Method for the single step introduction of a plurality of genes in microbial cells

摘要

The invention provides a method for producing a vector for introducing a plurality of genes into a host cell, the method comprising the steps of: a) providing for each gene of the plurality of genes, a DNA fragment comprising an expression cassette for expression of the gene in the host cell, wherein the DNA fragment has staggered 5' and 3' ends that are part of a restriction enzyme recognition sequence, which recognition sequence is not present elsewhere in the DNA fragment and which recognition sequence at the 5' end of the DNA fragment differs from the recognition sequence at the 3' end of the DNA fragment, and wherein the overhang of the restriction enzyme recognition sequence at the 5' end is complementary with the overhang of the restriction enzyme recognition sequence at the 3' end; and b) combining the DNA fragments as obtained in a) by insertion of the DNA fragments into a unique restriction enzyme recognition site in a suitable vector, wherein the unique restriction enzyme recognition site in the vector has the same sequence as one of the two restriction recognition sequences at the 5' and 3' ends of the fragments in a), and wherein combination of a DNA fragment with a further DNA fragment produces a dysfunctional restriction enzyme site at the fusion site of the fragments. The DNA fragment comprising the expression cassette is preferably assembled from subfragments comprising functional elements that together form the expression cassette, which subfragments have staggered 5' and 3' ends, the overhang at the 5' end of each subfragment differs from the overhang at its 3' end, and the overhang at the 3' end of the subfragments comprising one type of element is complementary with the overhang at the 5' end of the subfragments comprising another type of element to allow assembly and operable linkage of the subfragments in a 5' to 3'order to form the functional expression cassette. The invention further provides a method for introducing a plurality of genes into a host cell, preferably into a filamentous fungal host cell, the method comprising the step of transforming the host cell with a vector obtainable by the above methods, in a single step.