Archives of Microbiology

, Volume 148, Issue 4, pp 314–320 | Cite as

Glycerol metabolism in the methylotrophic yeast Hansenula polymorpha: phosphorylation as the initial step

  • W. de Koning
  • W. Harder
  • L. Dijkhuizen
Original Papers

Abstract

In hansenula polymorpha glycerol is metabolized via glycerol kinase and NAD(P)-independent glycerol-3-phosphate (G3P) dehydrogenase, enzymes which hitherto were reported to be absent in this methylotrophic yeast. Activity of glycerol kinase was readily detectable when cell-free extracts were incubated at pH 7–8 with glycerol/ATP/Mg2+ and a discontinuous assay for G3P formation was used. This glycerol kinase activity could be separated from dihydroxyacetone (DHA) kinase activity by ion exchange chromatography. Glycerol kinase showed relatively low affinities for glycerol (apparent Km=1.0 mM) and ATP (apparent Km=0.5 mM) and was not active with other substrates tested. No inhibition by fructose-1,6-bisphosphate (FBP) was observed. Both NAD-dependent and NAD(P)-independent G3P dehydrogenases were present. The latter enzyme could be assayed with PMS/MTT and cosedimented with the mitochondrial fraction. Glucose partly repressed synthesis of glycerol kinase and NAD(P)-independent G3P dehydrogenase, but compared to several other non-repressing carbon sources no clear induction of these enzymes by glycerol was apparent. Amongst glycerolnegative mutants of H. polymorpha strain 17B (a DHA kinase-negative mutant), strains blocked in either glycerol kinase or membrane-bound G3P dehydrogenase were identified. Crosses between representatives of the latter mutants and wild type resulted in the isolation of, amongst others, segregants which had regained DHA kinase but were still blocked in the membrane-bound G3P dehydrogenase. These strains, employing the oxidative pathway, were only able to grow very slowly in glycerol mineral medium.

Key words

Hansenula polymorpha Glycerol Glycerol kinase Dihydroxyacetone Dihydroxyacetone kinase Methanol Methylotrophy Regulation 

Abbreviations

DHA

dihydroxyacetone

G3P

glycerol-3-phosphate

EMS

ethyl methanesulphonate

MTT

3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide

PMS

phenazine methosulphate

FBP

fructose-1,6-bisphosphate

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Adler L, Blomberg A, Nilsson A (1985) Glycerol metabolism and osmoregulation in the salt-tolerant yeast Debaromyces hansenii. J Bacteriol 162:300–306Google Scholar
  2. Avigad G (1983) A simple spectrophotometric determination of formaldehyde and other aldehydes: Application to periodate-oxidized glycol systems. Anal Biochem 134:499–504Google Scholar
  3. Babel W, Hofmann KH (1982) The relation between the assimilation of methanol and glycerol in yeasts. Arch Microbiol 132:179–184Google Scholar
  4. Dijken JP van, Harder W, Beardsmore AJ, Quayle JR (1978) Dihydroxyacetone: an intermediate in the assimilation of methanol by yeasts? FEMS Microbiol Lett 4:97–102Google Scholar
  5. Douma AC, Veenhuis M, de Koning W, Evers M, Harder W (1985) Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha. Arch Microbiol 143:237–243Google Scholar
  6. Egli Th, Lindley ND (1984) Mitochondrial activities in the methylotrophic yeast Kloeckera sp. 2201 during growth with glucose and/or methanol. J Gen Microbiol 130:3239–3249Google Scholar
  7. Gancedo C, Gancedo JM, Sols A (1968) Glycerol metabolism in yeasts. Pathways of utilization and production. Eur J Biochem 5:165–172Google Scholar
  8. Gancedo C, Llobell A, Ribas JC, Luchi F (1986) Isolation and characterization of mutants from Schizosaccharomyces pombe defective in glycerol catabolism. Eur J Biochem 159:171–174Google Scholar
  9. Grunnet N, Lundquist F (1967) Kinetics of glycerol kinases from mammalian liver and Candida mycoderma. Eur J Biochem 3:78–84Google Scholar
  10. Hocking AD (1986) Effects of water activity and culture age on the glycerol accumulation patterns of five fungi. J Gen Microbiol 132:269–275Google Scholar
  11. Kato N, Kobayashi H, Shimao M, Sakazawa C (1986) Dihydroxyacetone production from methanol by a dihydroxyacetone kinase deficient mutant of Hansenulo polymorpha. Appl Microbiol Biotechnol 23:180–186Google Scholar
  12. Kawamoto S, Yamada T, Tanaka A, Fukui S (1979) Distinct subcellular localization of NAD-linked and FAD-linked glycerol-3-phosphate dehydrogenases in N-alkane-grown Candida tropicalis. FEBS Lett 97:253–256Google Scholar
  13. Koning W de, Dijkhuizen L (1986) The relationship between methanol, dihydroxyacetone and glycerol metabolism in the methylotrophic yeast Hansenula polymorpha. Abstr 5th Int Symp on Microbial Growth on C1 Compounds. Haren, The Netherlands, p 91Google Scholar
  14. Koning W de, Gleeson MAG, Harder W, Dijkhuizen L (1987) Regulation of methanol metabolism in the yeast Hansenula polymorpha. Isolation and characterization of mutants blocked in methanol assimilatory enzymes. Arch Microbiol 147:375–383Google Scholar
  15. Kremer DR, Hansen TA (1987) Glycerol and dihydroxyacetone dissimilation in Desulfovibrio strains. Arch Microbiol 147: 249–256Google Scholar
  16. Lin ECC (1976) Glycerol dissimilation and its regulation in bacteria. Ann Rev Microbiol 30:535–578Google Scholar
  17. Martin EJ St, Freedberg WB, Lin ECC (1977) Kinase replacement by a dehydrogenase for Escherichia coli glycerol utilization. J Bacteriol 131:1026–1028Google Scholar
  18. May JW, Sloan J (1981) Glycerol utilization by Schizosaccharomyces pombe: Dehydrogenation as the initial step. J Gen Microbiol 123:183–185Google Scholar
  19. May JW, Marshall JH, Sloan J (1982) Glycerol utilization by Schizosaccharomyces pombe: Phosphorylation of dihydroxyacetone by a specific kinase as the second step. J Gen Microbiol 128:1763–1766Google Scholar
  20. Nishise H, Tani Y, Yamada H (1985) Alternation of the dissimilation pathway for glycerol and amplification of glycerol dehydrogenase in mutant strains of Cellulomonas sp. NT3060. Agric Biol Chem 49:3115–3122Google Scholar
  21. O'Connor ML, Quayle JR (1979) Mutants of Hansenula polymorpha and Candida boidinii impaired in their ability to grow on methanol. J Gen Microbiol 113:203–208Google Scholar
  22. Schütz H, Radler F (1984) Propanediol-1,2-dehydratase and metabolism of glycerol of Lactobacillus brevis. Arch Microbiol 139:366–370Google Scholar
  23. Sprague GF, Jr, Cronan JE, Jr (1977) Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism. J Bacteriol 129:1335–1342Google Scholar
  24. Tani Y, Yamada K (1987) Diversity of glycerol metabolism of methylotrophic yeasts. FEMS Microbiol Lett 40:151–153Google Scholar
  25. Thorner JW (1975) Glycerol kinase. In: Wood WA (ed) Methods in enzymology, vol 42C. Academic Press, New York, pp 148–156Google Scholar
  26. Thorner JW, Paulus H (1973) Glycerol and glycerate kinases. In: Boyer PO (ed) The enzymes, vol 8. Academic Press, New York, pp 487–508Google Scholar
  27. Walt JP van der, Yarrow D (1984) Methods for the isolation, maintenance, classification and identification of yeasts. In: Kreger-van Rij NJW (ed) The yeasts, a taxonomic study. Elsevier Science Publishers BV, Amsterdam, pp 45–104Google Scholar
  28. Wieland O (1974) Glycerol. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol 3. Academic Press, New York, pp 1404–1409Google Scholar
  29. Wieland O, Suyter M (1957) Glycerokinase: Isolierung und Eigenschaften des Enzyms. Biochem Z 329:320–331Google Scholar

Copyright information

© Springer-Verlag 1987

Authors and Affiliations

  • W. de Koning
    • 1
  • W. Harder
    • 1
  • L. Dijkhuizen
    • 1
  1. 1.Department of MicrobiologyUniversity of GroningenHarenThe Netherlands

Personalised recommendations