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Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cerevisiae

Abstract

The glucose/xylose facilitator Gxf1 from Candida intermedia was expressed in the recombinant xylose-fermenting Saccharomyces cerevisiae strain TMB 3057. The new strain, TMB 3411, displayed approximately two times lower K m for xylose transport compared to a control strain not expressing Gxf1. In aerobic batch cultivation, the specific growth rate was significantly higher at low xylose concentration, 4 g/L, when Gxf1 was expressed, whereas it remained unchanged at high xylose concentration, 40 g/L. Similarly, in aerobic-xylose-limited chemostat culture, the Gxf1-expressing strain consumed more xylose than the control strain at low dilution rates (low xylose concentration), whereas the situation was reversed at higher dilution rates (high xylose concentration). Also, under anaerobic conditions, the Gxf1-expressing strain showed faster xylose uptake and ethanol formation at low substrate concentrations. The results are discussed in relation to previous observations, which suggested that transport controlled xylose utilization in recombinant xylose-utilizing S. cerevisiae only at low xylose concentrations.

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References

  1. Andreasen AA, Stier TJ (1953) Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. J Cell Physiol 41:23–36

  2. Dagert M, Ehrlich SD (1979) Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6:23–28

  3. 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 66:3381–3386

  4. Fell DA (1992) Metabolic control analysis—a survey of its theoretical and experimental development. Biochem J 286:313–330

  5. Fell DA (1997) Understanding the control of metabolism. Portland, London

  6. Fonseca C, Romao R, De Sousa HR, Hahn-Hägerdal B, Spencer-Martins I (2007) l-Arabinose transport and catabolism in yeast. FEBS J 274:3589–3600

  7. Gardonyi M, Jeppsson M, Liden G, Gorwa-Grausland MF, Hahn-Hägerdal B (2003a) Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae. Biotechnol Bioeng 82:818–824

  8. Gardonyi M, Osterberg M, Rodrigues C, Spencer-Martins I, Hahn-Hägerdal B (2003b) High capacity xylose transport in Candida intermedia PYCC 4715. FEMS Yeast Res 3:45–52

  9. Gietz RD, Sugino A (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking 6-base pair restriction sites. Gene 74:527–534

  10. Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/S-DNA/Peg procedure. Yeast 11:355–360

  11. Gorgens JF, Van Zyl WH, Knoetze JH, Hahn-Hägerdal B (2001) The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium. Biotechnol Bioeng 73:238–245

  12. Hahn-Hägerdal B, Karhumaa K, Fonseca C, Spencer-Martins I, Gorwa-Grauslund MF (2007a) Towards industrial pentose-fermenting yeast strains. Appl Microbiol Biotechnol 74:937–953

  13. Hahn-Hägerdal B, Karhumaa K, Jeppsson M, Gorwa-Grauslund MF (2007b) Metabolic engineering for pentose utilization in Saccharomyces cerevisiae. In: Olssen L (ed) Biofuels, 1st edn. Springer, Berlin, pp 147–177

  14. Hamacher T, Becker J, Gardonyi M, Hahn-Hägerdal B, Boles E (2002) Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization. Microbiology 148:2783–2788

  15. Hector RE, Qureshi N, Hughes SR, Cotta MA (2008) Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption. Appl Microbiol Biotechnol 80:675–684

  16. Karhumaa K, Hahn-Hägerdal B, Gorwa-Grauslund MF (2005) Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering. Yeast 22:359–368

  17. Katahira S, Ito M, Takema H, Fujita Y, Tanino T, Tanaka T, Fukuda H, Kondo A (2008) Improvement of ethanol productivity during xylose and glucose co-fermentation by xylose-assimilating S. cerevisiae via expression of glucose transporter Sut1. Enzyme Microb Technol 43:115–119

  18. Kuyper M, Harhangi HR, Stave AK, Winkler AA, Jetten MSM, De Laat W, Den Ridder JJJ, Op Den Camp HJM, Van Dijken JP, Pronk JT (2003) High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae. FEMS Yeast Res 4:69–78

  19. Leandro MJ, Goncalves 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 395:543–549

  20. Leandro MJ, Spencer-Martins I, Goncalves P (2008) The expression in Saccharomyces cerevisiae of a glucose/xylose symporter from Candida intermedia is affected by the presence of a glucose/xylose facilitator. Microbiology 154:1646–1655

  21. Mumberg D, Muller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122

  22. Ohgren K, Bengtsson O, Gorwa-Grauslund MF, Galbe M, Hahn-Hägerdal B, Zacchi G (2006) Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400. J Biotechnol 126:488–498

  23. Olofsson K, Rudolf A, Liden G (2008) Designing simultaneous saccharification and fermentation for improved xylose conversion by a recombinant strain of Saccharomyces cerevisiae. J Biotechnol 134:112–120

  24. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45

  25. Saloheimo A, Rauta J, Stasyk OV, Sibirny AA, Penttila M, Ruohonen L (2007) Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases. Appl Microbiol Biotechnol 74:1041–1052

  26. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

  27. Sedlak M, Ho NW (2004) Characterization of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co-fermentation by a recombinant Saccharomyces yeast. Yeast 21:671–684

  28. Verduyn C, Postma E, Scheffers WA, Van Dijken JP (1992) Effect of benzoic acid on metabolic fluxes in yeasts - a continuous culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501–517

  29. Walfridsson M, Anderlund M, Bao X, Hahn-Hägerdal B (1997) Expression of different levels of enzymes from the Pichia stipitis XYL1 and XYL2 genes in Saccharomyces cerevisiae and its effects on product formation during xylose utilisation. Appl Microbiol Biotechnol 48:218–224

  30. Wenzel TJ, Teunissen AWRH, Steensma HY (1995) PDA1 messenger-RNA - a standard for quantitation of messenger-RNA in Saccharomyces cerevisiae superior to ACT1 messenger-RNA. Nucleic Acids Res 23:883–884

  31. Xue XD, Ho NWY (1990) Xylulokinase activity in various yeasts including Saccharomyces cerevisiae containing the cloned xylulokinase gene. Appl Biochem Biotechnol 24-5:193–199

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Acknowledgments

This work was supported by the European Union (NILE, EU contract no 019882). Lina Thornblad is acknowledged for excellent technical assistance.

Author information

Correspondence to B. Hahn-Hägerdal.

Additional information

Prof. Isabel Spencer-Martins passed away during the final preparation of this manuscript.

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Runquist, D., Fonseca, C., Rådström, P. et al. Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cerevisiae . Appl Microbiol Biotechnol 82, 123–130 (2009). https://doi.org/10.1007/s00253-008-1773-y

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Keywords

  • Saccharomyces cerevisiae
  • Xylose transport
  • GXF1
  • Candida intermedia
  • Lignocellulose