Abstract
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.
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Andrianopoulos, A. and Hynes, M.J. (1990) Sequence and functional analysis of the positively acting regulatory geneamdR fromAspergillus nidulans.Mol. Cell. Biol. 10, 3194–203.
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.
Boel, E., Hansen, M.T., Hjort, I., Hoegh, I. and Fiil, N.P. (1984) Two different types of intervening sequences in the glucoamylase gene fromAspergillus niger.EMBO J 3, 1581–5.
Borsuk, P.A., Nagieć, M.M., Stepień, P.P. and Bartnik, E. (1982) Organization of the ribosomal RNA gene cluster inAspergillus nidulans.Gene 17, 147–52.
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.
Chambers, S.P., Prior, S.E., Barstow, D.A. and Minton, N.P. (1988) The pMTLnic − cloning vectors. I. Improved pUC polylinker region to facilitate the use of sonicated DNA for nucleotide sequencing.Gene 68, 139–49.
Cami, B. and Kourilsky, P. (1978) Screening of cloned recombinant DNA in bacteria byin situ colony hybridization.Nucl. Acid Res. 5, 2381.
Debets, A.M.J., Holub, E.F., Swart, K., van den Broek, H.W.J. and Bos, C.J. (1990a) An electrophoretic karyotype ofAspergillus niger.Mol. Gen. Genet. 224, 264–8.
Debets, A.J.M., Swart, K. and Bos, C.J. (1990b) Genetic analysis ofAspergillus niger: isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eight linkage group.Mol. Gen. Genet. 221, 453–8.
Debets, A.M.J., Swart, K., Holub, E.F., Goosen, T. and Bos, C.J. (1990c) Genetic analysis ofamdS transformants ofAspergillus niger and their use in chromosome mapping.Mol. Gen. Genet. 222, 284–90.
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.
Fowler, T., Berka, R.M. and Ward, M. (1990) Regulation of theglaA gene ofAspergillus niger.Curr. Genet. 18, 537–45.
Goosen, T., Bloemheuvel, G., Gysler, C., de Bie, D.A., van den Broek, H.W.J. and Swart, K. (1987) Transformation ofAspergillus niger using the homologous orotidine-5′-phosphate decarboxylase gene.Curr. Genet. 11, 499–503.
Goyon, C. and Faugeron, G. (1989) Targeted transformation ofAscobolus immersus andde novo methylation of resulting duplicated DNA sequences.Mol. Cell. Biol. 9, 2818–27.
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. Liss
Hanahan D. (1983) Studies on transformation ofEscherichia coli with plasmids.J. Mol. Biol. 166, 557–80.
Hata, Y., Tsuchiya, K., Kitamoto, K., Gomi, K., Kumagai, C., Tamura, G. and Hara, S. (1991) Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) fromAspergillus oryzae.Gene 108, 145–50.
Hynes, M.J., Corrick, C.M. and King, J.A. (1983) Isolation of genomic clones containing theamdS gene ofAspergillus nidulans and their use in the analysis of the structural and regulatory mutations.Mol. Cell. Biol. 3, 1430–39.
Hynes, M.J. and Davis, M.A. (1986) TheamdS gene ofAspergillus nidulans: control by multiple regulatory signals.BioEssays 5, 123.
Ish-Horowicz, D. and Burke, J.F. (1981) Rapid and efficient cosmid cloning.Nucl. Acids Res. 9, 2989–98.
Kelly, J.M. and Hynes, M.J. (1985) Transformation ofAspergillus niger by theamdS gene ofAspergillus nidulans.EMBO J. 4, 475–79.
Kelly, J.M. and Hynes, M.J. (1987) Multiple copies of theamdS gene ofAspergillus nidulans cause titration oftrans-acting regulatory proteins.Curr. Genet. 12, 21–31.
Metwally, M., el Sayed, M., Osman, M., Hanegraaf, P.P.F., Stouthamer, A.H. and van Verseveld, H.W. (1991) Bioenergetic consequences of glucoamylase production in carbon-limited chemostat cultures ofAspergillus niger.Antonie van Leeuwenhoek 59, 35–43.
Miller, B.L., Miller, K.Y., Roberti, K.A. and Timberlake W.E. (1987) Position-dependent and independent mechanisms regulate cell-specific expression of theSpoCl gene cluster ofAspergillus nidulans.Mol. Cell. Biol. 7, 427–34.
Mooibroek, H., Kuipers, A.G.J., Sietsma, J.H., Punt, P.J. and Wessels, J.G.H. (1990) Introduction of hygromycin B resistance intoSchizophyllum commune: preferential methylation of donor DNA.Mol. Gen. Genet. 222, 41–8.
Nunberg, J.H., Meade, J.H., Cole, G., Lawyer, F.C., McCabe, P., Schweickart, V., Tal, R., Witmann, V.P., Flatgaard, J.E. and Innis, M.A. (1984) Molecular cloning and characterization of the glucoamylase gene ofAspergillus awamori.Mol. Cell. Biol. 4, 2306–15.
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.
Punt, P.J., Oliver, R.P., Dingemanse, M.A., Pouwels, P.H. and van den Hondel, C.A.M.J.J. (1987) Transformation ofAspergillus based on the hygromycin B resistance marker fromEscherichia coli.Gene 56, 117–24.
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.
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.
Selker, E.U. and Garrett, P.W. (1988) DNA sequence duplications trigger inactivation inNeurospora crassa.Proc. Natl Acad. Sci. USA 85, 6870–4.
Timberlake, W.E. (1978) Low repetitive DNA content inAspergillus nidulans.Science 202, 973–5.
Van den Hondel, C.A.M.J.J., Punt, P.J. and van Gorcom, R.F.M. (1992) Production of extracellular proteins by the filamentous fungusAspergillus.Antonie van Leeuwenhoek 61, 153–60.
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.
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.
Wernars, K. (1986) DNA-mediated tranformation of filamentous fungusAspergillus nidulans. Thesis, Agricultural University, Wageningen, the Netherlands.
Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of M13mp18 and pUC19 vectors.Gene 33, 103–19.
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Verdoes, J.C., Punt, P.J., Schrickx, J.M. et al. Glucoamylase overexpression inAspergillus niger: Molecular genetic analysis of strains containing multiple copies of theglaA gene. Transgenic Research 2, 84–92 (1993). https://doi.org/10.1007/BF01969381
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DOI: https://doi.org/10.1007/BF01969381