D-amino acid-inducible gene expression system for
The present invention relates to the field of fungal biotechnology, more particularly to a strong inducible gene expression system in fungal species, such as a species of the Rhodosporidium genus or the Rhodotorula genus.
1. A D-amino acid inducible gene expression system operable in a transgenic fungal cell comprising a nucleic acid construct comprising a D-amino acid inducible promoter operably linked to a heterologous polynucleotide, wherein the promoter is selected from the group consisting of:
(a) a promoter having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:94;
(b) a promoter having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:95;
(c) a promoter having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:96;
(d) a promoter having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:97; and
(e) a promoter having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:5, and
wherein the promoter comprises a promoter motif comprising the nucleotide sequence set forth in SEQ ID NO:6.
2. The expression system of claim 1 , wherein the fungal cell is a transgenic fungal cell of a species of Rhodsporidium genus or Rodottorula genus.
3. The expression system of claim 1 , wherein the transgenic fungal cell comprises a mutant D-amino acid oxidase gene having no or reduced D-amino acid oxidase activity compared to a fungal cell comprising a wild type D-amino acid oxidase gene.
4. The expression system of claim 1 , wherein the heterologous polynucleotide is operably linked to a transcription terminator.
5. The expression system of claim 1 , wherein the nucleic acid construct contains a nucleotide sequence having at least 75% identity to the nucleotide sequence set forth in SEQ ID NO:7.
6. The expression system of claim 5 , wherein the nucleotide sequence is an intron operably linked to the inducible promoter and operably linked to the heterologous polynucleotide.
7. The expression system of claim 5 , wherein the nucleotide sequence is an intron inserted into the 5′ region of the heterologous polynucleotide.
8. The expression system of claim 5 , wherein the nucleotide sequence is an intron inserted into the 5′ UTR of the inducible promoter.
9. The expression system of claim 5 , wherein the nucleotide sequence is part of the coding sequence of the heterologous polynucleotide.
10. The expression system of claim 5 , wherein the nucleotide sequence is selected from the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59.
11. The expression system of claim 1 , wherein the heterologous polynucleotide encodes a protein of interest.
12. The expression system of claim 1 , wherein the heterologous polynucleotide encodes an RNA molecule for down regulating a target gene of interest.
13. A transgenic fungal cell comprising the expression system of claim 1 .
14. A composition comprising the transgenic fungal cell of claim 13 and a culture medium comprising a D-amino acid.
15. The composition of claim 14 , wherein the D-amino acid is D-alanine, D-threonine, D-serine, D-valine or D-proline.
16. The composition of claim 14 , wherein the concentration of the D-amino acid in the culture medium is from about 0.1 mM to about 100 mM.
17. A method for preparing a transgenic fungal cell comprising:
(a) introducing the expression system of claim 1 into a fungal cell and
(b) selecting a transgenic fungal cell which comprises the nucleic acid construct.
18. A method of inducible expression of a polynucleotide in a fungal cell comprising culturing the transgenic fungal cell of claim 13 in a culture medium comprising a D-amino acid to induce expression of the heterologous polynucleotide.
19. The method of claim 18 , wherein the D-amino acid is D-alanine, D-threonine, D-serine, D-valine or D-proline.
20. The method of claim 18 , wherein the concentration of the D-amino acid in the culture medium is from about 0.1 mM to about 100 mM.