Applied Microbiology and Biotechnology

, Volume 89, Issue 2, pp 375–385 | Cite as

Heterologous expression of a thermophilic esterase in Kluyveromyces yeasts

  • Saul Nitsche Rocha
  • José Abrahão-Neto
  • María Esperanza Cerdán
  • Andreas Karoly Gombert
  • María Isabel González-SisoEmail author
Applied Microbial and Cell Physiology


In the present work, a thermophilic esterase from Thermus thermophilus HB27 was cloned into Kluyveromyces marxianus and into Kluyveromyces lactis using two different expression systems, yielding four recombinant strains. K. lactis showed the highest esterase expression levels (294 units per gram dry cell weight, with 65% of cell-bound enzyme) using an episomal system with the PGK promoter and terminator from Saccharomyces cerevisiae combined with the K. lactis k1 secretion signal. K. marxianus showed higher secretion efficiency of the heterologous esterase (56.9 units per gram dry cell weight, with 34% of cell-bound enzyme) than K. lactis. Hydrolytic activities for the heterologous esterases were maximum at pH values between 8.0 and 9.0 for both yeast species and at temperatures of 50 °C and 45 °C for K. marxianus and K. lactis, respectively. When compared to previously published data on this same esterase produced in the original host or in S. cerevisiae, our results indicate that Kluyveromyces yeasts can be considered good hosts for the heterologous secretion of thermophilic esterases, which have a potential application in biodiesel production or in resolving racemates.


Thermophilic esterase Kluyveromyces marxianus Kluyveromyces lactis Heterologous expression 



We would like to thank Dr. Nancy da Silva (University of California at Irvine) for kindly providing us the pNADFL11 plasmid, Dr. Hiroshi Fukuhara for providing the pSPGK1 plasmid, and Dr. Wésolowski-Louvel for kindly donating the K. lactis PM5-3C strain. SNR acknowledges grants received from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, São Paulo, Brazil) and from Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil), which made possible a 1-year internship for the researcher at the Biochemistry and Molecular Biology Laboratory of Universidade da Coruña (Spain), where the DNA work was carried out. This work was financially supported by FAPESP and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil), and by Xunta de Galicia (Proyecto PGIDIT06REM38302PR, Spain). General support to the group of the UDC was funded by “Consolidación” program from C.E.O.U. Xunta de Galicia cofinanced by FEDER.

Supplementary material

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ESM 1 (PDF 27 kb)


  1. Bellaver LH, de Carvalho NM, Abrahao-Neto J, Gombert AK (2004) Ethanol formation and enzyme activities around glucose-6-phosphate in Kluyveromyces marxianus CBS 6556 exposed to glucose or lactose excess. FEMS Yeast Res 4:691–698CrossRefGoogle Scholar
  2. Bergkamp RJ, Bootsman TC, Toschka HY, Mooren AT, Kox L, Verbakel JM, Geerse RH, Planta RJ (1993) Expression of an alpha-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus. Appl Microbiol Biotechnol 40:309–317CrossRefGoogle Scholar
  3. Bornscheuer UT (2002) Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev 26:73–81CrossRefGoogle Scholar
  4. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115–132CrossRefGoogle Scholar
  5. Chahinian H, Sarda L (2009) Distinction between esterases and lipases: comparative biochemical properties of sequence-related carboxylesterases. Protein Pept Lett 16:1149–1161CrossRefGoogle Scholar
  6. Chang A, Scheer M, Grote A, Schomburg I, Schomburg D (2009) BRENDA, AMENDA and FRENDA the enzyme information system: new content and tools in 2009. Nucleic Acids Res 37:D588–D592CrossRefGoogle Scholar
  7. De Pourcq K, De Schutter K, Callewaert N (2010) Engineering of glycosylation in yeast and other fungi: current state and perspectives. Appl Microbiol Biotechnol 87:1617–1631CrossRefGoogle Scholar
  8. Demirjian DC, Moris-Varas F, Cassidy CS (2001) Enzymes from extremophiles. Curr Opin Chem Biol 5:144–151CrossRefGoogle Scholar
  9. Du W, Li W, Sun T, Chen X, Liu D (2008) Perspectives for biotechnological production of biodiesel and impacts. Appl Microbiol Biotechnol 79:331–337CrossRefGoogle Scholar
  10. Fonseca GG, Gombert AK, Heinzle E, Wittmann C (2007) Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source. FEMS Yeast Res 7:422–435CrossRefGoogle Scholar
  11. Fonseca GG, Heinzle E, Wittmann C, Gombert AK (2008) The yeast Kluyveromyces marxianus and its biotechnological potential. Appl Microbiol Biotechnol 79:339–354CrossRefGoogle Scholar
  12. Fuciños P, Abadin CM, Sanroman A, Longo MA, Pastrana L, Rua ML (2005) Identification of extracellular lipases/esterases produced by Thermus thermophilus HB27: partial purification and preliminary biochemical characterisation. J Biotechnol 117:233–241CrossRefGoogle Scholar
  13. Giardina P, Palmieri G, Scaloni A, Fontanella B, Faraco V, Cennamo G, Sannia G (1999) Protein and gene structure of a blue laccase from Pleurotus ostreatus1. Biochem J 341(Pt 3):655–663CrossRefGoogle Scholar
  14. Gupta R, Rathi P, Bradoo S (2003) Lipase mediated upgradation of dietary fats and oils. Crit Rev Food Sci Nutr 43:635–644CrossRefGoogle Scholar
  15. Harman G, Hayes C, Lorito M, Broadway R, Di Pietro A, Peterbauer C, Tronsmo A (1993) Chitinolytic enzymes of Trichoderma harzianum: purification of chitobiosidase and endochitinase. Phytopathology 83:313–318CrossRefGoogle Scholar
  16. Hong J, Wang Y, Kumagai H, Tamaki H (2007) Construction of thermotolerant yeast expressing thermostable cellulase genes. J Biotechnol 130:114–123CrossRefGoogle Scholar
  17. Idiris A, Todha H, Kumagai H, Takegawa K (2010) Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 86:403–417CrossRefGoogle Scholar
  18. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168Google Scholar
  19. Kiers J, Zeeman AM, Luttik M, Thiele C, Castrillo JI, Steensma HY, van Dijken JP, Pronk JT (1998) Regulation of alcoholic fermentation in batch and chemostat cultures of Kluyveromyces lactis CBS 2359. Yeast 14:459–469CrossRefGoogle Scholar
  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  21. López-López O, Fuciños P, Pastrana L, Rua ML, Cerdán ME, Gonzalez-Siso MI (2010) Heterologous expression of an esterase from Thermus thermophilus HB27 in Saccharomyces cerevisiae. J Biotechnol 145:226–232CrossRefGoogle Scholar
  22. Moehle CM, Aynardi MW, Kolodny MR, Park FJ, Jones EW (1987) Protease B of Saccharomyces cerevisiae: isolation and regulation of the PRB1 structural gene. Genetics 115:255–263Google Scholar
  23. Müller S, Sandal T, Kamp-Hansen P, Dalboge H (1998) Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis. Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica. Yeast 14:1267–1283CrossRefGoogle Scholar
  24. Olsson L, Nielsen J (1997) On-line and in situ monitoring of biomass in submerged cultivations. TIBTECH 15:517–522Google Scholar
  25. Rao CN, Reddy P, Liu Y, O’Toole E, Reeder D, Foster DC, Kisiel W, Woodley DT (1996) Extracellular matrix-associated serine protease inhibitors (Mr 33, 000, 31, 000, and 27, 000) are single-gene products with differential glycosylation: cDNA cloning of the 33-kDa inhibitor reveals its identity to tissue factor pathway inhibitor-2. Arch Biochem Biophys 335:82–92CrossRefGoogle Scholar
  26. Rocha SN, Abrahao-Neto J, Cerdán ME, González-Siso MI, Gombert AK (2010) Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus. Microb Cell Fact 9:4CrossRefGoogle Scholar
  27. Rouwenhorst RJ, Visser LE, Van Der Baan AA, Scheffers WA, Van Dijken JP (1988) Production, distribution, and kinetic properties of inulinase in continuous cultures of Kluyveromyces marxianus CBS 6556. Appl Environ Microbiol 54:1131–1137Google Scholar
  28. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  29. Schmidt-Dannert C, Rua ML, Atomi H, Schmid RD (1996) Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. molecular cloning, nucleotide sequence, purification and some properties. Biochim Biophys Acta 1301:105–114Google Scholar
  30. van Ooyen AJ, Dekker P, Huang M, Olsthoorn MM, Jacobs DI, Colussi PA, Taron CH (2006) Heterologous protein production in the yeast Kluyveromyces lactis. FEMS Yeast Res 6:381–392CrossRefGoogle Scholar
  31. 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 8:501–517CrossRefGoogle Scholar
  32. Walsh DJ, Bergquist PL (1997) Expression and secretion of a thermostable bacterial xylanase in Kluyveromyces lactis. Appl Environ Microbiol 63:3297–3300Google Scholar
  33. Walsh DJ, Gibbs MD, Bergquist PL (1998) Expression and secretion of a xylanase from the extreme thermophile, thermotoga strain FjSS3B.1, in Kluyveromyces lactis. Extremophiles 2:9–14CrossRefGoogle Scholar
  34. Zitomer RS, Hall BD (1976) Yeast cytochrome c messenger RNA. In vitro translation and specific immunoprecipitation of the CYC1 gene product. J Biol Chem 251:6320–6326Google Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Saul Nitsche Rocha
    • 1
    • 4
  • José Abrahão-Neto
    • 2
  • María Esperanza Cerdán
    • 3
  • Andreas Karoly Gombert
    • 1
  • María Isabel González-Siso
    • 3
    Email author
  1. 1.Department of Chemical EngineeringUniversity of São PauloSão PauloBrazil
  2. 2.School of Pharmaceutical SciencesUniversity of São PauloSão PauloBrazil
  3. 3.Departamento de Bioloxía Celular e Molecular, Facultade de CienciasUniversidade da CoruñaCoruñaSpain
  4. 4.Universidade PositivoRua Pedro Viriato Parigot de SouzaCuritibaBrasil

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