Patent ID: 6703200
Filing Date: 2004-03-09
Classification: H01G,H01M,Y02E,Y02T

Abstract:
A method for facilitating site directed homologous recombination in a filamentous fungus to produce mutants comprising:a) providing a large insert vector library comprising one or more large insert vectors, wherein each of said large insert vectors comprises more than 20 kilobase pairs genomic DNA from a target filamentous fungus and a first selectable marker functional for selection in bacteria; b) providing a second vector comprising a transposable element, said transposable element comprising a nucleotide sequence coding for a second selectable marker flanked on each side by an inverted repeat sequence, wherein said second selectable marker is bifunctional for selection in bacteria and the target filamentous fungus and wherein said inverted repeat sequences are functional as a binding site for a transposase; c) incubating one or more of said large insert vectors with said second vector, in the presence of a transposase specific for the inverted terminal repeat sequences on said second vector, such that the transposable element transposes into said genomic DNA to produce a disrupted large insert vector; d) optionally, amplifying the disrupted large insert vectors resulting from step (c) in a bacterial cell and selecting for the presence of said first and second selectable markers in said bacterial cell; e) introducing at least one of said disrupted large insert vectors into a target host cell from said target filamentous fungus so that homologous recombination occurs between said genomic DNA in said disrupted large insert vector and the genome of said target filamentous fungus and thereby produce a mutated target cell; and f) selecting for the presence of said second selectable marker and screening for successful homologous recombination produced by step (e) in said mutated target cell.