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) replacing an endogenous first gene by targeted integration by introducing into the filamentous fungal strain a first tandem construct comprising (i) a homologous 5′ region of the first gene, 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 gene, a homologous flanking region thereof, or a combination thereof;
• and
• (b) replacing an endogenous second gene by targeted integration by introducing into the filamentous fungal strain a second tandem construct comprising (i) a homologous 5′ region of the second gene, 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 gene, a homologous flanking region thereof, or a combination thereof;
• wherein each of the tandem constructs integrates by homologous recombination into the chromosome of the filamentous fungal strain.