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Metabolic engineering of the yeast Hansenula polymorpha for the construction of efficient ethanol producers

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Abstract

Until recently, the methylotrophic yeast has not been considered as a potential producer of biofuels, particularly, ethanol from lignocellulosic hydrolysates. The first work published 10 years ago revealed the ability of the thermotolerant methylotrophic yeast Hansenula polymorpha to ferment xylose—one of the main sugars of lignocellulosic hydrolysates—which has made the yeast a promising organism for high-temperature alcoholic fermentation. Such a feature of H. polymorpha could be used in the implementation of a potentially effective process of simultaneous saccharification and fermentation (SSF) of raw materials. SSF makes it possible to combine enzymatic hydrolysis of raw materials with the conversion of the sugars produced into ethanol: enzymes hydrolyze polysaccharides to monomers, which are immediately consumed by microorganisms (producers of ethanol). However, the efficiency of alcoholic fermentation of major sugars produced via hydrolysis of lignocellulosic raw materials and, especially, xylose by wild strains of H. polymorpha requires significant improvements. In this review, the main results of metabolic engineering of H. polymorpha for the construction of improved producers of ethanol from xylose, starch, xylan, and glycerol, as well as that of strains with increased tolerance to high temperatures and ethanol, are represented.

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References

  1. Ryabova, O.B., Chmil, O.M., and Sibirny, A.A., Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha, FEMS Yeast Res., 2003, vol. 3, pp. 157–164.

    Article  Google Scholar 

  2. Schubert, C., Can biofuels finally take center stage? Nat. Biotechnol., 2006, vol. 24, no. 7, pp. 777–784.

    Article  PubMed  CAS  Google Scholar 

  3. Hahn-Hägerdal, B., Galbe, M., Gorwa-Grauslund, M.F., et al., Bio-ethanol—the fuel of tomorrow from the residues of today, Trends Biotechnol., 2006, vol. 24, no. 12, pp. 549–556.

    Article  PubMed  Google Scholar 

  4. Hill, J., Nelson, E., Tilman, D., et al., Environmental, economic and ethanol biofuels, Proc. Natl. Acad. Sci. U.S.A., 2006, vol. 103, no. 30, pp. 11206–11210.

    Article  PubMed  CAS  Google Scholar 

  5. Olofsson, K., Bertilsson, M., and Liden, G., A short review on SSF—an interesting process option for ethanol production from lignocellulosic feedstocks, Biotechnol. Biofules, 2008, vol. 1, no. 1, p. 7.

    Article  Google Scholar 

  6. Abdel-Banat, B.M., Hoshida, H., Ano, A., et al., High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl. Microbiol. Biotechnol., 2010, vol. 85, no. 4, pp. 861–867.

    Article  PubMed  CAS  Google Scholar 

  7. Ishchuk, O.P., Voronovsky, A.Y., Abbas, C.A., and Sibirny, A.A., Construction of Hansenula polymorpha strains with improved thermotolerance, Biotechnol. Bioeng., 2009, vol. 104, no. 5, pp. 911–919.

    Article  PubMed  CAS  Google Scholar 

  8. Du, Preez J.C. and van der Walt, J.P., Fermentation of D-xylose to ethanol by a strain of Candida shehatae, Biotechnol. Lett., 1983, vol. 5, pp. 357–362.

    Article  Google Scholar 

  9. Toivola, A., Yarrow, D., Bosch, E., et al., Alcoholic fermentation of D-xylose by yeasts, Appl. Environ. Microbiol., 1984, vol. 47, no. 6, pp. 1221–1223.

    PubMed  CAS  Google Scholar 

  10. Du, PreezJ.C., Bosch, M., and Bernard, A., Prior xylose fermentation by Candida shehatae and Pichia stipitis: effects of pH, temperature and substrate concentration, Enzyme Microb. Technol., 1986, vol. 8, no. 6, pp. 360–364.

    Article  Google Scholar 

  11. Jeffries, T.W. and Jin, Y.-S., Metabolic engineering for improved fermentation of pentoses by yeasts, Appl. Microbiol. Biotechnol., 2004, vol. 63, pp. 495–509.

    Article  PubMed  CAS  Google Scholar 

  12. Zeng, Q.K., Du, H.L., Wang, J.F., et al., Reversal of coenzyme specificity and improvement of catalytic efficiency of Pichia stipitis xylose reductase by rational site-directed mutagenesis, Biotechnol. Lett., 2009, vol. 31, no. 7, pp. 1025–1029.

    Article  PubMed  CAS  Google Scholar 

  13. Matsushika, A., Inoue, H., Kodaki, T., and Sawayama, S., Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives, Appl. Microbiol. Biotechnol., 2009, vol. 84, no. 1, pp. 37–53.

    Article  PubMed  CAS  Google Scholar 

  14. Brat, D., Boles, E., and Wiedemann, B., Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae, Appl. Environ. Microbiol., 2009, vol. 75, no. 8, pp. 2304–2311.

    Article  PubMed  CAS  Google Scholar 

  15. Kuyper, M., Hartog, M.M., Toirkens, M.J., et al., Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation, FEMS Yeast Res., 2005, vol. 5, pp. 399–409.

    Article  PubMed  CAS  Google Scholar 

  16. Jeffries, T.W. and Shi, N.Q., US Patent 6071729, 2000.

  17. Shi, N.Q., Cruz, J., Sherman, F., and Jeffries, T.W., Sham-sensitive alternative respiration in the xylosemetabolizing yeast Pichia stipitis, Yeast, 2002, vol. 19, no. 14, pp. 1203–1220.

    Article  PubMed  CAS  Google Scholar 

  18. Hahn-Hagerdal, B., Karhumaa, K., Fonseca, C., et al., Towards industrial pentose-fermenting yeast strains, Appl. Microbiol. Biotechnol., 2007, vol. 74, no. 5, pp. 937–953.

    Article  PubMed  Google Scholar 

  19. Wisselink, H.W., Toirkens, M.J., Wu, Q., et al., Novel evolutionary engineering approach for accelerated utilization of glucose, xylose, and arabinose mixtures by engineered Saccharomyces cerevisiae strains, Appl. Environ. Microbiol., 2009, vol. 75, no. 4, pp. 907–914.

    Article  PubMed  CAS  Google Scholar 

  20. Grabek-Lejko, D., Ryabova, O.B., Oklejewicz, B., et al., Plate ethanol-screening assay for selection of the Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability for xylose alcoholic fermentation, J. Ind. Microbiol. Biotechnol., 2006, vol. 33, no. 11, pp. 934–940.

    Article  PubMed  CAS  Google Scholar 

  21. Dmytruk, O.V., Dmytruk, K.V., Abbas, C.A., et al., Engineering of xylose reductase and overexpression of xylitol dehydrogenase and xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha, Microb. Cell Fact., 2008, vol. 23, pp. 7–21.

    Google Scholar 

  22. Dmytruk, O.V., Voronovsky, A.Y., Abbas, C.A., et al., Overexpression of bacterial xylose isomerase and yeast host xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha, FEMS Yeast Res., 2008, vol. 8, pp. 165–173.

    Article  PubMed  CAS  Google Scholar 

  23. Voronovsky, A.Y., Ryabova, O.B., Verba, O.V., et al., Expression of xylA genes encoding xylose isomerases from Escherichia coli and Streptomyces coelicolor in the methylotrophic yeast Hansenula polymorpha, FEMS Yeasts Res., 2005, vol. 5, pp. 1055–1062.

    Article  CAS  Google Scholar 

  24. Pronk, J.T., Yde Steensma, H., and Van Dijken, J.P., Pyruvate metabolism in Saccharomyces cerevisiae, Yeast, 1996, vol. 12, no. 16, pp. 1607–1633.

    Article  PubMed  CAS  Google Scholar 

  25. Schaaff, I., Heinisch, J., and Zimmermann, F.K., Overproduction of glycolytic enzymes in yeast, Yeast, 1989, vol. 5, no. 4, pp. 285–290.

    Article  PubMed  CAS  Google Scholar 

  26. van Hoek, P., Flikweert, M.T., van der Aart, Q.J., et al., Effects of pyruvate decarboxylase overproduction on flux distribution at the pyruvate branch poing in Saccharomyces cerevisiae, Appl. Environ. Microbiol., 1998, vol. 64, no. 6, pp. 2133–2140.

    PubMed  Google Scholar 

  27. Ishchuk, O.P., Voronovsky, A.Y., Stasyk, O.V., et al., Overexpression of pyruvate decarboxylse in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose, FEMS Yeast Res., 2008, vol. 8, no. 7, pp. 1164–1174.

    Article  PubMed  CAS  Google Scholar 

  28. Suwannarangsee, S., Oh, D.B., Seo, J.W., et al., Characterization of alcohol dehydrogenase 1 of the thermo-tolerant methylotrophic yeast Hansenula polymorpha, Appl. Micribiol. Biotechnol., 2010, vol. 88, no. 2, pp. 497–507.

    Article  CAS  Google Scholar 

  29. Denis, C.L., Ferguson, J., and Young, E.T., mRNA levels for the fermentative alcohol dehydrogenase of Saccharomyces cerevisiae decrease upon growth on a non-fermentable carbon source, J. Biol. Chem., 1983, vol. 258, pp. 1165–1171.

    PubMed  CAS  Google Scholar 

  30. Lutstort, U. and Megnet, R., Multiple forms of alcohol dehydrogenase in Saccharomyces cerevisiae. 1. Physiological control of ADH-2 and properties of ADH-2 and ADH-4, Arch. Biochem. Biophys., 1968, vol. 126, pp. 933–944.

    Article  Google Scholar 

  31. Cho, J.-Y. and Jeffries, T.W., Pichia stipitis genes for alcohol dehydrogenase with fermentative and respiratory functions, Appl. Environ. Microbiol., 1998, vol. 64, pp. 1350–1358.

    PubMed  CAS  Google Scholar 

  32. Passoth, V., Schafer, B., Liebel, B., et al., Molecular cloning of alcohol dehydrogenase genes of the yeast Pichia stipitis and identification of the fermentative ADH, Yeast, 1998, vol. 14, pp. 1311–1325.

    Article  PubMed  CAS  Google Scholar 

  33. Reinders, A., Romano, I., Wiemken, A., and De Virgilio, C., The thermophilic yeast Hansenula polymorpha does not require trehalose synthesis for growth at high temperatures but does for normal acquisition of thermotolerance, J. Bacteriol., 1999, vol. 181, no. 15, pp. 4665–4668.

    PubMed  CAS  Google Scholar 

  34. Kim, J., Alizadeh, P., Harding, T., et al., Disruption of the yeast ATH1 gene confers better survival after dehydration, freezing, and ethanol shock: potential commercial applications, Appl. Environ. Microbiol., 1996, vol. 62, no. 5, pp. 1563–1569.

    PubMed  CAS  Google Scholar 

  35. Londesborough, J. and Varimo, K., Characterization of two trehalases in baker’s yeast, Biochem. J., 1984, vol. 219, no. 2, pp. 511–518.

    PubMed  CAS  Google Scholar 

  36. Nwaka, S., Mechler, B., and Holzer, H., Deletion of the ATH1 gene in Saccharomyces cerevisiae prevents growth on trehalose, FEBS Lett., 1996, vol. 386, nos. 2/3, pp. 235–238.

    Article  PubMed  CAS  Google Scholar 

  37. Parrou, J.L., Jules, M., Beltran, G., and Francois, J., Acid trehalase in yeasts and filamentous fungi: localization, regulation and physiological function, FEMS Yeast. Res., 2005, vol. 5, nos. 6/7, pp. 503–511.

    Article  PubMed  CAS  Google Scholar 

  38. Jung, Y.J. and Park, H.D., Antisense-mediated inhibition of acid trehalase (ATH1) gene expression promotes ethanol fermentation and tolerance in Saccharomyces cerevisiae, Biotechnol. Lett., 2005, vol. 27, nos. 23/24, pp. 1855–1859.

    Article  PubMed  CAS  Google Scholar 

  39. Haslbeck, M., Franzmann, T., Weinfurtner, D., and Buchner, J., Some like it hot: the structure and function of small heat-shock proteins, Nat. Struct. Mol. Biol., 2005, vol. 12, no. 10, pp. 842–846.

    Article  PubMed  CAS  Google Scholar 

  40. Kitagawa, M., Miyakawa, M., Matsumura, Y., and Tsuchido, T., Escherichia coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants, Eur. J. Biochem., 2002, vol. 269, no. 12, pp. 2907–2917.

    Article  PubMed  CAS  Google Scholar 

  41. Yoshida, J. and Tani, T., Hsp16p is required for thermotolerance in nuclear mRNA export in fission yeast Schizosaccharomyces pombe, Cell Struct. Funct., 2005, vol. 29, nos 5/6, pp. 125–138.

    Article  Google Scholar 

  42. Haslbeck, M., Braun, N., Stromer, T., et al., Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae, EMBO J., 2004, vol. 23, no. 3, pp. 638–649.

    Article  PubMed  CAS  Google Scholar 

  43. Cashikar, A.G., Duennwald, M., and Lindquist, S.L., A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104, J. Biol. Chem., 2005, vol. 280, no. 25, pp. 23869–23875.

    Article  PubMed  CAS  Google Scholar 

  44. Weibezahn, J., Tessarz, P., Schlieker, C., et al., Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB, Cell, 2004, vol. 119, no. 5, pp. 653–665.

    Article  PubMed  CAS  Google Scholar 

  45. Lindquist, S. and Kim, G., Heat-shock protein 104 expression is sufficient for thermotolerance in yeast, Proc. Natl. Acad. Sci. U.S.A., 1996, vol. 93, no. 11, pp. 5301–5306.

    Article  PubMed  CAS  Google Scholar 

  46. Parsell, D.A. and Lindquist, S., The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins, Annu. Rev. Genet., 1993, vol. 27, pp. 437–496.

    Article  PubMed  CAS  Google Scholar 

  47. Guerra, E., Chye, P.P., Berardi, E., and Piper, P.W., Hypoxia abolishes transience of the heat-shock response in the methylotrophic yeast Hansenula polymorpha, Microbiology, 2005, vol. 151, no. 3, pp. 805–811.

    Article  PubMed  CAS  Google Scholar 

  48. Pugh, D.J., Ab, E., Faro, A., et al., DWNN, a novel ubiquitin-like domain, implicates RBBP6 in mRNA processing and ubiquitin-like pathways, BMC Struct. Biol., 2006, vol. 5, p. 6:1.

  49. Weissman, A.M., Themes and variations on ubiquitylation, Nat. Rev. Mol. Cell Biol., 2001, vol. 2, no. 3, pp. 169–178.

    Article  PubMed  CAS  Google Scholar 

  50. Passmore, L.A. and Barford, D., Getting into position: the catalytic mechanisms of protein ubiquitylation, Biochem. J., 2004, vol. 379, no. 3, pp. 513–525.

    Article  PubMed  CAS  Google Scholar 

  51. Cardona, F., Aranda, A., and del Olmo, M., Ubiquitin ligase Rsp5p is involved in the gene expression changes during nutrient limitation in Saccharomyces cerevisiae, Yeast, 2009, vol. 26, no. 1, pp. 1–15.

    Article  PubMed  CAS  Google Scholar 

  52. Costa, V., Amorim, M.A., Reis, E., et al., Mitochondrial superoxide dismutase is essential for ethanol tolerance of Saccharomyces cerevisiae in the post-diauxic phase, Microbiology, 1997, vol. 143, no. 5, pp. 1649–1656.

    Article  PubMed  CAS  Google Scholar 

  53. Nomura, M. and Takagi, H., Role of the yeast acetyltransferase Mpr1 in oxidative stress: regulation of oxygen reactive species caused by a toxic proline catabolism intermediate, Proc. Natl. Acad. Sci. U.S.A., 2004, vol. 101, no. 34, pp. 12616–12621.

    Article  PubMed  CAS  Google Scholar 

  54. Kadam, K.L. and Schmidt, S.L., Evaluation of Candida acidothermophilum in ethanol production from lignocellulosic biomass, Appl. Microbiol. Biotechnol., 1997, vol. 48, pp. 709–713.

    Article  PubMed  CAS  Google Scholar 

  55. Fujita, Y. and Ito, J., Synergistic saccharofication, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme, Appl. Environ. Microbiol., 2004, vol. 70, pp. 1207–1212.

    Article  PubMed  CAS  Google Scholar 

  56. Gaspar, M., Kalman, G., and Reczey, K., Corn fiber as a raw material for hemicellulose and ethanol production, Process. Biochem., 2007, vol. 42, pp. 1135–1139.

    Article  CAS  Google Scholar 

  57. Eksteen, J.M., van Rensburg, P., Cordero, Otero R.R., and Pretorius, I.S., Starch fermentation by recombinant Saccharomyces cerevisiae strains expressing the α-amylase and glucoamylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera, Biotechnol. Bioeng., 2003, vol. 84, pp. 639–646.

    Article  PubMed  CAS  Google Scholar 

  58. Piontek, M., Hagedorn, J., Hollenberg, C.P., et al., Two novel gene expression systems based on the yeasts Schwanniomyces occidentalis and Pichia stipitis, Appl. Microbiol. Biotechnol., 1998, vol. 5, pp. 331–338.

    Article  Google Scholar 

  59. Shigechi, H., Koh, J., Fujita, Y., et al., Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisp0laying glucoamylase and α-amylase, Appl. Environ. Microbiol., 2004, vol. 70, pp. 5037–5040.

    Article  PubMed  CAS  Google Scholar 

  60. Wang, T.T., Lin, L.L., and Hsu, W.H., Cloning and expression of a Schwanniomyces occidentalis α-amylase gene in Saccharomyces cerevisiae, Appl. Environ. Microbiol., 1989, vol. 55, pp. 3167–3172.

    PubMed  CAS  Google Scholar 

  61. Gellissen, G., Janowicz, Z.A., Merckelbach, A., et al., Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase, Bio. Technology, 1991, vol. 9, pp. 291–295.

    Article  PubMed  CAS  Google Scholar 

  62. Voronovsky, A.Y., Rohulya, O.V., Abbas, C.A., and Sibirny, A.A., Development of strains of the thermotolerant yeast Hansenula polymorpha capable of alcoholic fermentation of starch and xylan, Metab. Eng., 2009, vol. 11, nos. 4/5, pp. 234–242.

    Article  PubMed  CAS  Google Scholar 

  63. Torronen, A., Mach, L.R., Massner, R., et al., The two major xylanases from Trichoderma reesei: characterization of both enzymes and genes, Biotechnology, 1992, vol. 10, pp. 1461–1465.

    Article  PubMed  CAS  Google Scholar 

  64. La Grange, D.C., Pretorius, I.S., and van Zyl, W.H., Expression of a Trichoderma reesei β-xylanase gene (XYN2) in Saccharomyces cerevisiae, Appl. Environ. Microbiol., 1996, vol. 62, pp. 1036–1044.

    PubMed  Google Scholar 

  65. Katahira, S., Fujita, Y., Mizuike, A., et al., Construction of a xylan-fermenting yeast strain through codisplay of xylanolytic enzymes on the surface of xyloseutilizing Saccharomyces cerevisiae cells, Appl. Enciron. Microbiol., 2004, vol. 70, pp. 5407–5414.

    Article  CAS  Google Scholar 

  66. Meister, A., Glutathione metabolism and its selective modification, J. Biol. Chem., 1988, vol. 263, no. 33, pp. 17205–17208.

    PubMed  CAS  Google Scholar 

  67. Alexandre, H., Ansanay-Galeote, V., Dequin, S., and Blondin, B., Global gene expression during short-term ethanol stress in Saccharomyces cerevisiae, FEBS Lett., 2001, vol. 498, pp. 98–103.

    Article  PubMed  CAS  Google Scholar 

  68. Ubiyvovk, V.M., Nazarko, T.Y., Stasyk, O.G., et al., GSH2, a gene encoding gamma-glutamylcysteine synthetase in the methylotrophic yeast Hansenula polymorpha, FEMS Yeast Res., 2002, vol. 2, pp. 327–332.

    PubMed  CAS  Google Scholar 

  69. Ubiyvovk, V.M., Ananin, V.M., Malyshev, A.Y., et al., Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast Hansenula polymorpha DL-1 BMC, Biotechnology, 2011, vol. 11, no. 1, p. 8.

    PubMed  CAS  Google Scholar 

  70. Grabek-Lejko, D., Kurylenko, O.O., Sibirny, V.A., et al., Alcoholic fermentation by wild-type Hansenula polymorpha and Saccharomyces cerevisiae versus recombinant strains with an elevated level of intracellular glutathione, J. Ind. Microbiol. Biotechnol., 2011 [Epub ahead of print].

    Google Scholar 

  71. Kiel, J.A., Rechinger, K.B., van der Klei, I.J., et al., The Hansenula polymorpha PDD1 gene product, essential for the selective degradation of peroxisomes, is a homologue of Saccharomyces cerevisiae Vps34p, Yeast, 1999, vol. 15, no. 9, pp. 741–754.

    Article  PubMed  CAS  Google Scholar 

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  1. Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine

    K. V. Dmytruk & A. A. Sibirny

  2. Rzeszow University, Rzeszow, Poland

    A. A. Sibirny

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Original Russian Text © K.V. Dmytruk, A.A. Sibirny, 2013, published in Tsitologiya i Genetika, 2013, Vol. 47, No. 6, pp. 3–21.

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Dmytruk, K.V., Sibirny, A.A. Metabolic engineering of the yeast Hansenula polymorpha for the construction of efficient ethanol producers. Cytol. Genet. 47, 329–342 (2013). https://doi.org/10.3103/S0095452713060029

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