Current Genetics

, Volume 51, Issue 3, pp 161–169 | Cite as

Functional analysis of Kluyveromyces lactis carboxylic acids permeases: heterologous expression of KlJEN1 and KlJEN2 genes

  • Odília Queirós
  • Leonor Pereira
  • Sandra Paiva
  • Pedro Moradas-Ferreira
  • Margarida Casal
Research Article

Abstract

The present work describes a detailed physiological and molecular characterization of the mechanisms of transport of carboxylic acids in Kluyveromyces lactis. This yeast species presents two homologue genes to JEN1 of Saccharomyces cerevisiae: KlJEN1 encodes a monocarboxylate permease and KlJEN2 encodes a dicarboxylic acid permease. In the strain K.lactis GG1888, expression of these genes does not require an inducer and activity for both transport systems was observed in glucose-grown cells. To confirm their key role for carboxylic acids transport in K. lactis, null mutants were analyzed. Heterologous expression in S. cerevisiae has been performed and chimeric fusions with GFP showed their proper localization in the plasma membrane. S. cerevisiae jen1Δ cells transformed with KlJEN1 recovered the capacity to use lactic acid, as well as to transport labeled lactic acid by a mediated mechanism. When KlJEN2 was heterologously expressed, S. cerevisiae transformants gained the ability to transport labeled succinic and malic acids by a mediated mechanism, exhibiting, however, a poor growth in malic acid containing media. The results confirmed the role of KlJen1p and KlJen2p as mono and dicarboxylic acids permeases, respectively, not subjected to glucose repression, being fully functional in S. cerevisiae.

Keywords

Yeast Kluyveromyces lactis  Carboxylate transport Heterologous expression GFP 

References

  1. Andrade RP, Casal M (2001) Expression of the lactate permease gene JEN1 from the yeast Saccharomyces cerevisiae. Fungal Genet Biol 32:105–111PubMedCrossRefGoogle Scholar
  2. Ausubel FA, Brent R, Kingston D, Moore D, Seidman JG, Smith JA, Struhl K (1998) In: Current protocols in molecular biology. Wiley, New York, pp 4.9.1–4.9.11Google Scholar
  3. Breunig KD, Bolotin-Fukuhara M, Bianchi MM, Bourgarel D, Falcone C, Ferrero I, Frontali L, Goffrini P, Krijger JJ, Mazzoni C, Milkowski C, Steensma HY, Wesolowski-Louvel M (2000) Regulation of primary carbon metabolism in Kluyveromyces lactis. Enzyme Microb Technol 26:771–780PubMedCrossRefGoogle Scholar
  4. Casal M, Cardoso H, Leão C (1996) Mechanisms regulating the transport of acetic acid in Saccharomyces cerevisiae. Microbiology 142:1385–1390PubMedCrossRefGoogle Scholar
  5. Casal M, Leão C (1995) Utilization of short-chain monocarboxylic acids by the yeast Torulaspora delbrueckii: specificity of the transport systems and their regulation. Biochim Biophys Acta 1267:122–130PubMedCrossRefGoogle Scholar
  6. Casal M, Paiva S, Andrade RP, Gancedo C, Leão C (1999) The lactate-proton symport of Saccharomyces cerevisiae is encoded by JEN1. J Bacteriol 181:2620–2623PubMedGoogle Scholar
  7. Cássio F, Leão C (1993) A comparative study on the transport of L(-)malic acid and other short-chain carboxylic acids in the yeast Candida utilis: evidence for a general organic acid permease. Yeast 9:743–752PubMedCrossRefGoogle Scholar
  8. Cássio F, Leão C, van Uden N (1987) Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae. Appl Environ Microbiol 53:509–513PubMedGoogle Scholar
  9. Côrte-Real M, Leão C (1989) Transport of L-malic acid and other dicarboxylic acids in the yeast Candida sphaerica. Appl Microbiol Biotechnol 31:551–555CrossRefGoogle Scholar
  10. Côrte-Real M, Leão C (1990) Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala. Appl Environ Microbiol 56:1109–1113PubMedGoogle Scholar
  11. De Deken RH (1966) The Crabtree effect: a regulatory system in yeast. J Gen Microbiol 44:149–156PubMedGoogle Scholar
  12. Ferrero I, Rossi C, Landini MP, Puglisi PP (1978) Role of the mithocondrial protein synthesis in the catabolite repression of the petite-negative yeast Kluyveromyces lactis. Biochem Biophys Res Commun 80:340–348PubMedCrossRefGoogle Scholar
  13. Fonseca A, Spencer-Martins I, van Uden N (1991) Transport of lactic acid in Kluyveromyces marxianus: evidence for a monocarboxylate uniport. Yeast 7:775–780PubMedCrossRefGoogle Scholar
  14. Gerós H, Cássio F, Leão C (2000) Utilization and transport of acetic acid in Dekkera anomala and their implications on the survival of the yeast in acidic environments. J Food Prot 63(1):96–101PubMedGoogle Scholar
  15. Goffrini P, Algeri AA, Donnini C, Wésolowski-Louvel M, Ferrero I (1989) RAG1 and RAG2: nuclear genes involved in the dependence/independence on mitochondrial respiratory function for growth on sugars. Yeast 5(2):99–106PubMedCrossRefGoogle Scholar
  16. Grobler J, Bauer F, Subden RE, van Vuuren HJJ (1995) The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids. Yeast 11:1485–1491PubMedCrossRefGoogle Scholar
  17. Güldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524PubMedCrossRefGoogle Scholar
  18. Leão C, van Uden N (1986) Transport of lactate and other short chain monocarboxylates in the yeast Candida utilis. Appl Microbiol Biotechnol 23:389–393CrossRefGoogle Scholar
  19. Lodi T, Fontanesi F, Ferrero I, Donnini C (2004) Carboxylic acids permeases in yeast: two genes in Kluyveromyces lactis. Gene 339:111–119PubMedCrossRefGoogle Scholar
  20. Milkowski C, Krampe S, Weirich J, Hasse V, Boles E, Breunig KD (2001) Feedback regulation of glucose transporter gene transcription in Kluyveromyces lactis by glucose uptake. J Bacteriol 183(18):5223–5229PubMedCrossRefGoogle Scholar
  21. Mumberg D, Mailer R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122PubMedCrossRefGoogle Scholar
  22. Osothsilp C, Subden RE (1986) Malate transport in Schizosaccharomyces pombe. J Bacteriol 168:1439–1443PubMedGoogle Scholar
  23. Paiva S, Devaux F, Barbosa S, Jacq C, Casal M (2004) Ady2p is essencial for the acetate permease activity in the yeast Saccharomyces cerevisiae. Yeast 21(3):201–210PubMedCrossRefGoogle Scholar
  24. Paiva S, Kruckeberg AL, Casal M (2002) Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae. Biochem J 363(Pt 3):737–744PubMedCrossRefGoogle Scholar
  25. Prior C, Mamessier P, Fukuhara H, Chen XJ, Wésolowski-Louvel M (1993) The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis. Mol Cell Biol 13(7):3882–3889PubMedGoogle Scholar
  26. Queirós O (2002) Transporte e utilização de ácidos dicarboxílicos nas leveduras Kluyveromyces sp. e Saccharomyces cerevisiae: uma abordagem fisiológica, bioquímica e genética. Ph.D. Thesis, University of MinhoGoogle Scholar
  27. Queirós O, Casal M, Althoff S, Moradas-Ferreira P, Leão C (1998) Isolation and characterization of Kluyveromyces marxianus mutants deficient in malate transport. Yeast 14:401–407PubMedCrossRefGoogle Scholar
  28. Queirós O, Paiva S, Moradas-Ferreira P, Casal M (2003) Molecular and physiological characterization of monocarboxylic acids permeases in the yeast Kluyveromyces lactis. Yeast 20:S237Google Scholar
  29. Radler F (1993) Yeasts—metabolism of organic acids. In: Fleet GH (ed) Wine microbiology and biotechnology. Harwood Academic Publishers, Switerzland, pp 165–182Google Scholar
  30. Salmon JM (1987) L-malic acid permeation in resting cells of anaerobically grown Saccharomyces cerevisiae. Biochim Biophys Acta 901:30–34PubMedCrossRefGoogle Scholar
  31. Sambrook J, Fritisch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  32. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedCrossRefGoogle Scholar
  33. Snoek IS, Steensma HY (2006) Why does Kluyveromyces lactis not grow under anaerobic conditions? Comparison of essential anaerobic genes of Saccharomyces cerevisiae with the Kluyveromyces lactis genome. FEMS Yeast Res 6(3):393–403PubMedCrossRefGoogle Scholar
  34. Soares-Silva I, Paiva S, Kötter P, Entian K-D, Casal M (2004) Disruption of JEN1 from Candida albicans impairs the transport of lactate. Mol Membr Biol 21:401–411CrossRefGoogle Scholar
  35. Soares-Silva I, Schüller D, Andrade RP, Baltazar F, Cássio F, Casal M (2003) Functional expression of the lactate permease Jen1p of Saccharomyces cerevisisiae in Pichia pastoris. Biochem J 376(Pt 3):781–787PubMedCrossRefGoogle Scholar
  36. Sousa MJ, Miranda L, Côrte-Real M, Leão C (1996) Transport of acetic acid in Zygosaccharomyces bailii: effects of ethanol and their implications on the resistance of the yeast to acidic environments. Appl Environ Microbiol 62:3152–3157PubMedGoogle Scholar
  37. Sousa MJ, Mota M, Leão C (1992) Transport of malic acid in the yeast Schizosaccharomyces pombe: evidence for a proton-dicarboxylate symport. Yeast 8:1025–1031PubMedCrossRefGoogle Scholar
  38. Steensma HY (2003) Removal of dominant markers from the Kluyveromyces lactis genome using Cre/loxP system. In: Wolf K (ed) Non-convencional yeasts in genetics biochemistry and biotechnology. Springer, Berlin Heidelberg New York pp 175–178Google Scholar
  39. Thomas BJ, Rothstein R (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56:619–630PubMedCrossRefGoogle Scholar
  40. Weirich J, Goffrini P, Kuger P, Ferrero I, Breunig KD (1997) Influence of mutations in hexose-transporter genes on glucose repression in Kluyveromyces lactis. Eur J Biochem 249(1):248–257PubMedCrossRefGoogle Scholar
  41. Wésolowski-Louvel M, Breunig KD, Fukuhara H (1996) Kluyveromyces lactis. In: Wolf K (ed) Nonconventional yeasts in biotechnology, a handbook. Springer, Berlin Heidelberg New York, pp 139–201Google Scholar
  42. Zmijewski MJJr, MacQuillan AM (1975) Dual effects of glucose on dicarboxylic acids transport in Kluyveromyces lactis. Can J Microbiol 21:473–480CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Odília Queirós
    • 1
    • 3
  • Leonor Pereira
    • 2
    • 3
  • Sandra Paiva
    • 2
  • Pedro Moradas-Ferreira
    • 3
    • 4
  • Margarida Casal
    • 2
  1. 1.Instituto Superior de Ciências da Saúde-Norte (ISCSN)Rua Central da Gandra 1317 4585-116 GandraParedesPortugal
  2. 2.Centro de Biologia, Departamento de BiologiaUniversidade do Minho, Campus de GualtarBragaPortugal
  3. 3.Instituto de Biologia Molecular e Celular (IBMC)Universidade do PortoPortoPortugal
  4. 4.Instituto de Ciências Biomédicas Abel Salazar (ICBAS)Universidade do PortoPortoPortugal

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