MD simulations

The first experiments we performed in the wet lab are the tests of the receptors we modelled via molecular dynamics. As soon as we finished building our constructs, we transformed them into PEX5 knock out yeast cells. The results of this experiments can be seen in the following figure.

The fluorescent signal of mTurquoise was detected in the whole cell of each modelled receptor−peptide combination. This indicates that our receptors were not able to recognize the PTS-variants tagged to mTurquoise and thus we did not see import.

PEX5 variant R19

Our second approach for the modification of the importer PEX5 was our so called receptor R19. Based on published literature we built this receptor by replacing three amino acids within the PEX5 protein sequence of the wild type yeast. The corresponding modified PTS1* is characterized by its -SYY sequence at the very end of the peptide. In the following figure one can see the microscopy image of this import system variant cloned in PEX5 knock-out yeast.

PEX13, as an integral protein of the peroxisomal membrane, provides perfect features to mark the membrane to validate proper location. As described in our experimental design, we used Pex13's transmembrane domain and fused mRuby to it. The figure above shows clearly the location of the peroxisomes and the fluorescent signal of mTurquoise is definitely located in the peroxisomes., which proves that R19 transport our cargo into the peroxisomes. On the contrary, mTurquoise is located in the whole cytosol in the wild type strain with PTS1* and the R19 strain, meaning that there is no functional import. On the other hand we observed similar fluorescent signals in wild type yeasts that possess the modified PTS tagged to mTurquoise, as can be seen in figure xx: In the same figure one can see the wild-type yeast expressing mTurquoise tagged to the natural PTS1. If one compares both pictures, it becomes directly clear that the wild-type receptor is not able to recognize our PTS1* and thus no import could be detected. So all our negative controls were not able to import mTurquoise into the peroxisome, which is exactly what we wanted to achieve.
The results show that our new designed import machinery works, but even more: Together with the negative controls this proves that we established a completely orthogonal import machinery that is fully independent from the natural yeast peroxisomal import machinery.

Violacein assay

The Violacein assay would have been an easy and fast application to identify possible yeast colonies that possess a functional new import machinery. It would have eased finding a fitting peroxisomal targeting signal for our PEX5 variants due to the huge number of different PTS1 variants we could have screened using this method. Because time ran out and we already found a fitting import machinery in our receptor R19 and our modified PTS1* P*, we decided to discontinue this experiment and focused on the validation of our previously generated results.

Conclusion

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.

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The biased mutagenesis of the PTS2 could be characterized with a split-variant of YFP ( yellow fluorescent protein) or a split-luciferase. YFP tends to self assemble, consequently appropriate internal controls have to be designed Horstman (2014). Luciferase is highly efficient because almost all energy is converted into light, the protein is thus very sensitive Azad (2014). It offers a suitable alternative to YFP as a single readout protein. We expected to detect luminescence as well in the actual samples as in the negative control containing no peroxisomal targeting signals due to split assembly in the cytoplasm. Unfortunately no suitable method to measure luminescence in the peroxisomes was established in this project. Prerequisite for detecting luminescence is the availability of the substrate luciferin. It does not diffuse into the peroxisome in concentrations high enough for the luminescence reaction and becomes the limiting factor Leskinen (2003).

An alternative step to verify the localization of the assembled split-luciferase in the peroxisome is to extract and purify the organelles. Prof. Ralph Erdmann established this method: a cell-free homogenate is created and the organelles are pelleted by centrifugation steps Cramer (2015). This workflow can be used to characterize the content of the purified peroxisomes by Western blot analysis.

To measure the import efficiency of a vast amount of targeting sequences via split-luciferase one needs to ensure a sufficient luciferin concentration in the peroxisome. Therefore luciferin importer have to be implemented in the peroxisomal membrane. Since this implies a huge cloning effort split-luciferase is not suitable for high throughput screening. ´

At the random mutagenesis approach one expected green and white colonies indicating varying import efficiencies. The colonies containing “DNK” or “NNN” substitutions in the variable PTS2 region show a wide range of colours between white and dark green. The wild type PTS2 colonies depict a constant light green colour. The negative control containing VioE without a PTS2 shows a dark green colour in every colony.

Therefore we were able to generate targeting sequences of different effectivities. Subsequently the OD₆₀₀ and the fluorescence with an excitation wavelength of 535 nm and emission wavelength of 585 nm were measured. According to XXX https://www.nature.com/articles/ncomms11152 zitieren XX production of PDV was associated with a yet unknown red fluorescent product, detectable at the described wavelength. The import efficiency can be defined as the fluorescence per OD₆₀₀. A wide distribution of different values were observed indicating a broad variety of different PTS2 versions.

A high value correlates with an inefficient targeting sequence since VioE is not imported into the peroxisome with the respective sequence. A low fluorescence per OD₆₀₀ indicates a strong targeting sequence resulting in a low VioE concentration in the cytoplasm and no conversion of Tryptophan to PDV.

The next step would be to isolate the plasmids of promising yeast strains and sequence them. Subsequently mutations leading to an increased import can be characterized and organized in a library consisting of different part for different import effiency.

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To check whether our membrane anchors localize in the peroxisomal membrane we used a Zeiss Elyra PS microscope. For Pex15 we observed localization using a construct with mVenus fused to the C-terminus of the Pex15 version we used. The fluorescence in the cells showed the typical shape of a peroxisomal localization (Figure 5). Shown in figure 4 is the localization of PEX26, which was highlighted using an N-terminal fusion with the fluorescent Protein mRuby. The microscopy pictures also indicate peroxisomal localization and even an co-localization with sfGFP-PTS1

Next we measured secretion of compounds that are inside our artificial compartment, using a liquid GUS-assay*needs to be change* . Towards this purpose we coexpressed GUS-PTS1 and Snc1 fused to different membrane anchors. For lysis controls, GUS with PTS1 was expressed in the Strains BY4742 and BY4742 with the gene Pex11 deleted.
The fluorescence increase over time of the samples which are decorated with snares*needs to be change* is higher in comparison to that of the lysis controls. The highest activity could be measured in the samples using the truncated Pex15 membrane anchor without a linker. The same construct in a background strain with a Pex11 deletion showed a lower GUS activity in the supernatant. The strains expressing Snc1 linked to PEX26 or Snc1 directly fused to the n-Terminus of Pex15 only showed minor increase of RFU over time. (Figure 6.)

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