Molecular and General Genetics MGG

, Volume 248, Issue 3, pp 270–277 | Cite as

TheAspergillus parasiticus polyketide synthase genepksA, a homolog ofAspergillus nidulans wA, is required for aflatoxin B1 biosynthesis

  • Perng-Kuang Chang
  • Jeffrey W. Cary
  • Jiujiang Yu
  • Deepak Bhatnagar
  • Thomas E. Cleveland
Original Paper


Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced byAspergillus parasiticus andAspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway inA. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipientA. parasiticus genome at a specific locus, designatedpksA. Sequence analysis suggest thatpksA is a homolog of theAspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conservedβ-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of thepksA product. Noβ-ketoacyl reductase and enoyl reductase domains were found, suggesting thatpksA does not encode catalytic activities for processingβ-carbon similar to those required for long chain fatty acid synthesis. ThepksA gene is located in the aflatoxin pathway gene cluster and is linked to thenor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose thatpksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.

Key words

Polyketide synthase gene Aflatoxin Aspergillus parasiticus Gene cluster Anthraquinone 


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  1. Adye, J, Mateles RI (1964) Incorporation of labelled compounds into aflatoxin. Biochim Biophys Acta 86:418–420PubMedGoogle Scholar
  2. Aguilar F, Harris CC, Sun T, Hollstein M, Cerutti P (1994) Geographic variation of p53 mutational profile in nonmalignant human liver. Science 264:1317–1319PubMedGoogle Scholar
  3. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  4. Amy CM, Witkowski A, Naggert J, Williams B, Randhawa Z, Smith S (1989) Molecular cloning and sequencing of cDNAs encoding the entire rat fatty acid synthase. Proc Natl Acad Sci USA 86:3114–3118PubMedGoogle Scholar
  5. Aramayo R, Timberlake WE (1990) Sequence and molecular structure of theAspergillus nidulans yA (laccase I) gene. Nucleic Acids Res 18: 3415PubMedGoogle Scholar
  6. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1987) Current protocols in molecular biology, vol 1. John Wiley and Sons, New YorkGoogle Scholar
  7. Beck J, Ripka S, Siegner A, Schiltz E, Schweizer E (1990) The multifunctional 6-methylsalicylic acid synthase gene ofPenicillium patulum. Eur J Biochem 192:487–498PubMedGoogle Scholar
  8. Bhatnagar D, Cleveland TE, Kingston DGI (1991) Enzymological evidence for separate pathways for aflatoxin B1 and B2 biosynthesis. Biochemistry 30:4343–4350PubMedGoogle Scholar
  9. Bhatnagar D, Ehrlich KC, Cleveland TE (1992) Oxidation-reduction reactions in biosynthesis of secondary metabolites. In: Bhatnagar D, Lillehoj EB, Arora DK (eds) Handbook of applied mycology, vol 5. Mycotoxins in ecological systems. Marcel Dekker, New York pp 255–286Google Scholar
  10. Bhatnagar D, McCormick SP, Lee LS, Hill RA (1987) Identification ofO-methylsterigmatocystin as an aflatoxin B1 and G1 precursor inAspergillus parasiticus. Appl Environ Microbiol 53:1028–1033PubMedGoogle Scholar
  11. Bressac B, Kew M, Wands J, Ozturk M (1991) Selective G to T mutations of p53 gene in hepatocellular carcinoma from South Africa. Nature 350:429–431PubMedGoogle Scholar
  12. Brobst SW, Townsend CA (1994) The potential role of fatty acid initiation in the biosynthesis of the fungal aromatic polyketide aflatoxin B1. Can J Chem 72:200–207Google Scholar
  13. Chang P-K, Skory CD, Linz JE (1992) Cloning of a gene associated with aflatoxin B1 biosynthesis inAspergillus parasiticus. Curr Genet 21:231–233PubMedGoogle Scholar
  14. Chang P-K, Bhatnagar D, Cleveland TE (1993a) Disruption of the aflatoxin biosynthetic pathway through genetic complementation inAspergillus parasiticus. FASEB J 7: A1229Google Scholar
  15. Chang P-K, Cary JW, Bhatnagar D, Cleveland TE, Bennett JW, Linz JE, Woloshuk CP, Payne GA (1993b) Cloning of theAspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis. Appl Environ Microbiol 59: 3273–3279PubMedGoogle Scholar
  16. Chirala SS, Kasturi R, Pazirandeh M, Stolow DT, Huang W-Y, Wakil SJ (1989) A novel cDNA extension procedure. J Biol Chem 264:3750–3757PubMedGoogle Scholar
  17. Cleveland TE, Bhatnagar D, Brown RL (1991) Aflatoxin production via cross-feeding of pathway intermediates during cofermentation of aflatoxin pathway-blockedAspergillus parasiticus mutants. Appl Environ Microbiol 57:2907–2911PubMedGoogle Scholar
  18. Davis ND, Lyer SK, Diener UL (1987) Improved method of screening for aflatoxin with a coconut agar medium. Appl Environ Microbiol 53:1593–1595PubMedGoogle Scholar
  19. Donadio S, Staver MJ, McAlpine JB, Swanson S, Katz L (1991) Modular organization of genes required for complex polyketide biosynthesis. Science 252:675–679PubMedGoogle Scholar
  20. Dutton MF (1988) Enzymes and aflatoxin biosynthesis. Microbiol Rev 53:274–295Google Scholar
  21. Eaton DL, Groopman JD (1994) The toxicology of aflatoxins. Academic Press, San DiegoGoogle Scholar
  22. Gish W, States DJ (1993) Identification of protein coding regions by database similarity search. Nature Genet 3:266–272PubMedGoogle Scholar
  23. Heathcote JG, Hibbert JR (1978) Aflatoxins: chemical and biological aspects. Elsevier-Scientific, AmsterdamGoogle Scholar
  24. Hopwood DA, Sherman DH (1990) Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Genet 24: 37–66PubMedGoogle Scholar
  25. Horng JS, Chang P-K, Pestka JJ, Linz JE (1990) Development of a homologous transformation system forAspergillus parasiticus with the gene encoding nitrate reductase. Mol Gen Genet 224:294–296PubMedGoogle Scholar
  26. Hutchinson CR, Decker H, Motamedi H, Shen B, Summers RG (1993) Molecular genetics and biochemistry of a bacterial type II polyketide synthase. In: Baltz RH, Ingolia T, Hageman G (eds) Genetics and molecular biology of industrial microorganisms. American Society for Microbiology, Washington DC, pp 203–216Google Scholar
  27. Jelinek CF, Pohland AE, Wood GE (1989) Worldwide occurrence of mycotoxins in foods and feeds—an update. J Assoc Off Anal Chem 72:223–230PubMedGoogle Scholar
  28. Katz L, Donadio S (1993) Polyketide synthesis: prospects for hybrid antibiotics. Annu Rev Microbiol 47:875–912PubMedGoogle Scholar
  29. Keller NP, Dischinger HC, Bhatnagar D, Cleveland TE, Ullah AHL (1993) Purification of a 40-kilodalton methyltransferase active in the aflatoxin biosynthetic pathway. Appl Environ Microbiol 59:479–484PubMedGoogle Scholar
  30. Keller NP, Kantz NJ, Adams TH (1994)Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin. Appl Environ Microbiol 60:1444–1450PubMedGoogle Scholar
  31. Kottig H, Rottner G, Beck K-F, Schweizer M, Schweizer E (1991) The pentafunctionalFASI genes ofSaccharomyces cerevisiae andYarrowia lipolytica are co-linear and considerably longer than previously estimated. Mol Gen Genet 226: 310–314PubMedGoogle Scholar
  32. Lin BK, Anderson JA (1992) Purification and properties of versicolonal cyclase fromAspergillus parasiticus. Arch Biochem Biophy 293:67–70Google Scholar
  33. Mahanti N, Bhatnagar D, Cary J, Linz JE (1995) Structure and function ofuvm8, a gene involved in polyketide backbone synthesis in the aflatoxin pathway inAspergillus parasiticus. Appl Environ Microbiol, in pressGoogle Scholar
  34. Mathur M, Kolattukudy PE (1992) Molecular cloning and sequencing of the gene for mycocerosic acid synthase, a novel fatty acid elongation multifunctional enzyme, fromMycobacterium tuberculosis var.bovis Bacillus Calmette-Guerin. J Biol Chem 267:19388–19395PubMedGoogle Scholar
  35. Mayorga ME, Timberlake WE (1992) The developmentally regulatedAspergillus nidulans wA gene encodes a polypeptide homologous to polyketide and fatty acid synthases. Mol Gen Genet 235:205–212PubMedGoogle Scholar
  36. Payne GA, Nystorm GJ, Bhatnagar D, Cleveland TE, Woloshuk CP (1993) Cloning of theafl-2 gene involved in aflatoxin biosynthesis fromAspergillus flavus. Appl Environ Microbiol 59:156–162PubMedGoogle Scholar
  37. Pontecorvo G, Roper JA, Hemmons LM, MacDonald KD, Bufton WJ (1953) The genetics ofAspergillus nidulans. Adv Genet 5:141–238PubMedGoogle Scholar
  38. Scotti C, Piatti M, Cuzzoni A, Perani P, Tognoni A, Grandi G, Galizzi A, Albertine AM (1993) ABacillus subtilis ORF coding for a polypeptide highly similar to polyketide synthases. Gene 130:65–71PubMedGoogle Scholar
  39. Skory CD, Chang P-K, Cary J, Linz JE (1992) Isolation and characterization of a gene fromAspergillus parasiticus associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis. Appl Environ Microbiol 58:3527–3537PubMedGoogle Scholar
  40. Townsend CA, Christensen SB (1983) Stable isotope studies of anthraquinone intermediates in the aflatoxin pathway. Tetrahedron 39:3575–3582Google Scholar
  41. Trail F, Chang P-K, Cary J, Linz JE (1994) Structural and functional analysis of thenor-1 gene involved in the biosynthesis of aflatoxins byAspergillus parasiticus. Appl Environ Microbiol 60:4078–4085PubMedGoogle Scholar
  42. Trail F, Mahanti N, Mehigh R, Liang S-H, Zhou R, Rarich M, Linz JE (1995) A physical and transcriptional map of the aflatoxin gene cluster inAspergillus parasiticus and the functional disruption of a gene involved early in the aflatoxin pathway. Appl Environ Microbiol, in pressGoogle Scholar
  43. Vidal-Gros A, Viviani F, Labesse G, Boccara M, Gaudry M (1994) Polyhydroxynaphthalene reductase involved in melanin biosynthesis inMagnaporthe grisea. Eur J Biochem 219:985–992PubMedGoogle Scholar
  44. Wakil AJ (1989) Fatty acid synthase, a proficient multifunctional enzyme. Biochemistry 28:4523–4530PubMedGoogle Scholar
  45. Wang IK, Reeves C, Gaucher GM (1991) Isolation and sequencing of a genomic DNA clone containing the 3′ terminus of the 6-methylsalicylic acid polyketide synthetase gene ofPenicillium urticae. Can J Microbiol 37: 86–95PubMedGoogle Scholar
  46. Yabe K, Ando Y, Hashimoto J, Hamasaki T (1989) Two distinct0-methyltransferases in aflatoxin biosynthesis. Appl Environ Microbiol 55:2172–2177PubMedGoogle Scholar
  47. Yu J, Cary JW, Bhatnagar D, Cleveland TE, Keller NP, Chu FS (1992) Molecular cloning and characterization of anAspergillus parasiticus gene involved in the conversion of sterigmatocystin to0-methylsterigmatocystin in aflatoxin biosynthesis. Appl Environ Microbiol 59:3564–3571Google Scholar
  48. Yu J, Chang P-K, Cary JW, Wright M, Bhatnagar D, Cleveland TE, Payne GA, Linz JE (1995) Comparative mapping of aflatoxin pathway gene clusters inAspergillus parasiticus andAspergillus flavus. Appl Environ Microbiol, 61:2365–2371PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Perng-Kuang Chang
    • 1
  • Jeffrey W. Cary
    • 2
  • Jiujiang Yu
    • 2
  • Deepak Bhatnagar
    • 2
  • Thomas E. Cleveland
    • 2
  1. 1.Department of Cell and Molecular BiologyTulane UniversityNew OrleansUSA
  2. 2.Southern Regional Research Center, Agricultural Research ServiceU.S. Department of AgricultureNew OrleansUSA

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