1887

Microbiology Society logo

Volume 148, Issue 4

Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis

The GenBank accession number for the sequence reported in this paper is AY046926.

Free

Abstract

Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches ’bottle-necks’ are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected ’rate-limiting’ steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): (abbreviations used in rate reactions and equations are defined in Table 1) , ALS, acetolactate synthase , in silico modelling , Lactococcus lactis , LDH, L-lactate dehydrogenase , MCA, metabolic control analysis , Metabolic control analysis , NOX, NADH oxidase and pyruvate distribution
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-4-1003
2002-04-01
2024-04-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/4/1481003a.html?itemId=/content/journal/micro/10.1099/00221287-148-4-1003&mimeType=html&fmt=ahah

References

  1. Abbe K., Takahashi S., Yamada T. 1982; Involvement of oxygen-sensitive pyruvate formate-lyase in mixed-acid fermentation by Streptococcus mutans under strictly anaerobic conditions. J Bacteriol 152:175–182
     [Google Scholar]
  2. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254 [CrossRef]
     [Google Scholar]
  3. Bresters T. W., De Kok A., Veeger C. 1975; The pyruvate-dehydrogenase complex from Azotobacter vinelandii . 2. Regulation of the activity. Eur J Biochem 59:347–353 [CrossRef]
     [Google Scholar]
  4. Cachon R., Divies C. 1993; Localization of Lactococcus lactis ssp. lactis bv. diacetylactis in alginate gel beads affects biomass density and synthesis of several enzymes involved in lactose and citrate metabolism. Biotechnol Tech 7:453–456
     [Google Scholar]
  5. Carballo J., Martin R., Bernardo A., Gonzalez J. 1991; Purification, characterization and some properties of diacetyl (acetoin) reductase from Enterobacter aerogenes . Eur J Biochem 198:327–332 [CrossRef]
     [Google Scholar]
  6. Chopin A., Chopin M. C., Moillo-Batt A., Langella P. 1984; Two plasmid-determined restriction and modification systems in Streptococcus lactis . Plasmid 11:260–263 [CrossRef]
     [Google Scholar]
  7. Crow V. L., Pritchard G. G. 1977; Fructose-1,6-diphosphate-activated l-lactate dehydrogenase from Streptococcus lactis : kinetic properties and factors affecting activation. J Bacteriol 131:82–91
     [Google Scholar]
  8. de Ruyter P. G., Kuipers O. P., de Vos W. M. 1996; Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62:3662–3667
     [Google Scholar]
  9. Fox D. K., Roseman S. 1986; Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli . J Biol Chem 261:13487–13497
     [Google Scholar]
  10. Gibson T. D., Parker S. M., Woodward J. R. 1991; Purification and characterization of diacetyl reductase from chicken liver and Streptococcus lactis and enzymic determination of diacetyl and diketones. Enzyme Microb Technol 13:171–178 [CrossRef]
     [Google Scholar]
  11. Henkin J., Abeles R. H. 1976; Evidence against an acyl-enzyme intermediate in the reaction catalyzed by clostridial phosphotransacetylase. Biochemistry 15:3472–3479 [CrossRef]
     [Google Scholar]
  12. Hillier A. J., Jago G. R. 1982; l-Lactate dehydrogenase, FDP-activated, from Streptococcus cremoris . Methods Enzymol 89 Pt D:362–367
     [Google Scholar]
  13. Hofmeyr J. H., Cornish-Bowden A. 1997; The reversible Hill equation: how to incorporate cooperative enzymes into metabolic models. Comput Appl Biosci 13:377–385
     [Google Scholar]
  14. Hugenholtz J., Starrenburg M. J. C. 1992; Diacetyl production by different strains of Lactococcus lactis ssp . lactis var . diacetylactis and Leuconostoc spp. Appl Microbiol Biotechnol 38:17–22
     [Google Scholar]
  15. Kacser H. 1983; The control of enzyme systems in vivo : elasticity analysis of the steady state. Biochem Soc Trans 11:35–40
     [Google Scholar]
  16. Kacser H., Burns J. A. 1973; The control of flux. Symp Soc Exp Biol 27:65–104
     [Google Scholar]
  17. Kashket E. R., Wilson T. H. 1973; Proton-coupled accumulation of galactoside in Streptococcus lactis 7962. Proc Natl Acad Sci USA 70:2866–2869 [CrossRef]
     [Google Scholar]
  18. Kholodenko B. N., Cascante M., Hoek J. B., Westerhoff H. V., Schwaber J. 1998; Metabolic design: how to engineer a living cell to desired metabolite concentrations and fluxes. Biotechnol Bioeng 59:239–247 [CrossRef]
     [Google Scholar]
  19. Kim S. G., Batt C. A. 1993; Cloning and sequencing of the Lactococcus lactis subsp . lactis groESL operon. Gene 127:121–126 [CrossRef]
     [Google Scholar]
  20. Kuipers O. P., Beerthuyzen M. M., de Ruyter P. G., Luesink E. J., de Vos W. M. 1995; Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction. J Biol Chem 270:27299–27304 [CrossRef]
     [Google Scholar]
  21. Kuipers O. P., de Ruyter P. G. G. A., Kleerebezem M., De Vos W. M. 1997; Controlled overproduction of proteins by lactic acid bacteria. Trends Biotechnol 15:135–140 [CrossRef]
     [Google Scholar]
  22. Lopez de Felipe F., Hugenholtz J. 1999; Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions. FEMS Microbiol Lett 179:461–466 [CrossRef]
     [Google Scholar]
  23. Lopez de Felipe F., Hugenholtz J. 2001; Purification and characterization of the water forming NADH oxidase of Lactococcus lactis . Int Dairy J 11:37–44 [CrossRef]
     [Google Scholar]
  24. Lopez de Felipe F., Kleerebezem M., De Vos W. M., Hugenholtz J. 1998; Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase. J Bacteriol 180:3804–3808
     [Google Scholar]
  25. Mendes P. 1993; gepasi: a software package for modelling the dynamics, steady states and control of biochemical and other systems. Comput Appl Biosci 9:563–571
     [Google Scholar]
  26. Meyer R. R., Laine P. S. 1990; The single-stranded DNA-binding protein of Escherichia coli . Microbiol Rev 54:342–380
     [Google Scholar]
  27. Monnet C., Phalip V., Schmitt P., Divies C. 1994a; Comparison of alpha-acetolactate synthase and alpha-acetolactate decarboxylase in Lactococcus spp. and Leuconostoc spp. Biotechnol Lett 16:257–262 [CrossRef]
     [Google Scholar]
  28. Monnet C., Schmitt P., Divies C. 1994b; Diacetyl production in milk by an alpha-acetolactic acid accumulating strain of Lactococcus lactis ssp . lactis biovar diacetylactis . J Dairy Sci 77:2916–2924 [CrossRef]
     [Google Scholar]
  29. Neidhardt F. C., Umbarger H. E. others 1996; Chemical composition of Escherichia coli . In Escherichia coli and Salmonella, Cellular and Molecular Biology . , 2nd edn. pp 13–16 Edited by Neidhardt F. C. Washington, DC: American Society for Microbiology;
  30. Novak L., Cocaign-Bousquet M., Lindley N. D., Loubiere P. 1998; Cometabolism sugar–amino acids in Lactococcus lactis . Lait 78:17–22 [CrossRef]
     [Google Scholar]
  31. Petit C., Vilchez F., Marczak R. 1989; Influence of citrate on the diacetyl and acetoin production by fully grown cells of Streptococcus lactis ssp. diacetylactis . Curr Microbiol 19:319–324 [CrossRef]
     [Google Scholar]
  32. Platteeuw C., Hugenholtz J., Starrenburg M. J. C., Van Alen-Boerrigter I., De Vos W. M. 1995; Metabolic engineering of Lactococcus lactis : influence of the overproduction of alpha-acetolactate synthase in strains deficient in lactate dehydrogenase as a function of culture conditions. Appl Environ Microbiol 61:3967–3971
     [Google Scholar]
  33. Savageau M. A. 1991; Biochemical systems theory: operational differences among variant representations and their significance. J Theor Biol 151:509–530 [CrossRef]
     [Google Scholar]
  34. Schmidt H. L., Stocklein W., Danzer J., Kirch P., Limbach B. 1986; Isolation and properties of an H2O-forming NADH oxidase from Streptococcus faecalis . Eur J Biochem 156:149–155 [CrossRef]
     [Google Scholar]
  35. Shone C. C., Fromm H. J. 1981; Steady-state and pre-steady-state kinetics of coenzyme A linked aldehyde dehydrogenase from Escherichia coli . Biochemistry 20:7494–7501 [CrossRef]
     [Google Scholar]
  36. Snoep J. L., Teixeira De Mattos M. J., Starrenburg M. J. C., Hugenholtz J. 1992a; Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis ssp. lactis bv. diacetylactis . J Bacteriol 174:4838–4841
     [Google Scholar]
  37. Snoep J. L., Westphal A. H., Benen J.-A. E., Teixeira De Mattos M. J., Neijssel O. M., De Kok A. 1992b; Isolation and characterization of the pyruvate dehydrogenase complex of anaerobically grown Enterococcus faecalis NCTC 775. Eur J Biochem 203:245–250 [CrossRef]
     [Google Scholar]
  38. Snoep J. L., De Graef M. R., Westphal A. H., De Kok A., Teixeira De Mattos M. J., Neijssel O. M. 1993; Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis , Lactococcus lactis and Escherichia coli : implications for their activity in vivo . FEMS Microbiol Lett 114:279–283 [CrossRef]
     [Google Scholar]
  39. Starrenburg M. J. C., Hugenholtz J. 1991; Citrate fermentation by Lactococcus and Leuconostoc spp. Appl Environ Microbiol 57:3535–3540
     [Google Scholar]
  40. Strecker H. J., Harary I. 1954; Bacterial butylene glycol dehydrogenase and diacetyl reductase. J Biol Chem 211:263–270
     [Google Scholar]
  41. Swindell S. R., Benson K. H., Griffin H. G., Renault P., Ehrlich S. D., Gasson M. J. 1996; Genetic manipulation of the pathway for diacetyl metabolism in Lactococcus lactis . Appl Environ Microbiol 62:2641–2643
     [Google Scholar]
  42. Thauer R. K., Jungermann K., Decker K. 1977; Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180
     [Google Scholar]
  43. Wills C., Kratofil P., Londo D., Martin T. 1981; Characterization of the two alcohol dehydrogenases of Zymomonas mobilis . Arch Biochem Biophys 210:775–785 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-4-1003
Loading
Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysisThe GenBank accession number for the sequence reported in this paper is AY046926.
Microbiology 148, 1003 (2002); https://doi.org/10.1099/00221287-148-4-1003
/content/journal/micro/10.1099/00221287-148-4-1003
/content/journal/micro/10.1099/00221287-148-4-1003
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error