Integrated Human practice - Support by Prof. Dr. Ralf Erdmann
In order to gain fundamental insights into yeast peroxisomes we contacted Prof. Dr. Ralf Erdmann and his research fellows Prof. Dr. Wolfgang Schliebs and Dr. Wolfgang Girzalsky from the university of Bochum who are experts in peroxisomal research. Thanks to their research experience and huge knowledge regarding yeast peroxisomes we were able to significantly improve our project design and lab work.
The group´s research topics comprise biogenesis of the peroxisomal membrane, peroxisomal metabolism and metabolite transport, proliferation of peroxisomes and peroxisomal import. In our first encounter on the 6th of April we introduced them to the essentials of our artificial cell compartment developed from yeast peroxisomes and our blueprint to create and control it. The conversation soon became very scientific, discussing protein import, peroxisomal membrane proteins and peroxisomal metabolism and lasted roughly four hours. On the one hand we received great feedback for our idea and additional interesting knowledge about the lab work with peroxisomes. On the other hand we were able to convince all leaders of the working group of our project’s great potential and they appeared to be very interested in the iGEM competition itself.
In order to maintain our contact to the working group from Bochum and gather further useful hints, we invited them to a private meeting with our team in Duesseldorf. In the meantime, we developed our sub projects and achieved first results which we discussed together on the 1st of june. On that day, we welcomed them with coffee and cake and started discussing right away. We talked about our latest results and further possible experimental designs for controlling our compartment more precisely. We received tremendous feedback, also referring to their own research, to other experts or unsolved questions. They pointed out one potential weakness of our project regarding the orthogonal peroxisomal protein import. They said that the cargo release inside the peroxisomal lumen could be a possible flaw since the natural PTS1 sequence is typically recognized by Pex8 at the luminal side of the peroxisomal membrane. Subsequently, Pex8 binds to the PTS1 and facilitates the cargo release of the protein of interest and PTS1 by competitive inhibition. Since we planned to create an artificial PTS1 peptide, the process of competitive binding could be affected by decreased binding affinity of Pex8.
Considering that, we started an alternative project for creating an orthogonal import machinery targeting the second protein import mechanism via Pex7 and the N-terminal peroxisomal targeting signal 2 (PTS2) with a random mutagenesis approach. Interestingly, the peroxisomal protein import facilitated by Pex7 does not include Pex8. Moreover, the exact mechanism of cargo release of PTS2 from Pex7 could not be clarified yet and remains to be elucidated. Nevertheless, Prof. Dr. Erdmann advised us to try the PTS2 mutagenesis approach since the mechanism works under countless conditions although its precise function is still unclear.
Furthermore, we discussed whether the precursor for the desired nootkatone pathway farnesyl pyrophosphate (FPP) is already present in the peroxisome. There is evidence that FPP is imported into plant and mammalian peroxisomes, but the import mechanism in yeast peroxisomes itself remains unrevealed. However, no measurements for FFP in yeast were performed yet but other experts like Dr. Florian David from biopatiolia who is working on metabolic engineering suggests that FPP is abundant in yeast peroxisomes.
Moreover, we gathered important clues about the cultivation of yeast strains which are deficient for the Pex11 protein as proliferation factor, learned how to design truncated peroxisomal membrane protein anchors in order to localize several fluorescent marker proteins or enzymes at the luminal peroxisomal membrane and obtained the opportunity to learn a peroxisome purification method performed by the working group of Prof. Dr. Erdmann, which is not trivial since the membrane properties are very similar to other cell organelles.
Conclusively, the advice from Prof. Dr. Erdmann boosted our knowledge on yeast peroxisomes and greatly improved our experimental designs. We are very grateful that he and his colleagues took the time for answering our questions and contributing to our discussions.
