Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1990 Aug;56(8):2378–2383. doi: 10.1128/aem.56.8.2378-2383.1990

Catabolite Inactivation in the Methylotrophic Yeast Pichia pastoris†

William D Murray 1,*, Sheldon J B Duff 1, Terry J Beveridge 1
PMCID: PMC184737  PMID: 16348251

Abstract

Inactivation of the alcohol oxidase enzyme system of Pichia pastoris, during the whole-cell bioconversion of ethanol to acetaldehyde, was due to catabolite inactivation. Electron microscopy showed that methanol-grown cells contained peroxisomes but were devoid of these microbodies after the bioconversion. Acetaldehyde in the presence of O2 was the effector of catabolite inactivation. The process was initiated by the appearance of free acetaldehyde, and was characterized by an increase in the level of cyclic AMP, that coincided with a rapid 55% drop in alcohol oxidase activity. Further enzyme inactivation, believed to be due to proteolytic degradation, then proceeded at a constant but slower rate and was complete 21 h after acetaldehyde appearance. The rate of catabolite inactivation was dependent on acetaldehyde concentration up to 0.14 mM. It was temperature dependent and occurred within 24 h at 37°C and by 6 days at 15°C but not at 3°C. Alcohol oxidase activity was psychrotolerant, with only a 17% decrease in initial specific activity over a temperature drop from 37 to 3°C. In contrast, protease activity was inhibited at temperatures below 15°C. When the bioconversion was run at 3°C, catabolite inactivation was prevented. In the presence of 3 M Tris hydrochloride buffer, 123 g of acetaldehyde per liter was produced at 3°C, compared with 58 g/liter at 30°C. By using 0.5 M Tris in a cyclic-batch procedure, 140.6 g of acetaldehyde was produced.

Full text

PDF
2378

Images in this article

2380Image
on p.2380

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bormann C., Sahm H. Degradation of microbodies in relation to activities of alcohol oxidase and catalase in Candida boidinii. Arch Microbiol. 1978 Apr 27;117(1):67–72. doi: 10.1007/BF00689353. [DOI] [PubMed] [Google Scholar]
  2. Corbin J. D., Gettys T. W., Blackmore P. F., Beebe S. J., Francis S. H., Glass D. B., Redmon J. B., Sheorain V. S., Landiss L. R. Purification and assay of cAMP, cGMP, and cyclic nucleotide analogs in cells treated with cyclic nucleotide analogs. Methods Enzymol. 1988;159:74–82. doi: 10.1016/0076-6879(88)59009-2. [DOI] [PubMed] [Google Scholar]
  3. DUELL E. A., INOUE S., UTTER M. F. ISOLATION AND PROPERTIES OF INTACT MITOCHONDRIA FROM SPHEROPLASTS OF YEAST. J Bacteriol. 1964 Dec;88:1762–1773. doi: 10.1128/jb.88.6.1762-1773.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Edelman A. M., Blumenthal D. K., Krebs E. G. Protein serine/threonine kinases. Annu Rev Biochem. 1987;56:567–613. doi: 10.1146/annurev.bi.56.070187.003031. [DOI] [PubMed] [Google Scholar]
  5. Funaguma T., Toyoda Y., Sy J. Catabolite inactivation of fructose 1,6-bisphosphatase and cytoplasmic malate dehydrogenase in yeast. Biochem Biophys Res Commun. 1985 Jul 16;130(1):467–471. doi: 10.1016/0006-291x(85)90440-1. [DOI] [PubMed] [Google Scholar]
  6. Lenz A. G., Holzer H. Rapid reversible inactivation of fructose-1,6-bisphosphatase in Saccharomyces cerivisiae by glucose. FEBS Lett. 1980 Jan 14;109(2):271–274. doi: 10.1016/0014-5793(80)81103-3. [DOI] [PubMed] [Google Scholar]
  7. López-Boado Y. S., Herrero P., Gascón S., Moreno F. Catabolite inactivation of isocitrate lyase from Saccharomyces cerevisiae. Arch Microbiol. 1987 Apr;147(3):231–234. doi: 10.1007/BF00463480. [DOI] [PubMed] [Google Scholar]
  8. Mazón M. J., Gancedo J. M., Gancedo C. Inactivation of yeast fructose-1,6-bisphosphatase. In vivo phosphorylation of the enzyme. J Biol Chem. 1982 Feb 10;257(3):1128–1130. [PubMed] [Google Scholar]
  9. Müller D., Holzer H. Regulation of fructose-1,6-bisphosphatase in yeast by phosphorylation/dephosphorylation. Biochem Biophys Res Commun. 1981 Dec 15;103(3):926–933. doi: 10.1016/0006-291x(81)90899-8. [DOI] [PubMed] [Google Scholar]
  10. Polnisch E., Hofmann K. Cyclic AMP, fructose-2,6-bisphosphate and catabolite inactivation of enzymes in the hydrocarbon-assimilating yeast Candida maltosa. Arch Microbiol. 1989;152(3):269–272. doi: 10.1007/BF00409662. [DOI] [PubMed] [Google Scholar]
  11. Purwin C., Leidig F., Holzer H. Cyclic AMP-dependent phosphorylation of fructose-1,6-bisphosphatase in yeast. Biochem Biophys Res Commun. 1982 Aug 31;107(4):1482–1489. doi: 10.1016/s0006-291x(82)80166-6. [DOI] [PubMed] [Google Scholar]
  12. Ramos J., Cirillo V. P. Role of cyclic-AMP-dependent protein kinase in catabolite inactivation of the glucose and galactose transporters in Saccharomyces cerevisiae. J Bacteriol. 1989 Jun;171(6):3545–3548. doi: 10.1128/jb.171.6.3545-3548.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Saheki T., Holzer H. Comparisons of the tryptophan synthase inactivating enzymes with proteinases from yeast. Eur J Biochem. 1974 Mar 1;42(2):621–626. doi: 10.1111/j.1432-1033.1974.tb03377.x. [DOI] [PubMed] [Google Scholar]
  14. Tortora P., Birtel M., Lenz A. G., Holzer H. Glucose-dependent metabolic interconversion of fructose-1, 6-bisphosphatase in yeast. Biochem Biophys Res Commun. 1981 May 29;100(2):688–695. doi: 10.1016/s0006-291x(81)80230-6. [DOI] [PubMed] [Google Scholar]
  15. Tortora P., Burlini N., Hanozet G. M., Guerritore A. Effect of caffeine on glucose-induced inactivation of gluconeogenetic enzymes in Saccharomyces cerevisiae. A possible role of cyclic AMP. Eur J Biochem. 1982 Sep 1;126(3):617–622. doi: 10.1111/j.1432-1033.1982.tb06825.x. [DOI] [PubMed] [Google Scholar]
  16. Veenhuis M., Douma A., Harder W., Osumi M. Degradation and turnover of peroxisomes in the yeast Hansenula polymorpha induced by selective inactivation of peroxisomal enzymes. Arch Microbiol. 1983 Jun;134(3):193–203. doi: 10.1007/BF00407757. [DOI] [PubMed] [Google Scholar]
  17. Veenhuis M., Van Dijken J. P., Harder W. The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv Microb Physiol. 1983;24:1–82. doi: 10.1016/s0065-2911(08)60384-7. [DOI] [PubMed] [Google Scholar]
  18. Veenhuis M., van Dijken J. P., Harder W. Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha. Arch Microbiol. 1976 Dec 1;111(1-2):123–135. doi: 10.1007/BF00446559. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES