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Secretion of calf chymosin from the filamentous fungus Aspergillus oryzae

  • Applied Genetics and Regulation
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Abstract

Active calf chymosin was secreted from Aspergillus oryzae transformants when the chymosin cDNA was expressed under the control of glucoamylase gene (glaA) promoter. Secreted prochymosin was autocatalytically activated to the chymosin (0.07– 0.16 mg/l). Western blot analysis showed that a secreted protein immunoreactive with an anti-chymosin antibody was of similar size to authentic chymosin. Northern blot analysis revealed that mRNA of the chymosin cDNA was expressed at as high level as that of the glaA gene. The size and the level of the transcript were different among transformants, due to the intergration position of the plasmid on the chromosome.

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References

  • Barbesgaard P, Heldt-Hansen HP, Diderichsen B (1992) On the safety of Aspergillus oryzae: a review. Appl Microbiol Biotechnol 36:569–572

    Google Scholar 

  • Berg JA van der, Lanken KJ van der, Ooyen AJJ van, Renniers TCHM, Rietveld K, Schaap A, Brake AJ, Bishop RJ, Schultz K, Moyer D, Richman M, Shuster JR (1990) Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin. Bio/Technology 8:135–139

    Google Scholar 

  • Cathala G, Savouret JF, Mendez B, West BL, Karin M, Martial JA, Baxter JD (1983) A method for isolation of intact, translationally active ribonucleic acid. DNA 2:329–335

    Google Scholar 

  • Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299

    CAS  PubMed  Google Scholar 

  • Christensen T, Woeldike H, Boel E, Mortensen SB, Hjortshoej K, Thim L, Hansen MT (1988) High level expression of recombinant genes in Aspergillus oryzae. Bio/Technology 6:1419–1422

    CAS  Google Scholar 

  • Cullen D, Gray GL, Wilson LJ, Hayenga KJ, Lamsa MH, Rey MW, Norton S, Berka RM (1987) Controlled expression and secretion of bovine chymosin in Aspergillus nidulans. Bio/Technology 5:369–376

    Google Scholar 

  • Dunn-Coleman NS, Bloebaum P, Berka RM, Bodie E, Robinson N, Armstrong G, Ward M, Pretak M, Carter GL, LaCost R, Wilson LJ, Kodama KH, Balin EF, Bower B, Lamsa M, Heinsohn H (1991) Commercial levels of chymosin production by Aspergillus. Bio/Technology 9:976–981

    Google Scholar 

  • Emtage JS, Angal S, Doel MT, Harris TJR, Jenkins B, Lilley G, Lowe PA (1983) Synthesis of calf prochymosin (prorennin) in Escherichia coli. Proc Natl Acad Sci USA 80:3671–3675

    Google Scholar 

  • Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragment to high specific activity. Anal Biochem 132:6–13

    CAS  PubMed  Google Scholar 

  • Fincham JRS (1989) Transformation in fungi. Microbiol Rev 53:148–170

    Google Scholar 

  • Foltmann B (1970) Prochymosin and chymosin (prorennin and rennin). Methods Enzymol 19:421–436

    Google Scholar 

  • Goff CG, Moir DT, Kohno T, Gravius TC, Smith RA, Yamasaki E, Taunton-Rigby A (1984) Expression of calf prochymosin in Sacchatromyces cerevisiae. Gene 27:35–46

    Google Scholar 

  • Gomi K, Iimura Y, Hara S (1987) Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric Biol Chem 51:2549–2555

    Google Scholar 

  • Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

    CAS  PubMed  Google Scholar 

  • Harkki A, Uusitalo J, Bailey M, Penttila M, Knowles JKC (1989) A novel fungal expression system: secretion of active calf chymosin from the filamentous fungus Trichoderma reesei. Bio/Technology 7:596–603

    Google Scholar 

  • Hata Y, Kitamoto K, Gomi K, Kumagai C, Tamura G (1992) Functional elements of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase. Curr Genet 22:85–91

    CAS  PubMed  Google Scholar 

  • Hondel CAMJJ van den, Punt PJ, Gorcom RFM van (1991) Heterologous gene expression in filamentous fungi. In: Bennett JW, Lasure LL (eds) More gene manipulations in fungi. Academic Press, New York, pp 396–428

    Google Scholar 

  • Huge-Jensen B, Andreasen F, Christensen T, Christensen M, Thim L, Boel E (1989) Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae. Lipids 24:781–785

    CAS  PubMed  Google Scholar 

  • Jeenes DJ, Mackenzie DA, Roberts IN, Archer DB (1991) Heterologous protein production by filamentous fungi. In: Tombs MP (ed) Biotechnology and genetic engineering reviews, vol 9. Intercept, UK, pp 327–367

    Google Scholar 

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

    PubMed  Google Scholar 

  • Marston FAO, Lowe PA, Doel MT, Schoemaker JM (1984) Purification of calf prochymosin (prorennin) synthesized in Escherichia coli. Bio/Technology 2:800–804

    Google Scholar 

  • Mellor J, Dobson MJ, Roberts NA, Tuite MF, Emtage JS, White S, Lowe PA, Patel T, Kingsman AJ, Kingsman SM (1983) Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene 24:1–14

    Google Scholar 

  • Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78

    Article  CAS  PubMed  Google Scholar 

  • Moir D, Mao J, Schumm JW, Vovis GF, Alford BL, Taunton-Rigby A (1982) Molecular cloning and characterization of doube-stranded cDNA coding for bovine chymosin. Gene 19:127–138

    Google Scholar 

  • Nishimori K, Kawaguchi Y, Hidaka M, Uozumi T, Beppu T (1982) Expression of cloned calf prochymosin gene sequence in Escherichia coli. Gene 19:337–344

    Google Scholar 

  • Sambrook J, Fritsh EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.

    Google Scholar 

  • Saunders G, Picknett TM, Tuite MF, Ward M (1989) Heterologous gene expression in filamentous fungi. Trends Biotechnol 7:283–287

    Google Scholar 

  • Shibuya I, Tsuchiya K, Tamura G, Ishikawa T, Hara S (1992) Overproduction of an α-amylase/glucoamylase fusion protein in Aspergillus oryzae using a high expression vector. Biosci Biotechnol Biochem 56:1674–1675

    Google Scholar 

  • Tada S, Iimura Y, Gomi K, Takahashi K, Hara S, Yoshizawa K (1989) Cloning and nucleotide sequence of the genomic Taka-amylase A gene of Aspergillus oryzae. Agric Biol Chem 53:593–599

    Google Scholar 

  • Tsuchiya K, Tada S, Gomi K, Kitamoto K, Jigami Y, Tamura G (1992) High level expression of the synthetic human lysozyme gene in Aspergillus oryzae. Appl Microbiol Biotechnol 38:109–114

    Google Scholar 

  • Ward M (1989) Production of calf chymosin by Aspergillus awamori. In: Hershbrger CL, Queener SW, Hegeman G (eds) Genetics and molecular biology of industrial microorganisms. American Society for Microbiology, Washington DC, pp 288–294

    Google Scholar 

  • Ward PP, Lo J, Duke M, May GS, Headon DR, Conneely OM (1992) Production of biologically active recombinant human lactoferrin in Aspergillus oryzae. Bio/Technology 10:784–789

    Google Scholar 

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Correspondence to: K. Kitamoto

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Tsuchiya, K., Gomi, K., Kitamoto, K. et al. Secretion of calf chymosin from the filamentous fungus Aspergillus oryzae . Appl Microbiol Biotechnol 40, 327–332 (1993). https://doi.org/10.1007/BF00170388

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  • DOI: https://doi.org/10.1007/BF00170388

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