Project Plan Discussion with 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. The group´s research topics comprise biogenesis of the peroxisomal membrane, peroxisomal metabolism and metabolite transport, proliferation of peroxisomes and peroxisomal import. During 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, discussing protein import, peroxisomal membrane proteins and peroxisomal metabolism 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. To maintain 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. We talked about our latest results and further possible experimental designs for controlling our compartment more precisely. They pointed out one potential weakness of our project regarding the orthogonal peroxisomal protein import. They mentioned 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 sequence facilitating 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 working on similar fields of metabolic engineering suggest 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 conduct a peroxisomal 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.

Brainstorming with John Dueber and Will DeLoache

As we started with our idea of creating an artificial compartment, we came up with different design concepts. When we chose peroxisomes as our chassis we found the work of John Dueber, who characterized the import of proteins and metabolites into the peroxisome. With these tools he set the foundation for utilizing yeast peroxisome in order to build a synthetic organelle. Since we also used his Yeast Toolkit for modular, multipart assembly, we decided that he is the right person to discuss our project designs and contacted him for a Skype call. During the talk we discussed the import efficiency of the Pex7 import mechanism, problems regarding the secretion of the compartments, the importance of membrane permeability and the final goal of controlling the chemical environment of the peroxisomal lumen by integrating new membrane proteins. Due to the low import efficiency of the Pex7 import he expressed concerns using it as the import mechanism of our artificial compartments and instead focuses on the much more stable Pex5 import.

Regarding our secretion project he told us that he realized that the peroxisomes seem to be fixed to the cytoskeleton or at least are very immobile. Prof Dueber proposed that if we can immobilize the peroxisome we would get an increased amount of secreted protein or metabolite. Therefore we went back to design and constructed an INP1 knockout yeast strain with the Cas9. INP1 is a protein that tethers the peroxisome to the ER by binding to Pex3 and the knockout leads to immobilized peroxisomes.

Furthermore we discussed that one of the biggest bottlenecks of engineering artificial compartments, especially for metabolic engineering, is the control of the peroxisomal lumen properties, such as pH, redox state or cofactor balance. To overcome this problem we aimed at integrating new membrane proteins into the peroxisomes. We chose the proton pump bacteriorhodopsin and measured the change of pH in the artificial compartment with a pH dependent GFP. Additionally we designed and integrated different plant transporters for several metabolites in our toolbox. Long-term goals like these could be achieved with our toolbox and will provide unprecedented control over the chemistry and physiology of the cell.

Discussing common results with Alison Baker

After the first modeled receptors and peptides were tested negative, we started a second design cycle for an orthogonal import of proteins in our artificial compartment. Therefore we had a Skype call with Professor Alison Baker who is working on basic research aspects of the membrane transport processes in plant cells with a focus on transport of proteins and metabolites into peroxisomes. She gave us a more detailed view of the protein-protein interactions between the pex5 and pts1 in P. patens and A. thaliana. Based on the new knowledge we modeled the interactions between the modified receptor and pts1 sequence for our artificial compartment. After the second design phase, the new receptor pts1 pair was tested and could be validated to be fully orthogonal.

Interviewing Dr. Florian David – Biopetrolia

Having acquired an enlightening amount of foundational knowledge about peroxisome basics, compartmentation, and synthetic biology toolboxes, we set out to speak to experts from companies who are already using synthetic biology applications to replace inefficient production cycles by overcoming natural pathway complexity. During our research, we came across Dr. Florian David from Biopetrolia, a Swedish company that works on yeast strain engineering to find resource-efficient ways to produce valuable compounds such as cosmetic bioingredients, specialty chemicals, nutraceuticals, pharmaceuticals, biofuels, etc. [1]. Florian kindly agreed to a Skype meeting to listen to our project plans and discuss opportunities and issues.

So far, controlling the size and number of our compartment via Pex11 knockout or overexpression was considered a mere additional feature of our toolbox. Pex11 knockouts were used to create a reduced amount of giant peroxisomes, whereas Pex11 overexpression should provide a large amount of smaller ones. Since Florian and his team are constantly working on finding ways to create resource-efficient and highly productive pathways, he suggested working on the peroxisome’s morphology in order to positively influence the yield of metabolic reactions. One of their research groups had previously worked on optimizing the production fatty-acid-derived fatty alcohols, alkanes and olefins in peroxisomes, while at the same time decreasing the accumulation of byproducts. In order to achieve their goal, they worked with both, a Pex31, 32 knockout, as well as Pex34 overexpression, both of which positively affect the size and number of peroxisomes while at the same time lead to higher metabolic yield. For our purpose he recommended working with a Pex34 overexpression, since they found out that a knockout of Pex31, 32 leads to higher permeability of the peroxisomal membrane. After the conversation, we successfully implemented his suggestions into our project.

Finally, since speaking to experts should also help us to understand the potential of synthetic biology applications, Florian further discussed the benefits of both, their work and our compartmentation approach. These novel applications foster the introduction of tailor-made properties into molecules, making it easier to synthesize substances that are currently obtained from rare natural resources and mostly in environmentally-unfriendly ways. Economically, working with these novel approaches helps gaining independence from market fluctuation of traditional production processes. This means that products like pharmaceuticals, with a universal benefit could be produced at a low cost and therefore made available to a larger amount of the population. Furthermore, producing through compartmentation could ensure stable, scalable and on-purpose production through selective biological processes, bypassing chemical processing.

The conversation with Florian David was valuable in many ways. Not only did he foster our understanding of compartment applications, he also broadened our horizon regarding the potential of our work and common approaches.