Unilever Patent Holdings B.v.

Marcellus Johannes Augustinus De Groot

Alida Godelieve Maria Beijersbergen

Wouter Musters

Robertus Johannes Gouka

Paul Jan Jacob Hooykaas

Paul Bundock

A process for site-directed integration of multiple copies of a gene in a mould is provided, which comprises transforming a mould cell containing in its chromosomal DNA a restriction site for a rare-cutting endonuclease, e.g., I-Scel, preferably introduced at a desired locus, e.g., within a selectable marker gene or in the neighborhood thereof, with a piece of DNA comprising multiple copies of at least one expressible gene comprising at least one structural gene encoding a desired protein, surrounded by two DNA fragments homologous to part of the DNA upstream and downstream, and in the neighborhood, of said restriction site, while during the transformation of the mould the presence of the rare-cutting endonuclease is provided, followed by selecting or screening for a mould cell in which the multiple gene copies of said expressible gene are inserted into the chromosomal DNA of the mould. The piece of DNA can comprise a third DNA fragment completing any disrupted or partially deleted selectable marker gene in the chromosomal DNA. Preferably the mould belongs to the genus Aspergillus, especially to the species . Also provided are a transformed mould obtainable by a process according to the invention, a process for culturing such transformed mould, and a process for producing and optionally secreting a desired protein by culturing such transformed mould under conditions whereby the structural gene encoding said desired protein is expressed, and optionally isolating or concentrating the desired protein.

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Vlaardingen, Netherlands

Marcellus Johannes Augustinus De Groot