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Xplor® 2—an optimized transformation/expression system for recombinant protein production in the yeast Arxula adeninivorans

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Abstract

Combining ease of genetic manipulation and fermentation with the ability to secrete and to glycosylate proteins in the basic eukaryotic manner, Arxula adeninivorans provides an attractive expression platform. Based on a redesign of the basic vector, a new Arxula vector system, Xplor® 2, for heterologous gene expression was established, which allows (1) the construction of expression plasmids for supertransformation of A. adeninivorans strains secreting target proteins of biotechnological interest and (2) the integration of small vector cassettes consisting of yeast DNA sequences only. For this purpose, a set of modules including the ATRP1m selection-marker module, expression modules for constitutive expression of the genes phyK (Klebsiella-derived phytase) and IFNα2a (human interferon α), the HARS (Hansenula polymorpha autonomous replication sequence) for autonomous replication and the chaperone module AHSB4 promoter –HpCNE1 gene (calnexin) –PHO5 terminator to improve secretion efficiency were constructed and integrated in various combinations in the basic vector Xplor® 2. After removal of the complete Escherichia coli-based plasmid parts (resistance marker, ColE1 ori and f1(−) origin), the remaining yeast-based linear vector fragment with or without rDNA targeting sequences were transformed as yeast rDNA integrative expression cassettes and yeast integrative expression cassettes (YICs), respectively, and the resulting strains were tested for their capacity to secrete PhyK or IFNα2a. Maximal expression levels were consistently obtained using YICs for transformation irrespective of whether or not they carry HARS and/or calnexin modules. It is recommended that at least 50 such transformants be analyzed to ensure selection of the best transformants.

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References

  • Böer E, Mock HP, Bode R, Gellissen G, Kunze G (2005) An extracellular lipase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ALIP1 gene and characterization of the purified recombinant enzyme. Yeast 22:523–535

    Article  Google Scholar 

  • Böer E, Steinborn G, Kunze G, Gellissen G (2007a) Yeast expression platforms. Appl Microbiol Biotechnol 77:513–523

    Article  Google Scholar 

  • Böer E, Steinborn G, Matros A, Mock HP, Gellissen G, Kunze G (2007b) Production of interleukin-6 in Arxula adeninivorans, Hansenula polymorpha, and Saccharomyces cerevisiae applying the wide range vector (CoMed™) system to simultaneous comparative assessment. FEMS Yeast Res 7:1181–1187

    Article  Google Scholar 

  • Böer E, Steinborn G, Florschütz K, Körner M, Gellissen G, Kunze G (2009a) Arxula adeninivorans (Blastobotrys adeninivorans)—a dimorphic yeast of great biotechnological potential. In: Satyanarayana T, Kunze G (eds) Yeast biotechnology—diversity and application. Springer, The Netherlands, pp 453–468

    Google Scholar 

  • Böer E, Bode R, Mock HP, Piontek M, Kunze G (2009b) Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme. Yeast 26:323–337

    Article  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Gienow U, Kunze G, Schauer F, Bode R, Hofemeister J (1990) The yeast genus Trichosporon spec. LS3; molecular characterization of genomic complexity. Zbl Mikrobiol 145:3–12

    CAS  Google Scholar 

  • Hahn T, Tag K, Riedel K, Uhlig S, Baronian K, Gellissen G, Kunze G (2006) A novel estrogen sensor based on recombinant Arxula adeninivorans cells. Biosens Bioelectron 21:2078–2085

    Article  CAS  Google Scholar 

  • Kang HA, Gellissen G (2005) Hansenula polymorpha. In: Gellissen G (ed) Production of recombinant proteins—novel microbial and eukaryotic expression systems. Wiley-VCH, Weinheim, pp 111–142

    Chapter  Google Scholar 

  • Kaur P, Lingner A, Singh B, Böer E, Polajeva J, Steinborn G, Bode R, Gellissen G, Kunze G (2007) APHO1 from the yeast Arxula adeninivorans encodes an acid phosphatase of broad substrate specificity. Antonie Van Leuuwenhoek 91:45–55

    Article  CAS  Google Scholar 

  • Klabunde J, Kunze G, Gellissen G, Hollenberg CP (2003) Integration of heterologous genes in several yeast species using vectors containing a Hansenula polymorpha-derived rDNA-targeting element. FEMS Yeast Res 4:185–193

    Article  CAS  Google Scholar 

  • Klabunde J, Kleebank S, Piontek M, Hollenberg CP, Hellwig S, Degelmann A (2007) Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorpha. FEMS Yeast Res 7:1168–1180

    Article  CAS  Google Scholar 

  • Knoll A, Bartsch S, Husemann B, Engel P, Schroer K, Ribeiro B, Stöckmann C, Seletzky J, Büchs J (2007) High cell density cultivation of recombinant yeasts and bacteria under non-pressurized and pressurized conditions in stirred tank bioreactors. J Biotechnol 132:167–179

    Article  CAS  Google Scholar 

  • Kunze I, Hensel G, Adler K, Bernard J, Neubohn B, Nilssen C, Stoltenburg R, Kohlwein SD, Kunze G (1999) The green flourescent protein targets secretory proteins to the yeast vacuole. Biochim Biophys Acta 1410:287–298

    Article  CAS  Google Scholar 

  • Kurtzmann CP, Robnett JC (2007) Multigene phylogenetic analysis of the Trichomonascus, Wickerhammiella and Zygoascus yeast clades, and the proposal of Sugiyamaella gen. nov. and 14 new species combinations. FEMS Yeast Res 7:141–151

    Article  Google Scholar 

  • Middelhoven JW, Hoogkamer-Te Niet MC, Kreger van Rij NJW (1984) Trichosporon adeninovorans sp. nov., a yeast species utilizing adenine, xanthine, uric acid, putrescine and primary n-alkylamines as the sole source of carbon, nitrogen and energy. Antonie Van Leeuwenhoek 50:369–387

    Article  CAS  Google Scholar 

  • Middelhoven WJ, de Jonge IM, Winter M (1991) Arxula adeninivorans, a yeast assimilating many nitrogenous and aromatic compounds. Antonie Van Leeuwenhoek 60:129–137

    Article  Google Scholar 

  • Middelhoven WJ, Coenen A, Kraakman B, Gelpke MDS (1992) Degradation of some phenols and hydroxybenzoates by the imperfect ascomycetous yeasts Candida parapsilosis and Arxula adeninivorans: evidence for an operative gentisate pathway. Antonie Van Leeuwenhoek 62:181–187

    Article  CAS  Google Scholar 

  • Rose MD, Winston F, Hieter P (1990) Methods in yeast genetics. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Rösel H, Kunze G (1998) Integrative transformation of the dimorphic yeast Arxula adeninivorans LS3 based on hygromycin B resistance. Curr Genet 33:157–163

    Article  Google Scholar 

  • Sajidan A, Farouk A, Greiner R, Jungblut P, Müller EC, Borriss R (2004) Molecular and physiological characterization of a 3-phytase from soil bacterium Klebsiella sp. ASR1. Appl Microbiol Biotechnol 65:110–118

    Article  CAS  Google Scholar 

  • Sohn JH, Choi ES, Kim CH, Agaphonov MO, Ter-Avanesyan MD, Rhee JS, Rhee SK (1996) A novel autonomously replication sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-1. J Bacteriol 178:4420–4428

    Article  CAS  Google Scholar 

  • Steinborn G, Gellissen G, Kunze G (2005) Assessment of Hansenula polymorpha and Arxula adeninivorans-derived rDNA-targeting elements for the design of Arxula adeninivorans expression vectors. FEMS Yeast Res 5:1047–1054

    Article  CAS  Google Scholar 

  • Steinborn G, Böer E, Scholz A, Tag K, Kunze G, Gellissen G (2006) Application of a wide-range yeast vector (CoMed™) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts. Microb Cell Factories 5:33

    Article  Google Scholar 

  • Steinborn G, Gellissen G, Kunze G (2007a) A novel vector element providing multicopy vector integration in Arxula adeninivorans. FEMS Yeast Res 7:1197–1205

    Article  CAS  Google Scholar 

  • Steinborn G, Wartmann T, Gellissen G, Kunze G (2007b) Construction of an Arxula adeninivorans host–vector system based on trp1 complementation. J Biotechnol 127:392–401

    Article  CAS  Google Scholar 

  • Tanaka A, Ohnishi N, Fukui S (1967) Studies on the formation of vitamins and their function in hydrocarbon fermentation. Production of vitamin B6 by Candida albicans in hydrocarbon medium. J Ferment Technol 45:617–623

    CAS  Google Scholar 

  • Terentiev Y, Pico AH, Böer E, Wartmann T, Klabunde J, Breuer U, Babel W, Suckow M, Gellissen G, Kunze G (2004a) A wide-range integrative yeast expression vector system based on Arxula adeninivorans-derived elements. J Ind Microbiol Biotechnol 31:223–228

    Article  CAS  Google Scholar 

  • Terentiev Y, Breuer U, Babel W, Kunze G (2004b) Non-conventional yeasts as producers of polyhydroxyalkanoates—genetic engineering of Arxula adeninivorans. Appl Microbiol Biotechnol 64:376–381

    Article  CAS  Google Scholar 

  • Van der Walt JP, Smith MT, Yamada Y (1990) Arxula gen. nov. (Candidaceae), a new anamorphic, arthroconidial yeast genus. Antonie Van Leeuwenhoek 57:59–61

    Article  Google Scholar 

  • Wartmann T, Krüger A, Adler K, Bui MD, Kunze I, Kunze G (1995) Temperature dependent dimorphism of the yeast Arxula adeninivorans LS3. Antonie Van Leeuwenhoek 68:215–223

    Article  CAS  Google Scholar 

  • Wartmann T, Böer E, Huarto-Pico A, Sieber H, Bartelsen O, Gellissen G, Kunze G (2002) High-level production and secretion of recombinant proteins by the dimorphic yeast Arxula adeninivorans. FEMS Yeast Res 2:363–369

    CAS  PubMed  Google Scholar 

  • Wartmann T, Stoltenburg R, Böer E, Sieber H, Bartelsen O, Gellissen G, Kunze G (2003a) The ALEU2 gene—a new component for an Arxula adeninivorans-based expression platform. FEMS Yeast Res 3:223–232

    Article  CAS  Google Scholar 

  • Wartmann T, Bellebna C, Böer E, Gellissen G, Kunze G (2003b) The constitutive AHSB4 promoter—a novel component of the Arxula adeninivorans-based expression platform. Appl Microbiol Biotechnol 62:528–535

    Article  CAS  Google Scholar 

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Acknowledgements

The authors are grateful to Dr. P. Hardy for helpful discussions and critical reading of the manuscript and R. Franz and H. Bohlmann for excellent technical assistance. The research work was supported by grants from the German national Ministry of Education and Research (BMBF) within the CLIB2021 cluster (Wettbewerb Industrielle Biotechnologie 2021) and the Fonds der chemischen Industrie (GK).

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  1. Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstr. 3, 06466, Gatersleben, Germany

    Erik Böer & Gotthard Kunze

  2. ARTES Biotechnology GmbH, Elisabeth-Selbert-Str. 9, 40764, Langenfeld, Germany

    Michael Piontek

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  1. Erik Böer
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  2. Michael Piontek
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  3. Gotthard Kunze
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Correspondence to Gotthard Kunze.

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Böer, E., Piontek, M. & Kunze, G. Xplor® 2—an optimized transformation/expression system for recombinant protein production in the yeast Arxula adeninivorans . Appl Microbiol Biotechnol 84, 583–594 (2009). https://doi.org/10.1007/s00253-009-2167-5

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