Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Molecular characterization of StcI esterase from Aspergillus nidulans

  • 83 Accesses

Abstract

Aspergillus nidulans produces StcI esterase, which is involved in the biosynthesis of sterigmatocystin, a precursor of aflatoxins. Previous reports of this esterase in A. nidulans suggest that it is composed of 286 amino acid residues with a theoretical molecular mass of 31 kDa. Various conditions were evaluated to determine the optimal expression conditions for StcI; the highest level was observed when A. nidulans was cultured in solid oat media. Various esterases were expressed differentially according to the culture media used. However, specific antibodies designed to detect StcI reacted with a protein with an unexpected molecular mass of 35 kDa in cell extracts from all expression conditions. Analysis of the gene sequence and already reported expressed sequence tags indicated the presence of an additional 29-amino-acid N-terminal region of StcI, which is not a signal peptide and which has not been previously reported. We also detected the presence of this additional N-terminal region using reverse-transcriptase polymerase chain reaction. The complete protein (NStcI) was cloned and successfully expressed in Pichia pastoris.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Anderson JA, Chung CH (1990) Conversion of versiconal acetate to versiconal and versicolorin C in extracts from Aspergillus parasiticus. Mycophatologia 110:31–35

  2. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795

  3. Bennett JW, Henderberg A, Grossman K (1989) Sterigmatocystin production on complex and defined substrates. Mycopathologia 105(1):35–38

  4. Branch KR, Bennett JW, Bhatnagar D (1993) Sterigmatocystin production by Aspergillus nidulans. Fungal Genet Newsl 40:20–21

  5. Brown DW, Yu JH, Kelkar HS, Fernandes M, Nesbitt TC, Keller NP, Adams TH, Leonard TJ (1996) Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans. Proc Natl Acad Sci U S A 93(4):1418–1422

  6. Burge CB, Karlin S (1998) Finding the genes in genomic DNA. Curr Opin Struct Biol 8:346–354

  7. Chang PK, Yabe K, Yu J (2004) The Aspergillus parasiticus estA-encoded esterase converts versiconal hemiacetal acetate to versiconal and versiconol acetate to versiconol in aflatoxin biosynthesis. Appl Environ Microbiol 70:3593–3599

  8. DeCaprio D, Vinson JP, Pearson MD, Montgomery P, Doherty M, Galagan JE (2007) Conrad: gene prediction using conditional random fields. Genome Res 17(9):1389–1398

  9. Douglas SE (1994) DNA Strider: a Macintosh program for handling protein and nucleic acid sequences. Methods Mol Biol 25:181–194

  10. Feng GH, Leonard TJ (1998) Culture conditions control expression of the genes for aflatoxin and sterigmatocystin biosynthesis in Aspergillus parasiticus and A. nidulans. Appl Environ Microbiol 64(6):2275–2277

  11. Fischer M, Pleiss J (2003) The Lipase Engineering Database: a navigation and analysis tool for protein families. Nucleic Acids Research 31(1):319–321

  12. Galagan JE, Calvo SE, Cuomo C, Ma LJ, Wortman JR, Batzoglou S, Lee SI, Baştürkmen M, Spevak CC, Clutterbuck J, Kapitonov V, Jurka J, Scazzocchio C, Farman M, Butler J, Purcell S, Harris S, Braus GH, Draht O, Busch S, D’Enfert C, Bouchier C, Goldman GH, Bell-Pedersen D, Griffiths-Jones S, Doonan JH, Yu J, Vienken K, Pain A, Freitag M, Selker EU, Archer DB, Peñalva MA, Oakley BR, Momany M, Tanaka T, Kumagai T, Asai K, Machida M, Nierman WC, Denning DW, Caddick M, Hynes M, Paoletti M, Fischer R, Miller B, Dyer P, Sachs MS, Osmani SA, Birren BW (2005) Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438(7071):1105–1115

  13. Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784–3788

  14. Hall T (2007) BioEdit Biological Sequence Alignment Editor for Win95/98/NT/2K/XP. http://www.mbio.ncsu.edu/BioEdit/bioedit.html. Accessed 29 Jan 2007

  15. Hotelier T, Renault L, Cousin X, Negre V, Marchot P, Chatonnet A (2004) ESTHER, the database of the alpha/beta-hydrolase fold superfamily of proteins. Nucleic Acids Research 32:D145–D147

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

  17. Kakariari E, Georgalaki MD, Kalantzopoulos G, Tsakalidou E (2000) Purification and characterization of an intracellular esterase from Propionibacterium freudenreichii ssp. freudenreichii ITG 14. Le Lait 80:491–501

  18. Kato N, Brooks W, Calvo AM (2003) The expression of sterigmatocystin and penicillin genes in Aspergillus nidulans is controlled by veA, a gene required for sexual development. Eukaryot Cell 2(6):1178–1186

  19. Kawasaki L, Farrés A, Aguirre J (1995) Aspergillus nidulans mutants affected in acetate metabolism isolated as lipid nonutilizers. Exp Mycol 19(1):81–85

  20. Kelkar HS, Skloss TW, Haw JF, Keller NP, Adams TH (1997) Aspergillus nidulans stcL encodes a putative cytochrome P-450 monooxygenase required for bisfuran desaturation during aflatoxin/sterigmatocystin biosynthesis. J Biol Chem 272(3):1589–1594

  21. Keller NP, Kantz NJ, Adams TH (1994) Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin. Appl Environ Microbiol 60(5):1444–1450

  22. Keller NP, Nesbitt C, Sarr B, Phillips TD, Burow GB (1997) pH Regulation of sterigmatocystin and aflatoxin biosynthesis in Aspergillus spp. Phytopathology 87(6):643–648

  23. Koseki T, Miwa Y, Fushinobu S, Hashizume K (2005) Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: mutational analysis of catalytic residues. Biochim Biophys Acta 1749(1):7–13

  24. Kozak M (1987) An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 15(20):8125–8148

  25. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinformatics 5:150–163

  26. Kusumoto K, Hsieh DPH (1996) Purification and characterization of the esterases involved in aflatoxin biosynthesis in Aspergillus parasiticus. Can J Microbiol 42(8):804–810

  27. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  28. Larkin MA, Blackshields G, Brown NP, Chenna R et al (2007) ClustalW and Clustal X version 2. Bioinformatics 23(21):2947–2948

  29. Mandrich L, Merone L, Pezzullo M, Cipolla L, Nicotra F, Rossi M, Manco G (2005) Role of the N terminus in enzyme activity, stability and specificity in thermophilic esterases belonging to the HSL family. J Mol Biol 345(3):501–512

  30. Minning S, Serrano A, Ferrer P, Solá C, Schmid RD, Valero F (2001) Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris. J Biothecnol 86(1):59–70

  31. Motomura MN, Chihaya T, Shinozawa T, Hamazaki T, Yabe K (1999) Cloning and characterization of the O-methyltransferase I gene (dmtA) from Aspergillus parasiticus associated with the conversions of demethylsterigmatocystin to sterigmatocystin and dihydrodemethylsterigmatocystin to dihydrosterigmatocystin in aflatoxin biosynthesis. Appl Environ Microbiol 65(11):4987–4994

  32. Paukstelis PJ, Lambowitz AM (2008) Identification and evolution of fungal mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity. Proc Natl Acad Sci U S A 105(16):6010–6015

  33. Peña-Montes C, Gonzalez A, Castro-Ochoa D, Farrés A (2008) Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans. Appl Microbial Biotechnol 78(4):603–612

  34. Price MS, Conners SB, Tachdjian S, Kelly RM, Payne GA (2005) Aflatoxin conducive and non-conducive growth conditions reveal new gene associations with aflatoxin production. Fungal Genet Biol 42(6):506–518

  35. Roe BA, Kupfer D, Zhu H, Gray S, Clifton R, Prade J, Loros J, Dunlap J, Nelson M (2001) The Aspergillus nidulans and the Neurospora crassa cDNA Sequencing Project. University of Oklahoma Aspergillus nidulans cDNA Blast Server. http://www.genome.ou.edu/asper_blast.html. Accessed 23 Jan 2008

  36. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

  37. Sambrook JE, Fritsch F, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

  38. Sayari AF, Frikha F, Miled N, Mtibaa H, Ben Ali Y, Verger R, Gargouri Y (2005) N-terminal peptide of Rhizopus oryzae lipase is important for its catalytic properties. FEBS Lett 579(5):976–982

  39. Schneider P, Weber M, Hoffmeister D (2008) The Aspergillus nidulans enzyme TdiB catalyzes prenyltransfer to the precursor of bioactive asterriquinones. Fungal Genet Biol 45(3):302–309

  40. Spröte P, Hynes MJ, Hortschansky P, Shelesty E, Scharf DH, Wolke SM, Brakhage AA (2008) Identification of the novel penicillin biosynthesis gene aatB of Aspergillus nidulans and its putative evolutionary relationship to this fungal secondary metabolism gene cluster. Mol Microbiol 70(2):445–461

  41. Sugihara A, Tani T, Tominaga Y (1991) Purification and characterization of a novel thermostable lipase from Bacillus sp. J Biochem 109:211–216

  42. Wu J, Miller BL (1997) Aspergillus asexual reproduction and sexual reproduction are differentially affected by transcriptional and translational mechanisms regulating stunted gene expression. Mol Cell Biol 17(10):6191–6201

  43. Xiong AS, Yao QH, Peng RH, Han PL, Cheng ZM, Li Y (2005) High level expression of a recombinant acid phytase gene in Pichia pastoris. J Appl Microbiol 98(2):418–428

  44. Yabe K, Nakajima H (2004) Enzyme reactions and genes in aflatoxin biosynthesis. Appl Microbiol Biotechnol 64(6):745–755

  45. Yabe K, Ando Y, Hamasaki T (1991) A metabolic grid among versiconal hemiacetal acetate, versiconol acetate, versiconol and versiconal during aflatoxin biosynthesis. J Gen Microbiol 137:2469–2475

  46. Yu J, Leonard TJ (1995) Sterigmatocystin biosynthesis in A. nidulans requires a novel type I polyketide synthase. J Bacteriol 177(16):4792–4800

  47. Yu J, Chang PK, Cary JW, Wright M, Bhatnagar D, Cleveland TE, Payne GA, Linz JE (1995) Comparative mapping of aflatoxin pathway gene clusters in Aspergillus parasiticus and Aspergillus flavus. Appl Environ Microbiol 61:2365–2371

  48. Yu J, Woloshuk CP, Bhatnagar D, Cleveland TE (2000) Cloning and characterization of avfA and omtB genes involved in aflatoxin biosynthesis in three Aspergillus species. Gene 248(1–2):157–167

  49. Yu J, Chang PK, Bhatnagar D, Cleveland TE (2002) Cloning and functional expression of an esterase gene in Aspergillus parasiticus. Mycophatologia 156(3):227–234

Download references

Acknowledgments

CPM received a scholarship from DAAD (German Academic Exchange Service) that allowed collaboration with the ITB, University of Stuttgart, Germany. We appreciate the help of American Journal Experts with the English revision of this manuscript.

Author information

Correspondence to Amelia Farrés.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Peña-Montes, C., Lange, S., Flores, I. et al. Molecular characterization of StcI esterase from Aspergillus nidulans . Appl Microbiol Biotechnol 84, 917–926 (2009). https://doi.org/10.1007/s00253-009-2005-9

Download citation

Keywords

  • Aspergillus nidulans
  • Esterase
  • Cloning
  • Sterigmatocystin
  • N terminus