Increased production of secreted proteins by recombinant eukaryotic cells
Described herein are methods for increasing the amount of protein secreted by a cell. In one case, a cell is provided which contains a heterologous nucleic acid encoding a protein having unfolded protein response modulating activity and a heterologous nucleic acid encoding a protein of interest to be secreted. In one case, the protein having unfolded protein response modulating activity is selected from the proteins selected from the group consisting of HAC1, PTC2 and IRE1. The protein of interest can be any secreted protein such as a therapeutic or an industrial enzyme. For example the protein can be selected from the group consisting of lipase, cellulase, endo-glucosidase H, protease, carbohydrase, reductase, oxidase, isomerase, transferase, kinase, phosphatase, alpha-amylase, glucoamylase, lignocellulose hemicellulase, pectinase and ligninase.
1. A method of increasing the secretion of a heterologous protein in a fungal cell, comprising
inducing an unfolded protein response (UPR) by increasing the presence of a HAC1 UPR-modulating protein in said fungal cell, comprising transforming the fungal cell with a nucleic acid encoding a yeast or filamentous fungal HAC1 UPR-modulating protein comprising a DNA binding domain consisting of:
a) at least 90% sequence identity to the DNA binding domain of amino acid residues 84-147 of SEQ ID NO: 5;
b) at least 90% sequence identity to the DNA binding domain of amino acid residues 53-116 of SEQ ID NO: 6 or
c) the DNA binding domain of amino acid residues 45-109 of SEQ ID No:19, and
increasing secretion of the heterologous protein relative to secretion of the heterologous protein in a parental cell, wherein said fungal cell is a yeast or filamentous fungal cell.
2. The method of claim 1 wherein said HAC1 protein is constitutively produced.
3. The method of claim 1 wherein said HAC1 protein is encoded by a nucleic acid isolated from a cell selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Fusarium, Neurospora , and Penicillium.
4. The method of claim 1 wherein said HAC1 protein is encoded by a nucleic acid isolated from yeast.
5. The method of claim 4 wherein said yeast is Saccharomyces cerevislae.
6. The method of claim 1 wherein said HAC1 protein is encoded by a nucleic acid isolated from filamentous fungi.
7. The method of claim 6 wherein said fungi is from Trichoderma.
8. The method of claim 6 wherein said fungi is Trichoderma reesei.
9. The method of claim 6 wherein said fungi is from Aspergillus.
10. The method of claim 6 wherein said fungi is Aspergillus nidulans.
11. The method of claim 6 wherein said fungi is Aspergillus niger.
12. The method of claim 1 wherein said yeast or filamentous fungal cell is selected from the group consisting of Aspergillus spp., Trichoderma spp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces ssp., Pichia spp., Hansenula polymorpha, Fusarium spp., Neurospora spp., and Penicillium spp.
13. The method of claim 1 wherein said fungal cell is a yeast cell.
14. The method of claim 13 wherein said yeast is Saccharomyces cerevisiae.
15. The method of claim 1 wherein said fungal cell is a filamentous fungi.
16. The method of claim 15 wherein said fungi is from Trichoderma.
17. The method of claim 15 wherein said fungi is Trichoderma reesei.
18. The method of claim 15 wherein said fungi is from Aspergillus.
19. The method of claim 15 wherein said fungi is Aspergillus nidulans.
20. The method of claim 15 wherein said fungi is Aspergillus niger.
21. The method of claim 1 wherein said UPR-modulating protein comprises a DNA binding domain consisting of at least 90% identity to the DNA binding domain of a) amino acid residues 84-147 of SEQ ID NO: 5 or b) amino acid residues 53-116 of SEQ ID NO: 6.
22. The method of claim 1 wherein said UPR-modulating protein comprises a DNA binding domain consisting of at least 95% identity to the DNA binding domain of a) amino acid residues 84-147 of SEQ ID No: 5 or b) amino acid residues 53-116 of SEQ ID No: 6.
23. The method of claim 1 wherein said UPR-modulating protein comprises a DNA binding domain consisting of the DNA binding domain of amino acid residue positions 84 to 147 of SEQ ID NO: 5.
24. The method of claim 1 wherein said UPR-modulating protein comprises a DNA binding domain consisting of the DNA binding domain of amino acid residue positions of 53 to 116 of SEQ ID NO: 6.
25. The method of claim 1 , wherein said heterologous protein is selected from the group consisting of lipases, cellulases, endo-glucosidase H, proteases, carbohydrases, reductases, oxidases, isomerases, transferases, kinases, phosphatases, alpha-amylases, glucoamylases, hemicellulases, pectinases and ligninases.
26. The method of claim 25 , wherein the heterologous protein is a protease, cellulase, glucoamylase or alpha amylase.
27. The method of claim 1 , wherein the fungal cell is a Trichoderma or Aspergillus fungal cell, the UPR-modulating protein comprising a DNA binding domain consisting of at least 90% sequence identity to the DNA binding domain of a) amino acid residues 84-147 of SEQ ID NO: 5 or b) amino acid residues 53-116 of SEQ ID NO: 6 and the heterologous protein is selected from the group consisting of proteases, cellulases, glucoamylases, and alpha amylases.
28. The method of claim 27 , wherein the fungal cell is a Trichoderma cell and the UPR-modulating protein comprises a DNA binding domain consisting of at least 95% sequence identity to the DNA binding domain of a) amino acid residues 84-147 of SEQ ID NO: 5 or b) amino acid residues 53-116 of SEQ ID NO: 6.
29. The method of claim 27 , wherein the fungal cell is an Aspergillus cell and the UPR-modulating protein comprises a DNA binding domain consisting of at least 95% sequence similarity to the DNA binding domain of a) amino acid residues 84-147 of SEQ ID NO: 5; b) amino acid residues 53-116 of SEQ ID NO: 6.
30. The method of claim 1 , further comprising a promoter operably linked to the nucleic acid encoding the HAVC1 UPR-modulating protein, said promoter selected from the group consisting of cbh 1, gpdA, adh1 and pgk1.
31. The method of claim 1 wherein said UPR-modulating protein comprises a DNA binding domain consisting of the DNA binding domain of amino acid residue positions 45 to 109 of SEQ ID NO: 19.