Results
Introduction
While there still is much that can be done to improve our toolbox further, we are nonetheless extremely proud of our achievements. The many months of lab work definitely payed off! Below you can find the results of our efforts.
Sub-projects
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 obtain any evidences regarding orthogonal peroxisomal protein import.
PEX5 variant R19
Our second approach for the modification of the importer PEX5 was our designed 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. Figure 1.2 displays a fluorescence microscopy image proving our artificial protein import system in a PEX5 deficient yeast strain.
Our first results show that coexpression of R19 with mTurquoise tagged to PTS1* leads to import of the fluorescent reporter protein, indicated by localized fluorescence areas . The negative control consists of the wild type yeast strain carrying mTurquoise tagged with our designed PTS1* shows exactly the opposite: Fluorescence was detected in the whole cell, indicating that R19 is not capable to recognize and import the modified peroxisomal targeting signal with its cargo. Though, this figure does not prove that the reporter protein is located in the peroxisomes. Therefore we validated this results by coexpressing this import machinery with an peroxisomal marker protein as can be seen in the following.
PEX13, as an integral protein of the peroxisomal membrane, provides perfect features to mark the membrane in order to clarify whether the localized areas which were shown in figure 1.2 are indeed the peroxisomes As described in our experimental design, we used Pex13's transmembrane domain and fused mRuby to it. Figure 1.3 above shows clearly the location of the peroxisomes as the fluorescent signal of mTurquoise is definitely located in the peroxisomes, which proves that R19 transports 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, indicating that there is no functional protein import.
On the other hand we observed similar fluorescent signals in wild type yeasts that possess the modified PTS tagged to mTurquoise, as displayed in figure xx: Moreover, the figure shows the wild type yeast expressing mTurquoise tagged to the natural PTS1. Comparing both pictures, the wild type receptor is not capable to recognize our artificial PTS1* peptide and thus no import could be detected.
Conclusively, all our negative controls were not able to import mTurquoise into the peroxisomes, confirming the orthogonality of our artificial protein import mechanism.
Finally, our results clarify that we established a synthetic protein import machinery, which works fully independent from the natural yeast peroxisomal protein import system. We were able to demonstrate the recognition of an artificial PTS1* sequence by our designed PEX5 receptor R19 and that the same receptor does no longer recognize the wild type PTS1 sequence. That means we accomplished to modify a highly conserved protein import mechanism without destroying its function but changing its affinity for our distinguished peptide sequence. This facilitates the possibility to utilize the primordial peroxisomes as an artificial cell compartment.
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 indicate 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.
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.
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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 DeLoache, 2016 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.)
In order to have full control over the amount of expressed protein, we designed our plasmids with the inducible galactose promoter "pGAL1". Not only were we able to see that our fluorescent marked protein anchors from Pex3 and PEX26 would localize at specific points inside our cells but also to show that it was in deed the peroxisome they were accumulating at. For that we coexpressed each of our fluorescent membrane anchors together with a GFP protein that was fused to a PTS1 sequence and thus imported into the peroxisome. Under the fluorescent microscope the colocalization of both, the green fluorescing GFP and the red fluorescing mRuby is clearly visible, showing that our anchors integrated into the peroxisomal membrane.
Finally we used the same approach to send a mRuby-tagged bacteriorhodopsin to our compartment. In coexpressing it with the same GFP as in the previous steps, we could show that the bacteriorhodopsin as well as Pex3 and PEX26 were successfully integrated into the membrane of our compartment. Since bacteriorhodopsin is a rather complex protein, we're very optimistic about integrating other proteins into the membrane using the same approach.
Outlook
The ultimate goal of this subproject is, to have a complete set of ready to transform membrane proteins that could be combined with any promoter to create the optimal conditions for each desired situation. Besides bacteriorhodopsin, we also started to work with sugar translocators, since yeast does not posses the ability to import it into or export it from the peroxisome. This would open up a whole new chapter of peroxisomal usage, from example as a temporary storage compartment.
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In order to verify the cytosolic expression of ValS, BM3 and ADH we performed a Western blot analysis for each of the enzymes. We were able to verify the expression of ValS, BM3 and ADH with and without PTS1 in the yeast cytoplasm and peroxisomes, respectively.
Since protein abundance of ValS, BM3 and ADH, both with and without a peroxisome targeting signal, was verified in the cytosol, a mass spectrometry analysis (MS analysis) of nootkatone and its precursor valencene was performed.
There are three approaches in MS analysis. The first one is the qualitative approach in which is only determined if the substance is present or not. The second and third kinds are the quantitative or semi-quantitative approach in which the absolute or relative amount of a substance is investigated.
First, we tried to validate nootkatone and valencene with the first approach and screened our samples for the existence of the first intermediate valencene and our final product nootkatone.
We could not show the synthesis of Nootkatone nor valencene in our yeast yet, but we could smell its characteristic scent. The lack of proof via MS could result from an inefficient sample extraction or to low concentrations of product in the sample. The latter could also explain a peak in the MS analysis where nootkatone was expected. Unfortunately the peak is below detection limits and therefore can not be assumed to be a definite proof of nootkatone production.
Outlook
For further investigation we plan to do a semi-quantitative analysis by comparing the yield of samples of cytosolic synthesized nootkatone and peroxisomal nootkatone. To do so we need to perform a peroxisome purification in order to compare the amount of product produced. With this comparison we hope to proof that compartmentation, and thereby bypassing the problem of toxicity of substances for yeast, is the key for better yield of Nootkatone. Also we intend to do a quantitative MS analysis to clarify if the yield of our Nootkatone pathway is anywhere near the yield pathways with other enzymes/ enzyme-combinations could achieve. There was also the idea of exchanging cytosolic enzymes with membrane bound ones to see if there is any change in yield.
Already planned but not implemented due to lack of time, we have also a proof of localization of the pathway enzymes. Therefore we exchange the 3a part (Dueber Toolbox) 3xFlag/6xHis of the plasmid with an other fluorescent 3a part, namely mRuby2. We can then show the localization of the enzymes via microscopy.
Another factor to consider in further studies will be the available amount of FPP in peroxisomes. FPP is the essential precursor of nootkatone synthesis. But we cannot say yet if there is enough FPP in the peroxisome to justify an expression of the pathway in it. If there is not enough FPP available to generate Nootkatone over the concentration of 100 mg/L it does not matter that beta-nootkatol and nootkatone is toxic to the cell.
To tackle this problem there are three methods reported to increase the amount of FPP in the peroxisome for nootkatone production. The first one is to introduce a knockout mutation of squalene synthase and obtaining a mutant that is capable of efficient, aerobic uptake of ergosterol to limit the use of FPP for the sterol biosynthesis. The second approach is to knock out a phosphatase activity to limit the endogenous dephosphorylation of FPP. Third is to upregulate the catalytic activity of HMGR. Takahashi et al. (2007) .
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