Methods for obtaining positive transformants of a filamentous fungal host cell

Patent metadata

Field	Value
Publication number	US-9404101-B2
Application number	US-201214238681-A
Country	US
Kind code	B2
Filing date	Aug 23, 2012
Priority date	Aug 24, 2011
Publication date	Aug 2, 2016
Grant date	Aug 2, 2016

Abstract

The present invention relates to methods for obtaining positive transformants of a filamentous fungal host cell, comprising: transforming a tandem construct into a population of cells of the filamentous fungal host a tandem construct and isolating a transformant of the filamentous fungal host cell comprising the tandem construct. The present invention also relates to such tandem constructs, filamentous fungal host cells comprising such tandem constructs, and methods of producing multiple recombinant proteins.

First claim

What is claimed is:

1. A method for obtaining positive transformants of a filamentous fungal host cell, comprising:
(a) transforming into a population of cells of the filamentous fungal host a tandem construct comprising (i) one or more selectable markers, (ii) a first polynucleotide encoding a first polypeptide having biological activity operably linked to a first promoter and a first terminator, and (iii) a second polynucleotide encoding a second polypeptide having biological activity operably linked to a second promoter and a second terminator, wherein the tandem construct integrates by ectopic integration into the chromosome of the filamentous fungal host cell;
(b) selecting transformants based on the one or more selectable markers, wherein the number of positive transformants for the first and second polypeptides having biological activity obtained by transformation of the tandem construct is higher compared to the number of positive transformants obtained by co-transformation of separate constructs for each of the first and second polynucleotides; and
(c) isolating a transformant of the filamentous fungal host cell comprising the tandem construct expressing the first and second polypeptides having biological activity.

2. The method of claim 1 , wherein the number of positive transformants for the first and second polypeptides having biological activity obtained by transformation of the tandem construct is increased at least 1.1-fold compared to the number of positive transformants obtained by co-transformation of separate constructs for each of the first and second polynucleotides.

3. The method of claim 1 , wherein the tandem construct further comprises a first homologous repeat flanking 5′ of the one or more selectable markers and a second homologous repeat flanking 3′ of the one or more selectable markers, wherein the first homologous repeat and the second homologous repeat undergo homologous recombination to excise the one or more selectable markers.

4. The method of claim 3 , wherein the first and second homologous repeats are identical or have a sequence identity of at least 70% to each other.

5. The method of claim 3 , wherein upon the excision of the one or more selectable markers, the one or more selectable markers can be reused for introducing another tandem construct into the filamentous fungal host cell.

6. The method of claim 1 , wherein the number of positive transformants for the first and second polypeptides having biological activity obtained by transformation of the tandem construct is increased at least 2-fold compared to the number of positive transformants obtained by co-transformation of separate constructs for each of the first and second polynucleotides.

7. The method of claim 1 , wherein the number of positive transformants for the first and second polypeptides having biological activity obtained by transformation of the tandem construct is increased at least 5-fold compared to the number of positive transformants obtained by co-transformation of separate constructs for each of the first and second polynucleotides.

8. The method of claim 1 , wherein the number of positive transformants for the first and second polypeptides having biological activity obtained by transformation of the tandem construct is increased at least 10-fold compared to the number of positive transformants obtained by co-transformation of separate constructs for each of the first and second polynucleotides.

9. The method of claim 3 , wherein the first and second homologous repeats have a sequence identity of at least 80% to each other.

10. The method of claim 3 , wherein the first and second homologous repeats have a sequence identity of at least 90% to each other.

11. The method of claim 3 , wherein the first and second homologous repeats are each at least 50 bp.

12. The method of claim 1 , wherein the polypeptides having biological activity are different polypeptides.

13. The method of claim 1 , wherein the polypeptides having biological activity are the same polypeptide.

14. The method of claim 1 , wherein the promoters are different promoters.

15. The method of claim 1 , wherein the promoters are the same promoter.

16. The method of claim 1 , wherein the terminators are different terminators.

17. The method of claim 1 , wherein the terminators are the same terminator.

18. The method of claim 1 , wherein the filamentous fungal cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , or Trichoderma cell.

19. The method of claim 18 , wherein the Trichoderma strain is selected from the group consisting of Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei , and Trichoderma viride.

20. The method of claim 18 , wherein the Trichoderma strain is Trichoderma reesei .

Assignees

• Moyer Donna
• Novozymes Inc

Inventors

• Moyer Donna

Classifications

• C12Y302/01

  Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1) · CPC title

• C12N9/2445 Primary

  Beta-glucosidase (3.2.1.21) · CPC title

• C12N15/80

  for fungi · CPC title

• C12Y302/01021

  Beta-glucosidase (3.2.1.21) · CPC title

• C12N9/2402

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title