Skip to main content
Log in

The C2H2-type transcription factor, FlbC, is involved in the transcriptional regulation of Aspergillus oryzae glucoamylase and protease genes specifically expressed in solid-state culture

  • Applied genetics and molecular biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Aspergillus oryzae produces a large amount of secreted proteins in solid-state culture, and some proteins such as glucoamylase (GlaB) and acid protease (PepA) are specifically produced in solid-state culture, but rarely in submerged culture. From the disruption mutant library of A. oryzae transcriptional regulators, we successfully identified a disruption mutant showing an extremely low production level of GlaB but a normal level of α-amylase production. This strain was a disruption mutant of the C2H2-type transcription factor, FlbC, which is reported to be involved in the regulation of conidiospore development. Disruption mutants of other upstream regulators comprising a conidiation regulatory network had no apparent effect on GlaB production in solid-state culture. In addition to GlaB, the production of acid protease in solid-state culture was also markedly decreased by flbC disruption. Northern blot analyses revealed that transcripts of glaB and pepA were significantly decreased in the flbC disruption strain. These results suggested that FlbC is involved in the transcriptional regulation of genes specifically expressed under solid-state cultivation conditions, possibly independent of the conidiation regulatory network.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Akao T, Gomi K, Goto K, Okazaki N, Akita O (2002) Subtractive cloning of cDNA from Aspergillus oryzae differentially regulated between solid-state culture and liquid (submerged) culture. Curr Genet 41:275–281

    Article  CAS  PubMed  Google Scholar 

  • Anson ML (1938) Estimation of pepsin, papain and cathepsin with hemoglobin. J Gen Physiol 22:79–89

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cheevadhanarak S, Renno DV, Saunders G, Holt G (1991) Cloning and selective overexpression of an alkaline protease-encoding gene from Aspergillus oryzae. Gene 108:151–155

    Article  CAS  PubMed  Google Scholar 

  • Fling M, Horowitz NH, Heinemann SF (1963) The isolation and properties of crystalline tyrosinase from Neurospora. J Biol Chem 238:2045–2053

    CAS  PubMed  Google Scholar 

  • Fujieda N, Murata M, Yabuta S, Ikeda T, Shimokawa C, Nakamura Y, Hata Y, Itoh S (2012) Multifunctions of MelB, a fungal tyrosinase from Aspergillus oryzae. Chembiochem 13:193–201

    Article  CAS  PubMed  Google Scholar 

  • Gomi K, Iimura Y, Hara S (1987) Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric Biol Chem 51:2549–2555

    CAS  Google Scholar 

  • Gomi K, Arikawa K, Kamiya N, Kitamoto K, Kumagai C (1993) Cloning and nucleotide sequence of the acid protease-encoding gene (pepA) from Aspergillus oryzae. Biosci Biotechnol Biochem 57:1095–1100

    Article  CAS  PubMed  Google Scholar 

  • Gomi K, Akeno T, Minetoki T, Ozeki K, Kumagai C, Okazaki N, Iimura Y (2000) Molecular cloning and characterization of a transcriptional activator gene, amyR, involved in the amylolytic gene expression in Aspergillus oryzae. Biosci Biotechnol Biochem 64:816–827

    Article  CAS  PubMed  Google Scholar 

  • Guo JP, Ma Y (2008) High-level expression, purification and characterization of recombinant Aspergillus oryzae alkaline protease in Pichia pastoris. Protein Expr Purif 58:301–308

    Article  CAS  PubMed  Google Scholar 

  • Hata Y, Ishida H, Kojima Y, Ichikawa E, Kawano A, Suginami K, Imayasu S (1997) Comparison of two glucoamylase produced by Aspergillus oryzae in solid-state culture (koji) and in submerged culture. J Ferment Bioeng 84:532–537

    Article  CAS  Google Scholar 

  • Hata Y, Ishida H, Ichikawa E, Kawato A, Suginami K, Imayasu S (1998) Nucleotide sequence of an alternative glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae. Gene 207:127–134

    Article  CAS  PubMed  Google Scholar 

  • Hatamoto O, Umitsüki G, Machida M, Sano M, Tanaka A, Oka C, Maeda H, Tainaka H, Ito T, Uchikawa T, Masuda T, Matsushima K (2010) Recombinant vector capable of increasing secretion of Koji mold protease. U.S. patent US7842799 B2

  • Hisada H, Sano M, Ishida H, Hata Y, Machida M (2013) Identification of regulatory elements in the glucoamylase-encoding gene (glaB) promoter from Aspergillus oryzae. Appl Microbiol Biotechnol 97:4951–4956

    Article  CAS  PubMed  Google Scholar 

  • Ichinose S, Tanaka M, Shintani T, Gomi K (2014) Improved α-amylase production by Aspergillus oryzae after a double deletion of genes involved in carbon catabolite repression. Appl Microbiol Biotechnol 98:335–343

    Article  CAS  PubMed  Google Scholar 

  • Ishida H, Hata Y, Ichikawa E, Kawato A, Abe Y, Suginami K, Imayasu S (1998) Regulation of the glucoamylase-encoding gene (glaB), expressed in solid-state culture (koji) of Aspergillus oryzae. J Ferment Bioeng 86:301–307

    Article  CAS  Google Scholar 

  • Ishida H, Hata Y, Kawato A, Abe Y, Suginami K, Imayasu S (2000) Identification of functional elements that regulate the glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae. Curr Genet 37:373–379

    Article  CAS  PubMed  Google Scholar 

  • Jin FJ, Nishida M, Hara S, Koyama Y (2011) Identification and characterization of a putative basic helix-loop-helix transcription factor involved in the early stage of conidiophore development in Aspergillus oryzae. Fungal Genet Biol 48:1108–1115

    Article  CAS  PubMed  Google Scholar 

  • Kitano H, Kataoka K, Furukawa K, Hara S (2002) Specific expression and temperature-dependent expression of the acid protease-encoding gene (pepA) in Aspergillus oryzae in solid-state culture (Rice-Koji). J Biosci Bioeng 93:563–567

    Article  CAS  PubMed  Google Scholar 

  • Kwon NJ, Garzia A, Espeso EA, Ugalde U, Yu JH (2010) FlbC is a putative nuclear C2H2 transcription factor regulating development in Aspergillus nidulans. Mol Microbiol 77:1203–1219

    Article  CAS  PubMed  Google Scholar 

  • 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 438:1157–1161

    Article  PubMed  Google Scholar 

  • Machida M, Yamada O, Gomi K (2008) Genomics of Aspergillus oryzae: learning from the history of koji mold and exploration of its future. DNA Res 15:173–183

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mizutani O, Masaki K, Gomi K, Iefuji H (2012) Modified cre-loxP recombination in Aspergillus oryzae by direct introduction of cre recombinase for marker gene rescue. Appl Environ Microbiol 78:4126–4133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Murakami K, Ishida Y, Masaki A, Tatsumi H, Murakami S, Nakano E, Motai H, Kawabe H, Arimura H (1991) Isolation and characterization of the alkaline protease gene of Aspergillus oryzae. Agric Biol Chem 55:2807–2811

    CAS  PubMed  Google Scholar 

  • Obata H, Ishida H, Hata Y, Kawato A, Abe Y, Akao T, Akita O, Ichishima E (2004) Cloning of a novel tyrosinase-encoding gene (melB) from Aspergillus oryzae and its overexpression in solid-state culture (Rice Koji). J Biosci Bioeng 97:400–405

    Article  CAS  PubMed  Google Scholar 

  • Oda K, Kakizono D, Yamada O, Iefuji H, Akita O, Iwashita K (2006) Proteomic analysis of extracellular proteins from Aspergillus oryzae grown under submerged and solid-state culture conditions. Appl Environ Microbiol 72:3448–3457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ogawa M, Tokuoka M, Jin FJ, Takahashi T, Koyama Y (2010) Genetic analysis of conidiation regulatory pathways in koji-mold Aspergillus oryzae. Fungal Genet Biol 47:10–18

    Article  CAS  PubMed  Google Scholar 

  • Ogawa M, Kobayashi T, Koyama Y (2012) ManR, A novel Zn(II)2Cys6 transcriptional activator, controls the β-mannan utilization system in Aspergillus oryzae. Fungal Genet Biol 49:987–995

    Article  CAS  PubMed  Google Scholar 

  • Park HS, Yu JH (2012) Genetic control of asexual sporulation in filamentous fungi. Curr Opin Microbiol 15:669–677

    Article  CAS  PubMed  Google Scholar 

  • Petersen KL, Lehmbeck J, Christensen J (1999) A new transcriptional activator for amylase genes in Aspergillus. Mol Gen Genet 262:668–676

    Article  CAS  PubMed  Google Scholar 

  • Sato H, Toyoshima Y, Shintani T, Gomi K (2011) Identification of potential cell wall component that allows taka-amylase A adsorption in submerged cultures of Aspergillus oryzae. Appl Microbiol Biotechnol 92:961–969

    Article  CAS  PubMed  Google Scholar 

  • Suzuki K, Tanaka M, Konno Y, Ichikawa T, Ichinose S, Hasegawa-Shiro S, Shintani T, Gomi K (2015) Distinct mechanism of activation of two transcription factors, AmyR and MalR, involved in amylolytic enzyme production in Aspergillus oryzae. Appl Microbiol Biotechnol 99:1805–1815

    Article  CAS  PubMed  Google Scholar 

  • Takahashi T, Jin FJ, Sunagawa M, Machida M, Koyama Y (2008) Generation of large chromosomal deletions in koji molds Aspergillus oryzae and Aspergillus sojae via a loop-out recombination. Appl Environ Microbiol 74:7684–7693

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tanaka M, Tokuoka M, Shintani T, Gomi K (2012) Transcripts of a heterologous gene encoding mite allergen der f 7 are stabilized by codon optimization in Aspergillus oryzae. Appl Microbiol Biotechnol 96:1275–1282

    Article  CAS  PubMed  Google Scholar 

  • Tatsumi H, Murakami S, Tsuji RF, Ishida Y, Murakami K, Masaki A, Kawabe H, Arimura H, Nakano E, Motai H (1991) Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease II, a unique metalloprotease. Mol Gen Genet 228:97–103

    Article  CAS  PubMed  Google Scholar 

  • Te Biesebeke R, van Biezen N, de Vos WM, van den Hondel CAMJJ, Punt PJ (2005) Different control mechanisms regulate glucoamylase and protease gene transcription in Aspergillus oryzae in solid-state and submerged fermentation. Appl Microbiol Biotechnol 67:75–82

    Article  Google Scholar 

  • Watanabe J, Tanaka H, Mogi Y, Yamazaki T, Suzuki K, Watanabe T, Yamada O, Akita O (2011) Loss of Aspergillus oryzae amyR function indirectly affects hemicellulolytic and cellulolytic enzyme production. J Biosci Bioeng 111:408–413

    Article  CAS  PubMed  Google Scholar 

  • Yamada O, Lee BR, Gomi K (1997) Transformation system for Aspergillus oryzae with double auxortophic mutations, niaD and sC. Biosci Biotechnol Biochem 61:1367–1369

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Yoji Hata for kindly providing the anti-GlaB antibody and Yukihiro Nakamura for technical advice about the tyrosinase activity assay. We also thank Takeshi Akao for technical advice about total RNA extraction from A. oryzae cultured in solid-state culture and Osamu Mizutani for kindly providing the ∆ligD::loxP pyrG strain. This study was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry and the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Katsuya Gomi.

Ethics declarations

Ethical approval

This article does not contain any study with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Mizuki Tanaka and Midori Yoshimura contributed equally to this article.

Electronic supplementary materials

ESM 1

(PDF 371 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tanaka, M., Yoshimura, M., Ogawa, M. et al. The C2H2-type transcription factor, FlbC, is involved in the transcriptional regulation of Aspergillus oryzae glucoamylase and protease genes specifically expressed in solid-state culture. Appl Microbiol Biotechnol 100, 5859–5868 (2016). https://doi.org/10.1007/s00253-016-7419-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-016-7419-6

Keywords

Navigation