Current Genetics

, Volume 17, Issue 3, pp 203–212 | Cite as

Cloning, characterization, and expression of two α-amylase genes from Aspergillus niger var. awamori

  • David R. Korman
  • Frank T. Bayliss
  • Christopher C. Barnett
  • Cynthia L. Carmona
  • Katherine H. Kodama
  • Theresa J. Royer
  • Sheryl A. Thompson
  • Michael Ward
  • Lori J. Wilson
  • Randy M. Berka
Original Articles

Summary

Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an α-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two α-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (≥98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.

Key words

α-amylase Filamentous fungi Gene duplication 

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References

  1. Adelman JP, Hayflick JS, Vasser M, Seeburg PH (1983) DNA 2:183–193Google Scholar
  2. Bennett JW (1985) Molds, manufacturing and molecular genetics. In: Timberlake WE (ed) Molecular genetics of filamentous fungi. Alan Liss, New York, pp 345–366Google Scholar
  3. Berka RM, Ward M, Wilson LJ, Hayenga KJ, Kodama KH, Carlomagno LP, Thompson SA (1990) Gene 86:153–162Google Scholar
  4. Bhella RS, Altosaar I (1984) Can J Microbiol 31:149–153Google Scholar
  5. Bhella RS, Altosaar I (1987) Biotechnol Appl Biochem 9:287–293Google Scholar
  6. Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL (1977) Gene 2:95–113Google Scholar
  7. Christensen T, Woeldike H, Boel E, Mortensen SB, Hjortshoej K, Thim L, Hansen MT (1988) Bio/Technol 6:1419–1422Google Scholar
  8. Crea R, Kraszewski A, Hirose T, Itakura K (1978) Proc Natl Acad Sci USA 75:5765–5769Google Scholar
  9. Cullen D, Gray GL, Wilson LJ, Hayenga KJ, Lamsa MH, Rey MW, Norton S, Berka RM (1987) Bio/Technol 5:369–376Google Scholar
  10. Davis RW, Botstein D, Roth JR (1980) Advanced bacterial genetics, a manual for genetic engineering. Cold Spring Harbor Press, Cold Spring Harbor, New YorkGoogle Scholar
  11. Erratt JA, Douglas PE, Maranelli F, Seligy VL (1984) Can J Biochem 62:678–690Google Scholar
  12. Gines MJ, Dove MJ, Seligy VL (1989) Gene 79:107–117Google Scholar
  13. Gurr SJ, Unkles SE, Kinghorn JR (1987) The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn JR (ed) Gene structure in eukaryotic microbes. IRL Press, Oxford, pp 93–139Google Scholar
  14. Kinnaird JM, Keighren MA, Kinsey JA, Eaton M, Fincham JRS (1982) Gene 20:387–396Google Scholar
  15. Lamsa M, Bloebaum P (1989) J Industr Microbiol (in press)Google Scholar
  16. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, New YorkGoogle Scholar
  17. Matsuura Y, Kusunoki M, Harada W, Kakudo M (1984) J Biochem 95:697–702Google Scholar
  18. Meade JH, White TJ, Shoemaker SP, Gelfand DH, Chang S, Innis MA (1987) Molecular cloning of carbohydrases for the food industry. In: Knoor D (ed) Food biotechnology. Marcel Dekker, New York, pp 393–411Google Scholar
  19. Messing J, Crea, Seeburg PH (1981) Nucleic Acids Res 9:309–321Google Scholar
  20. Mullaney EJ, Hamer JE, Roberti KA, Yelton MM, Timberlake WE (1985) Mol Gen Genet 199:37–45Google Scholar
  21. Nunberg, JH, Meade JH, Cole G, Lawyer FC, McCabe P, Schweickart V, Tal R, Whitman VP, Flatgaard JE, Innis MA (1984) Mol Cell Biol 4:2306–2315Google Scholar
  22. Pool EL, Underkofler LA (1953) J Agric Food Chem 1:87–90Google Scholar
  23. Raper KB, Fennell DI (1977) The genus Aspergillus. Robert E Krieger Publishing, Huntington, New YorkGoogle Scholar
  24. Sanger F, Nicklen S, Coulson AR (1977) Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  25. Shoemaker S, Schweickert V, Ladner M, Gelfand D, Kwok S, Myambo K, Innis MA (1983) Bio/Technol 1:691–696Google Scholar
  26. Tada S, Iimura Y, Gomi K, Takahashi K, Hara S, Yoshizawa K (1989) Agric Biol Chem 53:593–599Google Scholar
  27. Takagi, T, Toda H, Isemura T (1971) Bacterial and mold enzymes. In: Boyer PD (ed) The enzymes, vol 5. Academic Press, New York, pp 235–271Google Scholar
  28. Timberlake WE, Barnard EC (1981) Cell 26:29–37Google Scholar
  29. Toda H, Kondo K, Narita K (1982) Proc Jpn Acad Ser B Phys Bil Sci 58:208–212Google Scholar
  30. Vieira J, Messing J (1982) Gene 19:259–268Google Scholar
  31. Ward M, Kodama KH, Wilson LJ (1989) Exp Mycol 13:289–293Google Scholar
  32. Wilson EM, Franke CA, Black ME, Hruby DE (1989) Gene 77:69–78Google Scholar
  33. Wilson LJ, Carmona CL, Ward M (1988) Nucleic Acids Res 16:2339Google Scholar
  34. Wirsel S, Lachmund A, Wildhardt G, Ruttkowski E (1989) Mol Microbiol 3:3–14Google Scholar
  35. Yabuki M, Ono N, Hoshino K, Fukui S (1977) Appl Environ Microbiol 34:1–6Google Scholar
  36. Yanisch-Perron C, Vieira J, Messing J (1985) Gene 33:103–119Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • David R. Korman
    • 1
    • 2
  • Frank T. Bayliss
    • 1
    • 2
  • Christopher C. Barnett
    • 1
  • Cynthia L. Carmona
    • 1
  • Katherine H. Kodama
    • 1
  • Theresa J. Royer
    • 1
  • Sheryl A. Thompson
    • 1
  • Michael Ward
    • 1
  • Lori J. Wilson
    • 1
  • Randy M. Berka
    • 1
  1. 1.Genencor, Inc.South San FranciscoUSA
  2. 2.San Francisco State UniversitySan FranciscoUSA

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