Skip to main content
Log in

Metabolism associated with raised metabolic flux to sugar nucleotide precursors of exopolysaccharides in Lactobacillus delbrueckii subsp. bulgaricus

  • Original Paper
  • Published:
Journal of Industrial Microbiology and Biotechnology

Abstract

Exopolysaccharide (EPS) metabolism was studied in a galactose-negative strain of Lactobacillus delbrueckii subsp. bulgaricus, using two different approaches. Firstly, using both the parent strain and a chemically induced mutant with higher yield and specific productivity of EPS than the parent, comparative information was obtained relating to enzyme activities and metabolite levels associated with EPS formation when grown on lactose. Under continuous culture conditions (D=0.10 h−1), the higher metabolic flux towards EPS formation in the mutant strain relative to the parent appeared to be mediated by raised levels of UDP-glucose pyrophosphorylase (UGP). Marginally raised UDP-galactose 4-epimerase (UGE) activity in the mutant strain suggested that this enzyme could also play a role in EPS overproduction. The second approach involved investigating the effect of growth rate on sugar nucleotide metabolism in the parent, as it is known that EPS production is growth-associated in this strain. UGE activity in the parent strain appeared to increase when the growth rate was elevated from 0.05 to 0.10 h−1, and further to 0.35 h−1, conditions that can be associated with higher levels of metabolic flux to EPS formation. Concurrent with these increments, intracellular ATP levels in the cell were raised. In both investigations glucose-6-phosphate accumulated pointing to a constriction at this branch-point, and a limitation in the flow of carbon towards fructose-6-phosphate or glucose-1-phosphate. The changes in metabolism associated with enhanced flux to EPS provide guidance as to how the yield of Lactobacillus delbrueckii subsp. bulgaricus EPS can be improved.

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

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Bernstein RL (1965) Control aspects of uridine 5′-diphosphate and thymidine 5′-phosphate glucose synthesis by microbial enzymes. J Biol Chem 240:391–397

    CAS  Google Scholar 

  2. Bevill RD (1974) Deoxythymidine-5′-diphosphoglucose (dTDPG) (Metabolites: Nucleic Acids, Purines, Pyrimidines, Nucleosides, Coenzymes). In: Bergmeyer HU (ed) Methods of enzymatic analysis, 2nd English edn, vol 3. Academic, London, New York (translated from the 3rd German edn, Verlag Chemie, Weinheim, Germany), pp 2217–2220

  3. Boels IC, Ramos A, Kleerebezem M, de Vos WM (2001) Functional analysis of the Lactococcus lactis galU and galE genes and their impact on sugar nucleotide and exopolysaccharide biosynthesis. Appl Environ Microbiol 67:3033–3040

    Article  CAS  Google Scholar 

  4. Boels IC, Kleerebezem M, de Vos WM (2003) Engineering of carbon distribution between glycolysis and sugar nucleotide biosynthesis in Lactococcus lactis. Appl Environ Microbiol 69:1129–1135

    Article  CAS  Google Scholar 

  5. Cook AM, Urban E, Schlegel HG (1976) Measuring the concentrations of metabolites in bacteria. Anal Biochem 72:191–201

    Article  CAS  Google Scholar 

  6. De Vos WM, Vaughan EE (1994) Genetics of lactose utilization in lactic acid bacteria. FEMS Microbiol Rev 15:216–237

    Article  Google Scholar 

  7. Degeest B, De Vuyst L (2000) Correlation of activities of the enzymes α-phosphoglucomutase, UDP-galactose 4-epimerase, and UGP with exopolysaccharide biosynthesis by Streptococcus thermophilus LY03. Appl Environ Microbiol 66:3519–3527

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Escalante A, Wacher-Rodarte C, Garcia-Garibay M, Farres A (1998) Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus. J Appl Microbiol 84:108–114

    Article  CAS  Google Scholar 

  10. Fordyce AM, Crow VL, Thomas TD (1984) Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis. Appl Environ Microbiol 48:332–337

    CAS  Google Scholar 

  11. Garrigues C, Loubierre P, Lindley ND, Cocaign-Bousquet M (1997) Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio. J Bacteriol 179:5282–5287

    CAS  Google Scholar 

  12. Gassem MA, Schmidt KA, Frank JA (1997) Exopolysaccharide production from whey lactose by fermentation with Lactobacillus delbrueckii subsp. bulgaricus. J Food Sci 62:171–173

    Article  Google Scholar 

  13. Gilbert JM, Matsuhashi M, Strominger JL (1965) Thymidine diphosphate 4-acetamido-4,6-dideoxyhexoses. II. Purification and properties of thymidine diphosphate d-glucose oxidoreductase. J Biol Chem 240:1305–1308

    CAS  Google Scholar 

  14. Goh KT, Haisman DR, Singh H (2004) Development of an improved procedure for isolation and purification of exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483. Appl Microbiol Biotechnol 67:202–208

    Article  CAS  Google Scholar 

  15. Grobben GJ, Smith MR, Sikkema J, De Bont JAM (1996) Influence of fructose and glucose on the production of exopolysaccharides and the activities of enzymes involved in the sugar metabolism and the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772. Appl Environ Microbiol 46:279–284

    CAS  Google Scholar 

  16. Guedon E, Payot S, Desvaux M, Petitdemange H (1999) Carbon and electron flow in Clostridium cellulolyticum grown in chemostat culture on synthetic medium. J Bact 181:3262–3269

    CAS  Google Scholar 

  17. Guedon E, Desvaux M, Petitdemange H (2000) Kinetic analysis of Clostridium cellulolyticum carbohydrate metabolism: importance of glucose 1-phosphate and glucose 6-phosphate branch points for distribution of carbon fluxes inside and outside cells as revealed by steady-state continuous culture. J Bact 182:2010–2017

    Article  CAS  Google Scholar 

  18. Jensen PR, Hammer K (1998) Artificial promoters for metabolic optimization. Biotechnol Bioeng 58:191–195

    Article  CAS  Google Scholar 

  19. Keppler D, Decker K (1985) Uridine-5′-diphosphoglucose; uridine-5′-diphosphogalactose (Metabolites 2: Tri-and Dicarboxylic Acids, Purines, Pyrimidines and Derivatives, Coenzymes, Inorganic Compounds). In: Bergmeyer HU, Bergmeyer J, Graßl M (eds) Methods of enzymatic analysis, 3rd edn, vol VII. Verlagsgesellschaft, Weinheim, pp 524–530

  20. Kimmel SA, Roberts RF (1998) Development of a growth medium for exopolysaccharide production by Lactobacillus delbrueckii ssp. bulgaricus RR. Int J Food Microbiol 40:87–92

    Article  CAS  Google Scholar 

  21. Kitazawa H, Toba T, Itoh T, Kumano N, Adachi S, Yamaguchi T (1991) Antitumoral activity of slime-forming encapsulated Lactococcus lactis ssp. cremoris isolated from Scandinavian ropy sour milk “viili”. Animal Sci Technol 62:277–283

    Google Scholar 

  22. Kitazawa H, Itoh T, Tomioka Y, Mizugaki M, Yamaguchi T (1996) Induction of IFN-γ and IL-α production in macrophages stimulated with phosphopolysaccharide produced by Lactococcus lactis ssp. cremoris. Int J Food Microbiol 31:99–106

    Article  CAS  Google Scholar 

  23. Kitazawa H, Harata T, Uemura J, Saito T, Kaneko T, Itoh T (1998) Phosphate group requirement for mitogenic activation of lymphocytes by an extracellular phosphopolysaccharide from Lactobacillus delbrueckii ssp. bulgaricus. Int J Food Microbiol 40:169–175

    Article  CAS  Google Scholar 

  24. Laws A, Gu Y, Marshall V (2001) Biosynthesis, characterisation, and design of bacterial exopolysaccharides from lactic acid bacteria. Biotechnol Adv 19:597–625

    Article  CAS  Google Scholar 

  25. Looijesteijn PJ, Boels IC, Kleerebezem M, Hugenholtz J (1999) Regulation of exopolysaccharide production by Lactococcus lactis subsp. cremoris by the sugar source. Appl Environ Microbiol 65:5003–5008

    CAS  Google Scholar 

  26. Looijesteijn PJ, van Casteren WHM, Tuinier R, Doeswijk-Voragen CHL, Hugenholtz J (2000) Influence of different substrate limitations on the yield, composition, and molecular mass of exopolysaccharides produced by Lactococcus lactis subsp. cremoris in continuous cultures. J Appl Microbiol 89:116–122

    Article  CAS  Google Scholar 

  27. Lopez de Felipe F, Hugenholtz J (2001) Purification and characterization of the water forming NADH-oxidase from Lactococcus lactis. Int Dairy J 11:37–44

    Article  Google Scholar 

  28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with the folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  29. Marshall VM, Laws AP, Gu Y, Levander F, Radström P, De Vuyst L, Degeest B, Vaningelgem F, Dunn H, Elvin M (2001) Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure. Lett Appl Microbiol 32:433–437

    Article  CAS  Google Scholar 

  30. Martins LO, Sá-Correia I (1993) Temperature profiles of gellan gum synthesis and activities of biosynthetic enzymes. Biotechnol Appl Biochem 20:385–395

    Google Scholar 

  31. Marty-Teysset C, De la Torre F, Garel J-R (2000) Increased production of hydrogen peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon aeration: involvement of an NADH oxidase in oxidative stress. Appl Environ Microbiol 66:262–267

    CAS  Google Scholar 

  32. Nakajima H, Suzuki Y, Kaizu H, Hirota T (1992) Cholesterol lowering activity of ropy fermented milk. J Food Sci 57:1327–1329

    Article  CAS  Google Scholar 

  33. Neves AR, Ramos A, Nunes MC, Kleerebezem M, Hugenholtz J, de Vos WM, Almeida J, Santos H (1999) In vivo nuclear magnetic resonance studies of glycolytic kinetics in Lactococcus lactis. Biotechnol Bioeng 64:200–212

    Article  CAS  Google Scholar 

  34. Qian N, Stanley GA, Hahn-Hägerdal B, Rådström P (1994) Purification and characterisation of two phosphoglucomutases from Lactococcus lactis subsp. lactis and their regulation in maltose- and glucose-utilizing cells. J Bacteriol 176:5304–5311

    CAS  Google Scholar 

  35. Ramos A, Boels IC, de Vos WM, Santos H (2001) Relationship between glycolysis and exopolysaccharide biosynthesis in Lactococcus lactis. Appl Environ Microbiol 67:33–41

    Article  CAS  Google Scholar 

  36. Ross LF, Chapital DC (1987) Simultaneous determination of carbohydrates and products of carbohydrate metabolism in fermentation mixtures by HPLC. J Chromatogr Sci 25:112–117

    CAS  Google Scholar 

  37. Sjöberg A, Hahn-Hägerdal B (1989) β-Glucose-1-phosphate, a possible mediator for polysaccharide formation in maltose-assimilating Lactococcus lactis. Appl Environ Microbiol 55:1549–1554

    Google Scholar 

  38. Sutherland IW (1972) Bacterial exopolysaccharides. Adv Microb Physiol 8:143–213

    Article  CAS  Google Scholar 

  39. Thomas TD, Ellwood DC, Longyear VMC (1979) Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures. J Bacteriol 138:109–117

    CAS  Google Scholar 

  40. Tuesink B, Walsh MC, Van Dam K, Westerhoff HV (1998) The danger of metabolic pathways with turbo design. Trends Biochem Sci 23:162–169

    Article  Google Scholar 

  41. Van Kranenburg R, Marugg JD, Van Swam II, Willem NJ, de Vos WM (1997) Molecular characterization of the plasmid-encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis. Mol Microbiol 24:387–397

    Article  Google Scholar 

  42. Wang S-F, Gabriel O (1969) Biological mechanisms involved in the formation of deoxy sugars. V. Isolation and crystallization of thymidine diphosphate-d-glucose oxidoreductase from Escherichia coli B. J Biol Chem 244:3430–3437

    CAS  Google Scholar 

  43. Welman AD, Maddox IS (2003a) Fermentation performance of an exopolysaccharide-producing strain of Lactobacillus delbrueckii subsp. bulgaricus. J Ind Microbiol Biotechnol 30:661–668

    Article  CAS  Google Scholar 

  44. Welman AD, Maddox IS, Archer RH (2003b) Exopolysaccharide and extracellular metabolite production by Lactobacillus delbrueckii subsp. bulgaricus, grown on lactose in continuous culture. Biotechnol Lett 25:1515–1520

    Article  CAS  Google Scholar 

  45. Welman AD, Maddox IS, Archer RH (2003c) Screening and selection of exopolysaccharide-producing strains of Lactobacillus delbrueckii subsp. bulgaricus. J Appl Microbiol 95:1200–1206

    Article  CAS  Google Scholar 

Download references

Acknowledgement

This study was supported in part by the New Zealand Foundation for Research, Science and Technology through the TBG program.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to A. D. Welman.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Welman, A.D., Maddox, I. & Archer, R.H. Metabolism associated with raised metabolic flux to sugar nucleotide precursors of exopolysaccharides in Lactobacillus delbrueckii subsp. bulgaricus . J IND MICROBIOL BIOTECHNOL 33, 391–400 (2006). https://doi.org/10.1007/s10295-005-0075-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-005-0075-y

Keywords

Navigation