This subproject was a big challenge but also a big opportunity for our project. As the relocalization of an enzymatic pathway like the nootkatone and violacein pathway depends on a working import machinery that selects specifically for certain cargo proteins, this subproject was and is a crucial part for our whole project.
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<h3>Heading 3</h3>
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Our results show that we designed and established a new and orthogonal peroxisomal import system in yeast. We modified one of the most conserved import machineries within the domain of eukaryotes - no matter if it is plants, mammals or fungi. This opens up new possibilities for biotechnological applications since this import system can be used to shift toxic compound reactions into the natural stress-resistant peroxisomes and thereby it can increase the yield and efficiency of rare biomolecule production <i> in vivo </i>.
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Furthermore, we managed to make a big step further towards a synthetic cell: While many research groups try to build up a synthetic cell from scratch, we decided to build it up from the inside by subverting its natural functional systems and making it fully customizable and controllable.
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This is why our new import machinery shows the potential for biotechnology and real world applications in general.
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<h3> Peroxicretion</h3>
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<p>Using microscopy we were able to show, that our membrane anchors localize in a typical Peroxisomal pattern [6].<br>
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The results of the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf"> GUS-assay*needs to be change* </a> 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 <i>S.Cerevisiae</i>, since to this point it has only been demonstrated in <a href="https://2017.igem.org/Team:Cologne-Duesseldorf"> <i>Aspargillus Niger</i>*needs to be change* </a>
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Even though our secretion is not as efficient as the unconventional secretion in other organisms [10], 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 <a href="https://2017.igem.org/Team:Cologne-Duesseldorf"> metabolic pathways*needs to be change* </a>. <br>
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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 <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#SizeAndNumber"> size and number</a> of the artificial compartments in the cell. <br>
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Due to the fact, that peroxisomes are linked to the endoplasmic reticulum (ER) in <i>S. cerevisiae</i> cells, the efficiency of our system could be inhibited. To overcome this problem one possible solution would be to delete <i>Inp1</i> in the background strain. Inp1 works as a molecular link between peroxisomal Pex3 and ER Pex3. Deletion of <i>Inp1</i> leads to mobile peroxisomes which should lead to more fusion events. <br>
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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 <a href="https://2017.igem.org/Team:Cologne-Duesseldorf"> optogenetics*needs to be change* </a>. </p>
This subproject was a big challenge but also a big opportunity for our project. As the relocalization of an enzymatic pathway like the nootkatone and violacein pathway depends on a working import machinery that selects specifically for certain cargo proteins, this subproject was and is a crucial part for our whole project.
Our results show that we designed and established a new and orthogonal peroxisomal import system in yeast. We modified one of the most conserved import machineries within the domain of eukaryotes - no matter if it is plants, mammals or fungi. This opens up new possibilities for biotechnological applications since this import system can be used to shift toxic compound reactions into the natural stress-resistant peroxisomes and thereby it can increase the yield and efficiency of rare biomolecule production in vivo .
Furthermore, we managed to make a big step further towards a synthetic cell: While many research groups try to build up a synthetic cell from scratch, we decided to build it up from the inside by subverting its natural functional systems and making it fully customizable and controllable.
This is why our new import machinery shows the potential for biotechnology and real world applications in general.
Peroxicretion
Using microscopy we were able to show, that our membrane anchors localize in a typical Peroxisomal pattern [6].
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 [10], 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* .
