Glucoamylase overexpression inAspergillus niger: Molecular genetic analysis of strains containing multiple copies of theglaA gene
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A strategy, based on the usage of theamdS selection marker and a cosmid vector containing four copies of the glucoamylase gene (glaA), was developed to obtain glucoamylase (GLA)-overproducingA. niger strains. With this strategy, fungal strains carrying up to 200 copies of theglaA gene could be isolated at a relatively high frequency. In each transformant analysed, integration occurred in a single chromosome. A significant increase in the extracellular GLA production was observed in most of the transformants carrying multiple copies of theglaA gene. Further analysis showed that the amount of GLA that is produced was not proportional to the number ofglaA copies in these transformants. However, the level of GLA production clearly correlated with the amount ofglaA mRNA produced in these transformants. From these results it is concluded that GLA production is limited at the level of transcription.
Keywordstranscription regulation regulatory protein amdS selection marker cosmid vector CHEF analysis
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- Bennett, J.W. and Lasure, L.L. (1991) Growth media. In Bennett, J.W. and Lasure, L.L. eds,More Gene Manipulations in Fungi, pp. 444–5, San Diego: Academic Press, Inc.Google Scholar
- Bos, C.J. (1986) Induced mutation and somatic recombination as tools for genetic analysis and breeding of imperfect fungi. Thesis, Agricultural University, Wageningen, the Netherlands.Google Scholar
- Cami, B. and Kourilsky, P. (1978) Screening of cloned recombinant DNA in bacteria byin situ colony hybridization.Nucl. Acid Res. 5, 2381.Google Scholar
- Durand, N., Reymond, P. and Fèvre, M. (1992) Transformation and modification of transformation markers during an induced parasexual cycle inPenicillium roqueforti.Curr. Genet. 21, 377–83.Google Scholar
- Gwynne, D.O., Buxton, F.P., Gleeson, M.A., and Davies, R.W. (1987) Genetically engineered secretion of foreign proteins fromAspergillus species. In Burgess, R., ed.,Protein Purification: Micro to Macro, pp. 355–65. New York: Alan R. LissGoogle Scholar
- O'Connel, M.J., Dowzer, C.E.A. and Kelly, J.M. (1990) The ribosomal repeat ofAspergillus niger and its effect on transformation frequency.Fung. Genet. Newslett. 37, 29–30.Google Scholar
- Punt, P.J., Zegers, N.D., Busscher, M., Pouwels, P.H. and van den Hondel, C.A.M.J.J. (1990) Intracellular and extracellular production of proteins inAspergillus under control of expression signals of the highly expressedAspergillus nidulans gpdA gene.J. Biotech. 17, 19–34.Google Scholar
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989)Molecular Cloning: a Laboratory Manual 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
- Van Hartingsveldt, W., van Zeijl, C.M.J., Veenstra, A.E., van den Berg, J.A., Pouwels, P.H., van Gorcom, R.F.M. and van den Hondel, C.A.M.J.J. (1990) Heterologous gene expression inAspergillus: analysis of chymosin production in single-copy transformants ofA. niger. In Heslot, H., Davies, J., Florent, J., Bobichon, L., Durand, G. and Penasse, L., eds.,Proceedings of the 6th International Symposium on Genetics of Industrial Microorganisms pp. 107–16, Strasbourg: Société Française de Microbiologie.Google Scholar
- Ward, M. (1989) Heterologous gene expression inAspergillus. In Nevalainen, H. and Penttilä, M. eds.,Proceedings of the EMBO-Alko Workshop on Molecular Biology of Filamentous Fungi, pp. 119–28. Helsinki: Foundation for Biotechnical and Industrial Fermentation Research.Google Scholar
- Wernars, K. (1986) DNA-mediated tranformation of filamentous fungusAspergillus nidulans. Thesis, Agricultural University, Wageningen, the Netherlands.Google Scholar