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Applied Microbiology and Biotechnology

, Volume 74, Issue 5, pp 1041–1052 | Cite as

Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases

  • Anu Saloheimo
  • Jenita Rauta
  • Oleh V. Stasyk
  • Andrei A. Sibirny
  • Merja Penttilä
  • Laura RuohonenEmail author
Applied Genetics and Molecular Biotechnology

Abstract

In the present study, we modified xylose uptake properties of a recombinant xylose-utilizing yeast Saccharomyces cerevisiae by expression of heterologous and homologous permease-encoding genes. In a mutant yeast strain with the main seven hexose transporter genes deleted, and engineered for xylose utilization, we screened an expression cDNA library of the filamentous fungus Trichoderma reesei (Hypocrea jecorina) for enhanced growth on xylose plates. One cDNA clone with significant homology to fungal sugar transporters was obtained, but when the clone was retransformed into the host, it did not support significant growth on xylose. However, during a long liquid culture of the strain carrying the cDNA clone, adaptive mutations apparently occurred in the host, which led to growth on xylose but not on glucose. The new transporter homologue, Trxlt1 thus appears to code for a protein specific for xylose uptake. In addition, xylose-transporting properties of some homologous hexose transporters were studied. All of them, i.e., Hxt1, Hxt2, Hxt4, and Hxt7 were capable of xylose uptake. Their affinities for xylose varied, K m values between 130 and 900 mM were observed. The single-Hxt strains showed a biphasic growth mode on xylose, alike the Trxlt1 harboring strain. The initial, slow growth was followed by a long lag and finally by exponential growth.

Keywords

Xylose uptake Saccharomyces cerevisiae Hexose transporters Trichoderma reesei transporter Adaptive mutation(s) 

Notes

Acknowledgements

We want to acknowledge John Londesborough for his advice on the uptake studies and analysis of the results, for instructive discussions, as well as important comments on the manuscript. Virve Vidgren is thanked for the help in cloning of the hexose transporter genes and Carmen Limon for the help in sequencing of the xylose transporter. Technical assistance of Seija Rissanen, Aila Siltala, Eila Leino, and Titta Manninen is warmly thanked. This work is part of the research program “VTT Industrial Biotechnology” (Academy of Finland; Finnish Centre of Excellence program, 2000–2005, Project no. 64330). The financial support of Technology Agency of Finland (Project no. Tekes 40416/01) is acknowledged.

References

  1. Billard P, Ménart S, Blaisonneau J, Bolotin-Fukuhara M, Fukuhara H, Wésolowski-Louvel M (1996) Glucose uptake in Kluyveromyces lactis: role of the HGT1 gene in glucose transport. J Bacteriol 178:5860–5866Google Scholar
  2. Boles E, Hollenberg C (1997) The molecular genetics of hexose transport in yeasts. FEMS Microbiol Rev 21:85–111CrossRefGoogle Scholar
  3. Boles E, Gohlmann HW, Zimmermann FK (1996) Cloning of a second gene encoding 5-phosphofructo-2-kinase in yeast, and characterization of mutant strains without fructose-2,6-bisphosphate. Mol Microbiol 20:65–76CrossRefGoogle Scholar
  4. Buziol S, Becker J, Baumeister A, Jung S, Mauch K, Reuss M, Boles E (2002) Determination of in vivo kinetics of the starvation-induced Hxt5 glucose transporter of Saccharomyces cerevisiae. FEMS Yeast Res 2:283–291Google Scholar
  5. Cornish-Bowden A, Eisenthal R (1974) Statistical considerations in the estimation of enzyme kinetic parameters by the direct linear plot and other methods. Biochem J 139:721–730Google Scholar
  6. Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890CrossRefGoogle Scholar
  7. Does AL, Bisson LF (1989) Characterization of xylose uptake in the yeasts Pichia heedie and Pichia stipitis. Appl Environ Microbiol 55:159–164Google Scholar
  8. Eisenthal R, Cornish-Bowden A (1974) The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem J 139:715–720Google Scholar
  9. Eliasson A, Christensson C, Wahlbom CF, Hahn-Hägerdal B (2000) Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures. Appl Environ Microbiol 66:3381–3386CrossRefGoogle Scholar
  10. Gárdonyi M, Jeppsson M, Lidén G, Gorwa-Grauslund MF, Hahn-Hägerdal B (2002) Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae. Biotechnol Bioeng 82:818–824CrossRefGoogle Scholar
  11. Gárdonyi M, Österberg M, Rodrigues C, Spencer-Martins I, Hahn-Hägerdal B (2003) High capacity xylose transport in Candida intermedia PYCC 4715. FEMS Yeast Res 3:45–52CrossRefGoogle Scholar
  12. Gietz D, St Jean A, Woods R, Schiestl R (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425CrossRefGoogle Scholar
  13. Goffrini P, Ferrero I, Donnini C (2002) Respiration-dependent utilization of sugars in yeasts: a determinant role for sugar transporters. J Bacteriol 184:427–432CrossRefGoogle Scholar
  14. Hamacher T, Becker J, Gárdonyi M, Hahn-Hägerdal B, Boles E (2002) Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization. Microbiology 148:2783–2788Google Scholar
  15. Hill J, Ian KA, Donald G, Griffith DE (1991) DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res 19:5791CrossRefGoogle Scholar
  16. Ho NWY, Chen Z, Brainard AP (1998) Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose. Appl Environ Microbiol 64:1852–1859Google Scholar
  17. Hoffman C, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272CrossRefGoogle Scholar
  18. Kotyk A (1967) Properties of the sugar carrier in baker’s yeast. Folia Microbiol 12:121–131Google Scholar
  19. Kruckeberg AL, Ye L, Berden JA, van Dam K (1999) Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein. Biochem J 339:299–307CrossRefGoogle Scholar
  20. Kuyper M, Hartog MMP, Toirkens MJ, Almering MJH, Winkler AA, van Dijken JP, Pronk JT (2005a) Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Res 5:399–409CrossRefGoogle Scholar
  21. Kuyper M, Toirkens MJ, Diderich JA, Winkler AA, van Dijken JP, Pronk JT (2005b) Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain. FEMS Yeast Res 5:925–934CrossRefGoogle Scholar
  22. Kötter P, Ciriacy M (1993) Xylose fermentation by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 38:776–783CrossRefGoogle Scholar
  23. Kötter P, Amore R, Hollenberg C, Ciriacy M (1990) Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant. Curr Genet 18:493–500CrossRefGoogle Scholar
  24. Leandro MJ, Conçalves P, Spencer-Martins I (2006) Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H+ symporter. Biochem J 395:543–549CrossRefGoogle Scholar
  25. Lee W-J, Kim M-D, Ryu Y-W, Bisson LF, Seo J-H (2002) Kinetic studies on glucose and xylose transport in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 60:186–191CrossRefGoogle Scholar
  26. Lichtenberg H, Heyer M, Höfer M (1999) Tpr1, a Schizosaccharomyces pombe protein involved in potassium transport. FEBS Lett 457:363–368CrossRefGoogle Scholar
  27. Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  28. Lucas C, van Uden N (1986) Transport of hemicellulose monomeres in the xylose-fermenting yeast Candida shehatae. Appl Microbiol Biotechnol 23:491–495CrossRefGoogle Scholar
  29. Maier A, Völker B, Boles E, Fuhrmann GF (2002) Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters. FEMS Yeast Res 2:539–550Google Scholar
  30. Makuc J, Cappellaro C, Boles E (2004) Co-expression of a mammalian accessory trafficking protein enables functional expression of the rat MCT1 monocarboxylate transporter in Saccharomyces cerevisiae. FEMS Yeast Res 4:795–801CrossRefGoogle Scholar
  31. Margolles-Clark E, Tenkanen M, Nakari-Setälä T, Penttilä M (1996) Cloning of genes encoding alpha-l-arabinofuranosidase and beta-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae. Appl Environ Microbiol 62:3840–3846Google Scholar
  32. Nobre A, Lucas C, Leão C (1999) Transport and utilization of hexoses and pentoses in the halotolerant yeast Debaryomyces hansenii. Appl Environ Microbiol 65:3594–3598Google Scholar
  33. Özcan S, Johnston M (1999) Function and regulation of yeast hexose transporters. Microbiol Mol Biol Rev 63:554–569Google Scholar
  34. Pitkänen J-P, Rintala E, Aristidou A, Ruohonen L, Penttilä M (2005) Xylose chemostat isolates of Saccharomyces cerevisiae show altered metabolite and enzyme levels compared with xylose, glucose, and ethanol metabolism of the original strain. Appl Microbiol Biotechnol 67:827–837CrossRefGoogle Scholar
  35. Prior C, Fukuhara H, Blaisonneau J, Wésolowski-Louvel M (1993) Low-affinity glucose carrier gene LGT1 of Saccharomyces cerevisiae, a homologue of the Kluyveromyces lactis RAG1 gene. Yeast 9:1373–1377CrossRefGoogle Scholar
  36. Reifenberger E, Freidel K, Ciriacy M (1995) Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on glycolytic flux. Mol Microbiol 16:157–167CrossRefGoogle Scholar
  37. Reifenberger E, Boles E, Ciriacy M (1997) Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. Eur J Biochem 245:324–333CrossRefGoogle Scholar
  38. Richard P, Toivari M, Penttilä M (2000) The role of xylulokinase in Saccharomyces cerevisiae xylulose catabolism. FEMS Microbiol Lett 190:39–43CrossRefGoogle Scholar
  39. Richard P, Verho R, Putkonen M, Londesborough J, Penttilä M (2003) Production of ethanol from l-arabinose by Saccharomyces cerevisiae containing a fungal L-arabinose pathway. FEMS Yeast Res 3:185–189CrossRefGoogle Scholar
  40. Ruohonen L, Aalto M, Keränen S (1995) Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins. J Biotechnol 39:193–203CrossRefGoogle Scholar
  41. Saloheimo A, Henrissat B, Hoffrén A, Teleman O, Penttilä M (1994) A novel, small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast. Mol Microbiol 13:219–228CrossRefGoogle Scholar
  42. Salusjärvi L, Pitkänen J-P, Aristidou A, Ruohonen L, Penttilä M (2006) Gene expression analysis of recombinant xylose-fermenting Saccharomyces cerevisiae reveals novel responses to xylose as a carbon source. Appl Biochem Biotechnol 128:237–261CrossRefGoogle Scholar
  43. Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USAGoogle Scholar
  44. Sedlak M, Ho NWY (2004) Characterisation of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co-fermentation by a recombinant Saccharomyces yeast. Yeast 21:671–684CrossRefGoogle Scholar
  45. Sherman F, Fink G, Hicks JB (1983) Methods in yeast genetics. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USAGoogle Scholar
  46. Sherwood PW, Katic I, Sanz P, Carlson M (2000) A glucose transporter chimera confers a dominant negative glucose starvation phenotype in Saccharomyces cerevisiae. Genetics 155:989–992Google Scholar
  47. Sonderegger M, Sauer U (2003) Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose. Appl Environ Microbiol 69:1990–1998CrossRefGoogle Scholar
  48. Stambuk BU, Franden MA, Singh A, Zhang M (2003) d-Xylose transport by Candida succiphila and Kluyveromyces marxianus. Appl Biochem Biotechnol 105–108:255–263CrossRefGoogle Scholar
  49. Toivari M, Aristidou A, Ruohonen L, Penttilä M (2001) Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability. Metab Eng 3:236–249CrossRefGoogle Scholar
  50. Tokai M, Kawasaki H, Kikuchi Y, Ouchi K (2000) Cloning and characterization of the CSF1 gene of Saccharomyces cerevisiae, which is required for nutrient uptake at low temperature. J Bacteriol 182:2865–2868CrossRefGoogle Scholar
  51. Varma A, Singh BB, Karnani N, Lichtenberg-Frate H, Hofer M, Magee BB, Prasad R (2000) Molecular cloning and functional characterisation of a glucose transporter, CaHGT1, of Candida albicans. FEMS Microbiol Lett 182:15–21CrossRefGoogle Scholar
  52. Wahlbom CF, Cordero Otero RR, van Zyl WH, Hahn-Hägerdal B, Jönsson LJ (2003) Molecular analysis of a Saccharomyces cerevisiae mutant with improved ability to utilize xylose shows enhanced expression of proteins involved in transport, initial xylose metabolism and the pentose phosphate pathway. Appl Environ Microbiol 69:740–746CrossRefGoogle Scholar
  53. Walfridsson M, Hallborn J, Penttilä M, Keränen S, Hahn-Hägerdal B (1995) Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase. Appl Environ Microbiol 61:4184–4190Google Scholar
  54. Weierstall T, Hollenberg CP, Boles E (1999) Cloning and characterization of three genes (SUT1-3) encoding glucose transporters of the yeast Pichia stipitis. Mol Microbiol 31:871–883CrossRefGoogle Scholar
  55. Wieczorke R, Krampe S, Weierstall T, Freidel K, Hollenberg CP, Boles E (1999) Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett 464:123–128CrossRefGoogle Scholar
  56. Ye L, Kruckeberg AL, Berden JA, van Dam K (1999) Growth and glucose repression are controlled by glucose transport in Saccharomyces cerevisiae cells containing only one glucose transporter. J Bacteriol 181:4673–4675Google Scholar
  57. Zhang Y, Lee H (1997) Site-directed mutagenesis of the cysteine residues in the Pichia stipitis xylose reductase. FEMS Microbiol Lett 147:227–232CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Anu Saloheimo
    • 1
  • Jenita Rauta
    • 1
    • 4
  • Oleh V. Stasyk
    • 2
  • Andrei A. Sibirny
    • 2
    • 3
  • Merja Penttilä
    • 1
  • Laura Ruohonen
    • 1
    Email author
  1. 1.VTT, Technical Research Centre of FinlandEspooFinland
  2. 2.Institute of Cell BiologyNational Academy of Sciences of UkraineLvivUkraine
  3. 3.Department of Metabolic EngineeringUniversity of RzeszówRzeszówPoland
  4. 4.Laboratory of Cancer Genetics, Institute of Medical TechnologyUniversity of TampereTampereFinland

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