Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Patent
1996-12-03
2000-02-15
Kemmerer, Elizabeth
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
435 691, 435 914, 4351721, 4352532, 435325, 536 2374, C12N 510
Patent
active
060251859
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to fungi, which do not produce proteases. The fungi of the invention are useful as hosts for the production of proteins susceptible to proteolytic degradation by the proteases usually produced, and the invention consequently encompasses processes for the production of proteins of interest in high yields by using the fungi of the invention. The invention also comprises methods for producing such fungi and DNA constructs to be used in these methods.
BACKGROUND OF THE INVENTION
Fungi, and especially filamentous fungi, are widely used commercially because of their ability to secrete remarkably high levels of proteins
Among the filamentous fungi species belonging to the genus Aspergillus have a long history of commercial use for the production of endogenous and lately also heterologous proteins.
One disadvantage with most microorganisms used for the production of proteins is the inherent production of proteases which may subject a protein product of interest to degradation due to proteolysis.
Various ways of avoiding this have been envisaged. Among other solutions it has been suggested to delete or disrupt the genes encoding the various proteases. Unfortunately the fungi produce a high number of proteases making such a solution more or less unrealistic.
A need is therefore persisting for strains of filamentous fungi exhibiting no or very low levels of protease production.
For a number of years it has been known that the regulatory gene areA which mediates nitrogen metabolite repression in A. nidulans influences the production of extracellular proteases (Arst & Cove, molec. gen. Genet. 126, (1973) 111-141).
The areA gene from A. nidulans has been cloned (Caddick et al., EMBO Journal 5, (1986) 1087-1090) and various modifications made to it to evaluate functions of different regions in the activator protein encoded by this gene (Stankovitch et al. Mol. Microbiol. 7, (1993) 81-87). Furthermore the gene coding the corresponding function in A. fumigatus apparently has been cloned recently (Hensel et al. 2nd European Conference on Fungal Genetics, Apr. 28 to May 1, 1994, Book of Abstracts, E11).
From the literature a single use is also known of a strain of A. nidulans of genotype argB areA1 as a host for the production of t-PA (Upshall et al. Biotechnology 5, (1987) 1301-1304). In this example only the argB genotype is used as a selection marker through its arginine prototrophy, while the area genotype is simply a coincidence.
The present invention has as an object the alleviation of the need for protease free filamentous fungi.
SUMMARY OF THE INVENTION
The present invention consequently relates to fungi, wherein the areA gene by recombinant DNA technology has been modified such that it cannot be expressed in a way providing for a functional AreA activator.
The invention furthermore relates to methods for producing such fungi, obtained by deletion of the areA gene.
This may be obtained through a method comprising part has been substituted, deleted, or extra DNA has been inserted,
The information obtained from the above mentioned cloning of the areA gene may also be used in connection with the well-known anti-sense technology, to construct an expression plasmid giving rise to synthesis of a RNA molecule complementary to the mRNA transcribed from the areA gene, and to transform the fungus of interest therewith.
The invention furthermore relates to DNA constructs intended for use in the above mentioned methods.
Furthermore the invention relates to methods of producing a desired protein or gene product, especially secreted proteins, whereby a fungal host modified and optionally transformed with a DNA construct comprising at least a DNA sequence coding for the protein or gene product of interest, is cultivated in a suitable growth medium at appropriate conditions and the desired gene product is recovered and purified.
When working with the invention it was surprisingly found that the fungi of the invention produces such secreted proteins in a much improved yield.
REFERENCES:
patent: 5179003 (1993-01-01), Wolf et al.
patent: 5190931 (1993-03-01), Inouye
Molec. gen. Genet. vol. 126, pp. 111-141 (1973).
The EMBO Journal, vol. 5, pp. 1087-1090 (1986).
Molecular Microbiology vol. 7(1), pp. 81-87 (1993).
Books of Abstracts, 2nd European Conference on Jungal Genetics Apr. 28-May 1, 1994 1 page.
Bio.backslash.Technology, vol. 5, pp. 1301-1304 (1987).
Gene, vol. 95, pp. 123-127 (1990).
The EMBO Journal, vol. 9, No. 5, pp. 1355-1364 (1990).
Upshall et al. (1987) Bio/technology 5:1301-134.
Stankovitch et al. (1993) 7:81-87 Molec Microbiol.
Y.H. Fu et al., Molecular Microbiology, (1990), 4 (11), pp. 1847-1852.
Xiao Dong Xiao et al., Current Genetics (1993) 24: pp. 212-218.
Ying-Hui Fu, Molecular And Cellular Biology, Mar. 1990, pp. 1056-1065.
B.L. Cohen, Trans. Br. Mycol. Soc. 76 (3) pp. 447-450 (1981).
B.L. Cohen, Journal of General Microbiology (1972), 71, pp. 293-299.
B.L. Cohen, Journal of General Microbiology (1973), 79, pp. 311-320.
Tove Christensen et al., Applied And Environmental Microbiology, Sep. 1998, pp. 3232-3237.
Christensen Tove
Hynes Michael J.
Gregg Valeta
Kemmerer Elizabeth
Novo Nordisk Als
Romeo David S.
Zelson Steve T.
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