Demonstrate
Pex5 Import
The achievement of an orthogonal import was a huge step towards an artificial compartment that could be utilized for all kinds of synthetic biological applications. The relocalization of pathways into an isolated space benefits biotechnological production, opens new doors and enriches possibilities.
Although, we could not demonstrate our concept by fully relocating complex metabolic pathways, importing fluorescent proteins is already a solid proof of concept. Our results show that the transport of fluorescent markers such as mTurquoise work really well, assuming our new import machinery to not alter the function of the cargo of interest. Thus, we provide that any protein of interest can be shifted fully intact into the peroxisome via our import system. This orthogonality emphasizes the importance of this system for synthetic biology as it contributes to the engineering of a synthetic cell.
The most important difference is that we conquered this big challenge by converting the cells functional systems from the inside. But what is of far more importance: the possibility of this system to fight world threatening diseases. Our system makes the proteome of the peroxisomes accessible and engineerable. Due to the natural function of peroxisomes as a stress-resistant compartment our import machinery enables the shift of metabolic pathways from the cytosol into the peroxisome and by this increases the productivity, yield and efficiency of the pathway’s end product. We chose nootkatone and violacein as exemplary pathways to demonstrate the potential of our project. Our system is not limited only for producing drugs, it is rather a versatile tool to efficiently maintain the toxicity and limited in vivo production of compounds from many different branches of industry.
As an outlook, we predict major yields for the two products, nootkatone, violacein and many more compounds of interest once we relocate their pathways into the peroxisome.
Pex7 import
We archived to generate various PTS2 sequences showing different import efficiencies. With this valuable tool one is able to alter protein concentrations in the peroxisome to further customize the artificial compartment. Essential proteins as the ubiquitin binding proteins regulate stability and localisation of ubiquitinated proteins (Wilkinson)>/abbr> QELLE. Therefore the C-terminal domain of the protein interacts with the ubiquitinated protein. To establish a pathway containing proteins like this in the compartment they must not be modified on their C-terminus.Nevertheless we can import them into the compartment with the modified PTS2 sequences.
Secretion
Using microscopy we were able to show, that our membrane anchors localize in a typical peroxisomal pattern
(Halbach et al, 2006)
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The results of the GUS-assay 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 this point it has only been demonstrated in Aspergillus niger.
Even though our secretion is not as efficient as the unconventional secretion in other organisms (Stock et al, 2012), the possibility to secrete the content 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.
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 is 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 is 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 leading 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 is 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.
