Stable Genomic Integration of Multiple Polynucleotide Copies

A method of constructing a recombinant cell comprising in its chromosome two or more copies of an open reading frame (ORF) or operon encoding at least one polypeptide of interest, each copy being under the transcriptional control of a promoter, said method comprising the steps of:

• (a) providing a cell comprising in its chromosome two or more copies of a first recognition sequence (RS1) of a site specific recombinase, wherein each copy of RS1 is located downstream of the promoter;
• (b) introducing into said cell a polynucleotide construct comprising the ORF or operon and a second recognition sequence (RS2) of the site specific recombinase, where RS2 is located and oriented with respect to the ORF or operon so that an in vivo recombination of RS2 with a copy of RS1 in the chromosome of the cell will integrate the construct into the chromosome and place the ORF or operon downstream of and in the same orientation as the promoter;
• and
• (c) recombining RS2 with the two or more copies of RS1 in the presence of the site specific recombinase, wherein two or more copies of the ORF or operon are integrated into the chromosome and each placed:
• (i) either directly under the transcriptional control of the promoter, or
• (ii) downstream of and in the same orientation as the promoter but separated from it by a region, which can be excised after one or more optional recombination events, whereby the ORF or operon of interest is placed under the transcriptional control of the promoter.