Fungal production system
The present invention provides a new fungal production system comprising a fungal host strain of Chrysosporium lucknowense wherein the endogenous cellulase secretion is less than 20% of the endogenous cellulase secretion of Chrysosporium lucknowense strain UV 18-25. Preferably, also the secretion of endogenous protease, endogenous β-glucanase and endogenous cellobiohydrolase is less than 20% of the secretion of Chrysosporium lucknowense strain UV 18-25. Furthermore, fungal host strains are provided wherein several genes have been disrupted. According to another aspect of the invention a method for homologous and/or heterologous production of a pure protein with a purity of higher than 75%, comprising expressing a gene encoding said protein in the strains according to the invention have been described. Furthermore, a method for production of artificial protein mixes comprising expressing a gene encoding each of said proteins in a strain according to the invention have been disclosed. Finally a method for simplified screening of strains functionally expressing a desired enzyme by application of said strains have been provided.
1. A strain of Chrysosporium lucknowense selected from the group consisting of W1L deposited at CBS under accession number 122189, W1L#100.1 deposited at CBS under accession number 122190, and a strain derived therefrom by gene disruption.
2. The strain of claim 1 in which the gene encoding endochitinase 1 (chi1) has been disrupted.
3. The strain of claim 1 in which one or more genes encoding alkaline protease 1 (alp1), alkaline protease 2 (alp2), proteinase A (pep4), glucoamylase (Gla1), exochitinase (Chi2) or laminarinase (Lam1) has been disrupted.
4. The strain of claim 1 , selected from the group consisting of:
(a) W1L#100.1Δalp1Δpyr5, and
(b) W1L#100.1Δalp1Δchi1Δpyr5.
5. A method for producing a homologous or heterologous protein comprising expressing a gene encoding said homologous or heterologous protein in the Chrysosporium lucknowense strain of claim 1 .
6. The method of claim 5 , wherein said homologous or heterologous protein is produced with a purity of higher than 75%.
7. A method for producing a protein mixture comprising homologous and/or heterologous proteins, which method comprises expressing genes encoding said proteins of said mixture in the Chrysosporium lucknowense strain of claim 1 .
8. A method for isolating a genetically modified fungal strain of Chrysosporium lucknowense strain UV 18-25, wherein cellulase and protease secretion is less than 20% of endogenous cellulase and protease secretion of unmodified Chrysosporium lucknowense strain UV 18-25, comprising the steps of:
(i) mutagenizing fungi of said Chrysosporium lucknowense strain UV 18-25,
(ii) plating the mutagenized fungi on ASC plates,
(iii) selecting at least one colony showing a reduced cellulose clearing zone, enhanced sporulation and white color,
(iv) plating the at least one colony selected in step (iii) on skim milk plates, and
(v) selecting from the plates of step (iv) at least one colony showing a reduced protein degradation halo,
thereby isolating said modified fungal strain.
9. The method of claim 8 , further comprising a step of mutagenizing the fungi prior to step (iv).
10. A method for isolating a genetically modified fungal strain of Chrysosporium lucknowense strain UV 18-25, wherein cellulase secretion is less than 20% of endogenous cellulase secretion of unmodified Chrysosporium lucknowense strain UV 18-25, comprising the steps of:
(i) mutagenizing fungi of said Chrysosporium lucknowense strain UV 18-25;
(ii) plating the mutagenized fungi on ASC plates; and
(iii) selecting a colony showing a reduced cellulose clearing zone, enhanced sporulation and white color from the plates of step (ii), thereby isolating said modified fungal strain.