The global regulator LaeA controls production of citric acid and endoglucanases in Aspergillus carbonarius

  • Tore Linde
  • Marta Zoglowek
  • Mette Lübeck
  • Jens Christian Frisvad
  • Peter Stephensen Lübeck
Genetics and Molecular Biology of Industrial Organisms
First Online:
Received:
Accepted:

Abstract

The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have investigated the regulatory effect of LaeA on production of citric acid and cellulolytic enzymes in A. carbonarius. Two types of A. carbonarius strains, having laeA knocked out or overexpressed, were constructed and tested in fermentation. The knockout of laeA significantly decreased the production of citric acid and endoglucanases, but did not reduce the production of beta-glucosidases or xylanases. The citric acid accumulation was reduced with 74–96 % compared to the wild type. The endoglucanase activity was reduced with 51–78 %. Overexpression of LaeA seemed not to have an effect on citric acid production or on cellulose or xylanase activity.

Keywords

LaeA Citric acid Cellulases Aspergillus carbonarius Metabolic engineering 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

The project was financed by Novozymes A/S and the Danish Strategic Research program MycoFuelChem (DSF Grant No. 11-116803). Technician Gitte Hinz-Berg is thanked for HPLC analysis.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

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

References

  1. 1.
    Abarca ML, Accensi F, Cano J, Cabañes FJ (2004) Taxonomy and significance of black aspergilli. Antonie Van Leeuwenhoek 86:33–49. doi: 10.1023/b:anto.0000024907.85688.05 CrossRefPubMedGoogle Scholar
  2. 2.
    Bayman P, Baker JL (2006) Ochratoxins: a global perspective. Mycopathologia 162:215–223. doi: 10.1007/s11046-006-0055-4 CrossRefPubMedGoogle Scholar
  3. 3.
    Bayram O, Krappmann S, Ni M, Bok JW, Helmstaedt K, Valerius O, Braus-Stromeyer S, Kwon NJ, Keller NP, Yu JH, Braus GH (2008) VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science 320:1504–1506. doi: 10.1126/science.1155888 CrossRefPubMedGoogle Scholar
  4. 4.
    Bayram OS, Bayram O, Valerius O, Park HS, Irniger S, Gerke J, Ni M, Han KH, Yu JH, Braus GH (2010) LaeA control of velvet family regulatory proteins for light-dependent development and fungal cell-type specificity. PLoS Genet 6:e1001226. doi: 10.1371/journal.pgen.1001226 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bayram Ö, Braus GH (2012) Coordination of secondary metabolism and development in fungi: the velvet family of regulatory proteins. FEMS Microbiol Rev 36:1–24. doi: 10.1111/j.1574-6976.2011.00285.x CrossRefPubMedGoogle Scholar
  6. 6.
    Bok JW, Balajee SA, Marr KA, Andes D, Nielsen KF, Frisvad JC, Keller NP (2005) LaeA, a regulator of morphogenic fungal virulence factors. Eukaryot Cell 4:1574–1582. doi: 10.1128/EC.4.9.1574-1582.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bok JW, Keller NP (2004) LaeA, a regulator of secondary metabolism in Aspergillus spp. Eukaryot Cell 3:527–535. doi: 10.1128/EC.3.2.527 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Brakhage AA (2012) Regulation of fungal secondary metabolism. Nat Rev Microbiol 11:21–32. doi: 10.1038/nrmicro2916 CrossRefPubMedGoogle Scholar
  9. 9.
    Crespo-Sempere A, Marín S, Sanchis V, Ramos AJ (2013) VeA and LaeA transcriptional factors regulate ochratoxin A biosynthesis in Aspergillus carbonarius. Int J Food Microbiol 166:479–486. doi: 10.1016/j.ijfoodmicro.2013.07.027 CrossRefPubMedGoogle Scholar
  10. 10.
    Dai Z, Baker SE (2015) Enhanced citric acid production in Aspergillus with inactivated asparagine-linked glycosylation protein 3 (Alg3), and/or increased LaeA expression. US Patent 9,023,637 B2, 5 May 2015Google Scholar
  11. 11.
    Dowd PF (1989) Toxicity of naturally occurring levels of the Penicillium mycotoxins citrinin, ochratoxin A, and penicillic acid to the corn earworm, Heliothis zea, and the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae). Environ Entomol 18:24–29CrossRefGoogle Scholar
  12. 12.
    Fernandes M, Keller NP, Adams TH (1998) Sequence-specific binding by Aspergillus nidulans AflR, a C6 zinc cluster protein regulating mycotoxin biosynthesis. Mol Microbiol 28:1355–1365. doi: 10.1046/j.1365-2958.1998.00907.x CrossRefPubMedGoogle Scholar
  13. 13.
    Frandsen RJN, Andersson JA, Kristensen MB, Giese H (2008) Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi. BMC Mol Biol. doi: 10.1186/1471-2199-9-70 PubMedPubMedCentralGoogle Scholar
  14. 14.
    Hansen NB, Lübeck M, Lübeck PS (2014) Advancing USER cloning into simpleUSER and nicking cloning. J Microbiol Methods 96:42–49CrossRefPubMedGoogle Scholar
  15. 15.
    Hoff B, Kamerewerd J, Sigl C, Mitterbauer R, Zadra I, Kürnsteiner H, Kück U (2010) Two components of a velvet-like complex control hyphal morphogenesis, conidiophore development, and penicillin biosynthesis in Penicillium chrysogenum. Eukaryot Cell 9:1236–1250. doi: 10.1128/EC.00077-10 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Houbraken J, Spierenburg H, Frisvad JC (2012) Rasamsonia, a new genus comprising thermotolerant and thermophilic Talaromyces and Geosmithia species. Antonie van Leeuwenhoek Int J Gen Mol Microbiol 101:403–421. doi: 10.1007/s10482-011-9647-1 CrossRefGoogle Scholar
  17. 17.
    Häkkinen M (2014) Transcriptional analysis of Trichoderma reesei under conditions inducing cellulase and hemicellulase production, and identification of factors influencing protein production. University of Helsinki, HelsinkiGoogle Scholar
  18. 18.
    Jain S, Keller N (2013) Insights to fungal biology through LaeA sleuthing. Fungal Biol Rev 27:51–59. doi: 10.1016/j.fbr.2013.05.004 CrossRefGoogle Scholar
  19. 19.
    Jørgensen TR, Nielsen KF, Arentshorst M, Park J, van den Hondel CA, Frisvad JC, Ram AF (2011) Submerged conidiation and product formation by Aspergillus niger at low specific growth rates are affected in aerial developmental mutants. Appl Environ Microbiol 77:5270–5277. doi: 10.1128/AEM.00118-11 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Kale S, Milde L, Trapp M, Frisvad J, Keller N, Bok J (2008) Requirement of LaeA for secondary metabolism and sclerotial production in Aspergillus flavus. Fungal Genet Biol 45:1422–1429CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Karaffa L, Sándor E, Fekete E, Szentirmai A (2001) The biochemistry of citric acid of accumulation by Aspergillus niger (a review). Acta Microbiol Immunol Hung 48:429–440CrossRefPubMedGoogle Scholar
  22. 22.
    Karimi-Aghcheh R, Bok JW, Phatale PA, Smith KM, Baker SE, Lichius A, Omann M, Zeilinger S, Seiboth B, Rhee C, Keller NP, Freitag M, Kubicek CP (2013) Functional analyses of Trichoderma reesei LAE1 reveal conserved and contrasting roles of this regulator. G3 Genes Genomes Genet 3:369–378. doi: 10.1534/g3.112.005140 Google Scholar
  23. 23.
    Keller NP, Turner G, Bennett JW (2005) Fungal secondary metabolism—from biochemistry to genomics. Nat Rev Microbiol 3:937–947. doi: 10.1038/nrmicro1286 CrossRefPubMedGoogle Scholar
  24. 24.
    Klitgaard A, Iversen A, Andersen MR, Larsen TO, Frisvad JC, Nielsen KF (2014) Aggressive dereplication using UHPLC-DAD-QTOF: screening extracts for up to 3000 fungal secondary metabolites. Anal Bioanal Chem 406:1933–1943. doi: 10.1007/s00216-013-7582-x CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Kolasa M, Ahring B, Lübeck P, Lübeck M (2014) Co-cultivation of Trichoderma reesei RutC30 with three black Aspergillus strains facilitates efficient hydrolysis of pretreated wheat straw and shows promises for on-site enzyme production. Bioresour Technol 169:143–148CrossRefPubMedGoogle Scholar
  26. 26.
    Lee SB, Milgroom MG, Taylor JW (1988) A rapid, high yield mini-prep method for isolation of total genomic DNA from fungi. Fungal Genet Newsl 35:23–24Google Scholar
  27. 27.
    Lim FY, Hou Y, Chen Y, Oh JH, Lee I, Bugni TS, Keller NP (2012) Genome-based cluster deletion reveals an endocrocin biosynthetic pathway in Aspergillus fumigatus. Appl Environ Microbiol 78:4117–4125. doi: 10.1128/AEM.07710-11 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Linde T, Hansen NB, Lübeck M, Lübeck PS (2014) Fermentation in 24-well plates is an efficient screening platform for filamentous fungi. Lett Appl Microbiol 59:224–230. doi: 10.1111/lam.12268 CrossRefPubMedGoogle Scholar
  29. 29.
    McDonagh A, Fedorova ND, Crabtree J, Yu Y, Kim S, Chen D, Loss O, Cairns T, Goldman G, Armstrong-James D, Haynes K, Haas H, Schrettl M, May G, Nierman WC, Bignell E (2008) Sub-telomere directed gene expression during initiation of invasive aspergillosis. PLoS Pathog 4:e1000154. doi: 10.1371/journal.ppat.1000154 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Niu J, Arentshorst M, Nair PDS, Dai Z, Baker S, Frisvad JC, Nielsen KF, Punt PJ, Ram AFJ (2016) Identification of a classical mutant in the industrial host Aspergillus niger by systems genetics: LaeA is required for citric acid production and regulates the formation of some secondary metabolites. G3 Genes Genomes Genet 6:193–204. doi: 10.1534/g3.115.024067 Google Scholar
  31. 31.
    Olsen LR, Hansen NB, Bonde MT, Genee HJ, Holm DK, Carlsen S, Hansen BG, Patil KR, Mortensen UH, Wernersson R (2011) PHUSER (Primer Help for USER): a novel tool for USER fusion primer design. Nucl Acids Res 39:W61–W67. doi: 10.1093/nar/gkr394 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Patananan AN, Palmer JM, Garvey GS, Keller NP, Clarke SG (2013) A novel automethylation reaction in the Aspergillus nidulans LaeA protein generates S-methylmethionine. J Biol Chem 288:14032–14045CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Pedersen M, Hollensted M, Lange L, Andersen B (2009) Screening for cellulose and hemicellulose degrading enzymes from the fungal genus Ulocladium. Int Biodeterioation Biodegrad 63:484–489CrossRefGoogle Scholar
  34. 34.
    Reyes-Dominguez Y, Bok JW, Berger H, Shwab EK, Basheer A, Gallmetzer A, Scazzocchio C, Keller N, Strauss J (2010) Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans. Mol Microbiol 76:1376–1386. doi: 10.1111/j.1365-2958.2010.07051.x CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. CSHL Press, Cold Spring Harbor, New YorkGoogle Scholar
  36. 36.
    Samson RA, Hoekstra ES, Frisvad JC (eds) (2004) Introduction to food and airborne fungi, 7th edn. Centraalbureau voor Schimmelcultures, Utrecht, NetherlandsGoogle Scholar
  37. 37.
    Seiboth B, Karimi RA, Phatale PA, Linke R, Hartl L, Sauer DG, Smith KM, Baker SE, Freitag M, Kubicek CP (2012) The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei. Mol Microbiol 84:1150–1164. doi: 10.1111/j.1365-2958.2012.08083.x CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Shu P, Johnson MJ (1948) Production by submerged fermentation with Aspergillus niger. Ind Eng Chem 40:1202–1205CrossRefGoogle Scholar
  39. 39.
    Smedsgaard J (1997) Terverticillate penicillia studied by direct electrospray mass spectrometric profiling of crude extracts. II. Database and identification. Biochem Syst Ecol 25:65–71. doi: 10.1016/S0305-1978(96)00087-7 CrossRefGoogle Scholar
  40. 40.
    Sunga AJ, Tolstorukov I, Cregg JM (2008) Posttransformational vector amplification in the yeast Pichia pastoris. FEMS Yeast Res 8:870–876. doi: 10.1111/j.1567-1364.2008.00410.x CrossRefPubMedGoogle Scholar
  41. 41.
    Sørensen A, Lübeck PS, Lübeck M, Teller PJ, Ahring BK (2011) β-Glucosidases from a new Aspergillus species can substitute commercial β-glucosidases for saccharification of lignocellulosic biomass. Can J Microbiol 57(8):638–650CrossRefPubMedGoogle Scholar
  42. 42.
    Tamayo-Ramos JA, Orejas M (2014) Enhanced glycosyl hydrolase production in Aspergillus nidulans using transcription factor engineering approaches. Biotechnol Biofuels 7:103. doi: 10.1186/1754-6834-7-103 CrossRefGoogle Scholar
  43. 43.
    Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3-new capabilities and interfaces. Nucl Acids Res 40:1–12. doi: 10.1093/nar/gks596 CrossRefGoogle Scholar
  44. 44.
    Wang S, Liu G, Wang J, Yu J, Huang B, Xing M (2013) Enhancing cellulase production in Trichoderma reesei RUT C30 through combined manipulation of activating and repressing genes. J Ind Microbiol Biotechnol 40:633–641. doi: 10.1007/s10295-013-1253-y CrossRefPubMedGoogle Scholar
  45. 45.
    Wiemann P, Brown D, Kleigrewe K, Bok J, Keller N, Humpf H, Tudzynski B (2010) FfVel1 and FfLae1, components of a velvet-like complex in Fusarium fujikuroi, affect differentiation, secondary metabolism and virulence. Mol Microbiol 77:972–994PubMedPubMedCentralGoogle Scholar
  46. 46.
    Woloshuk CP, Foutz KR, Brewer JF, Bhatnagar D, Cleveland TE, Payne G (1994) Molecular characterization of aflR, a regulatory locus for aflatoxin biosynthesis. Appl Environ Microbiol 60:2408–2414PubMedPubMedCentralGoogle Scholar
  47. 47.
    Wood TM, Bhat KM (1988) Methods for measuring cellulase activities. Meth Enzymol 160:87–112CrossRefGoogle Scholar
  48. 48.
    Wu ZH, Wang TH, Huang W, Qu YB (2001) A simplified method for chromosome DNA preparation from filamentous fungi. Mycosystema 20:575–577Google Scholar
  49. 49.
    Yang L, Lübeck M, Lübeck PS (2014) Deletion of glucose oxidase changes the pattern of organic acid production in Aspergillus carbonarius. AMB Express 4:54. doi: 10.1186/s13568-014-0054-7 CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Zoglowek M, Hansen GH, Lübeck PS, Lübeck M (2015) Fungal consortia for conversion of lignocellulose into bioproducts. In: Silva RD (ed) Mycology: Current and Future Developments, vol 1. Bentham Books, pp 329–365Google Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2016

Authors and Affiliations

  • Tore Linde
    • 1
  • Marta Zoglowek
    • 2
  • Mette Lübeck
    • 1
  • Jens Christian Frisvad
    • 3
  • Peter Stephensen Lübeck
    • 1
  1. 1.Section for Sustainable BiotechnologyAalborg UniversityCopenhagen SVDenmark
  2. 2.Carlsberg Research Laboratory, Yeast & Fermentation, Group CommercialCopenhagen VDenmark
  3. 3.DTU, Institute for System-biologi, Fungal ChemodiversityKgs. LyngbyDenmark

Personalised recommendations