Applied Microbiology and Biotechnology

, Volume 92, Issue 6, pp 1197–1206 | Cite as

A carrier fusion significantly induces unfolded protein response in heterologous protein production by Aspergillus oryzae

  • Ayako Ohno
  • Jun-ichi Maruyama
  • Takashi Nemoto
  • Manabu Arioka
  • Katsuhiko Kitamoto
Applied genetics and molecular biotechnology


In heterologous protein production by filamentous fungi, target proteins are expressed as fusions with homologous secretory proteins, called carriers, for higher production yields. Although carrier fusion is thought to overcome the bottleneck in transcriptional and (post)translational processes during heterologous protein production, there is limited knowledge of its physiological effects on the host strain. In this study, we performed DNA microarray analysis by comparing gene expression patterns of two Aspergillus oryzae strains expressing either carrier- or non-carrier-fused bovine chymosin (CHY). When CHY was expressed as a fusion with α-amylase (AmyB), the production level increased by approximately 2-fold as compared with the non-carrier-fused CHY. DNA microarray analysis revealed that the carrier fusion significantly up-regulated many genes involved in endoplasmic reticulum (ER) protein-folding and secretion. Consistently, hacA transcripts were efficiently spliced in the strain expressing the carrier-fused CHY, indicating an unfolded protein response (UPR). The carrier-fused CHY was detected intracellularly without processing at the Kex2 cleavage site, which is likely recognized in the Golgi, and the carrier fusion delayed extracellular CHY production in the early growth phase as compared with the non-carrier-fused expression. Taken together, our data suggest a proposal that the carrier fusion temporarily accumulates the carrier-fused CHY in the ER and significantly induces UPR.


Aspergillus oryzae Heterologous protein production Carrier UPR ER 



This study was supported by a Grant-in-Aid for Scientific Research (S) and a Grant-in-Aid for Scientific Research on Priority Areas “Applied Genomics” from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Supplementary material

253_2011_3487_MOESM1_ESM.pdf (316 kb)
ESM 1 (PDF 316 kb)


  1. Akao T, Sano M, Yamada O, Akeno T, Fujii K, Goto K, Ohashi-Kunihiro S, Takase K, Yasukawa-Watanabe M, Yamaguchi K, Kurihara Y, Maruyama J, Juvvadi PR, Tanaka A, Hata Y, Koyama Y, Yamaguchi S, Kitamoto N, Gomi K, Abe K, Takeuchi M, Kobayashi T, Horiuchi H, Kitamoto K, Kashiwagi Y, Machida M, Akita O (2007) Analysis of expressed sequence tags from the fungus Aspergillus oryzae cultured under different conditions. DNA Res 14:47–57CrossRefGoogle Scholar
  2. Arvas M, Pakula T, Lanthaler K, Saloheimo M, Valkonen M, Suortti T, Robson G, Penttilä M (2006) Common features and interesting differences in transcriptional responses to secretion stress in the fungi Trichoderma reesei and Saccharomyces cerevisiae. BMC Genomics 7:32CrossRefGoogle Scholar
  3. Bryant NJ, Boyd A (1993) Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartment. J Cell Sci 106:815–822Google Scholar
  4. Gouka RJ, Punt PJ, van den Hondel CA (1997) Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels. Appl Environ Microbiol 63:488–497Google Scholar
  5. Guillemette T, van Peij NN, Goosen T, Lanthaler K, Robson GD, van den Hondel CA, Stam H, Archer DB (2007) Genomic analysis of the secretion stress response in the enzyme-producing cell factory Aspergillus niger. BMC Genomics 8:158CrossRefGoogle Scholar
  6. Ito K, Asakura T, Morita Y, Nakajima K, Koizumi A, Shimizu-Ibuka A, Masuda K, Ishiguro M, Terada T, Maruyama J, Kitamoto K, Misaka T, Abe K (2007) Microbial production of sensory-active miraculin. Biochem Biophys Res Commun 360:407–411CrossRefGoogle Scholar
  7. Jalving R, van de Vondervoort PJ, Visser J, Schaap PJ (2000) Characterization of the kexin-like maturase of Aspergillus niger. Appl Environ Microbiol 66:363–336CrossRefGoogle Scholar
  8. Jin FJ, Maruyama J, Juvvadi PR, Arioka M, Kitamoto K (2004) Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzae. FEMS Microbiol Lett 239:79–85CrossRefGoogle Scholar
  9. Jin FJ, Watanabe T, Juvvadi PR, Maruyama J, Arioka M, Kitamoto K (2007) Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae. Appl Microbiol Biotechnol 76:1059–1068CrossRefGoogle Scholar
  10. Kasuya T, Nakajima H, Kitamoto K (1999) Cloning and characterization of the bipA gene encoding ER chaperone BiP from Aspergillus oryzae. J Biosci Bioeng 88:472–478CrossRefGoogle Scholar
  11. Kimura S, Maruyama J, Takeuchi M, Kitamoto K (2008) Monitoring global gene expression of proteases and improvement of human lysozyme production in nptB gene disruptant of Aspergillus oryzae. Biosci Biotechnol Biochem 72:499–502CrossRefGoogle Scholar
  12. Kimura S, Maruyama J, Kikuma T, Arioka M, Kitamoto K (2011) Autophagy delivers misfolded secretory proteins accumulated in endoplasmic reticulum to vacuoles in the filamentous fungus Aspergillus oryzae. Biochem Biophys Res Commun 406:464–470CrossRefGoogle Scholar
  13. Kitamoto K (2002) Molecular biology of the Koji molds. Adv Appl Microbiol 51:129–153CrossRefGoogle Scholar
  14. Lee BR, Yamada O, Kitamoto K, Takahashi K (1996) Cloning, characterization and overexpression of a gene (pdiA) encoding protein disulfide isomerase of Aspergillus oryzae. J Ferment Bioeng 82:538–543CrossRefGoogle Scholar
  15. Mabashi Y, Kikuma T, Maruyama J, Arioka M, Kitamoto K (2006) Development of a versatile expression plasmid construction system for Aspergillus oryzae and its application to visualization of mitochondria. Biosci Biotechnol Biochem 70:1882–1889CrossRefGoogle Scholar
  16. Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, Kusumoto K, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H, Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE, Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D, Cleveland TE, Fedorova ND, Gotoh O, Horikawa H, Hosoyama A, Ichinomiya M, Igarashi R, Iwashita K, Juvvadi PR, Kato M, Kato Y, Kin T, Kokubun A, Maeda H, Maeyama N, Maruyama J, Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I, Sakamoto K, Sawano T, Takahashi M, Takase K, Terabayashi Y, Wortman JR, Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide Y, Komori T, Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono K, Kuhara S, Ogasawara N, Kikuchi H (2005) Genome sequencing and analysis of Aspergillus oryzae. Nature 438:1157–1161CrossRefGoogle Scholar
  17. Maruyama J, Kitamoto K (2008) Multiple gene disruptions by marker recycling with highly efficient gene-targeting background (DeltaligD) in Aspergillus oryzae. Biotechnol Lett 30:1811–1817CrossRefGoogle Scholar
  18. Minetoki T, Nunokawa Y, Gomi K, Kitamoto K, Kumagai C, Tamura G (1996) Deletion analysis of promoter elements of the Aspergillus oryzae agdA gene encoding alpha-glucosidase. Curr Genet 30:432–438CrossRefGoogle Scholar
  19. Mulder HJ, Nikolaev I, Madrid SM (2006) HACA, the transcriptional activator of the unfolded protein response (UPR) in Aspergillus niger, binds to partly palindromic UPR elements of the consensus sequence 5′-CAN(G/A)NTGT/GCCT-3′. Fungal Genet Biol 43:560–572CrossRefGoogle Scholar
  20. Nakajima K, Asakura T, Maruyama J, Morita Y, Oike H, Shimizu-Ibuka A, Misaka T, Sorimachi H, Arai S, Kitamoto K, Abe K (2006) Extracellular production of neoculin, a sweet-tasting heterodimeric protein with taste-modifying activity, by Aspergillus oryzae. Appl Environ Microbiol 72:3716–3723CrossRefGoogle Scholar
  21. Nemoto T, Maruyama J, Kitamoto K (2009a) Improvement of heterologous protein production in Aspergillus oryzae by RNA interference of α-amylase genes. Biosci Biotech Biochem 73:2370–2373CrossRefGoogle Scholar
  22. Nemoto T, Watanabe T, Mizogami Y, Maruyama J, Kitamoto K (2009b) Isolation of Aspergillus oryzae mutants for heterologous protein production from a double proteinase gene disruptant. Appl Microbiol Biotechnol 82:1105–1114CrossRefGoogle Scholar
  23. Ngiam C, Jeenes DJ, Punt PJ, van den Hondel CA, Archer DB (2000) Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger. Appl Environ Microbiol 66:775–782CrossRefGoogle Scholar
  24. Pedersen VB, Christensen KA, Foltmann B (1979) Investigations on the activation of bovine prochymosin. Eur J Biochem 94:573–580CrossRefGoogle Scholar
  25. Punt PJ, van Gemeren IA, Drint-Kuijvenhoven J, Hessing JG, van Muijlwijk-Harteveld GM, Beijersbergen A, Verrips CT, van den Hondel CA (1998) Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli. Appl Microbiol Biotechnol 50:447–454CrossRefGoogle Scholar
  26. Punt PJ, Drint-Kuijvenhoven A, Lokman BC, Spencer JA, Jeenes D, Archer DA, van den Hondel CA (2003) The role of the Aspergillus niger furin-type protease gene in processing of fungal proproteins and fusion proteins. Evidence for alternative processing of recombinant (fusion-) proteins. J Biotechnol 106:23–32CrossRefGoogle Scholar
  27. Ron D, Walter P (2007) Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8:519–529CrossRefGoogle Scholar
  28. Sims AH, Gent ME, Lanthaler K, Dunn-Coleman NS, Oliver SG, Robson GD (2005) Transcriptome analysis of recombinant protein secretion by Aspergillus nidulans and the unfolded-protein response in vivo. Appl Environ Microbiol 71:2737–2747CrossRefGoogle Scholar
  29. Tsuchiya K, Nagashima T, Yamamoto Y, Gomi K, Kitamoto K, Kumagai C, Tamura G (1994) High level secretion of calf chymosin using a glucoamylase-prochymosin fusion gene in Aspergillus oryzae. Biosci Biotechnol Biochem 58:895–899CrossRefGoogle Scholar
  30. Valkonen M, Ward M, Wang H, Penttilä M, Saloheimo M (2003) Improvement of foreign-protein production in Aspergillus niger var. awamori by constitutive induction of the unfolded-protein response. Appl Environ Microbiol 69:6979–6986CrossRefGoogle Scholar
  31. van Gemeren IA, Punt PJ, Drint-Kuyvenhoven A, Broekhuijsen MP, van't Hoog A, Beijersbergen A, Verrips CT, van den Hondel CA (1997) The ER chaperone encoding bipA gene of black Aspergilli is induced by heat shock and unfolded proteins. Gene 198:43–52CrossRefGoogle Scholar
  32. Wang H, Ward M (2000) Molecular characterization of a PDI-related gene prpA in Aspergillus niger var. awamori. Curr Genet 37:57–64CrossRefGoogle Scholar
  33. Wang H, Entwistle J, Morlon E, Archer DB, Peberdy JF, Ward M, Jeenes DJ (2003) Isolation and characterisation of a calnexin homologue, clxA, from Aspergillus niger. Mol Genet Genomics 268:684–691Google Scholar
  34. Watanabe T, Matsuo I, Maruyama J, Kitamoto K, Ito Y (2007) Identification and characterization of an intracellular lectin, calnexin, from Aspergillus oryzae using N-glycan-conjugated beads. Biosci Biotechnol Biochem 71:2688–2696CrossRefGoogle Scholar
  35. Yamada O, Lee BR, Gomi K, Iimura Y (1999) Cloning and functional analysis of the Aspergillus oryzae conidiation regulator gene brlA by its disruption and misscheduled expression. J Biosci Bioeng 87:424–429CrossRefGoogle Scholar
  36. Yoon J, Kimura S, Maruyama J, Kitamoto K (2009) Construction of quintuple protease gene disruptant for heterologous protein production in Aspergillus oryzae. Appl Microbiol Biotechnol 82:691–701CrossRefGoogle Scholar
  37. Yoon J, Aishan T, Maruyama J, Kitamoto K (2010) Enhanced production and secretion of heterologous proteins by the filamentous fungus Aspergillus oryzae via disruption of vacuolar protein sorting receptor gene Aovps10. Appl Environ Microbiol 76:5718–5727CrossRefGoogle Scholar
  38. Yoon J, Maruyama J, Kitamoto K (2011) Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins. Appl Microbiol Biotechnol 89:747–759CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Ayako Ohno
    • 1
  • Jun-ichi Maruyama
    • 1
  • Takashi Nemoto
    • 1
  • Manabu Arioka
    • 1
  • Katsuhiko Kitamoto
    • 1
  1. 1.Department of BiotechnologyThe University of TokyoTokyoJapan

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