Selection of the Optimal Yeast Host for the Synthesis of Recombinant Enzymes

  • Felix Bischoff
  • Martin Giersberg
  • Falko Matthes
  • Tobias Schwalenberg
  • Sebastian Worch
  • Gotthard KunzeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1923)


Yeasts, like Arxula adeninivorans, Hansenula polymorpha, Pichia pastoris, Debaryomyces hansenii, Debaryomyces polymorphus, Schwanniomyces occidentalis, Yarrowia lipolytica, and Saccharomyces cerevisiae are frequently used producers of recombinant enzymes, particularly when posttranslational modifications are mandatory to obtain full functionality. The wide-range transformation/expression platform presented in this chapter can be used to select the optimal yeast host for high-level synthesis of the desired enzyme with favorable biochemical properties. This platform is composed of a selection marker and up to four expression modules in a linearized cassette. Here we describe the protocols for the assembly as well as the transformation of yeast strains with the respective cassettes, screening of transformants, the isolation and biochemical characterization of the enzymes, and finally a simple fermentation strategy to achieve maximal yields of the chosen recombinant enzyme.

Key words

Wide-range expression module Gibson assembly Yeast transformation Transformant screening Recombinant enzymes Fermentation strategy 



The work was supported by grants from the German Federal Ministry of Economics and Technology (Bundesministerium für Wirtschaft und Technologie, BMWi) within its program “Zentrales Innovationsprogramm Mittelstand, ZIM” (Grant Numbers KF2131620MD2, KF2131628SA4).


  1. 1.
    Böer E, Steinborn G, Kunze G, Gellissen G (2007) Yeast expression platforms. Appl Microbiol Biotechnol 77:513–523. Scholar
  2. 2.
    Gellissen G, Hollenberg CP (1997) Application of yeasts in gene expression studies: a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis—a review. Gene 190:87–97. Scholar
  3. 3.
    Madzak C, Nicaud J-M, Gaillardin C (2005) Yarrowia lipolytica. In: Production of recombinant proteins: novel microbial and eukaryotic expression systems. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, FRG, pp 163–189CrossRefGoogle Scholar
  4. 4.
    Terentiev Y, Pico AH, Böer E et al (2004) A wide-range integrative yeast expression vector system based on Arxula adeninivorans-derived elements. J Ind Microbiol Biotechnol 31:223–228. Scholar
  5. 5.
    Ilgen C, Lin-Cereghino J, Cregg JM (2005) Pichia pastoris. In: Production of recombinant proteins. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, FRG, pp 143–162CrossRefGoogle Scholar
  6. 6.
    Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741–750. Scholar
  7. 7.
    Böer E, Gellissen G, Kunze G (2005) Arxula adeninivorans. In: Production recombinant proteins. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, FRG, pp 89–110CrossRefGoogle Scholar
  8. 8.
    Kunze G, Gaillardin C, Czernicka M et al (2014) The complete genome of Blastobotrys (Arxula) adeninivorans LS3—a yeast of biotechnological interest. Biotechnol Biofuels 7:66. Scholar
  9. 9.
    Gellissen G, Janowicz ZA, Weydemann U et al (1992) High-level expression of foreign genes in Hansenula polymorpha. Biotechnol Adv 10:179–189. Scholar
  10. 10.
    Stöckmann C, Scheidle M, Klee D et al (2009) Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment. Microb Cell Factories 8:22. Scholar
  11. 11.
    Chamas A, Pham HTM, Baronian K, Kunze G (2017) Biosensors based on yeast/fungal cells. In: Biotechnology of yeasts and filamentous fungi. Springer International Publishing, Cham, pp 351–371CrossRefGoogle Scholar
  12. 12.
    Bischoff F, Litwińska K, Cordes A et al (2015) Three new cutinases from the yeast Arxula adeninivorans that are suitable for biotechnological applications. Appl Environ Microbiol 81:5497–5510. Scholar
  13. 13.
    Rösel H, Kunze G (1998) Integrative transformation of the dimorphic yeast Arxula adeninivorans LS3 based on hygromycin B resistance. Curr Genet 33:157–163. Scholar
  14. 14.
    Böer E, Steinborn G, Matros A et al (2007) Production of interleukin-6 in Arxula adeninivorans, Hansenula polymorpha and Saccharomyces cerevisiae by applying the wide-range yeast vector (CoMed™) system to simultaneous comparative assessment. FEMS Yeast Res 7:1181–1187. Scholar
  15. 15.
    Biernacki M, Marzec M, Roick T et al (2017) Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) accumulation in Arxula adeninivorans by stabilization of production. Microb Cell Factories 16:144. Scholar
  16. 16.
    Kunze G, Kunze I (1994) Characterization of Arxula adeninivorans strains from different habitats. Antonie Van Leeuwenhoek 65:29–34. Scholar
  17. 17.
    Samsonova IA, Kunze G, Bode R, Böttcher F (1996) A set of genetic markers for the chromosomes of the imperfect yeast Arxula adeninivorans. Yeast 12:1209–1217.<1209::AID-YEA12>3.0.CO;2-WCrossRefPubMedGoogle Scholar
  18. 18.
    Böer E, Steinborn G, Florschütz K et al (2009) Arxula adeninivorans (Blastobotrys adeninivorans)—a dimorphic yeast of great biotechnological potential. In: Yeast biotechnology: diversity and applications. Springer Netherlands, Dordrecht, pp 615–634CrossRefGoogle Scholar
  19. 19.
    Álvaro-Benito M, Fernández-Lobato M, Baronian K, Kunze G (2013) Assessment of Schwanniomyces occidentalis as a host for protein production using the wide-range Xplor®2 expression platform. Appl Microbiol Biotechnol 97:4443–4456. Scholar
  20. 20.
    Robinson JS, Klionsky DJ, Banta LM, Emr SD (1988) Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol 8:4936–4948CrossRefGoogle Scholar
  21. 21.
    Bischoff F, Chamas A, Litwińska K et al (2017) Applications of Blastobotrys (Arxula) adeninivorans in biotechnology. In: Yeast diversity in human Welfare. Springer Singapore, Singapore, pp 455–479CrossRefGoogle Scholar
  22. 22.
    Tanaka A, Ohnishi N, Fukui S (1967) Studies on the formation of vitamins and their function in hydrocarbon fermentation. Production of vitamins and their function in hydrocarbon medium. J Ferment Technol 45:617–623Google Scholar
  23. 23.
    Elving PJ, Markowitz JM, Rosenthal I (1956) Preparation of buffer systems of constant ionic strength. Anal Chem 28:1179–1180. Scholar
  24. 24.
    Böer E, Piontek M, Kunze G (2009) Xplor® 2—an optimized transformation/expression system for recombinant protein production in the yeast Arxula adeninivorans. Appl Microbiol Biotechnol 84:583–594. Scholar
  25. 25.
    Gibson DG, Young L, Chuang R-Y et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345. Scholar
  26. 26.
    Gibson DG, Smith HO, Hutchison CA et al (2010) Chemical synthesis of the mouse mitochondrial genome. Nat Methods 7:901–903. Scholar
  27. 27.
    Dohmen RJ, Strasser AWM, Honer CB, Hollenberg CP (1991) An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7:691–692. Scholar
  28. 28.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. Scholar
  29. 29.
    Rauter M, Kasprzak J, Becker K et al (2014) ADH from Rhodococcus ruber expressed in Arxula adeninivorans for the synthesis of 1-(S)-phenylethanol. J Mol Catal B Enzym 104:8–16. Scholar
  30. 30.
    Kasprzak J, Bischoff F, Rauter M et al (2016) Synthesis of 1-(S)-phenylethanol and ethyl (R)-4-chloro-3-hydroxybutanoate using recombinant Rhodococcus erythropolis alcohol dehydrogenase produced by two yeast species. Biochem Eng J 106:107–117. Scholar
  31. 31.
    Pointing SB (1999) Qualitative methods for the determination of lignocellulolytic enzyme production by tropical fungi. Fungal Divers 2:17–33Google Scholar
  32. 32.
    Bleve G, Lezzi C, Mita G et al (2008) Molecular cloning and heterologous expression of a laccase gene from Pleurotus eryngii in free and immobilized Saccharomyces cerevisiae cells. Appl Microbiol Biotechnol 79:731–741. Scholar
  33. 33.
    Kiiskinen L-L, Ratto M, Kruus K (2004) Screening for novel laccase-producing microbes. J Appl Microbiol 97:640–646. Scholar
  34. 34.
    Traut TW, Ropp PA, Poma A (1991) Purine nucleoside phosphorylase: allosteric regulation of a dissociating enzyme. Adv Exp Med Biol 309B:177–180CrossRefGoogle Scholar
  35. 35.
    Glogauer A, Martini VP, Faoro H et al (2011) Identification and characterization of a new true lipase isolated through metagenomic approach. Microb Cell Factories 10:54. Scholar
  36. 36.
    Halonen P, Reinikainen T, Nyyssölä A, Buchert J (2009) A high throughput profiling method for cutinolytic esterases. Enzym Microb Technol 44:394–399. Scholar
  37. 37.
    Böer E, Breuer FS, Weniger M et al (2011) Large-scale production of tannase using the yeast Arxula adeninivorans. Appl Microbiol Biotechnol 92:105–114. Scholar
  38. 38.
    Bradoo S, Gupta R, Saxena RK (1996) Screening of extracellular tannase-producing fungi: development of a rapid and simple plate assay. J Gen Appl Microbiol 42:325–329. Scholar
  39. 39.
    Bae H, Yanke L, Cheng K-J, Selinger L (1999) A novel staining method for detecting phytase activity. J Microbiol Methods 39:17–22. Scholar
  40. 40.
    Shi H, Ding H, Huang Y et al (2014) Expression and characterization of a GH43 endo-arabinanase from Thermotoga thermarum. BMC Biotechnol 14:35. Scholar
  41. 41.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428. Scholar
  42. 42.
    Schiøtt M, De Fine Licht HH, Lange L, Boomsma JJ (2008) Towards a molecular understanding of symbiont function: identification of a fungal gene for the degradation of xylan in the fungus gardens of leaf-cutting ants. BMC Microbiol 8:40. Scholar
  43. 43.
    Giersberg M, Degelmann A, Bode R et al (2012) Production of a thermostable alcohol dehydrogenase from Rhodococcus ruber in three different yeast species using the Xplor®2 transformation/expression platform. J Ind Microbiol Biotechnol 39:1385–1396. Scholar
  44. 44.
    Bischoff F (2017) Identifizierung und Charakterisierung von drei Cutinasen der Hefe Arxula adeninivorans LS3 und deren Einsatz zum Abbau von Polyestern. PhD Thesis. Ernst-Moritz-Arndt-Universität GreifswaldGoogle Scholar
  45. 45.
    Peng B, Williams TC, Henry W et al (2015) Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities. Microb Cell Factories 14:91. Scholar
  46. 46.
    Liu Z, Tyo KEJ, Martínez JL et al (2012) Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae. Biotechnol Bioeng 109(5):1259–1268. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Felix Bischoff
    • 1
  • Martin Giersberg
    • 1
  • Falko Matthes
    • 1
  • Tobias Schwalenberg
    • 1
  • Sebastian Worch
    • 1
  • Gotthard Kunze
    • 1
    Email author
  1. 1.Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)GaterslebenGermany

Personalised recommendations