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Screening and Selection of Production Strains: Secretory Protein Expression and Analysis in Hansenula polymorpha

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Recombinant Protein Production in Yeast

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1923))

Abstract

The thermotolerant methylotrophic yeast Hansenula polymorpha has been used as a host for the high-level production of recombinant proteins from industrial enzymes to therapeutic proteins. Despite favorable characteristics of the H. polymorpha-based platform for application to heterologous gene expression, several problems and limitations, such as over-glycosylation and proteolytic degradation, can be encountered in the development of production strains for secretory proteins. Here, H. polymorpha genetic tools and host strains, developed for authentic processing and modification of secretory recombinant proteins, are introduced with the analytical protocols.

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References

  1. van der Klei IJ, Yurimoto H, Sakai Y, Veenhuis M (2006) The significance of peroxisomes in methanol metabolism in methylotrophic yeast. Biochim Biophys Acta 1763(12):1453–1462

    Article  Google Scholar 

  2. Kang HA, Gellissen G (2005) Hansenula polymorpha. In: Gellissen G (ed) Production of recombinant proteins-novel microbial and eukaryotic expression systems. Wiley-VCH, Weinheim, p 111

    Chapter  Google Scholar 

  3. Seo HS, Park JS, Han KY, Bae KD, Ahn SJ, Kang HA et al (2008) Analysis and characterization of hepatitis B vaccine particles synthesized from Hansenula polymorpha. Vaccine 26(33):4138–4144

    Article  CAS  Google Scholar 

  4. Weydemann U, Keup P, Piontek M, Strasser AW, Schweden J, Gellissen G et al (1995) High-level secretion of hirudin by Hansenula polymorpha—authentic processing of three different preprohirudins. Appl Microbiol Biotechnol 44(3-4):377–385

    Article  CAS  Google Scholar 

  5. Kang HA, Sohn JH, Choi ES, Chung BH, Yu MH, Rhee SK (1998) Glycosylation of human alpha 1-antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts. Yeast 14(4):371–381

    Article  CAS  Google Scholar 

  6. Melmer G (2005) Biopharmaceuticals and the industrial environment. In: Gellissen G (ed) Production of recombinant proteins—novel microbial and eukaryotic expression systems. Wiley-VCH, Weinheim, pp 361–383

    Chapter  Google Scholar 

  7. Degelmann A, Müller F, Sieber H, Jenzelewski V, Suckow M, Strasser AW et al (2002) Strain and process development for the production of human cytokines in Hansenula polymorpha. EMS Yeast Res 2(3):349–361

    CAS  Google Scholar 

  8. Kang HA, Kang W, Hong WK, Kim MW, Kim JY, Sohn JH et al (2001) Development of expression systems for the production of recombinant human serum albumin using the MOX promoter in Hansenula polymorpha DL-1. Biotechnol Bioeng 76(2):175–185

    Article  CAS  Google Scholar 

  9. Heo JH, Hong WK, Cho EY, Kim MW, Kim JY, Kim CH et al (2003) Properties of the Hansenula polymorpha-derived constitutive GAP promoter, assessed using an HSA reporter gene. FEMS Yeast Res 4(2):175–184

    Article  CAS  Google Scholar 

  10. Heo JH, Won HS, Kang HA, Rhee SK, Chung BH (2002) Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha. Protein Expr Purif 24(1):117–122

    Article  CAS  Google Scholar 

  11. Sohn MJ, Oh DB, Kim EJ, Cheon SA, Kwon O, Kim JY et al (2012) HpYPS1 and HpYPS7 encode functional aspartyl proteases localized at the cell surface in the thermotolerant methylotrophic yeast Hansenula polymorpha. Yeast 29(1):1–16

    Article  CAS  Google Scholar 

  12. Cook MW, Thygesen HV (2003) Safety evaluation of a hexose oxidase expressed in Hansenula polymorpha. Food Chem Toxicol 41(4):523–529

    Article  CAS  Google Scholar 

  13. Mayer AF, Hellmuth K, Schlieker H, Lopez-Ulibarri R, Oertel S, Dahlems U et al (1999) An expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha. Biotechnol Bioeng 63(3):373–381

    Article  CAS  Google Scholar 

  14. Park BS, Vladimir A, Kim CH, Rhee SK, Kang HA (2004) Secretory production of Zymomonas mobilis levansucrase by the methylotrophic yeast Hansenula polymorpha. Enzym Microb Technol 34(2):132–138

    Article  CAS  Google Scholar 

  15. Kim MW, Rhee SK, Kim JY, Shimma Y, Chiba Y, Jigami Y et al (2004) Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha. Glycobiology 14(3):243–251

    Article  CAS  Google Scholar 

  16. Blazhenko OV, Zimmermann M, Kang HA, Bartosz G, Penninckx MJ, Ubiyvovk VM et al (2006) Accumulation of cadmium ions in the methylotrophic yeast Hansenula polymorpha. Biometals 19(6):593–599

    Article  CAS  Google Scholar 

  17. Dmytruk OV, Dmytruk KV, Abbas CA, Voronovsky AY, Sibirny AA (2008) Engineering of xylose reductase and overexpression of xylitol dehydrogenase and xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha. Microb Cell Factories 7:21

    Article  Google Scholar 

  18. Suh SO, Zhou JJ (2010) Methylotrophic yeasts near Ogataea (Hansenula) polymorpha: a proposal of Ogataea angusta comb. nov. and Candida parapolymorpha sp. nov. FEMS Yeast Res 10(5):631–638

    CAS  PubMed  Google Scholar 

  19. Kurtzman CP (2011) A new methanol assimilating yeast, Ogataea parapolymorpha, the ascosporic state of Candida parapolymorpha. Antonie Van Leeuwenhoek 100(3):455–462

    Article  CAS  Google Scholar 

  20. Kang HA, Sohn JH, Agaphonov MO, Choi ES, Ter-Avanesyan MD, Rhee SK (2002) Development of expression systems for the production of recombinant proteins in Hansenula polymorpha DL-1. In: Gellissen G (ed) Hansenula polymorpha: biology and applications. Wiley-VCH, Weinheim, pp 124–146

    Google Scholar 

  21. Lahtchev KL, Semenova VD, Tolstorukov II, van der Klei I, Veenhuis M (2002) Isolation and properties of genetically defined strains of the methylotrophic yeast Hansenula polymorpha CBS4732. Arch Microbiol 177(2):150–158

    Article  CAS  Google Scholar 

  22. Kim H, Yoo SJ, Kang HA (2015) Yeast synthetic biology for the production of recombinant therapeutic proteins. FEMS Yeast Res 15(1):1–16

    Article  Google Scholar 

  23. Kim MW, Kim EJ, Kim JY, Park JS, Oh DB, Shimma Y et al (2006) Functional characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 genes as members of the yeast OCH1 mannosyltransferase family involved in protein glycosylation. J Biol Chem 281(10):6261–6272

    Article  CAS  Google Scholar 

  24. Oh DB, Park JS, Kim MW, Cheon SA, Kim EJ, Moon HY et al (2008) Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N-glycan by blocking core oligosaccharide assembly. Biotechnol J 3(5):659–668

    Article  CAS  Google Scholar 

  25. Cheon SA, Kim H, Oh DB, Kwon O, Kang HA (2012) Remodeling of the glycosylation pathway in the methylotrophic yeast Hansenula polymorpha to produce human hybrid-type N-glycans. J Microbiol 50(2):341–348

    Article  CAS  Google Scholar 

  26. Bae JH, Sohn JH, Rhee SK, Choi ES (2005) Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A. Yeast 22(1):13–19

    Article  CAS  Google Scholar 

  27. Kim SY, Sohn JH, Pyun YR, Choi ES (2002) A cell surface display system using novel GPI-anchored proteins in Hansenula polymorpha. Yeast 19(13):1153–1163

    Article  CAS  Google Scholar 

  28. Kim SY, Sohn JH, Pyun YR, Yang IS, Kim KH, Choi ES (2007) In vitro evolution of lipase B from Candida antarctica using surface display in Hansenula polymorpha. J Microbiol Biotechnol 17(8):1308–1315

    CAS  PubMed  Google Scholar 

  29. Cheon SA, Jung J, Choo JH, Oh DB, Kang HA (2014) Characterization of putative glycosylphosphatidylinositol-anchoring motifs for surface display in the methylotrophic yeast Hansenula polymorpha. Biotechnol Lett 36(10):2085–2094

    Article  CAS  Google Scholar 

  30. Moon HY, Cheon SA, Kim H, Agaphonov MO, Kwon O, Oh DB et al (2015) Hansenula polymorpha Hac1p is critical to protein N-glycosylation activity modulation, as revealed by functional and transcriptomic analyses. Appl Environ Microbiol 81(20):6982–6993

    Article  CAS  Google Scholar 

  31. Park JN, Sohn MJ, Oh DB, Kwon O, Rhee SK, Hur CG et al (2007) Identification of the cadmium-inducible Hansenula polymorpha SEO1 gene promoter by transcriptome analysis and its application to whole-cell heavy-metal detection systems. Appl Environ Microbiol 73(19):5990–6000

    Article  CAS  Google Scholar 

  32. Kang HA, Hong WK, Sohn JH, Choi ES, Rhee SK (2001) Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha. Appl Microbiol Biotechnol 55(6):734–741

    Article  CAS  Google Scholar 

  33. Yoo SJ, Chung SY, Lee DJ, Kim H, Cheon SA, Kang HA (2015) Use of the cysteine-repressible HpMET3 promoter as a novel tool to regulate gene expression in Hansenula polymorpha. Biotechnol Lett 37(11):2237–2245

    Article  CAS  Google Scholar 

  34. Agaphonov MO, Trushkina PM, Sohn JH, Choi ES, Rhee SK, Ter-Avanesyan MD (1999) Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1. Yeast 15(7):541–551

    Article  CAS  Google Scholar 

  35. Cheon SA, Choo J, Ubiyvovk VM, Park JN, Kim MW, Oh DB et al (2009) New selectable host-marker systems for multiple genetic manipulations based on TRP1, MET2 and ADE2 in the methylotrophic yeast Hansenula polymorpha. Yeast 26(9):507–521

    Article  CAS  Google Scholar 

  36. Sohn JH, Choi ES, Kang HA, Rhee JS, Agaphonov MO, Ter-Avanesyan MD et al (1999) A dominant selection system designed for copy-number-controlled gene integration in Hansenula polymorpha DL-1. Appl Microbiol Biotechnol 51(6):800–807

    Article  CAS  Google Scholar 

  37. Kim H, Moon HY, Lee DJ, Cheon SA, Yoo SJ, Park JN et al (2013) Functional and molecular characterization of novel Hansenula polymorpha genes, HpPMT5 and HpPMT6, encoding protein O-mannosyltransferases. Fungal Genet Biol 58-59:10–24

    Article  CAS  Google Scholar 

  38. Sohn JH, Choi ES, Kang HA, Rhee JS, Rhee SK (1999) A family of telomere-associated autonomously replicating sequences and their functions in targeted recombination in Hansenula polymorpha DL-1. J Bacteriol 181(3):1005–1013

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Levine DW, Cooney CL (1973) Isolation and characterization of a thermotolerant methanol-utilizing yeast. Appl Microbiol 26(6):982–990

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Hill J, Donald KA, Griffiths DE (1991) DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res 19(20):5791

    Article  CAS  Google Scholar 

  41. Thak EJ, Kim J, Lee DJ, Kim JY, Kang HA (2018) Structural analysis of N-/O-glycans assembled on proteins in yeasts. J Microbiol 56(1):11–23

    Article  CAS  Google Scholar 

  42. de Groot PW, de Boer AD, Cunningham J, Dekker HL, de Jong L, Hellingwerf KJ et al (2004) Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins. Eukaryot Cell 3(4):955–965

    Article  Google Scholar 

  43. Lee KJ, Jung JH, Lee JM, So Y, Kwon O, Callewaert N et al (2009) High-throughput quantitative analysis of plant N-glycan using a DNA sequencer. Biochem Biophys Res Commun 380(2):223–229

    Article  CAS  Google Scholar 

  44. Callewaert N, Geysens S, Molemans F, Contreras R (2001) Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment. Glycobiology 11(4):275–281

    Article  CAS  Google Scholar 

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Acknowledgment

The work was supported by the grant No. NRF-2013M3A6-A8073554 (Global Frontier Program for the Intelligent Synthetic Biology) from the National Research Foundation of Korea (NRF).

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Authors and Affiliations

  1. Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, Republic of Korea

    Su Jin Yoo, Hye Yun Moon & Hyun Ah Kang

Authors
  1. Su Jin Yoo
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  2. Hye Yun Moon
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  3. Hyun Ah Kang
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Corresponding author

Correspondence to Hyun Ah Kang .

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Editors and Affiliations

  1. Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU) and Austrian Centre of Industrial Biotechnology (acib), Vienna, Austria

    Brigitte Gasser

  2. Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU) and Austrian Centre of Industrial Biotechnology (acib), Vienna, Austria

    Diethard Mattanovich

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Yoo, S.J., Moon, H.Y., Kang, H.A. (2019). Screening and Selection of Production Strains: Secretory Protein Expression and Analysis in Hansenula polymorpha. In: Gasser, B., Mattanovich, D. (eds) Recombinant Protein Production in Yeast. Methods in Molecular Biology, vol 1923. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9024-5_5

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  • DOI: https://doi.org/10.1007/978-1-4939-9024-5_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9023-8

  • Online ISBN: 978-1-4939-9024-5

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