Demonstrate
PEX5 Import
The achievement of an orthogonal import was a huge step towards an fully artificial compartment that could be utilized for all kind of synthetic biological applications. The relocation of cytosolic pathways into an isolated space benefits biotechnological productions, opens new doors and enriches possibilities.
Unfortunately, we could not demonstrate our concept by relocating metabolic pathways but importing fluorescent proteins already is a proof of concept.
As an outlook, we predict major yields for the two products, Nootkatone and Violacein, once we relocated their pathways into the peroxisome.
Peroxicretion
Using microscopy we were able to show, that our membrane anchors localize in a typical Peroxisomal pattern
(Halbach N et al, 2006)
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The results of the GUS-assay*needs to be change* indicate, that the contents of the peroxisomes were successfully secreted into the supernatant. This is the first time it was shown that this system works in S.Cerevisiae, since to this point it has only been demonstrated in Aspargillus Niger*needs to be change*
Even though our secretion is not as efficient as the unconventional secretion in other organisms (Stock N et al, 2012), the possibility to secrete the compounds of our artificial compartment is still a substantial success with exciting implications. One could, for example, not only secrete proteins but also compounds from metabolic pathways*needs to be change* .
Here we have only shown our general proof of concept. The next steps would be to develop a more efficient system. An easy way to increase the yield of compounds in the supernatant would be to manipulate the size and number of the artificial compartments in the cell.
Due to the fact, that peroxisomes are linked to the endoplasmic reticulum (ER) in S. cerevisiae cells, the efficiency of our system could be inhibited. To overcome this problem one possible solution would be to delete Inp1 in the background strain. Inp1 works as a molecular link between peroxisomal Pex3 and ER Pex3. Deletion of Inp1 leads to mobile peroxisomes which should lead to more fusion events.
After the generation of an optimized background strain, the next goal would be stable genome integration of the secretion system. One of the problems of this integration would be the constitutive expression of the snare constructs. This would lead to a constant secretion and loss of the proteins in our compartment. For an optimal yield it would be better to use inducible promoters to control the secretion of the produced metabolites. Another approach could be to control the fusion of the compartment and the cell membrane by optogenetics*needs to be change* .
