Skip to main content
SpringerLink
Log in

Identification of functional cis-elements required for repression of the Taka-amylase A gene under secretion stress in Aspergillus oryzae

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

The expression of secreted proteins in filamentous fungi is down-regulated by a transcriptional feedback mechanism under endoplasmic reticulum stress, termed repression under secretion stress (RESS). To investigate the RESS mechanism, we analyzed the expression of the Taka-amylase A gene (amyB) in Aspergillus oryzae, which was depressed under secreted protein stress. We conducted a truncation and deletion analysis of the amyB promoter to identify cis-elements required for RESS. A nucleotide sequence (positions −378 to −291) without any binding sites for the transcriptional activator AmyR, which is involved in amylolytic gene expression, was required for RESS. The octamer sequence TCACGGGC (positions −307 to −300) constituted the core sequence of the upstream activating element essential for amyB down-regulation under secretion stress. Both the inactivation of AmyR and RESS contributed to the down-regulation of amyB expression under ER stress.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Al-Sheikh H, Watson AJ, Lacey GA, Punt PJ, MacKenzie DA, Jeenes DJ, Pakula T, Penttilä M, Alcocer MJ, Archer DB (2004) Endoplasmic reticulum stress leads to the selective transcriptional downregulation of the glucoamylase gene in Aspergillus niger. Mol Microbiol 53:1731–1742

    Article  CAS  PubMed  Google Scholar 

  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  PubMed  Google Scholar 

  • Carvalho ND, Jørgensen TR, Arentshorst M, Nitsche BM, van den Hondel CA, Archer DB, Ram AF (2012) Genome-wide expression analysis upon constitutive activation of the HacA bZIP transcription factor in Aspergillus niger reveals a coordinated cellular response to counteract ER stress. BMC Genomics 13:350

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Conesa A, Punt PJ, van Luijk N, van den Hondel CA (2001) The secretion pathway in filamentous fungi: a biotechnological view. Fungal Genet Biol 33:155–171

    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

    Article  CAS  Google Scholar 

  • Gouka RJ, Punt PJ, van den Hondel CA (1997) Efficient production of secreted proteins by Aspergillus: progress, limitations and prospects. Appl Microbiol Biotechnol 47:1–11

    Article  CAS  PubMed  Google Scholar 

  • Guillemette T, van Peij N, 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 8:158

    Article  PubMed Central  PubMed  Google Scholar 

  • Hisada H, Sano M, Hata Y, Abe Y, Machida M (2008) Deletion analysis of the catalase-encoding gene (catB) promoter from Aspergillus oryzae. Biosci Biotechnol Biochem 72:48–53

    Article  CAS  PubMed  Google Scholar 

  • Kanemori Y, Gomi K, Kitamoto K, Kumagai C, Tamura G (1999) Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system. Biosci Biotechnol Biochem 63:180–183

    Article  CAS  PubMed  Google Scholar 

  • Kubodera T, Yamashita N, Nishimura A (2000) Pyrithiamine resistance gene (ptrA) of Aspergillus oryzae: cloning, characterization and application as a dominant selectable marker for transformation. Biosci Biotechnol Biochem 64:1416–1421

    Article  CAS  PubMed  Google Scholar 

  • Kwon MJ, Jørgensen TR, Nitsche BM, Arentshorst M, Park J, Ram AF, Meyer V (2012) The transcriptomic fingerprint of glucoamylase over-expression in Aspergillus niger. BMC Genomics 13:701

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lee C, Kim J, Shin SG, Hwang S (2006) Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol 123:273–280

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Mori K (2000) Tripartite management of unfolded proteins in the endoplasmic reticulum. Cell 101:451–454

    Article  CAS  PubMed  Google Scholar 

  • Nakajima K, Asakura T, Maruyama J, Morita Y, Oike H, Shimizu-Ibuka A, Misaka T, Sorimache 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 72:3716–3723

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nemoto T, Maruyama J, Kitamoto K (2012) Contribution ratios of amyA, amyB, amyC genes to high-level alpha-amylase expression in Aspergillus oryzae. Biosci Biotechnol Biochem 76:1477–1483

    Article  CAS  PubMed  Google Scholar 

  • Nevalainen KM, Te’o VS, Bergquist PL (2005) Heterologous protein expression in filamentous fungi. Trends Biotechnol 23:468–474

    Article  CAS  PubMed  Google Scholar 

  • Pakula TM, Laxell M, Huuskonen A, Uusitalo J, Saloheimo M, Penttilä M (2003) The effects of drugs inhibiting protein secretion in the filamentous fungus Trichoderma reesei. Evidence for down-regulation of genes that encode secreted proteins in the stressed cells. J Biol Chem 278:45011–45020

    Article  CAS  PubMed  Google Scholar 

  • 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 71:2737–2747

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sun YF, Lin Y, Zhang JH, Zheng SP, Ye YR, Liang XX, Han SY (2012) Double Candida antarctica lipase B co-display on Pichia pastoris cell surface based on a self-processing foot-and-mouth disease virus 2A peptide. Appl Microbiol Biotechnol 96:1539–1550

    Article  CAS  PubMed  Google Scholar 

  • Tada S, Gomi K, Kitamoto K, Kumagai C, Tamura G, Hara S (1991) Identification of the promoter region of the Taka-amylase A gene required for starch induction. Agric Biol Chem 55:1939–1941

    Article  CAS  PubMed  Google Scholar 

  • Tanaka A, Kato M, Hashimoto H, Kamei K, Naruse F, Papagiannopoulos P, Davis MA, Hynes MJ, Kobayashi T, Tsukagoshi N (2000) An Aspergillus oryzae CCAAT-binding protein, AoCP, is involved in the high-level expression of the Taka-amylase A gene. Curr Genet 37:380–387

    Article  CAS  PubMed  Google Scholar 

  • Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101:249–258

    Article  CAS  PubMed  Google Scholar 

  • Tsuchiya K, Tada S, Gomi K, Kitamoto K, Kumagai C, Tamura G (1992) Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae. Biosci Biotechnol Biochem 56:1849–1853

    Article  CAS  PubMed  Google Scholar 

  • Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acid Res 35:W71–W74

    Article  PubMed Central  PubMed  Google Scholar 

  • Wang B, Pan L, Guo Y (2009) Construction of heterologous gene expression system for filamentous Aspergillus oryzae. J South China Univ Technol (Nat Sci Ed) 37:84–90

    CAS  Google Scholar 

  • Wang B, Guo G, Wang C, Lin Y, Wang X, Zhao M, Guo Y, He M, Zhang Y, Pan L (2010) Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing. Nucleic Acids Res 38:5075–5087

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ward OP, Qin WM, Dhanjoon J, Ye J, Singh A (2006) Physiology and biotechnology of Aspergillus. Adv Appl Microbiol 58:1–75

    Article  CAS  PubMed  Google Scholar 

  • Ye L, Pan L (2008) A comparison of the unfolded protein response in solid-state with submerged cultures of Aspergillus oryzae. Biosci Biotechnol Biochem 72:2998–3001

    Article  CAS  PubMed  Google Scholar 

  • Yoshino-Yasuda S, Fujino E, Matsui J, Ono N, Kato M, Kitamoto N (2013) Molecular analysis of AsamyR gene encoding transcriptional factor for amylolytic gene from Shoyu Koji Mold, Aspergillus sojae KBN1340. Food Sci Technol Res 19:505–511

    Article  CAS  Google Scholar 

  • Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Dominguez Y, Scazzocchio C (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41:973–981

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are grateful for financial support from the National Science and Technology Foundation during the 12th Five-Year Plan for Rural Development in China (Grant No. 2011AA100905), the Guangdong Provincial Department of Science and Technology Research Project (Grant No. 2012B010900028), and the National Natural Science Foundation of China (Grant No. 31100026).

Supporting information

Supplementary Table 1—Primers used for vector construction

Supplementary Table 2— Primers used for quantitative real-time PCR

Supplementary Fig. 1—Confirmation of PCR amplification specificity (a) and efficiency ( b) of the gapdh gene

Supplementary Fig. 2—Confirmation of PCR amplification specificity (a) and efficiency (b) of the hacAi gene

Supplementary Fig. 3—Confirmation of PCR amplification specificity (a) and efficiency (b) of the rml gene

Supplementary Fig. 4—Confirmation of PCR amplification specificity (a) and efficiency (b) of the amyB gene

Supplementary Fig. 5—Confirmation of PCR amplification specificity (a) and efficiency (b) of the amyR gene

Author information

Authors and Affiliations

  1. School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, People’s Republic of China

    Bin Zhou, Chao Wang, Bin Wang, Xiupeng Li, Jing Xiao & Li Pan

Authors
  1. Bin Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chao Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiupeng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jing Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Li Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Li Pan.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 1358 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhou, B., Wang, C., Wang, B. et al. Identification of functional cis-elements required for repression of the Taka-amylase A gene under secretion stress in Aspergillus oryzae . Biotechnol Lett 37, 333–341 (2015). https://doi.org/10.1007/s10529-014-1691-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-014-1691-2

Keywords

Search

Navigation