Methods For Producing Multiple Recombinant Polypeptides In A Filamentous Fungal Host Cell
AssigneeNovozymes, Inc.
InventorsDebbie Yaver, Qiming Jin
Life of this patent
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Abstract
The present invention relates to methods for constructing a filamentous fungal strain for production of multiple recombinant polypeptides having biological activity. The present invention also relates to methods for producing multiple recombinant polypeptides having biological activity in a filamentous fungal strain. The present invention also relates to filamentous fungal strains expressing multiple recombinant polypeptides having biological activity.
Claims24
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;
- (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;
- or
- (c) a combination of (a) and (b).
A filamentous fungal strain, comprising:
- (a) an endogenous first gene replaced by targeted integration with 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 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;
- (b) an endogenous second gene replaced by targeted integration with 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 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;
- or (c) a combination of (a) and (b).
A method for producing multiple recombinant polypeptides having biological activity in a filamentous fungal strain, comprising:
- cultivating a filamentous fungal host cell under conditions conducive for production of the polypeptides, wherein the filamentous fungal host cell comprises (a) an endogenous first gene replaced by targeted integration with 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 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;
- (b) an endogenous second gene replaced by targeted integration with 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 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;
- or (c) a combination of (a) and (b).
A tandem construct comprising (i) a homologous 5′ region of a 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 gene, a homologous flanking region thereof, or a combination thereof.
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 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;
- (b) inserting into an endogenous second locus by targeted integration 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;
- or
- (c) a combination of (a) and (b).
A filamentous fungal strain, comprising:
- (a) an endogenous first locus modified by insertion by targeted integration with 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 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;
- (b) an endogenous second locus modified by insertion by targeted integration with 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 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;
- or (c) a combination of (a) and (b).
A method for producing multiple recombinant polypeptides having biological activity in a filamentous fungal strain, comprising:
- cultivating a filamentous fungal host cell under conditions conducive for production of the polypeptides, wherein the filamentous fungal host cell comprises (a) an endogenous first locus modified by insertion by targeted integration with 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 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;
- (b) an endogenous second locus modified by insertion by targeted integration with 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 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;
- or (c) a combination of (a) and (b).
A tandem construct comprising (i) a homologous 5′ region of a 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 locus, a homologous flanking region thereof, or a combination thereof.
Dependencies are inferred from the claim text.