Methods For Producing Multiple Recombinant Polypeptides In A Filamentous Fungal Host Cell

A method for constructing a filamentous fungal strain for production of multiple recombinant polypeptides having biological activity, comprising:

• (a) inserting into an endogenous first locus by targeted integration by homologous recombination a first tandem construct comprising (i) a homologous 5′ region of the first locus, a homologous flanking region thereof, or a combination thereof, (ii) one or more first selectable markers, (iii) a first polynucleotide encoding a first polypeptide having biological activity operably linked to a first promoter and a first terminator, (iv) a second polynucleotide encoding a second polypeptide having biological activity operably linked to a second promoter and a second terminator, and (v) a homologous 3′ region of the first locus, a homologous flanking region thereof, or a combination thereof;
• and
• (b) inserting into an endogenous second locus by targeted integration by homologous recombination a second tandem construct comprising (i) a homologous 5′ region of the second locus, a homologous flanking region thereof, or a combination thereof, (ii) one or more second selectable markers, (iii) a third polynucleotide encoding a third polypeptide having biological activity operably linked to a third promoter and a third terminator, (iv) a fourth polynucleotide encoding a fourth polypeptide having biological activity operably linked to a fourth promoter and a fourth terminator, and (v) a homologous 3′ region of the second locus, a homologous flanking region thereof, or a combination thereof.