Regulation of genes encoding cellulolytic enzymes by Pal-PacC signaling in Aspergillus nidulans

  • 767 Accesses

  • 16 Citations


Cellulosic biomass represents a valuable potential substitute for fossil-based fuels. As such, there is a strong need to develop efficient biotechnological processes for the enzymatic hydrolysis of cellulosic biomass via the optimization of cellulase production by fungi. Ambient pH is an important factor affecting the industrial production of cellulase. In the present study, we demonstrate that several Aspergillus nidulans genes encoding cellulolytic enzymes are regulated by Pal-PacC-mediated pH signaling, as evidenced by the decreased cellulase productivity of the palC mutant and pacC deletants of A. nidulans. The deletion of pacC was observed to result in delayed induction and decreased expression of the cellulase genes based on time course expression analysis. The genome-wide identification of PacC-regulated genes under cellobiose-induced conditions demonstrated that genes expressed in a PacC-dependent manner included 82 % of ClrB (a transcriptional activator of the cellulase genes)-regulated genes, including orthologs of various transporter and β-glucosidase genes considered to be involved in cellobiose uptake or production of stronger inducer molecules. Together with the significant overlap between ClrB- and PacC-regulated genes, the results suggest that PacC-mediated regulation of the cellulase genes involves not only direct regulation by binding to their promoter regions but also indirect regulation via modulation of the expression of genes involved in ClrB-dependent transcriptional activation. Our findings are expected to contribute to the development of more efficient industrial cellulase production methods.

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

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Coradetti ST, Craig JP, Xiong Y, Shock T, Tian C, Glass NL (2012) Conserved and essential transcription factors for cellulase gene expression in ascomycete fungi. Proc Natl Acad Sci U S A 109:7397–7402

  2. Coradetti ST, Xiong Y, Glass NL (2013) Analysis of a conserved cellulase transcriptional regulator reveals inducer-independent production of cellulolytic enzymes in Neurospora crassa. Microbiology Open 2:595–609

  3. Eberhart BM, Beck RS, Goolsby KM (1977) Cellulase of Neurospora crassa. J Bacteriol 130:181–186

  4. Espeso EA, Arst HNJ (2000) On the mechanism by which alkaline pH prevents expression of an acid-expressed gene. Mol Cell Biol 20:3355–3363

  5. Espeso EA, Peñalva MA (1996) Three binding sites for the Aspergillus nidulans PacC zinc-finger transcription factor are necessary and sufficient for regulation by ambient pH of the isopenicillin N synthase gene promoter. J Biol Chem 271:28825–28830

  6. Espeso EA, Tilburn J, Sánchez-Pulido L, Brown CV, Valencia A, Arst HNJ, Peñalva MA (1997) Specific DNA recognition by the Aspergillus nidulans three zinc finger transcription factor PacC. J Mol Biol 274:466–480

  7. Galazka JMC, Tian C, Beeson WT, Martinez B, Glass NL, Cate JH (2010) Cellodextrin transport in yeast for improved biofuel production. Science 330:84–86

  8. Galindo A, Hervás-Aguilar A, Rodríguez-Galán O, Vincent O, Arst HNJ, Tilburn J, Peñalva MA (2007) PalC, one of two Bro1 domain proteins in the fungal pH signaling pathway, localizes to cortical structures and binds Vps32. Traffic 8:1346–1364

  9. Galindo A, Calcagno-Pizarelli AM, Arst HNJ, Peñalva MÁ (2012) An ordered pathway for the assembly of fungal ESCRT-containing ambient pH signalling complexes at the plasma membrane. J Cell Sci 125:1784–1795

  10. Gielkens M, González-Candelas L, Sánchez-Torres P, van de Vondervoort P, de Graaff L, Visser J, Ramón D (1999a) The abfB gene encoding the major α-L-arabinofuranosidase of Aspergillus nidulans: nucleotide sequence, regulation and construction of a disrupted strain. Microbiol 145:735–741

  11. Gielkens MM, Dekkers E, Visser J, de Graaff LH (1999b) Two cellobiohydrolase-encoding genes from Aspergillus niger require D-xylose and the xylanolytic transcriptional activator XlnR for their expression. Appl Environ Microbiol 65:4340–4345

  12. Häkkinen M, Sivasiddarthan D, Aro N, Saloheimo M, Pakula TM (2015) The effects of extracellular pH and of the transcriptional regulator PACI on the transcriptome of Trichoderma reesei. Microb Cell Factories 14:63

  13. He R, Ma L, Li C, Jia W, Li D, Zhang D, Chen S (2014) Trpac1, a pH response transcription regulator, is involved in cellulase gene expression in Trichoderma reesei. Enzym Microb Technol 67:17–26

  14. Käfer E (1977) Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv Genet 19:33–131

  15. Kato N, Murakoshi Y, Kato M, Kobayashi T, Tsukagoshi N (2002a) Isomaltose formed by α-glucosidases triggers amylase induction in Aspergillus nidulans. Curr Genet 42:43–50

  16. Kato N, Suyama S, Shirokane M, Kato M, Kobayashi T, Tsukagoshi N (2002b) Novel α-glucosidase from Aspergillus nidulans with strong transglycosylation activity. Appl Environ Microbiol 68:1250–1256

  17. Kunitake E, Tani S, Sumitani J, Kawaguchi T (2013) A novel transcriptional regulator, ClbR, controls the cellobiose- and cellulose-responsive induction of cellulase and xylanase genes regulated by two distinct signaling pathways in Aspergillus aculeatus. Appl Microbiol Biotechnol 97:2017–2028

  18. Kurasawa T, Yachi M, Suto M, Kamagata Y, Takao S, Tomita F (1992) Induction of cellulase by gentiobiose and its sulfur-containing analog in Penicillium purpurogenum. Appl Environ Microbiol 58:106–110

  19. Lockington RA, Rodbourn L, Barnett S, Carter CJ, Kelly JM (2002) Regulation by carbon and nitrogen sources of a family of cellulases in Aspergillus nidulans. Fungal Genet Biol 37:190–196

  20. MacCabe AP, Orejas M, Pérez-González CJ, Ramón D (1998) Opposite patterns of expression of two Aspergillus nidulans xylanase genes with respect to ambient pH. J Bacteriol 180:1331–1333

  21. Mach RL, Seiboth B, Myasnikov A, Gonzalez R, Strauss J, Harkki AM, Kubicek CP (1995) The bgl1 gene of Trichoderma reesei QM9414 encodes an extracellular, cellulose-inducible β-glucosidase involved in cellulase induction by sophorose. Mol Microbiol 16:687–697

  22. Makita T, Katsuyama Y, Tani S, Suzuki H, Kato N, Todd RB, Hynes MJ, Tsukagoshi N, Kato M, Kobayashi T (2009) Inducer-dependent nuclear localization of a Zn(II)2Cys6 transcriptional activator, AmyR, in Aspergillus nidulans. Biosci Biotechnol Biochem 73:391–399

  23. Marui J, Kitamoto N, Kato M, Kobayashi T, Tsukagoshi N (2002a) Transcriptional activator, AoXlnR, mediates cellulose-inductive expression of the xylanolytic and cellulolytic genes in Aspergillus oryzae. FEBS Lett 528:279–282

  24. Marui J, Tanaka A, Mimura S, de Graaff LH, Visser J, Kitamoto N, Kato M, Kobayashi T, Tsukagoshi N (2002b) A transcriptional activator, AoXlnR, controls the expression of genes encoding xylanolytic enzymes in Aspergillus oryzae. Fungal Genet Biol 35:157–169

  25. Mello-de-Sousa TM, Silva-Pereira I, Poças-Fonseca MJ (2011) Carbon source and pH-dependent transcriptional regulation of cellulase genes of Humicola grisea var. thermoidea grown on sugarcane bagasse. Enzym Microb Technol 48:19–26

  26. Motoyama T, Fujiwara M, Kojima N, Horiuchi H, Ohta A, Takagi M (1997) The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation. Mol Gen Genet 253:520–528

  27. Murakoshi Y, Makita T, Kato M, Kobayashi T (2012) Comparison and characterization of α-amylase inducers in Aspergillus nidulans based on nuclear localization of AmyR. Appl Microbiol Biotechnol 94:1629–1635

  28. Nakamura T, Makita T, Maeda Y, Tanoue N, Kato M, Kobayashi T (2006) Expression profile of amylolytic genes in Aspergillus nidulans. Biosci Biotechnol Biochem 70:2363–2370

  29. Noguchi Y, Sano M, Kanamaru K, Ko T, Takeuchi M, Kato M, Kobayashi T (2009) Genes regulated by AoXlnR, the xylanolytic and cellulolytic transcriptional regulator, in Aspergillus oryzae. Appl Microbiol Biotechnol 85:141–154

  30. 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

  31. Ogawa M, Kobayashi T, Koyama Y (2013) ManR, a transcriptional regulator of the β-mannan utilization system, controls the cellulose utilization system in Aspergillus oryzae. Biosci Biotechnol Biochem 77:426–429

  32. Peñalva MA, Lucena-Agell D, Arst HNJ (2014) Liaison alcaline: Pals entice non-endosomal ESCRTs to the plasma membrane for pH signaling. Curr Opin Microbiol 22:49–59

  33. Rowlands RT, Turner G (1973) Nuclear and extranuclear inheritance of oligomycin resistance in Aspergillus nidulans. Mol Gen Genet 126:201–216

  34. Saloheimo M, Kuja-Panula J, Ylösmäki E, Ward M, Penttilä M (2002) Enzymatic properties and intracellular localization of the novel Trichoderma reesei β-glucosidase BGLII (cel1A). Appl Environ Microbiol 68:4546–4553

  35. Sternberg D, Mandels GR (1979) Induction of cellulolytic enzymes in Trichoderma reesei by sophorose. J Bacteriol 139:761–769

  36. Sternberg D, Mandels GR (1980) Regulation of the cellulolytic system in Trichoderma reesei by sophorose: induction of cellulase and repression of β-glucosidase. J Bacteriol 144:1197–1199

  37. Stewart JC, Parry JB (1981) Factors influencing the production of cellulase by Aspergillus fumigatus (Fresenius). J Gen Microbiol 125:33–39

  38. Stricker AR, Grosstessner-Hain K, Würleitner E, Mach RL (2006) Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina. Eukaryot Cell 5:2128–2137

  39. Sun J, Nishiyama T, Shimizu K, Kadota K (2013) TCC: an R package for comparing tag count data with robust normalization strategies. BMC Bioinformatics 14:219

  40. Thomas-Chollier M, Sand O, Turatsinze JV, Janky R, Defrance M, Vervisch E, Brohée S, van Helden J (2008) RSAT: regulatory sequence analysis tools. Nucl Acids Res 36:W119–W127

  41. van Peij NN, Gielkens MM, de Vries RP, Visser J, de Graaff LH (1998a) The transcriptional activator XlnR regulates both xylanolytic and endoglucanase gene expression in Aspergillus niger. Appl Environ Microbiol 64:3615–3619

  42. van Peij NN, Visser J, de Graaff LH (1998b) Isolation and analysis of xlnR, encoding a transcriptional activator co-ordinating xylanolytic expression in Aspergillus niger. Mol Microbiol 27:131–142

  43. Yamakawa Y, Endo Y, Li N, Yoshizawa M, Aoyama M, Watanabe A, Kanamaru K, Kato M, Kobayashi T (2013) Regulation of cellulolytic genes by McmA, the SRF-MADS box protein in Aspergillus nidulans. Biochem Biophys Res Commun 431:777–782

  44. Zhang W, Kou Y, Xu J, Cao Y, Zhao G, Shao J, Wang H, Wang Z, Bao X, Chen G, Liu W (2013) Two major facilitator superfamily sugar transporters from Trichoderma reesei and their roles in induction of cellulase biosynthesis. J Biol Chem 288:32861–32872

  45. Znameroski EA, Li X, Tsai JC, Galazka JM, Glass NL, Cate JH (2014) Evidence for transceptor function of cellodextrin transporters in Neurospora crassa. J Biol Chem 289:2610–2619

Download references


This work was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry and by the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry.

Author information

Correspondence to Tetsuo Kobayashi.

Ethics declarations

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

Competing interests

The authors declare that they have no competing interests.

Electronic supplementary material


(PDF 823 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kunitake, E., Hagiwara, D., Miyamoto, K. et al. Regulation of genes encoding cellulolytic enzymes by Pal-PacC signaling in Aspergillus nidulans . Appl Microbiol Biotechnol 100, 3621–3635 (2016).

Download citation


  • Aspergillus nidulans
  • PalC
  • PacC
  • Cellulolytic enzymes
  • ClrB