Simultaneous site-specific integrations of multiple gene-copies

#This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

#The present invention relates to a method for the simultaneous site-specific integrations of multiple copies of a polynucleotide of interest into the genome of a fungal host cell using a transiently expressed recombinase together with suitable resident selection markers.

#A large number of naturally-occurring organisms have been found to produce useful polypeptide products, e.g., enzymes, the large scale production of which is desirable for research and commercial purposes. Once such a polypeptide product has been identified, efforts are often made to develop manufacturing methods having an improved productivity. One widely used method, which is based on recombinant DNA techniques, is to clone a gene encoding the product and insert the gene into a suitable expression system in order to express the product in a suitable host cell, either integrated in the chromosome or as an extrachromosomal entity, under conditions conducive for the expression of the product.

#FIG. 1 shows the basic scheme of the method of the invention, herein exemplified using the FRT/FLP recombinase system and pyrG as bi-directional selective marker together with the FLP-encoding gene flanked by two copies of the transcriptional terminator, labelled “Term” that enable the excision of the FLP and pyrG genes by double homologous recombination, which allows transient expression of FLP.

#FIG. 2 shows a plasmid map of pHUda981 (Pgpd, HSV1 tk, TtrpC are described in WO07045248).

#FIG. 3 shows a plasmid map of pHUda1019.

#Cytosine deaminase: Cytosine deaminase (EC 3.5.4.1) catalyzes the deamination of cytosine and 5-fluorocytosine (5FC) to form uracil and toxic 5-fluorouracil (5FU), respectively. When genetically modified cells comprising cytosine deaminase are combined with 5FC it is converted to toxic 5FU, so the cytosine deaminase-encoding gene is potentially a potent negative selection marker.

#It has also been shown that an inhibitor in the pyrimidine de novo synthesis pathway can be utilized to create a condition in which cells are dependent on the conversion of pyrimidine supplements to uracil by cytosine deaminase. Thus, only cells expressing the cytosine deaminase gene can be rescued in a positive selection medium comprising an inhibitor of the pyrimidine de novo synthesis as well as inosine and cytosine (See FIG. 1 of Wei and Huber, 1996, J Biol Chem 271(7): 3812). The inhibitor is preferably N-(phosphonacetyl)-L-aspartate (PALA), which inhibits aspartate carbamyl transferase.

#If necessary, cytosine deaminase activity may be quantitated by a genetic assay (Frederico L. A. et al, 1990, Biochemistry 29: 2532-2537).