Current Genetics

, Volume 18, Issue 6, pp 493–500 | Cite as

Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant

  • Peter Kötter
  • René Amore
  • Cornelis P. Hollenberg
  • Michael Ciriacy
Original Articles


A P. stipitis cDNA library in λgt11 was screened using antisera against P. stipitis xylose reductase and xylitol dehydrogenase, respectively. The resulting cDNA clones served as probes for screening a P. stipitis genomic library. The genomic XYL2 gene was isolated and the nucleotide sequence of the 1089 bp structural gene, and of adjacent non-coding regions, was determined. The XYL2 open-reading frame codes for a protein of 363 amino acids with a predicted molecular mass of 38.5 kDa. The XYL2 gene is actively expressed in S. cerevisiae transformants. S. cerevisiae cells transformed with a plasmid, pRD1, containing both the xylose reductase gene (XYL1) and the xylitol dehydrogenase gene (XYL2), were able to grow on xylose as a sole carbon source. In contrast to aerobic glucose metabolism, S. cerevisiae XYL1-XYL2 transformants utilize xylose almost entirely oxidatively.

Key words

Xylitol dehydrogenase gene Pichia stipitis Saccharomyces cerevisiae Xylose utilization 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Alexander NJ (1986) Appl Microbiol Biotechnol 25:203–207Google Scholar
  2. Amore R, Wilhelm M, Hollenberg CP (1989) Appl Microbiol Biotechnol 30:351–357Google Scholar
  3. Barnett JA (1976) The utilization of sugars by yeasts. In: Tipson RS, Horton D (eds) Advances in carbohydrate chemistry and biochemistry. Academic Press, New York, pp 125–235Google Scholar
  4. Batt CA, Carvallo S, Easson DD, Akedo MJr (1986) Biotechnol Bioeng 28:549–553Google Scholar
  5. Bennetzen JL, Hall BD (1982) J Biol Chem 257:3026–3031Google Scholar
  6. Bergmeyer HU, Bernt E (1974) D-Glucose, Bestimmung mit Glucose-Oxydase und Peroxydase. In: Bergmeyer HU (ed) Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim, 3rd edn., pp 1250–1259Google Scholar
  7. Bernt E, Gutmann I (1974) Äthanol, Bestimmung mit Alkohol-Dehydrogenase und NAD. In: Bergmeyer HU (ed) Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim, 3rd edn., pp 1545–1548Google Scholar
  8. Beutler H-O (1984) Xylitol. In: Bergmeyer HU (ed) Methods of enzymatic analysis, Volume VI, Metabolites 1: Carbohydrates. Verlag Chemie, Weinheim, 3rd edn., pp 484–490Google Scholar
  9. Bisson LF (1988) J Bacteriol 170:4838–4845Google Scholar
  10. Blake MS, Johnston KH, Russel-Jones GJ, Gottschlich EC (1984) Anal Biochem 136:175–179Google Scholar
  11. Broach JR (1983) Methods Enymol 101:307–325Google Scholar
  12. Bruinenberg PM, De Bot PHM, Van Dijken JP, Scheffers WA (1983a) Eur J Appl Microbiol Biotechnol 18:287–292Google Scholar
  13. Bruinenberg PM, Van Dijken JP, Scheffers WA (1983b) J Gen Microbiol 129:965–971Google Scholar
  14. Bruinenberg PM, De Bot PHM, Van Dijken JP, Scheffers WA (1984) Appl Microbiol Biotechnol 19:256–260Google Scholar
  15. Ciriacy M, Porep H (1986) Conversion of pentoses to ethanol by baker's yeast. In: Magnien E (ed) Biomolecular engineering in the european community. Martinus Nijhoff Publishers, Dordrecht, pp 675–681Google Scholar
  16. Cirillo VP (1968) J Bacteriol 95:603–611Google Scholar
  17. Dellweg H, Rizzi M, Mether H, Debus D (1984) Biotechnol Lett 6:395–400Google Scholar
  18. Dobson MJ, Tuite MF, Roberts NA, Kingsman AJ, Kingsman SM, Perkins RE, Conroy SC, Dunbar B, Fothergill LA (1982) Nucleic Acids Res 10:2625–2637Google Scholar
  19. Does AL, Bisson LF (1989) Appl Environ Microbiol 55:159–164Google Scholar
  20. Gong C-S, Chen LF, Flickinger MC, Chiang L-C, Tsao GT (1981) Appl Environ Microbiol 41:430–436Google Scholar
  21. Gong C-S, Claypool TA, McCracken LD, Maun CH, Ueng PP, Tsao GT (1983) Biotechnol Bioeng 25:85–102Google Scholar
  22. Gubler U, Hoffman BJ (1983) Gene 25:263–269Google Scholar
  23. Hagedorn J, Ciriacy M (1989) Curr Genet 16:27–33Google Scholar
  24. Halliwell G, Lovelady J (1981) J Gen Microbiol 126:211–217Google Scholar
  25. Hamilton R, Watanabe CK, de Boer HA (1987) Nucleic Acids Res 15:3581–3591Google Scholar
  26. Henikoff S (1984) Gene 28:351–359Google Scholar
  27. Hohn B, Murray K (1977) Proc Nat Acad Sci USA 74:3259–3263Google Scholar
  28. Huynh TV, Young RA, Davis RW (1985) Construction and screening cDNA libraries in λgt10 and λgt11. In: Glover D (ed) DNA cloning, a practical approach. IRL Press, Oxford, pp 49–78Google Scholar
  29. Jeffries TW (1983) Adv Biochem Eng 27:1–32Google Scholar
  30. Jeffries TW (1985) TIB 3:208–212Google Scholar
  31. Johnston M, Davis RW (1984) Mol Cell Biol 4:1440–1448Google Scholar
  32. Kilian SG, van Uden N (1988) Appl Microbiol Biotechnol 27:545–548Google Scholar
  33. Kotyk A (1967) Folia Microbiol 12:121–131Google Scholar
  34. Kozak M (1981) Nucleic Acids Res 9:5233–5252Google Scholar
  35. Laemmli UK (1970) Nature 227:680–685Google Scholar
  36. Lagunas R, Gancedo JM (1973) Eur J Biochem 37:90–94Google Scholar
  37. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  38. Mierendorf RC, Percy C, Young RA (1987) Methods Enymol 152:458–469Google Scholar
  39. Porep HJ (1987) Xyluloseverwertung bei Saccharomyces cerevisiae. PhD thesis, University of Düsseldorf, Federal Republic of GermanyGoogle Scholar
  40. Proudfoot NJ, Brownlee GG (1976) Nature 263:211–214Google Scholar
  41. Ramos J, Szkutnicka K, Cirillo VP (1988) J Bacteriol 170:5375–5377Google Scholar
  42. Rizzi M, Erlemann P, Bui-Thanh N-A, Dellweg H (1988) Appl Microbiol Biotechnol 29:148–154Google Scholar
  43. Sanger F, Nicklen S, Coulson AR (1977) Proc Nat Acad Sci USA 74:5463–5467Google Scholar
  44. Sarthy AV, McConaughy BL, Lobo Z, Sundstrom JA, Furlong CE, Hall BD (1987) Appl Environ Microbiol 53:1996–2000Google Scholar
  45. Senac T, Hahn-Hägerdal B (1990) Appl Environ Microbiol 56:120–126Google Scholar
  46. Skoog K, Hahn-Hägerdal B (1988) Enzyme Microb Technol 10:66–80Google Scholar
  47. Toivola A, Yarrow D, Van Den Bosch E, Van Dijken JP, Scheffers WA (1984) Appl Environ Microbiol 47:1221–1223Google Scholar
  48. Towbin H, Staelin T, Gordon J (1979) Proc Natl Acad Sci USA 76:4350–5354Google Scholar
  49. Ueng PP, Hunter CA, Gong C-S, Tsao GT (1981) Biotechnol Lett 3:315–320Google Scholar
  50. Verduyn C, van Kleef R, Frank J, Schreuder H, van Dijken JP, Scheffers WA (1985) Biochem J 226:669–677Google Scholar
  51. Wang PY, Schneider H (1980) Can J Microbiol 26:1165–1168Google Scholar
  52. Wieland OH (1984) Glycerol, UV-method. In: Bergmeyer HU (ed) Methods of enzymatic analysis, volume VI, Metabolites 1: Carbohydrates. Verlag Chemie, Weinheim, 3rd edn., pp 504–510Google Scholar
  53. Wierenga RK, De Maeyer MCH, Hol WGJ (1985) Biochemistry 24:1346–1357Google Scholar
  54. Wierenga RK, Terpstra P, Hol WGJ (1986) J Mol Biol 187:101–107Google Scholar
  55. Zalkin H, Yanofsky C (1982) J Biol Chem 257:1491–1500Google Scholar
  56. Zamenhoff S (1958) Methods Enymol 3:696–704Google Scholar
  57. Zaret KS, Sherman F (1982) Cell 28:563–573Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Peter Kötter
    • 1
  • René Amore
    • 1
  • Cornelis P. Hollenberg
    • 1
  • Michael Ciriacy
    • 1
  1. 1.Institut für MikrobiologieHeinrich-Heine-UniversitätDüsseldorfGermany

Personalised recommendations