Method for production of a compound in a eukaryotic cell
The present invention relates to a eukaryotic cell containing peroxisomes that are capable to fuse with a membrane-structure of the cell involved in the secretory pathway of the cell. In this way, the eukaryotic cell is able to release the peroxisomal content outside the cell. The invention also relates to a method for production of a compound of interest in said eukaryotic cell wherein said compound of interest is present in the peroxisome of the cell. Said compound of interest will accumulate in the peroxisome by a signal promoting peroxisome localization. Preferred host cells are filamentous fungal cells.
1. A method for production of a polypeptide of interest in a eukaryotic cell containing a peroxisome that expresses a chimeric protein comprising a domain of a v-SNARE polypeptide that is normally exposed at the cytosolic side of a donor membrane of the secretory pathway, a transmembrane domain of a peroxisomal membrane protein and a sequence that targets the transmembrane domain to the peroxisomal membrane wherein said peroxisome fuses via the v-SNARE domain on the cytosolic side of its peroxisomal membrane with a membrane-structure of the cell involved in the secretory pathway of the cell wherein the membrane-structure of the cell involved in the secretory pathway of the cell is selected from the group consisting of the plasma membrane, the Golgi complex and the Endoplasmic Reticulum, wherein said polypeptide of interest is present in the peroxisome of the cell, said method comprising culturing the eukaryotic cell in a suitable culture medium.
2. The method according to claim 1 , wherein the culture medium comprises material selected from the group consisting of an activator of ceramide activated protein phosphatase (CAPP) and a substance inducing peroxisome proliferation.
3. The method according to claim 1 , wherein at least 0.01 g/l of the polypeptide of interest is produced.
4. A method for production of a polypeptide of interest in a eukaryotic cell according to claim 1 , wherein the eukaryotic cell is cultured and a suitable amount of oxygen is fed to the culture to maintain the culture under conditions of oxygen limitation.
5. A method for production of a polypeptide of interest in a eukaryotic cell according to claim 1 , wherein the pH of the culture medium is altered during the culture process.
6. A method for production of a polypeptide of interest in a eukaryotic cell according to claim 1 , wherein the total duration of the culture process is 192 hours, which consists of:
a first phase of 72 hours wherein the pH of the culture medium is 6.0,
a transition phase of 24 hours wherein the pH of the culture medium is altered in a linear course from 6.0 to 6.7, and
a second phase of 96 hours wherein the pH of the culture medium is 6.7.
7. A method for production of a polypeptide of interest in a eukaryotic cell according to claim 1 , wherein the temperature of the culture medium is altered during the culture process.
8. A method for production of a polypeptide of interest in a eukaryotic cell according to claim 1 , wherein the temperature of the culture medium is altered from 30° C. to 36° C. during the culture process.
9. A method for production of a polypeptide of interest in a eukaryotic cell according to claim 4 , wherein the eukaryotic cell is a filamentous fungus.
10. The method according to claim 1 , further comprising purifying the polypeptide.
11. The method according to claim 2 , wherein the activator of CAPP is ceramide.
12. The method according to claim 2 , wherein the substance inducing peroxisome proliferation is oleate.
13. A method according to claim 9 , wherein the filamentous fungus is an Aspergillus species, and more preferably an Aspergillus niger strain.
14. A method according to claim 13 , wherein the Aspergillus species is an Aspergillus niger strain.