1887

Abstract

Summary: Isocitrate lyase (ICL) was assayed during batch cultivations of on soybean oil or glucose as carbon source. On soybean oil, a correlation between enzyme activity and riboflavin synthesis was observed. On glucose as carbon source, riboflavin overproduction started in the late growth phase when glucose was exhausted. ICL activity appeared in parallel and reached a maximum of 0.41 U (mg protein). This suggested synthesis of vitamin B from the intracellular reserve fat. ICL specific activity correlated with the enzyme concentration detected by specific antibodies. Itaconate, an efficient inhibitor of ICL, was used as an antimetabolite to screen mutants with enhanced ICL activity. Cultivations of an itaconate-resistant mutant on soybean oil revealed a 15% increase in enzyme specific activity and a 25-fold increase in riboflavin yield compared to the wild-type. On the other hand, growth experiments on glucose resulted in an eightfold increase in riboflavin yield but showed a 33% reduction in ICL specific activity compared to the wild-type grown on the same medium. These results support the idea of an ICL bottleneck in the riboflavin overproducer when plant oil is used as the substrate.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-142-2-419
1996-02-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/142/2/mic-142-2-419.html?itemId=/content/journal/micro/10.1099/13500872-142-2-419&mimeType=html&fmt=ahah

References

  1. Bacher A. 1990; Biosynthesis of flavins. Chemistry and Biochemistry of Flavoenzymes 1215–249 Edited by Müller F. Boca Raton: CRC Press;
    [Google Scholar]
  2. Barth G. 1985; Genetic regulation of isocitrate lyase in the yeast Yarrowia lipolytica. Curr Genet 10:119–124
    [Google Scholar]
  3. Bigelis R. 1989; Industrial products of biotechnology: application of gene technology. Biotechnology 7243 Edited by Rehm H. J., Reed G. Weinheim: VCH;
    [Google Scholar]
  4. Bowyer P., De Lucas J. R., Turner G. 1994; Regulation of the expression of isocitrate lyase gene (acuD) of Aspergillus nidulans. Mol & Gen Genet 242:484–489
    [Google Scholar]
  5. 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
    [Google Scholar]
  6. Cooper T. G. 1981 The Tools of Biochemistry Edited by Neumeier R., Maurer H. R. Berlin: Walter de Gruyter;
    [Google Scholar]
  7. Cooperman J. M., Lopez R. 1984; Riboflavin. Handbook of Vitamins299–327 Edited by Machlin L. J. New York: Marcel Dekker;
    [Google Scholar]
  8. De Lucas J. R., Valenciano S., Laborda F., Turner G. 1994; Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans. Arch Microbiol 162:409–413
    [Google Scholar]
  9. Demain A. L. 1972; Riboflavin oversynthesis. Annu Rev Microbiol 26:369–388
    [Google Scholar]
  10. Gainey L. D. S., Kolble K., Connerton I. F. 1991; Isolation and expression of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa: evidence for a second constitutive enzyme. Mol & Gen Genet 229:253–260
    [Google Scholar]
  11. Gancedo J. M. 1992; Carbon catabolite repression in yeast. Eur J Biochem 206:297–313
    [Google Scholar]
  12. Gancedo J. M., Gancedo C. 1986; Catabolite repression mutants of yeast. FEMS Microbiol Rev 32:179–187
    [Google Scholar]
  13. Gonzales E. 1977; Two-carbon assimilative capacity and the induction of isocitrate lyase in Saccharomyces cerevisiae. J Bacteriol 129:1343–1348
    [Google Scholar]
  14. Goodwin T. W. 1959; Production and biosynthesis of riboflavin in micro-organisms. Progress in Industrial Microbiology 1139–177 Edited by Hockenhull D. J. D. London: Heywood & Company;
    [Google Scholar]
  15. Hanson A. M. 1967; Microbial production of pigments and vitamins. Microbial Technology222–250 Edited by Peppier H. J. New York: Reinhold;
    [Google Scholar]
  16. Harlowe E., Lane D. 1988 Antibodies: a Laboratory Manual Cold Spring Harbor: Cold Spring Harbor Laboratory;
    [Google Scholar]
  17. Holzer H. 1976; Catabolite inactivation in yeast. Trends Biochem Sci I:178–181
    [Google Scholar]
  18. Hones I., Simon M., Weber H. 1991; Characterization of isocitrate lyase from the yeast Yarrowia lipolytica. J Basic Microbiol 31:251–258
    [Google Scholar]
  19. Hoyt J. C., Lin H.-P. P., Reeves H. C. 1994; In vivo phosphorylation of isocitrate lyase in Escherichia coli and Acinetobacter calcoaceticus. Curr Microbiol 28:67–69
    [Google Scholar]
  20. Khan F. R., McFadden B. A. 1979; Enzyme profiles in seedling development and the effect of itaconate, an isocitrate lyase-directed reagent. Plant Physiol 64:228–231
    [Google Scholar]
  21. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  22. Lago B. D., Kaplan L. 1981; Vitamin fermentations: B2 and B12. Adv Biotechnol 3:241–246
    [Google Scholar]
  23. Lopez-Boado Y. S., Herrero P., Gascon S., Moreno F. 1987; Catabolite inactivation of isocitrate lyase from Saccharomyces cerevisiae. Arch Microbiol 147:231–234
    [Google Scholar]
  24. Lopez-Boado Y. S., Herrero P., Fernandez T., Fernandez R., Moreno F. 1988; Glucose-stimulated phosphorylation of yeast isocitrate lyase in vivo. J Gen Microbiol 134:2499–2505
    [Google Scholar]
  25. Mazon M. J., Gancedo J. M., Gancedo C. 1982; Inactivation of yeast fructose-l,6-bisphosphatase. J Biol Chem 257:1128–1130
    [Google Scholar]
  26. McCammon M. T., Veenhuis M., Trapp S. B., Goodman J. M. 1990; Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae. J Bacteriol 172:5816–5827
    [Google Scholar]
  27. McFadden B. A., Purohit S. 1977; Itaconate, an isocitrate lyase-directed inhibitor in Pseudomonas indigofera. J Bacteriol 131:136–144
    [Google Scholar]
  28. Mehta S. M., Mattoo A. K., Modi V. V. 1972; Ribitol and flavinogenesis in Eremothecium ashbyii. Biochem J 130:159–166
    [Google Scholar]
  29. Müller D., Holzer H. 1981; Regulation of fructose-1,6-bisphosphatase in yeast by phosphorylation/dephosphorylation. Biochem Biophys Res Commun 103:926–933
    [Google Scholar]
  30. Nabeshima S., Tanaka A., Fukui S. 1977; Effect of carbon sources on the level of glyoxylate cycle enzymes in n-alkane-utilizable yeasts. Agric Biol Chem 41:275–279
    [Google Scholar]
  31. Okada H., Ueda M., Uchida M., Tanaka A. 1987; Isocitrate lyase and malate synthase of Candida tropicalis grown on different carbon sources. Agric Biol Chem 51:869–875
    [Google Scholar]
  32. Ozbas T., Kutsal T. 1986; Comparative study of riboflavin production from two microorganisms: Eremothecium ashbyii and Ashbya gossypii. Enzyme Microb Technol 8:593–596
    [Google Scholar]
  33. Perlman D. 1979; Microbial process for riboflavin production. Microbial Technology, Microbial Processes, 2nd edn. 1:521–527 Edited by Peppier H. J., Perlman D. New York: Academic Press;
    [Google Scholar]
  34. Pfeifer V. F., Tanner F. W. Jr, Vojnovich C., Traufler D. H. 1950; Riboflavin by fermentation with Ashbya gossypii. Ind Eng Chem 42:1776–1781
    [Google Scholar]
  35. Robertson E. F., Hoyt J. C., Reeves H. C. 1988; Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli. J Biol Chem 263:2477–2482
    [Google Scholar]
  36. Rua J., De Arriaga D., Busto F., Soler J. 1989; Effect of glucose on isocitrate lyase in Phycomyces blakesleeanus. J Bacteriol 171:6391–6393
    [Google Scholar]
  37. Rua J., De Arriaga D., Busto F., Soler J. 1990; Isocitrate lyase from Phycomyces blakesleeanus. Biochem J 272:359–367
    [Google Scholar]
  38. Schmidt G., Stahmann K.-P., Sahm H. 1996; Inhibition of purified isocitrate lyase identified itaconate and oxalate as potential antimetabolites for the riboflavin overproducer Ashbya gossypii. Microbiology 142:411–417
    [Google Scholar]
  39. Scholer A., Schuller H.-J. 1993; Structure and regulation of the isocitrate lyase gene JCL1 from the yeast Saccharomyces cerevisiae. Curr Genet 23:375–381
    [Google Scholar]
  40. Sjogren R. E., Romano A. H. 1967; Evidence for multiple forms of isocitrate lyase in Neurospora crassa. J Bacteriol 93:1638–1643
    [Google Scholar]
  41. Stahmann K.-P., Kupp C., Feldmann S. D., Sahm H. 1994; Formation and degradation of lipid bodies found in the riboflavin-producing fungus Ashbya gossypii. Appl Microbiol Biotechnol 42:121–127
    [Google Scholar]
  42. Vandamme E. J. 1992; Production of vitamins, coenzymes and related biochemicals by biotechnological processes. J Chem Technol Biotechnol 53:313–327
    [Google Scholar]
  43. Vanni P., Giachetti E., Pinzauti G., McFadden B. A. 1990; Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comp Biochem Physiol 95B:431–458
    [Google Scholar]
  44. Yoneda F. 1984; Riboflavin (B2). Kirk-Othmer Encyclopedia of Chemical Technology108–124 Edited by Grayson M., Ekroth D. New York: Wiley;
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-142-2-419
Loading
/content/journal/micro/10.1099/13500872-142-2-419
Loading

Data & Media loading...

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