Methods for producing secreted polypeptides
View Patent ↗The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding the polypeptide, wherein the first nucleotide sequence is foreign to the second nucleotide sequence and the 3′ end of the first nucleotide sequence is immediately upstream of the initiator codon of the second nucleotide sequence. The present invention also relates to the isolated signal peptide sequences and to constructs, vectors, and fungal host cells comprising the signal peptide sequences operably linked to nucleotide sequences encoding polypeptides.
1. A method for producing a secreted polypeptide, comprising:
(a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding the polypeptide, wherein the first nucleotide sequence is foreign to the second nucleotide sequence, the 3′ end of the first nucleotide sequence is immediately upstream of the initiator codon of the second nucleotide sequence, and the first, nucleotide sequence is selected from the group consisting of,
(i) a nucleotide sequence encoding a signal peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 37; and
(ii) a nucleotide sequence comprising or consisting of SEQ ID NO: 36; and
(b) isolating the secreted polypeptide from the cultivation medium,
wherein the fungal host cell produces at least about 25% more secreted polypeptide relative to the fungal host cell comprising a native signal peptide sequence operably linked naturally to the second nucleotide sequence encoding the polypeptide when cultured under identical production conditions.
2. The method of claim 1 , wherein the first nucleotide sequence encodes a signal peptide comprising the amino acid sequence of SEQ ID NO: 37.
3. The method of claim 2 , wherein the first nucleotide sequence encodes a signal peptide consisting of the amino acid sequence of SEQ ID NO: 37.
4. The method of claim 1 , wherein the first nucleotide sequence is contained in an endoglucanase V gene which is contained in Humicola insolens DSM 1800.
5. The method of claim 1 , wherein the second nucleotide sequence encodes a polypeptide native to the fungal host cell.
6. The method of claim 1 , wherein the second nucleotide sequence encodes a polypeptide heterologous to the fungal host cell.
7. The method of claim 1 , wherein the first nucleotide sequence comprises of SEQ ID NO: 36.
8. The method of claim 7 , wherein the first nucleotide sequence consists of SEQ ID NO: 36.
9. The method of claim 1 , wherein the fungal host cell contains one or more copies of the second nucleotide sequence.
10. The method of claim 1 , wherein the nucleic acid construct further comprises a promoter operably linked to the first and second nucleotide sequences.
11. The method of claim 10 , wherein the promoter is selected from the group consisting of a Trichoderma reesei cellobiohydroiase I gene promoter, Trichoderma reesei cellobiohydrolase II gene promoter, Trichoderma reesei xylanase I gene promoter, Trichoderma reesei xylanase II gene promoter, and Trichoderma reesei beta-xylosidase gene promoter.
12. The method of claim 1 , wherein the fungal host cell produces at least about 50% more polypeptide relative to the fungal host cell comprising a native signal peptide sequence operably linked to the second nucleotide sequence encoding the polypeptide when cultured under identical production conditions.
13. The method of claim 12 , wherein the fungal host cell produces at least about 75% more polypeptide relative to the fungal host cell comprising a native signal peptide sequence operably linked to the second nucleotide sequence encoding the polypeptide when cultured under identical production conditions.
14. The method of claim 13 , wherein the fungal host cell produces at least about 100% more polypeptide relative to the fungal host cell comprising a native signal peptide sequence operably linked to the second nucleotide sequence encoding the polypeptide when cultured under identical production conditions.
15. The method of claim 14 , wherein the fungal host cell produces at least about 200% more polypeptide relative to the fungal host cell comprising a native signal peptide sequence operably linked to the second nucleotide sequence encoding the polypeptide when cultured under identical production conditions.
16. The method of claim 15 , wherein the fungal host cell produces at least about 300% more polypeptide relative to the fungal host cell comprising a native signal peptide sequence operably linked to the second nucleotide sequence encoding the polypeptide when cultured under identical production conditions.
17. The method of claim 16 , wherein the fungal host cell produces at least about 400% more polypeptide relative to the fungal host cell comprising a native signal peptide sequence operably linked to the second nucleotide sequence encoding the polypeptide when cultured under identical production conditions.