Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Aug;177(16):4792–4800. doi: 10.1128/jb.177.16.4792-4800.1995

Sterigmatocystin biosynthesis in Aspergillus nidulans requires a novel type I polyketide synthase.

J H Yu 1, T J Leonard 1
PMCID: PMC177246  PMID: 7642507

Abstract

A filamentous fungus, Aspergillus nidulans, produces the carcinogenic mycotoxin sterigmatocystin (ST), which is a polyketide-derived secondary metabolite. A gene (pksST) encoding the ST polyketide synthase (PKSst) in A. nidulans was cloned, sequenced, and characterized. Large induced deletion mutants, which did not make ST or any ST intermediates, were used to identify genes associated with ST biosynthesis. Among the transcripts detected within the deletion region, which showed developmental expression with ST production, was a 7.2-kb transcript. Functional inactivation of the gene encoding the 7.2-kb transcript blocked production of ST and all ST intermediate substrates but did not affect transcription of the pathway genes, indicating that this gene was involved in a very early step of ST biosynthesis. These results also indicate that PKSst was not associated with activation of other ST genes. Sequencing of the region spanning this gene revealed that it encoded a polypeptide with a deduced length of 2,181 amino acids that had high levels of similarity to many of the known polyketide synthases and FASs. This gene, pksST, encodes a multifunctional novel type I polyketide synthase which has as active sites a beta-ketoacyl acyl carrier protein synthase, an acyltransferase, duplicated acyl carrier proteins, and a thioesterase, all of these catalytic sites may be multiply used. In addition, a 1.9-kb transcript, which also showed developmental expression, was mapped adjacent to pksST, and the sequence of this gene revealed that it encoded a cytochrome P-450 monooxygenase-like peptide.

Full Text

The Full Text of this article is available as a PDF (795.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adamson R. H. Induction of hepatocellular carcinoma in nonhuman primates by chemical carcinogens. Cancer Detect Prev. 1989;14(2):215–219. [PubMed] [Google Scholar]
  2. Aguilar F., Hussain S. P., Cerutti P. Aflatoxin B1 induces the transversion of G-->T in codon 249 of the p53 tumor suppressor gene in human hepatocytes. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8586–8590. doi: 10.1073/pnas.90.18.8586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amy C. M., Witkowski A., Naggert J., Williams B., Randhawa Z., Smith S. Molecular cloning and sequencing of cDNAs encoding the entire rat fatty acid synthase. Proc Natl Acad Sci U S A. 1989 May;86(9):3114–3118. doi: 10.1073/pnas.86.9.3114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aramayo R., Adams T. H., Timberlake W. E. A large cluster of highly expressed genes is dispensable for growth and development in Aspergillus nidulans. Genetics. 1989 May;122(1):65–71. doi: 10.1093/genetics/122.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baertschi S. W., Raney K. D., Shimada T., Harris T. M., Guengerich F. P. Comparison of rates of enzymatic oxidation of aflatoxin B1, aflatoxin G1, and sterigmatocystin and activities of the epoxides in forming guanyl-N7 adducts and inducing different genetic responses. Chem Res Toxicol. 1989 Mar-Apr;2(2):114–112. doi: 10.1021/tx00008a008. [DOI] [PubMed] [Google Scholar]
  6. Beck J., Ripka S., Siegner A., Schiltz E., Schweizer E. The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases. Eur J Biochem. 1990 Sep 11;192(2):487–498. doi: 10.1111/j.1432-1033.1990.tb19252.x. [DOI] [PubMed] [Google Scholar]
  7. Bennett J. W., Christensen S. B. New perspectives on aflatoxin biosynthesis. Adv Appl Microbiol. 1983;29:53–92. doi: 10.1016/s0065-2164(08)70354-x. [DOI] [PubMed] [Google Scholar]
  8. Bhatnagar D., Ullah A. H., Cleveland T. E. Purification and characterization of a methyltransferase from Aspergillus parasiticus SRRC 163 involved in aflatoxin biosynthetic pathway. Prep Biochem. 1988;18(3):321–349. doi: 10.1080/00327488808062532. [DOI] [PubMed] [Google Scholar]
  9. Brody H., Griffith J., Cuticchia A. J., Arnold J., Timberlake W. E. Chromosome-specific recombinant DNA libraries from the fungus Aspergillus nidulans. Nucleic Acids Res. 1991 Jun 11;19(11):3105–3109. doi: 10.1093/nar/19.11.3105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brown D. W., Salvo J. J. Isolation and Characterization of Sexual Spore Pigments from Aspergillus nidulans. Appl Environ Microbiol. 1994 Mar;60(3):979–983. doi: 10.1128/aem.60.3.979-983.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chang P. K., Cary J. W., Bhatnagar D., Cleveland T. E., Bennett J. W., Linz J. E., Woloshuk C. P., Payne G. A. Cloning of the Aspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis. Appl Environ Microbiol. 1993 Oct;59(10):3273–3279. doi: 10.1128/aem.59.10.3273-3279.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cortes J., Haydock S. F., Roberts G. A., Bevitt D. J., Leadlay P. F. An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. Nature. 1990 Nov 8;348(6297):176–178. doi: 10.1038/348176a0. [DOI] [PubMed] [Google Scholar]
  14. Davis N. K., Chater K. F. Spore colour in Streptomyces coelicolor A3(2) involves the developmentally regulated synthesis of a compound biosynthetically related to polyketide antibiotics. Mol Microbiol. 1990 Oct;4(10):1679–1691. doi: 10.1111/j.1365-2958.1990.tb00545.x. [DOI] [PubMed] [Google Scholar]
  15. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Donadio S., Staver M. J., McAlpine J. B., Swanson S. J., Katz L. Modular organization of genes required for complex polyketide biosynthesis. Science. 1991 May 3;252(5006):675–679. doi: 10.1126/science.2024119. [DOI] [PubMed] [Google Scholar]
  17. Donkersloot J. A., Mateles R. I., Yang S. S. Isolation of averufin from a mutant of Aspergillus parasiticus impaired in aflatoxin biosynthesis. Biochem Biophys Res Commun. 1972 Jun 9;47(5):1051–1055. doi: 10.1016/0006-291x(72)90939-4. [DOI] [PubMed] [Google Scholar]
  18. Dutton M. F. Enzymes and aflatoxin biosynthesis. Microbiol Rev. 1988 Jun;52(2):274–295. doi: 10.1128/mr.52.2.274-295.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fujii K., Kurata H., Odashima S., Hatsuda Y. Tumor induction by a single subcutaneous injection of sterigmatocystin in newborn mice. Cancer Res. 1976 May;36(5):1615–1618. [PubMed] [Google Scholar]
  20. Gibson T. J., Coulson A. R., Sulston J. E., Little P. F. Lorist2, a cosmid with transcriptional terminators insulating vector genes from interference by promoters within the insert: effect on DNA yield and cloned insert frequency. Gene. 1987;53(2-3):275–281. doi: 10.1016/0378-1119(87)90016-3. [DOI] [PubMed] [Google Scholar]
  21. Gutiérrez S., Díez B., Montenegro E., Martín J. F. Characterization of the Cephalosporium acremonium pcbAB gene encoding alpha-aminoadipyl-cysteinyl-valine synthetase, a large multidomain peptide synthetase: linkage to the pcbC gene as a cluster of early cephalosporin biosynthetic genes and evidence of multiple functional domains. J Bacteriol. 1991 Apr;173(7):2354–2365. doi: 10.1128/jb.173.7.2354-2365.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Harker C. L., Ellis T. H., Coen E. S. Identification and genetic regulation of the chalcone synthase multigene family in pea. Plant Cell. 1990 Mar;2(3):185–194. doi: 10.1105/tpc.2.3.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hitokoto H., Morozumi S., Wauke T., Sakai S., Yoshikawa S. Chemically defined medium for high yields of sterigmatocystin. Mycopathologia. 1982 May 22;78(2):99–105. doi: 10.1007/BF00442633. [DOI] [PubMed] [Google Scholar]
  24. Hohn T. M., McCormick S. P., Desjardins A. E. Evidence for a gene cluster involving trichothecene-pathway biosynthetic genes in Fusarium sporotrichioides. Curr Genet. 1993 Oct;24(4):291–295. doi: 10.1007/BF00336778. [DOI] [PubMed] [Google Scholar]
  25. Holzer K. P., Liu W., Hammes G. G. Molecular cloning and sequencing of chicken liver fatty acid synthase cDNA. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4387–4391. doi: 10.1073/pnas.86.12.4387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hopwood D. A., Sherman D. H. Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Genet. 1990;24:37–66. doi: 10.1146/annurev.ge.24.120190.000345. [DOI] [PubMed] [Google Scholar]
  27. Jelinek C. F., Pohland A. E., Wood G. E. Worldwide occurrence of mycotoxins in foods and feeds--an update. J Assoc Off Anal Chem. 1989 Mar-Apr;72(2):223–230. [PubMed] [Google Scholar]
  28. Katz L., Donadio S. Polyketide synthesis: prospects for hybrid antibiotics. Annu Rev Microbiol. 1993;47:875–912. doi: 10.1146/annurev.mi.47.100193.004303. [DOI] [PubMed] [Google Scholar]
  29. Keller N. P., Dischinger H. C., Jr, Bhatnagar D., Cleveland T. E., Ullah A. H. Purification of a 40-kilodalton methyltransferase active in the aflatoxin biosynthetic pathway. Appl Environ Microbiol. 1993 Feb;59(2):479–484. doi: 10.1128/aem.59.2.479-484.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Keller N. P., Kantz N. J., Adams T. H. Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin. Appl Environ Microbiol. 1994 May;60(5):1444–1450. doi: 10.1128/aem.60.5.1444-1450.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kimura N., Tsuge T. Gene cluster involved in melanin biosynthesis of the filamentous fungus Alternaria alternata. J Bacteriol. 1993 Jul;175(14):4427–4435. doi: 10.1128/jb.175.14.4427-4435.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. MacCabe A. P., van Liempt H., Palissa H., Unkles S. E., Riach M. B., Pfeifer E., von Döhren H., Kinghorn J. R. Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. Molecular characterization of the acvA gene encoding the first enzyme of the penicillin biosynthetic pathway. J Biol Chem. 1991 Jul 5;266(19):12646–12654. [PubMed] [Google Scholar]
  33. Maloney A. P., VanEtten H. D. A gene from the fungal plant pathogen Nectria haematococca that encodes the phytoalexin-detoxifying enzyme pisatin demethylase defines a new cytochrome P450 family. Mol Gen Genet. 1994 Jun 3;243(5):506–514. doi: 10.1007/BF00284198. [DOI] [PubMed] [Google Scholar]
  34. Marsden A. F., Caffrey P., Aparicio J. F., Loughran M. S., Staunton J., Leadlay P. F. Stereospecific acyl transfers on the erythromycin-producing polyketide synthase. Science. 1994 Jan 21;263(5145):378–380. doi: 10.1126/science.8278811. [DOI] [PubMed] [Google Scholar]
  35. Mathur M., Kolattukudy P. E. Molecular cloning and sequencing of the gene for mycocerosic acid synthase, a novel fatty acid elongating multifunctional enzyme, from Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guerin. J Biol Chem. 1992 Sep 25;267(27):19388–19395. [PubMed] [Google Scholar]
  36. Matsushima K., Ando Y., Hamasaki T., Yabe K. Purification and characterization of two versiconal hemiacetal acetate reductases involved in aflatoxin biosynthesis. Appl Environ Microbiol. 1994 Jul;60(7):2561–2567. doi: 10.1128/aem.60.7.2561-2567.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mayorga M. E., Timberlake W. E. The developmentally regulated Aspergillus nidulans wA gene encodes a polypeptide homologous to polyketide and fatty acid synthases. Mol Gen Genet. 1992 Nov;235(2-3):205–212. doi: 10.1007/BF00279362. [DOI] [PubMed] [Google Scholar]
  38. McDaniel R., Ebert-Khosla S., Hopwood D. A., Khosla C. Engineered biosynthesis of novel polyketides. Science. 1993 Dec 3;262(5139):1546–1550. doi: 10.1126/science.8248802. [DOI] [PubMed] [Google Scholar]
  39. Miller B. L., Miller K. Y., Timberlake W. E. Direct and indirect gene replacements in Aspergillus nidulans. Mol Cell Biol. 1985 Jul;5(7):1714–1721. doi: 10.1128/mcb.5.7.1714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Muench K. F., Misra R. P., Humayun M. Z. Sequence specificity in aflatoxin B1--DNA interactions. Proc Natl Acad Sci U S A. 1983 Jan;80(1):6–10. doi: 10.1073/pnas.80.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Northolt M. D., van Egmond H. P., Soentoro P., Deijll E. Fungal growth and the presence of sterigmatocystin in hard cheese. J Assoc Off Anal Chem. 1980 Jan;63(1):115–119. [PubMed] [Google Scholar]
  42. O'Hara E. B., Timberlake W. E. Molecular characterization of the Aspergillus nidulans yA locus. Genetics. 1989 Feb;121(2):249–254. doi: 10.1093/genetics/121.2.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ong T. M., De Serres F. J. Mutagenicity of chemical carcinogens in Neurospora crassa. Cancer Res. 1972 Sep;32(9):1890–1893. [PubMed] [Google Scholar]
  44. Ong T. M., Serres F. J. Mutation induction by difunctional alkylating agents in Neurospora crassa. Genetics. 1975 Jul;(3):475–482. doi: 10.1093/genetics/80.3.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Payne G. A., Nystrom G. J., Bhatnagar D., Cleveland T. E., Woloshuk C. P. Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus. Appl Environ Microbiol. 1993 Jan;59(1):156–162. doi: 10.1128/aem.59.1.156-162.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Scotti C., Piatti M., Cuzzoni A., Perani P., Tognoni A., Grandi G., Galizzi A., Albertini A. M. A Bacillus subtilis large ORF coding for a polypeptide highly similar to polyketide synthases. Gene. 1993 Aug 16;130(1):65–71. doi: 10.1016/0378-1119(93)90347-6. [DOI] [PubMed] [Google Scholar]
  48. Shen B., Hutchinson C. R. Enzymatic synthesis of a bacterial polyketide from acetyl and malonyl coenzyme A. Science. 1993 Dec 3;262(5139):1535–1540. doi: 10.1126/science.8248801. [DOI] [PubMed] [Google Scholar]
  49. Shimada T., Guengerich F. P. Evidence for cytochrome P-450NF, the nifedipine oxidase, being the principal enzyme involved in the bioactivation of aflatoxins in human liver. Proc Natl Acad Sci U S A. 1989 Jan;86(2):462–465. doi: 10.1073/pnas.86.2.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Skory C. D., Chang P. K., Cary J., Linz J. E. Isolation and characterization of a gene from Aspergillus parasiticus associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis. Appl Environ Microbiol. 1992 Nov;58(11):3527–3537. doi: 10.1128/aem.58.11.3527-3537.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Skory C. D., Chang P. K., Linz J. E. Regulated expression of the nor-1 and ver-1 genes associated with aflatoxin biosynthesis. Appl Environ Microbiol. 1993 May;59(5):1642–1646. doi: 10.1128/aem.59.5.1642-1646.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Smith D. J., Burnham M. K., Bull J. H., Hodgson J. E., Ward J. M., Browne P., Brown J., Barton B., Earl A. J., Turner G. Beta-lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes. EMBO J. 1990 Mar;9(3):741–747. doi: 10.1002/j.1460-2075.1990.tb08168.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stack M., Rodricks J. V. Method for analysis and chemical confirmation of sterigmatocystin. J Assoc Off Anal Chem. 1971 Jan;54(1):86–90. [PubMed] [Google Scholar]
  54. Summers R. G., Wendt-Pienkowski E., Motamedi H., Hutchinson C. R. Nucleotide sequence of the tcmII-tcmIV region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase. J Bacteriol. 1992 Mar;174(6):1810–1820. doi: 10.1128/jb.174.6.1810-1820.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tai M. H., Chirala S. S., Wakil S. J. Roles of Ser101, Asp236, and His237 in catalysis of thioesterase II and of the C-terminal region of the enzyme in its interaction with fatty acid synthase. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1852–1856. doi: 10.1073/pnas.90.5.1852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tang C. M., Cohen J., Holden D. W. An Aspergillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity. Mol Microbiol. 1992 Jun;6(12):1663–1671. doi: 10.1111/j.1365-2958.1992.tb00891.x. [DOI] [PubMed] [Google Scholar]
  57. Timberlake W. E. Molecular genetics of Aspergillus development. Annu Rev Genet. 1990;24:5–36. doi: 10.1146/annurev.ge.24.120190.000253. [DOI] [PubMed] [Google Scholar]
  58. Yabe K., Hamasaki T. Stereochemistry during aflatoxin biosynthesis: cyclase reaction in the conversion of versiconal to versicolorin B and racemization of versiconal hemiacetal acetate. Appl Environ Microbiol. 1993 Aug;59(8):2493–2500. doi: 10.1128/aem.59.8.2493-2500.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yager L. N., Kurtz M. B., Champe S. P. Temperature-shift analysis of conidial development in Aspergillus nidulans. Dev Biol. 1982 Sep;93(1):92–103. doi: 10.1016/0012-1606(82)90242-1. [DOI] [PubMed] [Google Scholar]
  60. Yu J., Cary J. W., Bhatnagar D., Cleveland T. E., Keller N. P., Chu F. S. Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis. Appl Environ Microbiol. 1993 Nov;59(11):3564–3571. doi: 10.1128/aem.59.11.3564-3571.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES