Development of the Thermotolerant Methylotrophic Yeast Hansenula polymorpha as Efficient Ethanol Producer

  • Kostyantyn Dmytruk
  • Olena Kurylenko
  • Justyna Ruchala
  • Olena Ishchuk
  • Andriy SibirnyEmail author


Until recently, the methylotrophic yeasts, including Hansenula polymorpha , have not been considered as a potential producer of biofuels, particularly, ethanol from lignocellulosics. However it is already known that the thermotolerant methylotrophic yeast H. polymorpha is capable to ferment xylose , glucose and cellobiose , the main sugars of lignocellulosic hydrolysates , under elevated temperature. These observations allow considering H. polymorpha as a promising organism for high temperature alcoholic fermentation in industrial applications. Although the amount of ethanol produced from xylose by the wild-type strains of H. polymorpha is extremely low, the successful approaches of metabolic engineering and classical selection had been developed during last decade, which permitted to increase ethanol accumulation from xylose 30-fold. The available strains accumulate 12.5 g of ethanol per liter from xylose at 45 °C. In this article, we present published and new approaches and main achievements on metabolic engineering and selection of H. polymorpha for improved producers of ethanol from xylose, starch , xylan , and glycerol, as well as that of strains with increased tolerance to high temperatures and ethanol.


Yeasts Ethanol H. polymorpha Metabolic engineering Methylotrophic yeasts 



This work was supported in part by National Academy of Sciences of Ukraine (Grant Nos 5–17, 6–17 and 35–17) and Science and Technology Center in Ukraine (STCU) (Grant 6188).


  1. Abdel-Banat, B.M., Hoshida, H., Ano, A., Nonklang, S. and Akada, R. 2010. Appl. Microbiol. Biotechnol. 85: 861–867.Google Scholar
  2. Alexandre, H., Ansanay-Galeote, V., Dequin, S. and Blondin, B. 2001. FEBS Lett. 498:98–103.Google Scholar
  3. Bergmeyer, H.U., Gawehn, K. and Grassl, M. (1974) In: Methods of Enzymatic Analysis (ed. H.U. Bergmeyer), Academic Press, Inc., New York, NY, Volume I, pp. 513–514.Google Scholar
  4. Brat, D., Boles, E., and Wiedemann, B. 2009. Appl. Environ. Microbiol. 75: 2304–2311.Google Scholar
  5. Cardona, F., Aranda, A. and del Olmo, M. 2009. Yeast 26: 1–15.Google Scholar
  6. Cashikar, A.G., Duennwald, M. and Lindquist, S.L. 2005. J. Biol. Chem. 280: 23869–23875.Google Scholar
  7. Cho, J.-Y. and Jeffries, T.W. 1998. Appl. Environ. Microbiol. 64: 1350–1358.Google Scholar
  8. Costa, V., Amorim, M.A., Reis, E., Quintanilha, A. and Moradas-Ferreira, P. 1997. Microbiology 143: 1649–1656.Google Scholar
  9. Denis, C.L., Ferguson, J. and Young, E.T. 1983. J. Biol. Chem. 258: 1165–1171.Google Scholar
  10. Dmytruk, K.V., Voronovsky, A.Y. and Sibirny, A.A. 2006 Curr. Genet. 50: 183–191.Google Scholar
  11. Dmytruk, O.V., Dmytruk, K.V., Abbas, C.A., Voronovsky, A.Y. and Sibirny, A.A. 2008a. Microb. Cell Fact. 23: 7–21.Google Scholar
  12. Dmytruk, O.V., Voronovsky, A.Y., Abbas, C.A., Dmytruk, K.V., Ishchuk, O.P. and Sibirny, A.A. 2008b. FEMS Yeast Res. 8: 165–173.Google Scholar
  13. Dmytruk, K., Kshanovska, B., Abbas, C. and Sibirny, A. 2016. Bioethanol 2: 24–31.Google Scholar
  14. Du Preez, J.C. and J.P. van der Walt. 1983. Biotechnol. Lett. 5: 357–362.Google Scholar
  15. Du Preez, J.C., Bosch, M. and Bernard, A. 1986. Enzyme and Microbial Technol. 8: 360–364.Google Scholar
  16. Eksteen, J.M., van Rensburg, P., Cordero, Otero, R.R. and Pretorius, I.S. 2003. Biotechnol. Bioengineer. 84: 639–646.Google Scholar
  17. Farwick, A., Bruder, S., Schadeweg, V., Oreb, M. and Boles, E. 2014. Proc. Natl. Acad. Sci. USA 111: 5159–5164.Google Scholar
  18. Fujita, Y. and Ito, J. 2004. Appl. Environ. Microbiol. 70: 1207–1212.Google Scholar
  19. Gaspar, M., Kalman, G. and Reczey, K. 2007. Process Biochem. 42: 1135–1139.Google Scholar
  20. Gellissen, G., Janowicz, Z.A., Merckelbach, A., Piontek, M., Keup, P., Weydemann, U., Hollenberg, C.P. and Strasser, A.W. 1991. BioTechnology 9: 291–295.Google Scholar
  21. Grabek-Lejko, D., Ryabova, O.B., Oklejewicz, B., Voronovsky, A.Y. and Sibirny, A.A. 2006. J. Ind. Microbiol. Biotechnol. 33: 934–940.Google Scholar
  22. Grabek-Lejko, D., Kurylenko, O.O., Sibirny, V.A., Ubiyvovk, V.M., Penninckx, M. and Sibirny, A.A. 2011. J. Ind. Microbiol. Biotechnol. 38: 1853–9.Google Scholar
  23. Guerra, E., Chye, P.P., Berardi, E. and Piper, P.W. 2005. Microbiology 151: 805–811.Google Scholar
  24. Hahn-Hägerdal, B., Galbe, M., Gorwa-Grauslund, M.F., Lidén, G. and Zacchi, G. 2006. Trends Biotechnol. 24: 549–556.Google Scholar
  25. Hahn-Hägerdal, B., Karhumaa, K., Fonseca, C., Spencer-Martins, I. and Gorwa-Grauslund, M.F. 2007. Appl. Microbiol. Biotechnol. 74: 937–953.Google Scholar
  26. Haslbeck, M., Braun, N., Stromer, T., Richter, B., Model, N., Weinkauf, S. and Buchner, J. 2004. EMBO J. 23: 638–649.Google Scholar
  27. Haslbeck, M., Franzmann, T., Weinfurtner, D. and Buchner, J. 2005. Nat. Struct. Mol. Biol. 12: 842–846.Google Scholar
  28. Haurie, V., Perrot, M., Mini, T., Jenö, P., Sagliocco, F. and Boucherie, H. 2001. J. Biol. Chem. 276: 76–85.Google Scholar
  29. Hedges, D., Proft, M. and Entian, K.D. 1995. Mol. Cell. Biol. 15: 1915–22.Google Scholar
  30. Hill, J., Nelson, E., Tilman, D., Polasky, S. and Tiffany, D. 2006. Proc. Natl. Acad. Sci. USA 103: 11206–11210.Google Scholar
  31. Ishchuk, O.P., Voronovsky, A.Y., Stasyk, O.V., Gayda, G.Z., Gonchar, M.V., Abbas, C.A. and Sibirny, A.A. 2008. FEMS Yeast Res. 8: 1164–1174.Google Scholar
  32. Ishchuk, O.P., Voronovsky, A.Y., Abbas, C.A. and Sibirny, A.A. 2009. Biotechnol. Bioeng. 104: 911–919.Google Scholar
  33. Ishchuk, O.P., Abbas, C.A. and Sibirny, A.A. 2010. J. Ind. Microbiol. Biotechnol. 37: 213–218.Google Scholar
  34. Jeffries, T.W. and Shi, N.Q. 2000. United States Patent; No 6071729.Google Scholar
  35. Jeffries, T.W. and Jin, Y.-S. 2004. Appl. Microbiol. Biotechnol. 63: 495–509.Google Scholar
  36. Jeffries, T.W., Grigoriev, I.V., Grimwood, J., Laplaza, J.M., Aerts, A., Salamov, A., Schmutz, J., Lindquist, E., Dehal, P., Shapiro, H., Jin, Y.-S., Passoth, V. and Richardson, P.M. 2007. Nat. Biotechnol. 25: 319–26.Google Scholar
  37. Jung, Y.J. and Park, H.D. 2005. Biotechnol. Lett. 27: 1855–1859.Google Scholar
  38. Kadam, K.L. and Schmidt, S.L. 1997. Appl. Microbiol. Biotechnol. 48: 709–713.Google Scholar
  39. Kiel, J.A. and Veenhuis, M. 2000. Cell. Biochem. Biophys. 32: 9–19.Google Scholar
  40. Kiel, J.A., Rechinger, K.B., van der Klei, I.J., Salomons, F.A., Titorenko, V.I. and Veenhuis, M. 1999. Yeast 15: 741–754.Google Scholar
  41. Kim, J., Alizadeh, P., Harding, T., Hefner-Gravink, A. and Klionsky, D.J. 1996. Appl. Environ. Microbiol. 62: 1563–1569.Google Scholar
  42. Kitagawa, M., Miyakawa, M., Matsumura, Y. and Tsuchido, T. 2002 Eur. J. Biochem. 269: 2907–2917.Google Scholar
  43. Kurylenko, O.O., Ruchala, J., Hryniv, O.B., Abbas, C.A., Dmytruk, K.V. and Sibirny, A.A. 2014. Microb. Cell. Fact. 13: 122.Google Scholar
  44. Kuyper, M., Hartog, M.M., Toirkens, M.J., Almering, M.J., Winkler, A.A., van Dijken, J.P. and Pronk, J.T. 2005. FEMS Yeast Res. 5: 399–409.Google Scholar
  45. La Grange, D.C., Pretorius, I.S. and van Zyl, W.H. 1996. Appl. Environ. Microbiol. 62: 1036–1044.Google Scholar
  46. Lindquist, S. and Kim, G. 1996. Proc. Natl. Acad. Sci. USA 93: 5301–5306.Google Scholar
  47. Londesborough, J. and Varimo, K. 1984. Biochem. J. 219: 511–518.Google Scholar
  48. Long, T.M., Su, Y.K., Headman, J., Higbee, A., Willis, L.B. and Jeffries, T.W. 2012. Appl. Environ. Microbiol. 78: 5492–500.Google Scholar
  49. Lutstorf, U. and Megnet, R. 1968. Arch. Biochem. Biophys. 126: 933–944.Google Scholar
  50. Matsushika, A., Inoue, H., Kodaki, T. and Sawayama, S. 2009. Appl. Microbiol. Biotechnol. 84: 37–53.Google Scholar
  51. Meister, A. 1988. J. Biol. Chem. 263: 17205–17208.Google Scholar
  52. Nomura, M. and Takagi, H. 2004. Proc. Natl. Acad. Sci. USA 101: 12616–12621.Google Scholar
  53. Nwaka, S., Mechler, B. and Holzer, H. 1996. FEBS Lett. 386: 235–238.Google Scholar
  54. Olofsson, K., Bertilsson, M. and Lidén, G. 2008. Biotechnol. Biofuels 1: 7.Google Scholar
  55. Parrou, J.L., Jules, M., Beltran, G. and François, J. 2005. FEMS Yeast Res. 5: 503–511.Google Scholar
  56. Parsell, D.A. and Lindquist, S. 1993. Annu. Rev. Genet. 27: 437–496.Google Scholar
  57. Passmore, L.A. and Barford, D. 2004. Biochem. J. 379: 513–525.Google Scholar
  58. Passoth, V., Schäfer, B., Liebel, B., Weierstall, T. and Klinner, U. 1998. Yeast 14: 1311–1325.Google Scholar
  59. Piontek, M., Hagedorn, J., Hollenberg, C.P., Gellissen, G. and Strasser, A.W.M. 1998. Appl. Microbiol. Biotechnol. 50: 331–338.Google Scholar
  60. Pugh, D.J., Ab, E., Faro, A., Lutya, P.T., Hoffmann, E. and Rees, D.J. 2006. BMC Struct. Biol. 6: 1.Google Scholar
  61. Qi, K., Zhong, J.J. and Xia, X.X. 2014. Appl. Environ. Microbiol. 80: 3879–3887.Google Scholar
  62. Reinders, A., Romano, I., Wiemken, A. and De Virgilio, C. 1999. J. Bacteriol. 181: 4665–4668.Google Scholar
  63. Ryabova, O.B., Chmil, O.M. and Sibirny, A.A. 2003. FEMS Yeast Res. 3: 157–164.Google Scholar
  64. Schaaff, I., Heinisch. J. and Zimmermann, F.K. 1989. Yeast 5: 285–290.Google Scholar
  65. Schubert, C. 2006. Nat. Biotechnol. 24: 777–784.Google Scholar
  66. Shi, N.Q., Cruz, J., Sherman, F. and Jeffries, T.W. 2002. Yeast 19: 1203–1220.Google Scholar
  67. Shigechi, H., Koh, J., Fujita, Y., Matsumoto, T., Bito, Y., Ueda, M., Satoh, E., Fukuda, H. and Kondo, A. 2004. Appl. Environ. Microbiol. 70: 5037–5040.Google Scholar
  68. Sohn, J., Choi, E., Kang, H., Rhee, J., Agaphonov, M., Ter-Avanesyan, M. and Rhee, S. 1999. Appl. Microbiol. Biotechnol. 51: 800–807.Google Scholar
  69. Suwannarangsee, S., Oh, D.B., Seo, J.W., Kim, C.H., Rhee, S.K., Kang, H.A., Chulalaksananukul, W. and Kwon, O. 2010. Appl. Microbiol. Biotechnol. 88: 497–507.Google Scholar
  70. Suwannarangsee, S., Kim, S., Kim, O.C., Oh, D.B., Seo, J.W., Kim, C.H., Rhee, S.K., Kang, H.A., Chulalaksananukul, W. and Kwon, O. 2012. Appl. Microbiol. Biotechnol. 96: 697–709.Google Scholar
  71. Tachibana, C., Yoo, J.Y., Tagne, J.B., Kacherovsky, N., Lee, T.I. and Young, E.T. 2005. Mol. Cell. Biol. 25: 2138–2146.Google Scholar
  72. Toivola, A., Yarrow, D., van den Bosch, E., van Dijken, J.P. and Scheffers, W.A. 1984. Appl. Environ. Microbiol. 47: 1221–1223.Google Scholar
  73. Torronen, A., Mach, L.R., Massner, R., Gonzales, R., Kalkkinen, N., Harkki, A. and Kubicek, C.P. 1992. Biothechnology 10: 1461–1465.Google Scholar
  74. Ubiyvovk, V.M., Nazarko, T.Y., Stasyk, O.G., Sohn, M.J., Kang, H.A. and Sibirny, A.A. 2002. FEMS Yeast Res. 2: 327–332.Google Scholar
  75. Ubiyvovk, V.M., Ananin, V.M., Malyshev, A.Y., Kang, H.A. and Sibirny, A.A. 2011. BMC Biotechnol. 11: 8.Google Scholar
  76. van Hoek, P., Flikweert, M.T., van der Aart, Q.J., Steensma, H.Y., van Dijken, J.P. and Pronk, J.T. 1998. Appl. Environ. Microbiol. 64: 2133–2140.Google Scholar
  77. Voronovsky, A.Y., Ryabova, O.B., Verba, O.V., Ishchuk, O.P., Dmytruk, K.V. and Sibirny, A.A. 2005. FEMS Yeast Res. 5: 1055–1062.Google Scholar
  78. Voronovsky, A.Y., Rohulya, O.V., Abbas, C.A. and Sibirny, A.A. 2009. Metab. Eng. 11: 234–242.Google Scholar
  79. Waites, M.J. and Quayle, J.R. 1981. J. Gen. Microbiol. 124: 309–316.Google Scholar
  80. Wang, T.T., Lin, L.L. and Hsu, W.H. 1989. Appl. Environ. Microbiol. 55: 3167–3172.Google Scholar
  81. Wang, R., Li, L., Zhang, B., Gao, X., Wang, D. and Hong, J. 2013. J. Ind. Microbiol. Biotechnol. 40: 841–854.Google Scholar
  82. Watanabe, D., Hashimoto, N., Mizuno, M., Zhou, Y., Akao, T. and Shimoi, H. 2013. Biosci. Biotechnol. Biochem. 77: 2255–2262.Google Scholar
  83. Weibezahn, J., Tessarz, P., Schlieker, C., Zahn, R., Maglica, Z., Lee, S., Zentgraf, H., Weber-Ban, E.U., Dougan, D.A., Tsai, F.T., Mogk, A. and Bukau, B. 2004. Cell 119: 653–665.Google Scholar
  84. Weissman, A.M. 2001. Nat. Rev. Mol. Cell Biol. 2: 169–178.Google Scholar
  85. Wisselink HW, Toirkens MJ, Wu Q, Pronk JT, van Maris AJ. 2009. Appl. Environ. Microbiol. 75: 907–914.Google Scholar
  86. Yoshida J, Tani T. 2005. Cell Struct. Funct. 29: 125–138.Google Scholar
  87. Zeng QK, Du HL, Wang JF, Wei DQ, Wang XN, Li YX, Lin Y. 2009. Biotechnol. Lett. 31: 1025–1029.Google Scholar

Copyright information

© Springer Science+Business Media Singapore 2017

Authors and Affiliations

  • Kostyantyn Dmytruk
    • 1
  • Olena Kurylenko
    • 1
  • Justyna Ruchala
    • 2
  • Olena Ishchuk
    • 3
  • Andriy Sibirny
    • 1
    • 2
    Email author
  1. 1.Institute of Cell BiologyNAS of UkraineLvivUkraine
  2. 2.Rzeszow UniversityRzeszowPoland
  3. 3.Department of BiologyLund UniversityLundSweden

Personalised recommendations