Promoter variants for expressing genes in a fungal cell
The present invention relates to methods for producing a biological substance, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the biological substance, wherein the fungal host cell comprises a first nucleic acid sequence encoding the biological substance operably linked to a second nucleic acid sequence comprising a promoter variant selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; and a subsequence thereof; and hybrid and tandem promoters thereof; and (b) isolating the biological substance from the cultivation medium. The present invention also relates to the isolated promoter variants and to constructs, vectors, and fungal host cells comprising the promoter variants operably linked to nucleic acid sequences encoding biological substances.
1. A method 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 first nucleic acid sequence encoding the polypeptide operably linked to a second nucleic acid sequence comprising a tandem promoter comprising at least SEQ ID NO: 3; and
(b) isolating the polypeptide from the cultivation medium.
2. The method of claim 1 , wherein the fungal host cell contains one or more copies of the first nucleic acid sequence.
3. The method of claim 1 , wherein the fungal host cell contains one copy of the first nucleic acid sequence.
4. The method of claim 1 , wherein the polypeptide is selected from the group consisting of an antigen, enzyme, growth factor, hormone, immunodilator, neurotransmitter, receptor, reporter protein, structural protein, and transcription factor.
5. The method of claim 1 , wherein the polypeptide is native or foreign to the fungal host cell.
6. The method of claim 1 , wherein the tandem promoter comprises SEQ ID NO: 3 and one or more promoters selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.
7. The method of claim 1 , wherein the tandem promoter comprises at least two copies of SEQ ID NO: 3.
8. An isolated tandem promoter comprising at least SEQ ID NO: 3.
9. The tandem promoter of claim 8 , wherein the tandem promoter comprises SEQ ID NO: 3 and one or more promoters selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.
10. The tandem promoter of claim 8 , wherein the tandem promoter comprises at least two copies of SEQ ID NO: 3.
11. A nucleic acid construct comprising a nucleic acid sequence encoding a polypeptide operably linked to the tandem promoter of claim 8 .
12. The tandem promoter of claim 11 , wherein the polypeptide is selected from the group consisting of an antigen, enzyme, growth factor, hormone, immunodilator, neurotransmitter, receptor, reporter protein, structural protein, and transcription factor.
13. A recombinant expression vector comprising the nucleic acid construct of claim 11 .
14. A recombinant host cell comprising the nucleic acid construct of claim 11 .
15. A method for producing a polypeptide, comprising (a) cultivating a homologously recombinant cell, having incorporated therein an introduced transcription unit comprising the tandem promoter of claim 8 , an exon, and/or a splice donor site operably linked to a second exon of an endogenous nucleic acid sequence encoding the polypeptide,
under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.