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Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe

  • Applied Genetics and Molecular Biotechnology
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Abstract

The creation of protease-deficient mutants to avoid product degradation is one of the current strategies employed to improve productivity and secretion efficiency of heterologous protein expression. We previously constructed a set of single protease-deficient mutants of the fission yeast Schizosaccharomyces pombe by respective disruption of 52 protease genes, and we succeeded in confirming useful disruptants (Idiris et al., Yeast 23:83–99, 2006). In the present study, we attempted multiple deletions of 13 protease genes, single deletions of which were previously confirmed as being beneficial for reducing extracellular product degradation. Using PCR-based gene replacement, a series of multiple deletion strains was constructed by multiple disruption of a maximum of seven protease genes. Effects of the resultant multiple deletion strains on heterologous expression were then measured by practical expression of a proteolytically sensitive model protein, the human growth hormone (hGH). Time profiles of hGH secretion from each resultant mutant demonstrated significantly enhanced hGH productivity with processing of the multiple protease deletions. The data clearly indicated that disruption of multiple protease genes in the fission yeast is an effective method for controlling proteolytic degradation of heterologous proteins particularly susceptible to proteases.

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References

  • Baneyx F, Georgiou G (1991) Construction and characterization of Escherichia coli strains deficient in multiple secreted proteases: protease III degrades high-molecular-weight substrates in vivo. J Bacteriol 173:2696–2703

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bähler J, Wu JQ, Longtine MS, Shah NG, McKenzie A 3rd, Steever AB, Wach A, Philippsen P, Pringle JR (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14:943–951

    PubMed  Google Scholar 

  • Bröker M, Ragg H, Karges HE (1987) Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Biochim Biophys Acta 908:203–213

    PubMed  Google Scholar 

  • Chapman GE, Rogers KM, Brittain T, Bradshaw RA, Bates OJ, Turner C, Cary PD, Crane-Robinson C (1981) The 20,000 molecular weight variant of human growth hormone. Preparation and some physical and chemical properties. J Biol Chem 256:2395–2401

    CAS  PubMed  Google Scholar 

  • Davey J, Davis K, Imai Y, Yamamoto M, Matthews G (1994) Isolation and characterization of krp, a dibasic endopeptidase required for cell viability in the fission yeast Schizosaccharomyces pombe. EMBO J 13:5910–5921

    CAS  PubMed  PubMed Central  Google Scholar 

  • Giga-Hama Y (1997) Fission yeast Schizosaccharomyces pombe: an attractive host for heterologous protein production. In: Giga-Hama Y, Kumagai H (eds) Foreign gene expression in fission yeast Schizosaccharomyces pombe. Springer, Berlin Heidelberg New York, pp 3–28

    Google Scholar 

  • Giga-Hama Y, Kumagai H (1999) Expression system for foreign genes using the fission yeast: Schizosaccharomyces pombe. Biotechnol Appl Biochem 30:235–244

    CAS  PubMed  Google Scholar 

  • Giga-Hama Y, Tohda H, Okada H, Owada MK, Okayama H, Kumagai H (1994) High level expression of human lipocortin I in the fission yeast Schizosaccharomyces pombe using a novel expression vector. Biotechnology (NY) 12:400–404

    CAS  Google Scholar 

  • Grimm C, Kohli J, Murray J, Maundrell K (1988) Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol Gen Genet 215:81–86

    CAS  PubMed  Google Scholar 

  • Hirano T, Funahashi S, Uemura T, Yanagida M (1986) Isolation and characterization of Schizosaccharomyces pombe cut mutants that block nuclear division but not cytokinesis. EMBO J 5:2973–2979

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hirashima K, Iwaki T, Takegawa K, Giga-Hama Y, Tohda H (2006) A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast. Nucleic Acids Res 34:e11

    PubMed  PubMed Central  Google Scholar 

  • Idiris A, Bi KW, Tohda H, Kumagai H, Giga-Hama Y (2006) Construction of a protease-deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease-sensitive heterologous proteins. Yeast 23:83–99

    CAS  PubMed  Google Scholar 

  • Ikeda S, Nikaido K, Araki K, Yoshitake A, Kumagai H, Isoai A (2004) Production of recombinant human lysosomal acid lipase in Schizosaccharomyces pombe: development of a fed-batch fermentation and purification process. J Biosci Bioeng 98:366–373

    CAS  PubMed  Google Scholar 

  • Imai Y, Yamamoto M (1992) Schizosaccharomyces pombe sxa1 + and sxa2 + encode putative proteases involved in the mating response. Mol Cell Biol 12:1827–1834

    CAS  PubMed  PubMed Central  Google Scholar 

  • Imai Y, Miyake S, Hughes DA, Yamamoto M (1991) Identification of a GTPase-activating protein homolog in Schizosaccharomyces pombe. Mol Cell Biol 11:3088–3094

    CAS  PubMed  PubMed Central  Google Scholar 

  • Isoai A, Kimura H, Reichert A, Schorgendorfer K, Nikaido K, Tohda H, Giga-Hama Y, Mutoh N, Kumagai H (2002) Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells. Biotechnol Bioeng 80:22–32

    CAS  PubMed  Google Scholar 

  • Jones EW (1991) Tackling the protease problem in Saccharomyces cerevisiae. Methods Enzymol 194: 428–453

    CAS  PubMed  Google Scholar 

  • Jønson L, Rehfeld JF, Johnsen AH (2004) Enhanced peptide secretion by gene disruption of CYM1, a novel protease in Saccharomyces cerevisiae. Eur J Biochem 271:4788–4797

    PubMed  Google Scholar 

  • Kang HA, Choi ES, Hong WK, Kim JY, Ko SM, Sohn JH, Rhee SK (2000) Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 53:575–582

    CAS  PubMed  Google Scholar 

  • Käser M, Kambacheld M, Kisters-Woike B, Langer T (2003) Oma1, a novel membrane-bound metallopeptidase in mitochondria with activities overlapping with the m-AAA protease. J Biol Chem 278:46414–46423

    PubMed  Google Scholar 

  • Kirisako T, Ichimura Y, Okada H, Kabeya Y, Mizushima N, Yoshimori T, Ohsumi M, Takao T, Noda T, Ohsumi Y (2000) The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J Cell Biol 151:263–276

    CAS  PubMed  PubMed Central  Google Scholar 

  • Komeda T, Sakai Y, Kato N, Kondo K (2002) Construction of protease-deficient Candida boidinii strains useful for recombinant protein production: cloning and disruption of proteinase A gene (PEP4) and proteinase B gene (PRBI). Biosci Biotechnol Biochem 66:628–631

    CAS  PubMed  Google Scholar 

  • Ladds G, Davey J (2000a) Sxa2 is a serine carboxypeptidase that degrades extracellular P-factor in the fission yeast Schizosaccharomyces pombe. Mol Microbiol 36:377–390

    CAS  PubMed  Google Scholar 

  • Ladds G, Davey J (2000b) Identification of proteases with shared functions to the proprotein processing protease krp1 in the fission yeast Schizosaccharomyces pombe. Mol Microbiol 38:839–853

    CAS  PubMed  Google Scholar 

  • Lang T, Schaefffeler E, Bernreuther D, Bredschneider M, Wolf DH, Thumm M (1998) Aut2p and Aut7p, two novel microtubule-associated proteins are essential for delivery of autophagic vesicles to the vacuole. EMBO J 17:3597–3607

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lee MG, Nurse P (1987) Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature 327:31–35

    CAS  PubMed  Google Scholar 

  • Lewis UJ (1984) Variants of growth hormone and prolactin and their posttranslational modifications. Annu Rev Physiol 46:33–42

    CAS  PubMed  Google Scholar 

  • Lewis UJ, Dunn JT, Bonewald LF, Seavey BK, Vanderlaan WP (1978) A naturally occurring structural variant of human growth hormone. J Biol Chem 253:2679–2687

    CAS  PubMed  Google Scholar 

  • Li CH (1982) Human growth hormone: 1974–1981. Mol Cell Biochem 46:31–41

    CAS  PubMed  Google Scholar 

  • Meerman HJ, Georgiou G (1994) Construction and characterization of a set of E. coli strains deficient in all known loci affecting the proteolytic stability of secreted recombinant proteins. Biotechnology (N Y) 12:1107–1110

    CAS  Google Scholar 

  • Moreno S, Klar A, Nurse P (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol 194:795–823

    CAS  PubMed  Google Scholar 

  • Okada H, Sekiya T, Yokoyama K, Tohda H, Kumagai H, Morikawa Y (1998a) Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products. Appl Microbiol Biotechnol 49:301–308

    CAS  PubMed  Google Scholar 

  • Okada H, Tada K, Sekiya T, Yokoyama K, Takahashi A, Tohda H, Kumagai H, Morikawa Y (1998b) Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414. Appl Environ Microbiol 64:555–563

    CAS  PubMed  PubMed Central  Google Scholar 

  • Okazaki K, Okazaki N, Kume K, Jinno S, Tanaka K, Okayama H (1990) High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe. Nucleic Acids Res 18:6485–6489

    CAS  PubMed  PubMed Central  Google Scholar 

  • Russell P (1989) Gene cloning and expression in fission yeast. In: Nasim A, Young P, Johnson BF (eds) Molecular biology of the fission yeast. Academic, San Diego, CA, pp 243–271

    Google Scholar 

  • Sander P, Grünewald S, Reiländer H, Michel H (1994) Expression of the human D2S dopamine receptor in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe: a comparative study. FEBS Lett 344:41–46

    CAS  PubMed  Google Scholar 

  • Sato S, Suzuki H, Widyastuti U, Hotta Y, Tabata S (1994) Identification and characterization of genes induced during sexual differentiation in Schizosaccharomyces pombe. Curr Genet 26:31–37

    CAS  PubMed  Google Scholar 

  • Spolaore B, Polverino de Laureto P, Zambonin M, Fontana A (2004) Limited proteolysis of human growth hormone at low pH: isolation, characterization, and complementation of the two biologically relevant fragments, 1–44 and 45–191. Biochemistry 43:6576–6586

    CAS  PubMed  Google Scholar 

  • Tokunaga M, Kawamura A, Yonekyu S, Kishida M, Hishinuma F (1993) Secretion of mouse α-amylase from fission yeast Schizosaccharomyces pombe: presence of chymostatin-sensitive protease activity in the culture medium. Yeast 9:379–387

    CAS  PubMed  Google Scholar 

  • Tommasino M, Contorni M, Scarlato V, Bugnoli M, Maundrell K, Cavalieri F (1990) Synthesis, phosphorylation, and nuclear localization of human papillomavirus E7 protein in Schizosaccharomyces pombe. Gene 93:265–270

    CAS  PubMed  Google Scholar 

  • Uzawa S, Samejima I, Hirano T, Tanaka K, Yanagida M (1990) The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene. Cell 62:913–925

    CAS  PubMed  Google Scholar 

  • Werten MW, de Wolf FA (2005) Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoriskex2 disruptant. Appl Environ Microbiol 71:2310–2317

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wu XC, Lee W, Tran L, Wong SL (1991) Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J Bacteriol 173:4952–4958

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yamada H, Mori H, Momoi H, Nakagawa Y, Ueguchi C, Mizuno T (1994) A fission yeast gene encoding a protein that preferentially associates with curved DNA. Yeast 10:883–894

    CAS  PubMed  Google Scholar 

  • Zhao Y, Lieberman HB (1995) Schizosaccharomyces pombe: a model for molecular studies of eukaryotic genes. DNA Cell Biol 14:359–371

    CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank Professor Kaoru Takegawa (Department of Life Sciences, Faculty of Agriculture, Kagawa University, Japan) for his helpful discussions. This study was carried out as part of the project “Development of Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and Technology Frontiers” of the Ministry of Economy, Trade and Industry (METI), Japan, and was supported by the New Energy and Industrial Technology Development Organization (NEDO), Japan.

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  1. Ke-wei Bi

    Present address: College of Pharmaceutical Sciences, Hebei University, Baoding, People’s Republic of China

Authors and Affiliations

  1. ASPEX Division, Research Center, Asahi Glass Co., Ltd., 1150 Hazawa-cho, Kanagawa-ku, Yokohama, 221-8755, Japan

    Alimjan Idiris, Hideki Tohda, Ke-wei Bi, Atsushi Isoai, Hiromichi Kumagai & Yuko Giga-Hama

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  1. Alimjan Idiris
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  2. Hideki Tohda
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Correspondence to Yuko Giga-Hama.

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Idiris, A., Tohda, H., Bi, Kw. et al. Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe . Appl Microbiol Biotechnol 73, 404–420 (2006). https://doi.org/10.1007/s00253-006-0489-0

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