun 331:1542–1547. doi:10.1016/j.bbrc.2005.04.073

    Article  CAS  PubMed  Google Scholar 

  22. Kiss RD, Stephanopoulos G (1991) Metabolic activity control of the l-lysine fermentation by restrained growth fed-batch strategies. Biotechnol Prog 7:501–509

    Article  CAS  Google Scholar 

  23. Klingenberg M, Pfaff E (1967) Means of terminating reactions. In: Estabroo RW, Pullman ME (eds) Methods in enzymology. Academic Press, New York, pp 680–684

    Google Scholar 

  24. Kuo CF, Mashino T, Fridovich I (1987) a, b-dihydroxyisovalerate dehydratase: a superoxide-sensitive enzyme. J Biol Chem 262:4724–4727

    CAS  PubMed  Google Scholar 

  25. Lange C, Rittman D, Wendisch VF, Bott M, Sahm H (2003) Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of l-valine. Appl Environ Microbiol 69:2521–2532. doi:10.1128/AEM.69.5.2521-2532.2003

    Article  CAS  PubMed  Google Scholar 

  26. Larisch C, Nakunst D, Huser AT, Tauch A, Kalinowski J (2007) The alternative sigma factor SigB of Corynebacterium glutamicum modulates global gene expression during transition from exponential growth to stationary phase. BMC Genomics 8:4. doi:10.1186/1471-2164-8-4

    Article  PubMed  Google Scholar 

  27. Leuchtenberger W, Huthmacher K, Drauz K (2005) Biotechnological production of amino acids and derivates: current status and prospects. Appl Microbiol Biotechnol 69:1–8. doi:10.1007/s00253-005-0155-y

    Article  CAS  PubMed  Google Scholar 

  28. Leyval D, Uy D, Delaunay S, Goergen JL, Engasser JM (2003) Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine producing strain of Corynebacterium glutamicum. J Biotechnol 104:241–252. doi:10.1016/S0168-1656(03)00162-7

    Article  CAS  PubMed  Google Scholar 

  29. Lowry OH, Rosebrough NJ, Farr LA, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  30. Marienhagen J, Kennerknecht N, Sahm H, Eggeling L (2005) Functional analysis of all aminotransferase proteins inferred from the genome sequence of Corynebacterium glutamicum. J Bacteriol 187:7639–7646. doi:10.1128/JB.187.22.7639-7646.2005

    Article  CAS  PubMed  Google Scholar 

  31. McHardy AC, Tauch A, Ruckert C, Puhler A, Kalinowski J (2003) Genome-based analysis of biosynthetic aminotransferase genes of Corynebacterium glutamicum. J Biotechnol 104:229–240. doi:10.1016/S0168-1656(03)00161-5

    Article  CAS  PubMed  Google Scholar 

  32. Morbach S, Junger C, Sahm H, Eggeling L (2000) Attenuation control of ilvBNC in Corynebacterium glutamicum: evidence of leader peptide formation without the presence of a ribosome binding site. J Biosci Bioeng 90:501–507. doi:10.1016/S1389-1723(01)80030-X

    CAS  PubMed  Google Scholar 

  33. Muniglia L (2001) Elaboration d’une stratégie expérimentale multicritère appliqué à la conception optimale d’un bioprocédé continu de production d’arômes laitiers. PhD thesis. Institut National Polytechnique de Lorraine, Nancy

  34. Nakunst D, Larisch C, Hüser AT, Tauch A, Pühler A, Kalinowski J (2007) The extracytoplasmic function-type sigma factor SigM of Corynebacterium glutamicum ATCC 13032 is involved in transcription of disulfide stress-related genes. J Bacteriol 189:4696–4707. doi:10.1128/JB.00382-07

    Article  CAS  PubMed  Google Scholar 

  35. Patek M, Krumbach K, Eggeling L, Sahm H (1994) Leucine synthesis in Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis. Appl Environ Microbiol 60:133–140

    CAS  PubMed  Google Scholar 

  36. Radmacher E, Vaitsikova A, Burger U, Krumbach K, Sahm H (2002) Linking central metabolism with increased pathway flux: l–valine accumulation by Corynebacterium glutamicum. Appl Environ Microbiol 68:2246–2250. doi:10.1128/AEM.68.5.2246-2250.2002

    Article  CAS  PubMed  Google Scholar 

  37. Ruklisha M, Shvinka J, Viesturs U (1992) Biotechnology of bacterial synthesis (in Russian, with 1993 annex in English). Zinatne, Riga

  38. Ruklisha M, Damberga B, Shvinka J (1993) Stringent control and ppGpp synthesis in Brevibacterium flavum during amino acid starvation. Proceed Acad Sci Latv 12:59–62

    Google Scholar 

  39. Ruklisha M, Viesturs U, Labane L (1995) Growth control and ppGpp synthesis in Brevibacterium flavum cells at various medium mixing rates and aeration intensities. Acta Biotechnol 15:41–48

    Article  CAS  Google Scholar 

  40. Ruklisha M, Ionina R, Paegle L, Petrovica G (2001) Metabolism and lysine biosynthesis control in Brevibacterium flavum: impact of stringent response. In: Simon JP, Durieux A (eds) Applied microbiology, vol 2. Kluwer, Dordrecht, pp 51–57

    Chapter  Google Scholar 

  41. Ruklisha M, Paegle L, Denina I (2007) l-valine biosynthesis during batch and fed-batch cultivations of Corynebacterium glutamicum: relationship between changes in bacterial growth rate and intracellular metabolism. Proc Biochem 42:634–640. doi:10.1016/j.procbio.2006.11.008

    Article  CAS  Google Scholar 

  42. Sahm H, Eggeling L (1999) D-pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding l-valine synthesis for d-pantothenate overproduction. Appl Environ Microbiol 65:1973–1979

    CAS  PubMed  Google Scholar 

  43. Shvinka JE, Toma MK, Galinina NI, Skards IV, Viesturs UE (1979) Production of superoxide radicals during bacterial respiration. J Gen Microbiol 113:377–382

    Google Scholar 

  44. Shvinka J, Viesturs U, Ruklisha M (1980) Yield regulation of lysine biosynthesis in Brevibacterium flavum. Biotechnol Bioeng 22:897–912

    Article  Google Scholar 

  45. Tauch A, Wehmeier L, Gotker S, Puhler A, Kalinowski J (2001) Relaxed rrn expression and amino acid requirement of a Corynebacterium glutamicum rel mutant defective in (p)ppGpp metabolism. FEMS Microbiol Lett 201:53–58

    Article  CAS  PubMed  Google Scholar 

  46. Tedin K, Norel F (2001) Comparison of ΔrelA strains of Escherichia coli and Salmonella enterica serovar typhimurium suggests a role for ppGpp in attenuation regulation of branched-chain amino acid biosynthesis. J Bacteriol 83:6184–6196. doi:10.1128/JB.183.21.6184-6196.2001

    Article  Google Scholar 

  47. Wehmeier L, Schafer A, Burkovski A, Kramer R, Mechold U, Malke H, Puhler A, Kalinowski J (1998) The role of the Corynebacterium glutamicum rel gene in (p)ppGpp metabolism. Microbiology 144:1853–1862

    Article  CAS  PubMed  Google Scholar 

  48. Westerfeld WW (1945) A colorimetric determination of blood acetoin. J Biol Chem 161:495–502

    CAS  Google Scholar 

  49. Zhang X, Dennis P, Ehrenberg M, Bremer H (2002) Kinetic properties of rrn promoters in Escherichia coli. Biochimie 84:981–996. doi:10.1016/S0300-9084(02)00010-X

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grant No. 04.1112 of the Research Council of the Academy of Sciences of the Republic of Latvia and by grant No. 204/07/J012 from the Scientific Council of the Czech Republic. I. Denina was supported by a grant from the European Social Fund (ESF).

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Authors and Affiliations

  1. Institute of Microbiology and Biotechnology, University of Latvia, Kronvalda Boulevard 4, 1586, Riga, Latvia

    Ilze Denina, Longina Paegle & Maija Ruklisha

  2. Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 14220, Prague 4, Czech Republic

    Marek Prouza, Jiři Holátko, Miroslav Pátek & Jan Nešvera

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  1. Ilze Denina
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  2. Longina Paegle
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Correspondence to Maija Ruklisha.

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Denina, I., Paegle, L., Prouza, M. et al. Factors enhancing l-valine production by the growth-limited l-isoleucine auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB ilvNM13 (pECKAilvBNC). J Ind Microbiol Biotechnol 37, 689–699 (2010). https://doi.org/10.1007/s10295-010-0712-y

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