Revision as of 20:07, 1 November 2017

Design

We designed a novel toolbox for complete control over all major functions of the peroxisome. The toolbox is our solution for improving the engineering workflow and predictability of synthetic constructs. Interested? Find out how.

Scientific background

The root problem

Synthetic biology is an engineering discipline. And while we are able to plan our constructs with tools like biobricks, a major difference to e.g. electrical engineering is that they are not nicely isolated on a chip, but surrounded by all types of interfering agents. One of the major issues regarding protein expression in a novel chassis is unwanted and unexpected crosstalk between engineered pathways and the native cellular processes of the production host. The other one is toxicity of the products or intermediates of the pathway. Both can greatly change our system’s behaviour which in some cases leads to us to having to trial-and-error find a solution, making our previously modeled optimization useless.

Our approach

The natural approach of organisms to deal with metabolic interference and toxic byproducts is subcellular compartmentalization. This has proven to be a functional solution in naturally occurring pathways in eukaryotes as well as new synthetic pathways for biotechnological application. Thus, the creation of a synthetic organelle presents a suitable strategy to increase the efficiency and yield of non-native pathways. A common approach is to build up artificial compartments from scratch. Many breakthroughs have been achieved in the last decade, however the creation of a fully synthetic compartment is yet a milestone to reach for. We on the contrary want to start by engineering artificial compartments through orthogonalization.

The peroxisome is the ideal starting candidate as it has many advantages over other compartments, including it being able to import fully folded proteins and not being essential in yeast under optimal growth conditions. Our project’s aim is to create a toolbox for manipulating and creating customizable peroxisomes as a first step towards synthetic organelles.

By modifying the import machinery of yeast peroxisomes only selected proteins will be imported into the peroxisomes leaving the researcher in full control over the content of the peroxisomal lumen. Furthermore our toolbox will include a secretion mechanism for the synthesized products, various intra-compartmental sensors, modules for the integration of proteins into the peroxisome membrane, as well as optogenetic control for some of these parts for a more precise spatiotemporal control.

As a proof of concept for the functionality of the toolbox and the customizable compartment two metabolic pathways will be integrated into the altered peroxisome: (i) Violacein biosynthesis and (ii) nootkatone biosynthesis. Violacein, a bisindole formed by condensation of two tryptophan molecules, is a violet pigment and thus easy to quantify in the cell. Nootkatone on the other side is a natural compound found inside the peel of the grapefruit, which gives it its characteristic taste and smell. In addition, nootkatone is a natural mosquito and tick repellent that is already being commercially used and industrially manufactured. Unfortunately, the production costs are extremely high. Furthermore, the production of nootkatone inside yeast is challenging as it is toxic for yeast and thus, the production efficiency is rather low. A successful implementation of the synthetic compartment will show increased yields in the production of these compounds and showcase the potential of this approach for similar future applications.

Cloning strategies and the Yeast Toolbox for Multipart-Assembly

While describing our cloning strategies we mentioned several levels, which stand for different stages of our plasmids. They are further described in the work of J.M. Dueber and colleagues, who designed the well established yeast toolkit we used in this project (Dueber). The toolkit offers the possibility to design plasmids with desired antibiotic resistances, promoters as well as terminators from standardized parts. It also provides fluorescence proteins, protein-tags and many more useful components as part plasmids. These part plasmids are distinguished in different part types due to their specific overhangs to ensure their combination in the correct order (e.g. promoter - gene of interest - terminator) all in a versatile one-pot Golden Gate reaction without time-consuming conventional cloning steps.

Figure 1: The yeast toolkit starter set comprises of 96 parts and vectors. The eight primary part types can be further divided into subtypes. (Dueber)

The cloning steps regarding the plasmid levels are implemented in E.coli in order to reduce the required time to generate the final plasmids. The different levels are therefore defined by their part content and their antibiotic resistances.

To generate a level 0 plasmid, the gene of interest is ligated into the provided level 0 backbone via Golden Gate assembly using the enzyme BsmBI. The backbone contains a resistance to Chloramphenicol, as well as an origin of replication, creating a very basic yet functional plasmid.

The level 1 plasmid contains a promoter and terminator suited for S. cerevisiae. There is the possibility of including a polyhistidine-tag if there is a need for Western blot analysis. The antibiotic resistance contained in the level 1 plasmid changes from chloramphenicol to ampicillin which enables filtering out residual level 0 plasmids contained in the Golden Gate product. Furthermore, the Dueber toolbox includes the possibility of designing GFP-Dropout cassettes. These are custom-built level 1 backbones whose inserts are sfGFP as well as promoter and terminator suited for E. coli. Upon a successful cloning step the GFP is replaced by the part(s) of interest, and correct colony shows a white colour. In case of a wrong ligation event colonies show a green fluorescence. This provides a very useful tool to detect unsuccessful cloned colonies. The enzyme used for level 1 changes from BsmBI to BsaI to avoid any interference between different steps.

The level 2 plasmid combines two or more genes of interest with their respective promoters, terminators and tags. The resistance changes from ampicillin to kanamycin. The enzyme of this step is BsmBI again. This level is useful, if the construct you are designing requires multiple genes to be transformed into one yeast strain.

Yeast nomenclature

To make it fast and easy to differentiate between endogenous and heterologous genes and gene products we decided to use S. cerevisiae nomenclature according to yeastgenome.org.

Below nomenclature at the example of your favorite gene 1, YFG1 is explained.

Letter code	Meaning
YFG1	Your favorite gene S. cerevisiae wild type allele
yfg1Δ	Gene deletion of your favorite gene
Yfg1	Protein product of YFG1
YFG2	A heterologous gene product from mammalian cells

Design of our sub-projects

Engineering of Pex5 and PTS1

Designing our receptors

To achieve the engineering of an orthogonal import pathway, we followed two approaches regarding Pex5. The first is based on targeted mutagenesis based on educated guesses which is first verified by molecular dynamics and later experimentally in the laboratory. The second approach is based on a recently published paper: We searched for literature dealing with the modification of the peroxisomal import machinery. During our research we came across a paper of Alison Baker et al., published in 2017, in which they present a synthetic construct of the Pex5 protein, partly Arabidopsis thaliana and partly Physcomitrella patens. Compared to the wild type Pex5, this one shows different binding affinities since it interacts with a PTS1* variant that does not interact with the wild type Pex5. Since the protein sequences of yeast's and plants's Pex5 differ quite a lot, we aligned both sequences to understand where the mutations were set.

**Figure 1.1:** Alignment of the *Arabidopsis thaliana's* PEX5 and *Saccharomyces cerevisiae*Pex5

The alignment shows three red marked amino acids we changed in our receptor sequence. Interestingly, these mutations are located within the TPR motifs of our Pex5 protein and this persuaded us to try out this receptor, we call it R19. Due to lack of time we tested this Pex5 variant in silico and in vivo simultaneously. We started molecular dynamics simulations with a couple of PTS variants that we already tested with our previous designs − one of them was actually the variant they used in the paper (YQSYY). The details and results of our structural modeling can be found in the modeling section.
Furthermore, we synthesized this variant and together with two receptors we designed based on educated guesses we got three receptors for our experimental work.

Experimental design

The easiest way to verify successful import is the localization of a fluorescent protein within the peroxisome. Due to that, we decided to tag mTurquoise with our PTS variants. Additionally, we wanted to mark the peroxisomal membrane, to be absolutely sure about the localization within the peroxisome. For that cause we chose the transmembrane domain of Pex13, tagged with the fluorescent protein mRuby.

Pex13−mRuby

We used the peroxisomal membrane protein Pex13 as a fluorescent marker − by just using the transmembrane domain of Pex13 with a short linker, we make sure that it has no influence on the peroxisomal features. To obtain a higher differentiation from mTurquoise, which we use for another construct, we chose to work with mRuby. Literature research revealed that such constructs have been tested before − Erdmann et al. (2004) described a construct containing only PEX13_200-310 with a C-terminal GFP.

**Figure 1.3:** PEX13 construct with C-terminal mRuby.

Pex5 variant

Alles nur Vorschläge, war so auch schon okay, übernimm was du magst, bin ir teilweise bei meinen sätzen auch unsicher :D

In order to achieve an orthogonal peroxisomal protein import machinery we used a Pex5 knockout yeast strain in which we and transformed our artificial it with a plasmid based Pex5 variant containing a modified PTS1* binding region. Our variation facilitates that is supposed to the detection of detect a non native PTS1* variant instead of the wild type PTS1one. The construct contains a medium strength promotor, the Pex5 gene and a terminator. The whole remaining plasmid parts can be seen in the plasmid map below. is displayed in figure X.

mTurquoise−PTS

Our approach for import verification of the protein import is based on 3D sim microscopy. a For that reason, the fluorescent protein mTurquoise including our modified tagged with the PTS1* variants was used in order to detect potential localisation of fluorescence signal. After several promotor tests with different strengths, we decided to express this construct only in low amounts, since this was the most suitable possibility to detect potential mTurquoise localization. Moreover, mTurquoise . This is advantageous sincebecause we detect only a low signal if the import does not work, hence small amounts of the protein are distributed in the whole cytosol. In contrast we see a clear signal if the import does work due to the relative high concentration inside the peroxisome.
Our construct is depicted in figure Xthe figure below.

**Figure 1.5:** Fluorescent protein mTurquoise tagged with the PTS variant.

Combination of our constructs

To combine our constructs, we cloned our PEX5 and mTurquoise constructs into a level 2 plasmid portrayed below.

**Figure 1.6:** Level 2 plasmid containing the Pex5 gene and the fluorescent protein.

Subsequently, We then did a co-transformation of with the PEX13−mRuby plasmid and the level 2 plasmid was performed in order to verify peroxisomal colocalizationcombine everything that is needed into our yeast.

PTS screening

Trusting on our targeted approach alone seemed risky − that is why we planned a In addition to our side directed approach in which we changed amino acids in the Pex5 binding pocket, we planned to perform a PTS screening in order to find the most favorable PTS for our three receptors. Dueber et al. (2016) used the Violacein assay for a similar purpose. In this study, They screening screened for the most qualified best PTS sequence for suitable for its recognition by the wild type receptor was performed successfully. r and were successful. Hence another subproject of our team is the integration of the Violacein pathway into the peroxisome (Violacein), we were already supplied with all necessary enzymes − VioA, VioB and VioE.

**Figure 1.8:** Pathway leading to the production of prodeoxyviolacein − the assay is based on the green color to identify colonies without functional import mechanism.

The figure above shows the principles of the assay. VioA and VioB are localized in the cytosol and lead to the production of the IPA imine dimer while VioE is tagged with a PTS1 variant. Successful import leads to white colonies whereas missing import results in green colonies due to the cytosolic production of prodeoxyviolacein.
Our rests upon the following two plasmids which are co-transformed into yeast.

**Figure 1.9:** Plasmids used for Violacein assay. The left one carries the coding sequence for the Pex5 variant, VioA and VioB whereas the right one contains the gene for VioE plus the attached PTS1 variant.

As shown above, we created one plasmid containing VioA, VioB and one of our Pex5 variants while the other plasmid only contained VioE. We then designed primers which bind to the VioE plasmid to amplify the whole plasmid except the terminator − random PTS1 variants were attached to VioE with the help of a random primer library. Following up, we did the ligation with the corresponding terminator and obtained a mix of several different VioE-PTS1 plasmids.
After plasmid amplification in Escherichia coli we then co-transformed yeast with the two constructs and waited for the colonies to grow. With the yeast growing, prodeoxyviolacein should be produced in yeast cells with absent import (green color) and the IPA imine dimer (white color) should be produced in those with functional import.
Plasmid preparation of those with white color and subsequent sequencing leads to the identification of functional PTS1 variants. Afterwards, we repeat the cloning steps described before to obtain a mTurquoise−PTS1* construct and co-transform it with the corresponding Pex5 variant. Eventually, the correct localization of mTurquoise tagged with these PTS1 variants provides proof for its function.

Mutagenesis of PTS2

To characterize the import efficiency for the site-directed PTS2 firefly luciferase was used. Luciferase is a luminescent protein which can be split in a C- and a N-terminal part. Only when combined, luminescence can be detected. To measure the import efficiency the two parts will be expressed and imported into the peroxisome in a separated way. The smaller part (Split2) of the split luciferase will be brought into the peroxisome first via the PTS1 dependent pathway. The other part is imported via the respective modified PTS2 sequence. The better this sequence is recognized by Pex7, the stronger the luminescence of the assembled luciferase can be detected in the peroxisome. There is a chance of split parts of the luciferase assembling in the cytosol if the import is too slow. To avoid wrong conclusions of the luciferase localisation, we designed a negative control experiment. It includes a split luciferase similar to the one used in the initial experiment, but without the peroxisomal targeting sequence. Consequently there will be no import into the peroxisome. If we subtract the luminescence of the negative control experiment from the luminescence of the main experiment we can define the degree of import.

In addition to a directed approach according to Kunze and colleagues we also want to perform a random mutagenesis experiment to alter the five variable amino acids of the core region of the PTS2 sequence in an unbiased manner. The aim is to generate a library of different peroxisomal PTS2. The 15 nucleotides are assembled by chance. In the DNA synthesis this sequence will either be described as [NNN]₅ or [DNK]₅. N stands for all four nucleotides mixed, K for either G or T and D for A,G or T. The “DNK” composition prohibits two out of three termination codons. Additionally with this library the amino acid frequency is improved towards a balanced ratio in between the different kinds (Dueber).

Each approach could generate up to 415 DNA sequences, which is roughly 1,07 billion. On the level of the amino acid sequence there are 3,2 million possibilities, since each residue can be taken by 20 different amino acids. For the assay we therefore need a high throughput method.

We adapted work of DeLoache, Russ and Dueber using the violacein pathway to measure the import effectiveness of tripeptides. The pathway consists of Violacein A (VioA), Violacein B (VioB) and Violacein E (VioE). It converts tryptophan into the green product prodeoxyviolacein (PDV). The first two enzymes, VioA and VioB, are expressed in the cytosol, and the third one, VioE, is targeted to the peroxisome with a PTS1 sequence. The degree of import can be measured by the intensity of green colour of the colonies. An efficient import signal leads to a strong import of the VioE into the peroxisome and subsequently to white colonies, because the intermediates cannot diffuse into the peroxisome to its respective enzyme. DeLoache et al. showed that there is a proportional correlation between the concentration of the green product PDV and a red fluorescent substance. The concentration of this product displays the import efficiency of the respective sequence.

This assay has been used for the evaluation of the generated PTS2 sequences. The VioE-PTS2 plasmids are harvested and cotransformed with a VioA-VioB plasmid. Each plasmid contains a specific auxotrophy marker. Consequently every growing colony contains both plasmids. To evaluate the respective sequence the concentration of the red fluorescence is measured. The more fluorescence is detected the more VioE is in the cytosol. Therefore the respective PTS2 is not that efficient. The other way around a low concentration of the fluorescent substance correlates with an efficient import via the respective PTS2.

Experimental Work/Design

In order to test our hypothesis we fused the last 59 amino acids of the C-terminus of human PEX26 (AA 246-305) to a red fluorescent protein, to further elucidate the Pex3/Pex19-dependent import. mRuby is generally used as a marker in combination with a fluorescent microscope to visualize the localization of the fusion protein. The C-terminus of PEX26 contains a helical signal-anchor, which serves as both, a mPTS and transmembrane domain. We designed our construct with mRuby2 fused to the N-terminal side of the PEX26-C-terminus, this way the mRuby should face the cytosolic side of the peroxisomal membrane. Quite similar to our mRuby-PEX26 approach, we designed a construct for the ER-dependent import. Therefore, we fused the mRuby2 fluorescent protein to the N-terminus of Pex3 (AA 1-39). This construct should be N-terminally anchored in the peroxisomal membrane, with mRuby2 again facing the cellular lumen.

Our main goal is to introduce a rather complex membrane protein to the peroxisome that can alter specific traits. For that we fused the Pex3 N-terminus (AA 1-39) to a Halobacterium salinarum bacteriorhodopsin protein (AA 16-262), replacing the first 16 amino acids (Pex3-BacR). The original archaeal bacteriorhodopsin acts as a proton pump by capturing light energy to move protons across the membrane out of the cell. The resulting proton gradient is subsequently converted into chemical energy. Our assumption is that the first transmembrane segment determines the orientation of the following protein and that therefore due to the N-terminal anchoring signal the bacteriorhodopsin will be inserted in reverse orientation, pumping the protons into the peroxisome. This way the pH of the peroxisomal lumen could actively be controlled and adjusted.

Finally, our project involved combining the work of other subteams to verify the localization of our constructs in the peroxisome and analyze the effects they have on the import. Therefore, we are using the superfolded-GFP protein, another fluorescent marker, which is in our case fused to the peroxisomal import sequence PT1, and a version of Pex11 that is fused to the fluorescent marker Venus. Both markers emit light in the green light spectrum, were as mRuby2 emits light in the red part of the spectrum, giving us a strong contrast and an easy way of differentiating between the two under the fluorescent microscope.

To physically create our constructs, we researched the DNA sequences of bacteriorhodopsin, Pex3 and PEX26 via UniProt and pre-designed our fusion constructs with the software tool „Geneious“. We ordered the synthesis of three separate parts ( Pex3, PEX26 and Pex3-BacR) from IDT. To ease out the process of assembling our plasmids, we used the „Dueber Toolbox", containing various parts such as promoters and terminators, to tailor the plasmids specific to your needs. Finally, to combine all the selected parts, we used the „Golden Gate” assembly method.

Experimental Design

We will adapt the system of Sagt and colleagues to secrete the content of our modified compartments (Sagt et al, 2009) .
For the application of this system in S. cerevisiae we use a truncated version of the v-SNARE Snc1 to decorate our compartments(Figure 3.1) (Gerst et al, 1997) .

**Figure 3.1 A diagram of the general domain structure of Snc1.** V is a variable domain which is not important for the binding to the t-SNARE. TM is the transmembrane domain. H1 and H2 are the a-helical segments, forming the SNAREpin with the t-SNARE (Gerst *et al*, 1997)

To decorate the compartments with the SNARE we use a peroxisomal transmembrane protein . In our case we use the proteins Pex15 or PEX26, which were further investigated in another sub project, and fuse Snc1 to the N-terminus. We expressed these constructs of membrane anchor and Snc1 constitutively under control of the RPL18B promotor. In case of Pex15 we used a truncated version, lacking a large part of the N-terminus, only consisting of the transmembrane domain (315-383) (Figure 3.1). For PEX26 we use the truncated version published in Halbach et al. (Halbach et al, 2006) .

**Figure 3.2 Concept of secreting peroxisomal contents to the supernatant.** For the secretion, the membrane anchor Pex15 or Pex26 is used. This anchor is used to decorate peroxisomes or our modified compartments with the v-SNARE Snc1. For the secretion Snc1 interacts with the t-SNAREs in the cell membrane. Induced from this interaction the vesicle and cell membrane fuse and the content of the compartment is secreted to the supernatant.

We verified our secretion using Beta-glucuronidase (GUS) as a reporter protein. In 2012 Stock and colleagues described the GUS reporter assay for unconventional secretion (Stock et al, 2012) . With it, it is possible to determine whether a protein is secreted conventional and is N-glycosylated or secreted unconventional and not N-glycosylated. GUS is a bacterial protein with an N-glycosylation-site, which is active only if the protein is not N-glycosylated. The GUS-activity can be measured with different reagents in plate or liquid assays. Liquid assays can be applied qualitatively as well as quantitatively to measure differences in activity. If GUS is secreted by the conventional pathway the N-glycosylation leads to inactivation of the enzyme (Stock et al, 2012) (Fig 3.2).

**Figure 3.3 The GUS Assay.** GUS secreted with an unconventional secreted protein like Cts1 from Ustilago maydis active in the supernatant. GUS secreted with a conventional Signal peptide (Sp) inactive in the supernatant. If GUS is in the cytoplasm there is also no activity (Lysis control) (Feldbrügge *et al*, 2013) .

GUS will be imported to the peroxisome with the PTS1 sequence and measured quantitatively in the supernatant. We will use a coexpression of GUS-PTS1 and Snc1-Pex15 or Snc1-PEX26 to identify the secretion of the compounds. Furthermore, we will use GUS-PTS1 expressed in S. cerevisiae without Snc1 fused to a membrane anchor for a control. We will measure the active GUS in the supernatant with a liquid assay based on the turnover of 4-methylumbelliferyl-beta-D-glucuronide to 4-methyl umbelliferone (4-MU) (Blázquez et al, 2007) . Here we expect a higher activity of GUS in the supernatant of cultures with Snc1 decorated peroxisomes.
To increase the variability of our constructs we also designed vectors with and without a GS-Linker connecting the Snc1 with the Pex15. Additionally we tested our constructs in strains with a deletion of Pex11 . This deletion leads to formation of larger peroxisomes and may increase the efficiency of our secretion mechanism.

Heading

Sensors

To enrich our toolbox we decided to measure four essential physiological factors: ATP, NADPH, Glutathione and the pH.
ATP/ADP conversion is used as an energy currency in many cellular processes like translocation of proteins and metabolites or anabolic and catabolic turnovers. It is assumed that ANT1P an ATP/AMP antiporter is located in the peroxisomal membrane and that the re shuttle mechanism of the peroxisomal protein import machinery is ATP dependent (Palmieri L. et al., 2001). NADPH plays an important role in anabolic pathways and is also indispensable to our desired pathways of nootkatone and violacein. Glutathione is an antioxidant and redox buffer which is also found in yeast peroxisomes. It is used as cofactor by at least two types of peroxisomal proteins the glutathione peroxidases and glutathione transferases, which reduce lipid- and hydrogen peroxides or transfer glutathione to lipid peroxides for the purpose of detoxification (Horiguchi H. et al., 2001). Furthermore, it partly represents the redox state of the peroxisome. Knowing the pH of a compartment is important to predict the activities of almost all enzymatic processes inside of it and to follow up acidification and basification upon conversion of metabolites.

We finally chose two ratiometric sensors to perform measurements with. The pH sensitive and glutathione redox state reporting green fluorescent proteins pHLuorin2 (Mahon M. J. et al., 2011) and roGFP2 (Schwarzländer M. et al., 2016). We aim to target them either in the peroxisomal lumen or the cytosol. To achieve peroxisomal targeting we attach the peroxisomal targeting signal 1 with Golden Gate cloning.

Figure 5.1 Level1 plasmids with promoters of medium strength and uracil auxotrophy. Top left roGFP2 cytosolic. Top right roGFP2 peroxisomal. Bottom left pHLourin2 cytosolic. Bottom right pHLourin2 peroxisomal.

Using promoters with different expression strength in order to find optimal measurement conditions is of high interest. There is a trade off between a high signal-to-noise ratio and self induced effects which are both dependent on expression levels (Schwarzländer M. et al., 2016) . This cannot be generalized for each sensor. For example, pHlourin2 has sparse influence on the existing pH because of the buffer effect of proteins.
Validation of the peroxisomal localization can be achieved via fluorescence overlap of the sensor and a peroxisomal marker in our case pex13-mRuby (import mechanism).

Figure 5.2 Level2 plasmids with peroxisomal marker Peroxin13-mRuby and uracil auxotrophy for colocalization. Left roGFP2 with strong promoter. Right pHLourin2 with strong promotor.

It can also be validated by transforming the sensors attached to the PTS1 sequence into Pex5 knockout yeast strain. The sensor is expected to show no specific localisation, because of the missing import sequence. We calibrate the sensors in living yeast cells and physiological ranges so that we can not only perform relative but also quantitative measurements. We aim to confirm our hypothesis of an more oxidized redox state of roGFP2 in peroxisomes with violacein pathway activity and want to measure differences in pH within yeast strains with peroxisomal membrane anchored pex3-bacteriorhodopsin protein (membrane proteins) .

Once expression and localization of the sensor is proven by microscopy, measurements with a plate reader or a fluorometer are acceptable. This allows a high number of replicates to be measured accurately in reasonable time. Microscopy is performed with a filter based Nikon Eclipse TI fluorescence microscope at 100-fold magnification and plate reader measurements are performed with a Tecan infinite plate reader (Mahon M. J. et al., 2011) . For pHLuorin2 emission intensity is measured at 535 nm upon excitation at 405 nm and 485 nm. Same settings were used for roGFP2 (Schwarzländer M. et al., 2016) . Evaluation of the reported signals is done by the excitation ratio of the the corresponding excitation wavelength.

The initial step is to find a reliable source to prove the abundance of our precursor Farnesyl pyrophosphate (FPP) in yeast peroxisomes. At this time there is no proof of existence of FPP inside yeast peroxisomes yet. However it is predicted to be present, as it was detected in mammalian and plant peroxisomes Olivier et al. (2000) .

The precursor FPP is converted into valencene by a valencene synthase (ValS). We chose the one from Callitropsis nootkatensis because of its comparably high efficiency in microorganisms. It achieves greater yields in yeast than the citrus valencene synthase. Furthermore, the product specificity is relatively high, while production of byproducts is low Beekwilder et al. (2014). The valencene synthase was also chosen because of its robustness towards pH and temperature changes Beekwilder et al. (2014) . Our modelling approach revealed that for optimal yields an overexpression of valencene synthase is necessary because of its slow conversion rate (Model). This is why we chose the strongest promoter of the yeast toolbox (Dueber Toolbox) for this attempt.

Figure 7.3 structure Valencene

The intermediate valencene is then converted into nootkatol by a P450 monooxygenase as well as into small amounts of our desired product nootkatone. The P450 monooxygenase we chose for this project was taken from the bacterium Bacillus megaterium. In this case it is not only a simple P450 monooxygenase, but an entire P450 system, consisting of a soluble P450 fused to a cytochrome P450 reductase (CPR) enzyme, making an additional reductase obsolete De Mot et al. (2002) . Unlike eukaryotic P450s, which are mostly membrane bound, this prokaryotic BM3-P450 is located in the cytosol facilitating an easier transport into the peroxisome, as membrane integration of proteins is a more difficult task to achieve than import of cytosolic proteins Girvan et al. (2006) . BM3 normally catalyzes the hydroxylation of long chain fatty acids Narhi et al. (1986) , which in our case could inhibit the conversion of valencene. For that reason we used a mutated version of BM3, which is called AIPLF. This variant is an enhanced version of the BM3 AIP version, which has a ten times better substrate oxidation rate for valencene than the wildtype BM3 and produces less byproduct when valencene concentration is saturated. Additionally to previous named benefits the AIPLF variant with 5 point mutations in the active side has a significantly lower binding affinity towards long chain fatty acids and therefore increase the transposition rate of valencene. Schulz et al. (2015) Lehmann (2016)

The alcohol dehydrogenase from Pichia pastoris subsequently converts nootkatol into nootkatone by oxidation. It uses NAD+ as a cofactor which is reduced in the reaction. The regeneration of this cofactor is facilitated by the BM3, which oxidizes NADH Schulz et al. (2015) .

The first milestone to achieve our goal is the separate integration of each of our three enzymes ValS, BM3 and ADH into level 1 vectors (Dueber Toolbox) in yeast and to verify their expression by Western Blot analysis. Therefore, a 3xFlag/6xHis-tag was added to the N-terminus of each of the proteins. It enables us to use an anti-His antibody followed by an anti-mouse-antibody to make protein abundances visible. Subsequently, the two enzymes ADH and ValS were combined in a level 2 cassette plasmid. BM3 is designed as a level 1 plasmid and will be co-expressed with the level 2 plasmid to achieve a nootkatone production. The expression of the enzymes in the cytoplasm is again verified by Western Blot analysis. The further approach aims to provide the enzymes with C-terminal peroxisomal targeting signals type one, which finally converts the nootkatone pathway into our artificial compartment.

Since the production of nootkatone does not lead to a change of colour we need to apply different methods for verifying substrates. Once we succeed with the qualitative validation via Western Blot analysis, we can verify the presence of nootkatone by using high performance liquid chromatography and mass spectrometry, respectively.

Model influence on Nootkatone expression

We modeled the nootkatone biosynthesis pathway using ordinary differential equations in order to optimize nootkatone production. We found two hard and an easy problem, all of which we could find a solution for. The easy problem is optimization of the enzyme concentrations of the biosynthesis pathway. According to our model of the Nootkatone pathway we found that overexpression of Valencene Synthase is necessary to maximize the Nootkatone yield, while both alcohol dehydrogenase and p450-BM3 have only minor effects on the yield.

One of the hard problems, as shown in our penalty model is the toxicity of nootkatone and nootkatol. Since the toxicity most likely stems from both nootkatone and nootkatol clogging up the cell wall we present our peroxisome as a solution for this problem. When comparing the cytosolic model to our peroxisomal model we found that, if our assumption that neither Nootkatone nor Nootkatol are able to pass the peroxisomal membrane holds up, we can greatly increase Nootkatone production.

The last hard problem is the influx of the pathway precursor farnesyl pyrophosphate (FPP). We used OptKnock analysis to design yeast strains with optimized FPP production. With this analysis we got hints that growing the yeast cells on a fatty acid medium might be a simple alternative to knocking out the desired genes.

Based on described of advantages of violacein this pathway was chosen. Violacein is naturally produced in numerous bacterial strains, most popular in the gram-negative Chromobacterium violaceum . It is related to biofilm production and shows typical activities of a secondary metabolite (Seong Yeol Choi et al., 2015) .

**Figure 8.1** The synthesis of Violacein requires five enzymes encoded by the VioABCDE operon. VioA, a flavin-dependent L-tryptophan oxidase and VioB, a heme protein, work in combination to oxidize and dimerize L-tryptophan to an IPA imine dimer. Hydrogen peroxide is released as a by-product of the VioA reaction. Next step by VioE is the rearrangement of the IPA imine dimer to protodeoxyviolaceinic acid, which can non-enzymatically oxidize to prodeoxyviolacein or, by VioC via deoxyviolaceinic acid, oxidize to pink deoxyviolacein. The flavin-dependent oxygenases VioC and VioD require interaction with the oxidized form of flavin-adenine dinucleotide (FAD) (uniprot, uniprot) . The two enzymes act sequentially: first, VioD hydroxylates protodeoxyviolaceinic acid, leading to protoviolaceinic acid. Second, VioC creates the oxindole at the 2-position of one indole ring, leading to violet violacein (Balibar CJ *et al.*, 2006) (Janis J. Füller *et al.*, 2016) .

Relocating the pathway into the peroxisome enables proximity of the enzymes and substrates. Furthermore the yeast cell is protected from the toxic substance hydrogen peroxide. Yeast peroxisomes have no problem with this as their main function is the beta-oxidation of fatty acids and the detoxification of the thereby produced H₂O₂ (Erdmann R. et al., 2007) . Because VioC and VioD are FAD-dependent, it is additionally an evidence for FAD availability inside of the peroxisome, if the synthesis of Violacein works. Otherwise the two enzymes would not be able to catalyze the reaction.
The genes for VioA, VioB and VioE were amplificated via PCR with Golden Gate compatible overhangs from the biobrick VioABCE (Part: BBa_K274004) .

By Golden Gate cloning the peroxisomal targeting sequence (PTS1) was attached to the C-terminus of every pathway protein. Combined with the other necessary parts of the toolbox they represent the level 1 plasmids.

The PTS-tag marks the proteins for the import into peroxisomes. This should first of all point out the functionality of the yeast’s natural import mechanism and also be the basis for demonstrating our own modeled PTS*, proving our designed orthogonal import mechanism. Furthermore we also aim to optimize the working conditions for the enzymes inside of the reaction room - the peroxisomes. For example to vary the pH with new membrane proteins such as bacteriorhodopsin. To secure this change, we can also check the current conditions by our designed sensors.

There are several methods to verify the pathway’s enzymes. First of all, violacein and several intermediates (prodeoxyviolacein, deoxyviolacein, proviolacein) are colorful and the production in yeast can be visualized easily. Furthermore we added a His-/Flag-tag to the N-terminus of every protein (see geneious plasmid cards) to confirm their expression via SDS page and western blot. After verifying the presence of the enzymes the next step is to test their functionality. Before performing in vivo experiments in yeast an in vitro assay was implemented. For this the three enzyme pathway leading to PDV was reconstructed, testing VioA, VioB and VioE. To enable the best conditions for the enzymes, the pathway was studied intensively and all needed cofactors were calculated and added to the in vitro reaction (see protocol prodeoxyviolacein in vitro assay). This included FAD, MgCl₂, catalase for decomposition of hydrogen peroxide, and the substrate L-tryptophan. The in vitro reaction was followed by qualitative analysis via HPLC and mass spectrometry.

Introduction

In order to achieve a fully controllable artificial compartment, the first step was to design a completely orthogonal import system. Next was the knockout of endogenous import systems. However, a few proteins are imported neither by the Pex5 nor the Pex7 import machinery. Therefore, specific genome engineering designs, such as knockouts, deleting or redirecting the protein localization could be utilized for the ultimate goal of creating a synthetic organelle.

Additionally, knockouts or genome integrations enable customization of the peroxisomal properties, such as membrane permeability, size/number, decoupling of peroxisomes from cytoskeleton and the peroxisomal metabolism.

All these strategies allow a rational design of an artificial compartment, which is fully engineerable regarding the proteome, metabolome and the entire peroxisomal environment.

Design of yeast multi -knockout strains

The Crispr Cas9 System

The demands on yeast engineering have significantly increased with the design of more complex systems and extensive metabolic pathways. Genetic techniques that have historically relied on marker recycling are unable to keep up with the ambitions of synthetic biologists. In recent years the Crispr Cas9 system has been used for several strain-engineering purposes, including:

Markerless integration of multiple genetic cassettes into selected genomic loci
Multiplexed and iterative gene knockouts without the need to recycle a marker
Precise genome editing – nucleotide substitutions, etc.

We utilized the Cas9 system as a tool for peroxisomal engineering and have adopted the existing toolbox from (Lee et al. 2015) and the complete cloning system which also provides the possibilities for genome integration and gene editing by Cas9. For this, two oligonucleotides have to be designed for targeting the Cas protein to the gene of interest.

Figure 1: Plasmid construction for the gRNA expression plasmid
Two oligos, containing the targeting sequence of the gRNA, have to be annealed and can then be integrated in the gRNA entry Vector by a Golden Gate reaction. Adapted from (Lee et al. 2015)

Several gRNA vectors can subsequently be assembled into one vector with a Cas9 expression cassette and then be transformed into yeast. The expression of Cas9 together with gene specific gRNA´s leads to double strand breakage followed by non-homologous end joining repair or homologous recombination, in case of added repair DNA (figure 3).

Figure 2: Plasmid construction for the expression plasmid containing Cas9 and gRNA´s
Vector for Cas9 and gRNA expression, assembled by a Golden Gate reaction, containing a URA marker, Cen6 yeast origin and a kanamycin resistance. Adapted from (Lee et al. 2015)

The combination of the Cas9 system with DNA repair sequences enables not only knockouts of peroxisomal proteins, but also allows redirecting protein localization by changing protein targeting signals or integration of linear DNA into yeast chromosomes. Genome engineering facilitates yeast strain development for customized peroxisomes.

Figure 3: Design of repair DNA sequences for homologous recombination after inducing double strand break by Cas9
Repair DNA sequences can be used to increase the efficiency for Cas9 guided knocking out of specific genes, but would also allow genomic integration of targeting signals or complete genes. Adapted from (Lee et al. 2015)

The peroxisomal proteome of yeast (saccharomyces cerevisiae)

The peroxisomal proteome is studied extensively for saccharomyces cerevisiae and contains exactly 67 proteins (Kohlwein et al. 2013). The function is characterized for the most of those proteins and it is known, that yeast peroxisomes are expendable under optimal growth conditions. Nevertheless, some knockouts are lethal under oleate or stress conditions.

Gene	Required for growth on oleate	Expression induced by oleate	Enzyme/activity	Molecular mass (kDa)	Isoelectric point	Molecules per cell	Localization	Function
ß-Oxidation enzymes
PCS60 (FAT2)	No	Yes	Medium chain fatty acyl-CoA synthetase	60.5	9.98	8.770	Peripheral peroxisomal membrane and matrix	Activates fatty acids with a preference for medium chain lengths, C9-C13
FAT1	No	-	Very long chain fatty acyl-CoA synthetase and long chain fatty acid transporter	77.1	8.47	16,900	Lipid droplet, ER, peroxisome Three predicted TM	Activates fatty acids with a preference for very long chain lengths, C20–C26
POX1	Yes	Yes	Acyl-CoA- oxidase	84.0	8.73	ND	Peroxisomal matrix	Oxidation of acyl-CoA
CTA1	No	Yes	Catalase	58.6	7.46	623	Peroxisomal matrix	Degrades hydrogen peroxide produced by Pox1
FOX2 (POX2)	Yes	Yes	Multifunctional enzyme; 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase	98.7	9.75	ND	Peroxisomal matrix	-
POT1 (FOX3, POX3)	Yes	Yes	3-Ketoacyl-CoA thiolase	44.7	7.56	ND	Peroxisomal matrix	Cleaves 3-ketoacyl-CoA into acyl-CoA and acetyl-CoA
DCI1 (ECI2)	-	-	Δ(3,5)-Δ(2,4)-dienoyl-CoA isomerase (putative)	30.1	8.83	ND	Peroxisomal matrix	Auxiliary enzyme of fatty acid β-oxidation; role in β-oxidation debated
SPS19 (SPX1)	Yes	Yes	2,4-Dienoyl-CoA reductase	31.1	9.67	ND	Peroxisomal matrix	Auxiliary enzyme of fatty acid β-oxidation
ECI1	Yes	Yes	Δ3, Δ2-enoyl-CoA isomerase	31.7	8.21	ND	Peroxisomal matrix	Auxiliary enzyme of fatty acid β-oxidation
TES1 (PTE1)	Yes	Yes	Acyl-CoA thioesterase	40.3	9.58	ND	Peroxisomal matrix	Auxiliary enzyme of fatty acid β-oxidation
MDH3	Yes	Yes	Malate dehydrogenase	37.3	10.00	3,300	Peroxisomal matrix	Required for the malate-oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+, part of the glyoxylate cycle
IDP3	Yes	Yes	NADP+ dependent isocitrate dehydrogenase	47.91	10.02	ND	Peroxisomal matrix	Required for the 2-ketoglutarate/isocitrate shuttle, exchanging peroxisomal NADP+ for cytosolic NADPH
CAT2	No	No	Carnitine acetyl-CoA transferase	77.2	8.34	470	Peroxisome mitochondria	Transfers activated acetyl groups to carnitine to form acetylcarnitine which can be shuttled across membranes
Glyoxylate cycle
CIT2	No	-	Citrate synthase	51.4	6.34	2,310	Peroxisomal matrix	Condensation of acetyl CoA and oxaloacetate to form citrate
MDH3	Yes	Yes	Malate dehydrogenase	37.3	10.00	3,300	Peroxisomal matrix	Required for the malate–oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+
MLS1	Yes	-	Malate synthase	62.8	7.18	ND	Peroxisomal protein	Required for utilization of nonfermentable carbon sources
Other peroxisome-associated enzyme activities
GPD1 (DAR1, HOR1, OSG1, OSR5	-	-	NAD-dependent glycerol-3-phosphate dehydrogenase	42.9	5.26	807	Peroxisome, cytosol, nucleus	Key enzyme of glycerol synthesis, essential for growth under osmotic stress
PNC1	-	-	Nicotinamidase	25.0	6.23	7,720	Peroxisome, cytosol	Converts nicotinamide to nicotinic acid as part of the NAD(+) salvage pathway
NPY1	-	-	NADH diphosphatase	43.5	6.26	846	Peroxisome cytosol	Hydrolyzes the pyrophosphate linkage in NADH and related nucleotides
STR3	-	-	Cystathionine β-lyase	51.8	7.96	ND	Peroxisome	Converts cystathionine into homocysteine
STR3	-	-	Cystathionine ß-lyase	51.8	7.96	ND	Peroxisome	Converts cystathionine into homocysteine
GTO1	-	-	ω-Class glutathione transferase	41.3	9.53	-	Peroxisome	Induced under oxidative stress
AAT2(ASP5)	-	Yes	Aspartate aminotransferase	46.1	8.50	7,700	Cytosol, peroxisome	Involved in nitrogen metabolism
PCD1	-	-	Nudix pyrophosphatase with specificity for coenzyme A and CoA derivatives	39.8	6.59	238	Peroxisome	May function to remove potentially toxic oxidized CoA disulfide from peroxisomes
LPX1	-	Yes	Triacylglycerol lipase	43.7	8.16	2,350	Peroxisomal matrix	-
Peroxisomal transporters
PXA1 (LPI1, PAL1, PAT2, SSH2	-	-	Subunit of a heterodimeric ATP-binding cassette transporter complex	100.0	10.34	ND	Peroxisomal membrane	Import of long-chain fatty acids into peroxisomes
PXA2 (PAT1)	-	-	Subunit of a heterodimeric ATP-binding cassette transporter complex	97.1	9.47	ND	Peroxisomal membrane	Import of long-chain fatty acids into peroxisomes
ANT1(YPR118C)	-	-	Adenine nucleotide transporter	36.4	10.6	2,250	Peroxisomal membrane	Involved in β-oxidation of medium-chain fatty acids
Peroxins
Pex1 (PAS1)	-	-	AAA ATPase	117.3	6.93	2,100	Peroxisomal membrane	Involved in recycling of Pex5, forms heterodimer with Pex6
Pex2 (RT1, PAS5)	-	-	E3 ubiquitin ligase	30.8	9.02	339	Peroxisomal membrane	RING finger protein, forms complex with Pex10 and Pex12. Involved in matrix protein import
Pex3 (PAS3)	-	-	-	50.7	6.29	1,400	Peroxisomal membrane	Required for proper localization of PMPs
Pex4 (PAS2, UBC10)	-	-	Ubiquitin conjugating enzyme	21.1	5.36	ND	Peroxisomal membrane	Involved in matrix protein import
Pex5 (PAS10)	-	-	Soluble PTS1 receptor	69.3	4.79	2,070	Cytosol and peroxisomal matrix	Required for import of PTS1-containing peroxisomal proteins, contains TPR domains
Pex6 (PAS8)	-	-	AAA ATPase	115.6	5.44	1,630	Peroxisomal membrane	Involved in recycling of Pex5, forms heterodimer with Pex1
Pex7 (PAS7, PEB1)	-	-	Soluble PTS2 receptor	42.3	8.34	589	Cytosol and peroxisomal matrix	Requires Pex18 and Pex21 for association to the receptor docking site, contains WD40 repeat
PEX8 (PAS6)	-	-	Intra peroxisomal organizer of the peroxisomal import machinery	68.2	7.62	538	Peroxisomal matrix and luminal membrane face	Pex5-cargo dissociation
Pex9	-	-	PTS-receptor	-	-	-	-	-
Pex10	-	-	E3 ubiquitin ligase	39.1	9.88	ND	Peroxisomal membrane	RING finger protein involved in Ubc4-dependent Pex5 ubiquitination. Forms complex with Pex2 and Pex12
PEX11 (PMP24, PMP 27)	-	-	-	26.9	10.65	1,630	Peroxisomal membrane	Involved in peroxisome fission, required for medium-chain fatty acid oxidation
Pex12 (PAS11)	-	-	E3 ubiquitin ligase	46.0	9.86	907	-	RING finger protein, forms complex with Pex2 and Pex10
Pex13 (PAS20)	-	-	Component of docking complex for Pex5 and Pex7	42.7	9.83	7,900	Peroxisomal membrane	Forms complex with Pex14 and Pex17
Pex14	-	-	Central component of the receptor docking complex	38.4	4.61	2,570	Peroxisomal membrane	Interacts with Pex13
Pex15 (PAS21)	-	-	-	43.7	8.42	1,070	Peroxisomal membrane	Recruitment of Pex6 to the peroxisomal membrane, tail anchored PMP
Pex17 (PAS9)	-	-	Component of docking complex for Pex5 and Pex7	23.2	10.24	656	Peroxisomal membrane	Forms complex with Pex13 and Pex14
Pex18	-	-	-	Required for PTS2 import	32.0	4.78	ND	Interacts with Pex7 partially redundant with Pex21
Pex19 (PAS12)	-	-	Chaperone and import receptor for newly synthesized PMP	38.7	4.08	5,350	Cytosol, peroxisome, farnesylated	Interacts with PMPs, involved in PMP sorting. Also interacts with Myo2 and contributes to peroxisome partitioning
Pex21	-	-	Required for PTS2 protein import	33.0	6.67	ND	Cytosol	Interacts with Pex7, partially redundant with Pex18
Pex22(YAF5)	-	-	Required for import of peroxisomal proteins	19.9	8.33	259	Peroxisomal membrane	Recruits Pex4 to the peroxisomal membrane
Pex25	-	-	Involved in the regulation of peroxisome size and maintenance, required for re-introduction of peroxisomes in peroxisome deficient cells	44.9	9.77	2,420	Peripheral peroxisomal membrane	Recruits GTPase RhoI to peroxisomes, interacts with homologous protein Pex27
Pex27	-	-	Involved in the regulation of peroxisome size and number	44.1	10.49	382	Peripheral peroxisomal membrane	Interacts with homologous protein Pex25
Pex28	-	-	Involved in the regulation of peroxisome size, number and distribution	66.1	7.09	ND	Peroxisomal membrane	May act upstream of Pex30, Pex31 and Pex 32
Pex29	-	-	63.5	6.8	5,040	Peroxisomal membrane	May act upstream of Pex30, Pex31 and Pex32
Pex30	-	-	Involved in the regulation of peroxisome number	59.5	5.59	4,570	Peroxisomal membrane	Negative regulator, partially functionally redundant with Pex31 and Pex32
Pex31	-	-	Involved in the regulation of peroxisome number	52.9	10.15	238	Peroxisomal membrane	Negative regulator, partially functionally redundant with Pex30 and Pex32
Pex32	-	-	Involved in the regulation of peroxisome number	48.6	9.14	ND	Peroxisomal membrane	Negative regulator partially functionally redundant with Pex30 and Pex31
PEX34	-	-	Involved in the regulation of peroxisome number	16.6	10.30	ND	Peroxisomal membrane	-
Peroxisome fission and inheritance
DYN2 (SLC1)	-	-	Light chain dynein	10.4	9.03	1,310	Cytosol	Microtubule motor protein
SEC20	-	-	v-SNARE	43.9	5.92	4,910	Golgi, ER	Involved in retrograde transport from the Golgi to the ER, interacts with the Dsl1 complex through Tip20
SEC39(DSL3)	-	-	Component of the Ds11p-tethering complex	82.4	4.65	1,840	ER, nuclear envelope	Proposed to be involved in protein secretion
DSL1 (RNS1)	-	-	Component of the ER target site that interacts with coatomer	88.1	4.69	8,970	Peripheral ER, Golgi membrane	Forms a complex with Sec39 and Tip20 that interacts with ER SNAREs, Sec20 and Use1
FIS1 (MDV2)	-	-	Required for peroxisome fission	17.7	9.87	2,410	Peroxisomal membrane mitochondria	Tail anchored protein recruits Dnm1 via Mdv1/Caf4; also involved in mitochondrial fission
DNM1	-	-	GTPase, dynamin like protein involved in peroxisome fission	85.0	5.25	9,620	-	Also involved in mitochondrial fission
VPS1 (GRD1, LAM1, SPO15, VPL1, VPT26)	-	-	GTPase, dynamin like protein involved in peroxisome fission	78.7	8.15	5,960	-	Also involved in vacuolar protein sorting
VPS34 (END12, PEP15, VPL7, VPT29, STT8, VPS7)	-	-	Phosphatidylinositol 3-kinase	100.9	7.79	1,080	-	Forms complex with Vps15
INP1	-	-	Involved in retention of peroxisomes in mother cells	47.3	8.34	639	Peroxisomal membrane	Recruited to the peroxisome by binding to Pex3
INP2	-	-	Myo2 receptor, involved in peroxisome inheritance	81.5	9.41	736	Peroxisomal membrane	-
RHO1	-	-	GTP binding protein of the Rho subfamily of Ras like proteins, involved in actin assembly at the peroxisome	23.2	6.07	ND	-	Involved in de novo peroxisome formation recruited to peroxisomes by Pex25

Knockout designs in our project

Pex 9

Pex9 is a recently discovered import receptor for PTS1 proteins, which is induced by oleate and is an import receptor for both malate synthase isoenzymes Mls1p and Mls2p. In order to get a completely empty reaction room, a Pex9 knockout was designed to prevent unintended protein import.

Pex 31 & Pex 32

It has been shown that knockouts of Pex31 and Pex32 leads to an increased Peroxisomal size, but additionally the membrane permeability was affected (Zhou et al. 2016). This effect can be used as a tool for engineering membrane permeability by knocking out or overexpress both genes. A knockout would lead to an increased permeability and one could think of an opposite effect in case of overexpression, but this has not been shown yet.

INP1

The INP1 knockout was designed after the skype call of Prof Dueber, who recommended us, decoupling of peroxisomes from cytoskeleton in order to improve the secretion efficiency. INP1 is responsible for the tethering of peroxisome, which would inhibit the secretion of peroxisomes.

POT1

The only protein, which is imported by the Pex7 import machinery in saccharomyces is the 3-ketoacyl-CoA thiolase (POT1). A knockout of POT1 would enable utilizing the Pex7 import for proteins of interest, which cannot be tagged at the C-terminus with pts1, without having unintended import of other enzymes.

Genomic integration of our novel Pex5 import receptor

After testing our new Pex5 import systems, which is completely orthogonal to the natural import, the next step would be to replace the endogenous system with our artificial import system. Therefore, an integration plasmid was designed with help of the previously described yeast toolbox, containing HO locus homologies and a hygromycin resistance (Figure 4). Afterwards the plasmid was transformed into the yeast strain which was created by our collaboration partner Aachen (double knockout strain Pex5 & Pex7).

Figure 4: Design of integration plasmid for integrating our orthogonal Pex5 import receptor.
Therefore, an integration plasmid was designed with help of the previously described yeast toolbox, containing HO locus homologies and a hygromycin resistance

The resulting yeast strain allows full control over the peroxisomal matrix proteome, by replacing the whole protein import machinery, which is the first step for creating our artificial compartment.

Outlook

Besides the genome engineering approaches, which were performed in our project one could think of more radical strategies for peroxisomal engineering. A final goal could be a “minimal peroxisome”, which contains only the proteins that are required for the biogenesis of the peroxisome and import of proteins and metabolites. On the one hand peroxisomal pathways could be redirected to cytosol or other organelles and one the other hand endogenous metabolic pathways could be redirected to our novel artificial compartment by changing the protein localization signal in the yeast genome with help of the Cas9 system. All these strategies would allow tremendous improvements for metabolic engineering applications by creating an artificial compartment, which can be rational designed and customized for specific metabolic pathways.

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-<h1>Project description</h1>
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+<h1>Design</h1>
+<p>We designed a novel toolbox for complete control over all major functions of the peroxisome. The toolbox is our solution for improving the engineering workflow and predictability of synthetic constructs. Interested? Find out how.</p>
+<h2 id="Background">Scientific background</h2>
+<h3 id="RootProblem">The root problem</h3>
+<p>Synthetic biology is an engineering discipline. And while we are able to plan our constructs with tools like biobricks, a major difference to e.g. electrical engineering is that they are not nicely isolated on a chip, but surrounded by all types of interfering agents. One of the major issues regarding protein expression in a novel chassis is unwanted and unexpected crosstalk between engineered pathways and the native cellular processes of the production host. The other one is toxicity of the products or intermediates of the pathway. Both can greatly change our system’s behaviour which in some cases leads to us to having to trial-and-error find a solution, making our previously modeled optimization useless. </p>
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+<h3 id="Approach">Our approach</h3>
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+<p>The natural approach of organisms to deal with metabolic interference and toxic byproducts is subcellular compartmentalization. This has proven to be a functional solution in naturally occurring pathways in eukaryotes as well as new synthetic pathways for biotechnological application. Thus, the creation of a synthetic organelle presents a suitable strategy to increase the efficiency and yield of non-native pathways. A common approach is to build up artificial compartments from scratch. Many breakthroughs have been achieved in the last decade, however the creation of a fully synthetic compartment is yet a milestone to reach for. We on the contrary want to start by engineering artificial compartments through orthogonalization.</p>
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+<p>The peroxisome is the ideal starting candidate as it has many advantages over other compartments, including it being able to import fully folded proteins and not being essential in yeast under optimal growth conditions. Our project’s aim is to create a toolbox for manipulating and creating customizable peroxisomes as a first step towards synthetic organelles.</p>
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+<p>By modifying the import machinery of yeast peroxisomes only selected proteins will be imported into the peroxisomes leaving the researcher in full control over the content of the peroxisomal lumen. Furthermore our toolbox will include a secretion mechanism for the synthesized products, various intra-compartmental sensors, modules for the integration of proteins into the peroxisome membrane, as well as optogenetic control for some of these parts for a more precise spatiotemporal control.</p>
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+<p>As a proof of concept for the functionality of the toolbox and the customizable compartment two metabolic pathways will be integrated into the altered peroxisome: (i) Violacein biosynthesis and (ii) nootkatone biosynthesis. Violacein, a bisindole formed by condensation of two tryptophan molecules, is a violet pigment and thus easy to quantify in the cell. Nootkatone on the other side is a natural compound found inside the peel of the grapefruit, which gives it its characteristic taste and smell. In addition, nootkatone is a natural mosquito and tick repellent that is already being commercially used and industrially manufactured. Unfortunately, the production costs are extremely high. Furthermore, the production of nootkatone inside yeast is challenging as it is toxic for yeast and thus, the production efficiency is rather low. A successful implementation of the synthetic compartment will show increased yields in the production of these compounds and showcase the potential of this approach for similar future applications.</p>
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+<h3>Scientific background</h3>
+<h4>Peroxisomal quick facts</h4>
+<
+ubiquitous single-membrane-bounded organelles
+assemble, multiply, or degrade in response to metabolic needs of the cell
+Can import folded and even oligomeric proteins
+Expendable in yeast under optimal growth conditions
+De novo biogenesis
+<h4>Peroxisome biogenesis and inheritance</h4>
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+<h2 id="CloningStrategies">Cloning strategies and the Yeast Toolbox for Multipart-Assembly</h2>
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+<p>While describing our cloning strategies we mentioned several <i>levels</i>, which stand for different stages of our plasmids. They are further described in the work of J.M. Dueber and colleagues, who designed the well established yeast toolkit we used in this project
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+  <a href="http://pubs.acs.org/doi/abs/10.1021/sb500366v ">  <abbr title="Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A highly characterized yeast toolkit for modular, multipart assembly. ACS synthetic biology, 4(9), 975-986.">
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+        (Dueber)</abbr></a>.
+The toolkit offers the possibility to design plasmids with desired antibiotic resistances, promoters as well as terminators from standardized parts. It also provides fluorescence proteins, protein-tags and many more useful components as part plasmids. These part plasmids are distinguished in different part types due to their specific overhangs to ensure their combination in the correct order (e.g. promoter - gene of interest - terminator) all in a versatile one-pot Golden Gate reaction without time-consuming conventional cloning steps.</p>
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+<div><img src="https://static.igem.org/mediawiki/2017/8/81/T--Cologne-Duesseldorf--Toolbox_assembly.jpeg
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-        			<stop offset="0.5" stop-color="#fff" stop-opacity="0.7" />
+    <figcaption>Figure 1: The yeast toolkit starter set comprises of 96 parts and vectors. The eight primary part types can be further divided into subtypes.
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+<a href="http://pubs.acs.org/doi/abs/10.1021/sb500366v "><abbr title="Lee, Michael E., et al. A highly characterized yeast toolkit for modular, multipart assembly. ACS synthetic biology 4.9 (2015): 975-986.">(Dueber)</abbr></a>
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+<p>The cloning steps regarding the plasmid <i>levels</i> are implemented in <i>E.coli</i> in order to reduce the required time to generate the final plasmids. The different <i>levels</i> are therefore defined by their part content and their antibiotic resistances. </p>
-    	</defs>
+<p>To generate a <i>level 0</i> plasmid, the gene of interest is ligated into the provided <i>level 0</i> backbone via Golden Gate assembly using the enzyme BsmBI. The backbone contains a resistance to Chloramphenicol, as well as an origin of replication, creating a very basic yet functional plasmid.</p>
-    	<title>artico</title>
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+<p>The <i>level 1</i> plasmid contains a promoter and terminator suited for <i>S. cerevisiae</i>. There is the possibility of including  a polyhistidine-tag if there is a need for Western blot analysis. The antibiotic resistance contained in the <i>level 1</i> plasmid changes from chloramphenicol to ampicillin which enables filtering out residual <i>level 0</i> plasmids contained in the Golden Gate product. Furthermore, the <i>Dueber toolbox</i> includes the possibility of designing GFP-Dropout cassettes. These are custom-built <i>level 1</i> backbones whose inserts are sfGFP as well as promoter and terminator suited for <i>E. coli</i>.  Upon a successful cloning step the GFP is replaced by the part(s) of interest, and correct colony shows a white colour. In case of a wrong ligation event colonies show a green fluorescence. This provides a very useful tool to detect unsuccessful cloned colonies. The enzyme used for <i>level 1</i> changes from BsmBI to BsaI to avoid any interference between different steps. </p>
+<p>The <i>level 2</i> plasmid combines two or more genes of interest with their respective promoters, terminators and tags. The resistance changes from ampicillin to kanamycin. The enzyme of this step is BsmBI again. This level is useful, if the construct you are designing requires multiple genes to be transformed into one yeast strain.</p>
-<h2>Introduction</h2>
-    <h3>Real world application</h3>
-    <p>As a proof of concept for our compartmentation strategy we intend to establish the Nootkatone pathway inside the peroxisome. Nootkatone is a natural compound found inside the peel of grapefruit, which gives it its characteristic taste and smell. In addition, Nootkatone is a natural repellent for mosquitoes and ticks that is already being commercially used and industrially manufactured. Unfortunately, the production costs are extremely high, because it has to either be extracted from the peel of millions of grapefruit or synthesized inside of yeast. The difficulties lie in the toxicity of the Nootkatone pathway towards yeast and the resulting low efficiency. Here our compartmentation comes into play: we plan to translocate the whole pathway into the modified peroxisome to prove that we have transformed the peroxisome into an independent compartment with all the features we require.</p>
+ <h3> Yeast nomenclature </h3>
+<p>To make it fast and easy to differentiate between endogenous and heterologous genes and gene products we decided to use <i> S. cerevisiae </i> nomenclature according to <a href="http://seq.yeastgenome.org/sgdpub/Saccharomyces_cerevisiae.pdf">yeastgenome.org</a>. </p>
+<p>Below nomenclature at the example of <b><u>y</u>our <u>f</u>avorite <u>g</u>ene 1, <i>YFG1</i></b> is explained. </p>
+<table>
+      <tr>
+        <th>Letter code</th>
+        <th>Meaning</th>
+      </tr>
+      <tr>
+        <td><i>YFG1</i></td>
+        <td><b>Y</b>our <b>f</b>avorite <b>g</b>ene <i>S. cerevisiae</i> wild type allele</td>
+       </tr>
+     <tr>
+        <td><i>yfg1</i>Δ</td>
+        <td>Gene deletion of <b>y</b>our <b>f</b>avorite <b>g</b>ene</td>
+       </tr>
+      <tr>
+        <td>Yfg1</td>
+        <td>Protein product of <i>YFG1</1></td>
+       </tr>
+      <tr>
+        <td>YFG2</td>
+        <td>A heterologous gene product from mammalian cells</td>
+       </tr>
+</table>
+<h2>Design of our sub-projects</h2>
 <button class="accordion">
-<h2 id="ProteinImport">Protein Import</h2>
+  <h2 id="ProteinImport">Protein Import</h2>
-<p>The peroxisome possesses two pathways for importing proteins with the main transport proteins being PEX5 and PEX7. We created an orthogonal PEX5 binding pocket and corresponding recognition peptide (PTS1) by structural modeling. We also created a library of PEX7 recognition sequences for import of proteins incompatible with the PTS1 peptide.</p>
+  <p>Control over the peroxisomal proteome is an essential requirement for our project. Our objective is the obtainment of a fully orthogonal import system by utilizing the two peroxins Pex5 and Pex7.</p>
-</button>
+  </button>
 <div class="panel">
-<h3>PTS1 Import</h3>
+ <h3 id="Pex5">Engineering of Pex5 and PTS1</h4>
+ <p>
+ </p><h4 id="h5-1">Designing our receptors</h5>
+ <p>To achieve the engineering of an orthogonal import pathway, we followed two approaches regarding Pex5. The first is based on targeted mutagenesis based on educated guesses which is first verified by <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#h2-1">molecular dynamics</a> and later experimentally in the laboratory.
+   The second approach is based on a recently published paper: We searched for literature dealing with the modification of the peroxisomal import machinery. During our research we came across a <a href="https://www.nature.com/articles/s41467-017-00487-7">paper</a> of Alison Baker <i>et al.</i>, published in 2017, in which they present a synthetic construct of the Pex5 protein, partly <i>Arabidopsis thaliana</i> and partly <i>Physcomitrella patens</i>. Compared to the wild type Pex5, this one shows different binding affinities since it interacts with a PTS1* variant that does not interact with the wild type Pex5. Since the protein sequences of yeast's and plants's Pex5 differ quite a lot, we aligned both sequences to understand where the mutations were set. </p>
+   <figure>
+<div class="flex-row-2">
+     <div>
+     <img src="https://static.igem.org/mediawiki/2017/b/be/Artico_atp5_yp5.png">
+     <figcaption>
+         <strong>Figure 1.1:</strong> Alignment of the <i>Arabidopsis thaliana's</i> PEX5 and <i>Saccharomyces cerevisiae</i>Pex5
+     </figcaption>
+	</div>
+     <div>
+     <img src="https://static.igem.org/mediawiki/2017/9/96/Artico_R19WT.png">
+     <figcaption>
+         <strong>Figure 1.2:</strong> Alignment of the yeast’s Pex5 with the Pex5 variant R19
+     </figcaption>
+	</div>
+</div>
+   </figure>
+<p>The alignment shows three red marked amino acids we changed in our receptor sequence. Interestingly, these mutations are located within the TPR motifs of our Pex5 protein and this persuaded us to try out this receptor, we call it R19. Due to lack of time we tested this Pex5 variant in silico and <i>in vivo</i> simultaneously. We started molecular dynamics simulations with a couple of PTS variants that we already tested with our previous designs &minus; one of them was actually the variant they used in the paper (<i>YQSYY</i>). The details and results of our structural modeling can be found in the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#h2-1">modeling section</a>.
+<br>
+Furthermore, we synthesized this variant and together with two receptors we designed based on educated guesses we got three receptors for our experimental work.</p>
+ <h4>Experimental design</h5>
+ <p>
+   The easiest way to verify successful import is the localization of a fluorescent protein within the peroxisome. Due to that, we decided to tag mTurquoise with our PTS variants. Additionally, we wanted to mark the peroxisomal membrane, to be absolutely sure about the localization within the peroxisome. For that cause we chose the transmembrane domain of Pex13, tagged with the fluorescent protein mRuby.
+ </p>
+ <h5>Pex13−mRuby</h6>
+ <p>We used  the peroxisomal membrane protein Pex13 as a fluorescent marker &minus; by just using the transmembrane domain of Pex13 with a short linker, we make sure that it has no influence on the peroxisomal features. To obtain a higher differentiation from mTurquoise, which we use for another construct, we chose to work with mRuby. Literature research revealed that such constructs have been tested before − <a href="https://www.ncbi.nlm.nih.gov/pubmed/15133130"><abbr title="Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals"> Erdmann et al. (2004)</abbr></a> described a construct containing only PEX13<sub>200-310</sub> with a C-terminal GFP.</p>
+   <figure>
+     <img src="https://static.igem.org/mediawiki/2017/d/d4/Artico_P13RUBY.png">
+     <figcaption>
+         <strong>Figure 1.3:</strong> PEX13 construct with C-terminal mRuby.
+     </figcaption>
+   </figure>
+   <h5>Pex5 variant</h6> Alles nur Vorschläge, war so auch schon okay, übernimm was du magst, bin ir teilweise bei meinen sätzen auch unsicher :D
+   <p>In order to achieve an orthogonal peroxisomal protein import machinery we used a Pex5 knockout yeast strain in which we and transformed our artificial it with a plasmid based Pex5 variant containing a modified PTS1* binding region. Our variation facilitates that is supposed to the detection of detect a non native PTS1* variant instead of the wild type PTS1one. The construct contains a medium strength promotor, the Pex5 gene and a terminator. The whole remaining plasmid parts can be seen in the plasmid map below. is displayed in figure X.
+     </p>
+     <figure>
+       <img src="https://static.igem.org/mediawiki/2017/d/da/Artico_pex5variant.png">
+       <figcaption><strong>Figure 1.4:</strong> Pex5 gene variant.</figcaption>
+     </figure>
+    <h5>mTurquoise−PTS</h6>
     <p>
-The vast majority of peroxisomal matrix proteins is imported by the PEX5 importer. PEX5 recognizes the C-terminal PTS1 peptide whose evolutionary conserved sequence is (S/A/C)-(K/R/H)-(L/M) (<a> Gould <i>et al.</i>, 1989 </a>).  PEX5 is a 612 amino acid protein which contains seven tetratrico peptide repeats (TPR). The TPR is a 34 amino acid motif which forms a structure of alpha-helices separated by one turn. A whole TPR domain consists of three of those structures (<a href="https://www.nature.com/nsmb/journal/v7/n12/full/nsb1200_1091.html"><abbr title="Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5.">Gatto Jr. et al 2000</abbr></a>).
+     Our approach for import verification of the protein import is based on 3D sim microscopy. a For that reason, the fluorescent protein mTurquoise including our modified  tagged with the PTS1* variants was used in order to detect potential localisation of fluorescence signal. After several promotor tests with different strengths, we decided to express this construct only in low amounts, since this was the most suitable possibility to detect potential mTurquoise localization. Moreover,  mTurquoise . This is advantageous sincebecause  we detect only a low signal if the import does not work, hence small amounts of the protein are distributed in the whole cytosol. In contrast we see a clear signal if the import does work due to the relative high concentration inside the peroxisome.
-TPR domains are often involved in protein&minus;protein interactions. As can be seen in the following figure, the TPR regions mediate the binding of the peroxisomal targeting signal.
+     <br>
-</p>
+     Our construct is depicted in figure Xthe figure below.
+		</p>
+     <figure>
+       <img src="https://static.igem.org/mediawiki/2017/b/bd/Artico_fppts.png">
+       <figcaption><strong>Figure 1.5:</strong> Fluorescent protein mTurquoise tagged with the PTS variant.</figcaption>
+     </figure>
+   <h5>Combination of our constructs</h6>
+   <p>To combine our constructs, we cloned our PEX5 and mTurquoise constructs into a level 2 plasmid portrayed below.</p>
+     <figure>
+       <img src="https://static.igem.org/mediawiki/2017/8/88/Artico_P5FP.png">
+       <figcaption><strong>Figure 1.6:</strong> Level 2 plasmid containing the Pex5 gene and the fluorescent protein.</figcaption>
+     </figure>
+     <p>Subsequently, We then did a co-transformation of with the PEX13−mRuby plasmid and the level 2 plasmid was performed in order to verify peroxisomal colocalizationcombine everything that is needed into our yeast.</p>
+ <h5>PTS screening</h6>
+<p>Trusting on our targeted approach alone seemed risky − that is why we planned a In addition to our side directed approach in which we changed amino acids in the Pex5 binding pocket, we planned to perform a PTS screening in order to find the most favorable PTS for our three receptors. <a href="https://www.nature.com/articles/ncomms11152"><abbr title="Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways.">Dueber et al. (2016)</abbr></a> used the Violacein assay for a similar purpose. In this study, They screening screened for the most qualified best PTS sequence for suitable for its recognition by the wild type receptor was performed successfully. r and were successful. Hence another subproject of our team is the integration of the Violacein pathway into the peroxisome (<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#Violacein">Violacein</a>), we were already supplied with all necessary enzymes − VioA, VioB and VioE.</p>
+<!-- eigenen pathway (danke violacein team) oder das aus dem paper?
+<div class="half-width">
+	<figure>
+		<img src="https://static.igem.org/mediawiki/2017/1/13/Artico_dueberassay.jpg">
+			<figcaption><strong>Figure 1.7:</strong> Violacein Assay (<a href="https://www.nature.com/articles/ncomms11152"><abbr="Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways.">Dueber et al., 2016</a>)</figcaption>
+	</figure>
+</div>
+-->
 <figure>
-<img src="https://static.igem.org/mediawiki/2017/b/b3/Artico_tpr.png">
+		<img src="https://static.igem.org/mediawiki/parts/a/a2/T--Cologne-Duesseldorf--PDVpathway.png">
 <figcaption>
-<strong>Figure 1.1: </strong>TPR domain of the human PEX5, with a pentapeptide in its binding pocket (<a href="https://www.nature.com/nsmb/journal/v7/n12/full/nsb1200_1091.html"><abbr title="Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5.">Gatto Jr. <i> et al. </i>, 2000</abbr></a>)
+<strong>Figure 1.8:</strong> Pathway leading to the production of prodeoxyviolacein &minus; the assay is based on the green color to identify colonies without functional import mechanism.
 </figcaption>
 </figure>
-<p>
+     <p>The figure above shows the principles of the assay. VioA and VioB are localized in the cytosol and lead to the production of the IPA imine dimer while VioE is tagged with a PTS1 variant. Successful import leads to white colonies whereas missing import results in green colonies due to the cytosolic production of prodeoxyviolacein.
-The following figure depicts the import mechanism of PTS1 tagged proteins via PEX5.
+     <br>
-</p>
+     Our rests upon the following two plasmids which are co-transformed into yeast.</p>
 <figure>
-   <img src="https://static.igem.org/mediawiki/2017/e/e7/Artico_p5shuttle.jpeg">
+     <div class="flex-row-2">
-   <figcaption><strong>Figure 1.2: </strong>Pex5 import mechanism (<a href="https://www.nature.com/articles/nrm1710"><abbr title="Peroxisomal matrix protein import: the transient pore model">Erdmann et al., 2005</abbr></a>)
+				<div>
-<br>
+         <img src="https://static.igem.org/mediawiki/2017/2/20/Artico_vioabr.png">
-Upon recognition of the PTS1 in the cytosol, PEX5 binds to its cargo (i). It docks to the peroxisomal membrane complex, consisting of PEX13, PEX14 and PEX17 (ii). This docking complex is connected to the RING-finger complex, consisting of PEX2, PEX10 and PEX12, via PEX8. This multi-protein complex is known as the importomer. PEX5 and PEX14 form a pore in the membrane, through which the cargo is translocated (iii). Due to competitive binding of PEX8's PTS1 motif, the receptor–cargo complex dissociates at the matrix site of the membrane (iv). The integral PTS1-receptor is either monoubiquitinated by the E2-enzyme PEX4 or polyubiquitinated by Ubc4 or Ubc5. The AAA peroxins PEX1 and PEX6, which are anchored to the peroxisomal membrane by PEX15, dislocate the ubiquitinated PEX5 from the membrane back to the cytosol (v). The polyubiquitinated PTS1-receptors are degraded by the proteasome, whereas the monoubiquitinated receptors are recycled for further rounds of import.
+				</div>
-</figcaption>
+				<div>
+         <img src="https://static.igem.org/mediawiki/2017/5/5c/Artico_vioeassay.png">
+				</div>
+     </div>
+       <figcaption>
+         <strong>Figure 1.9:</strong> Plasmids used for Violacein assay. The left one carries the coding sequence for the Pex5 variant, VioA and VioB whereas the right one contains the gene for VioE plus the attached PTS1 variant.
+       </figcaption>
 </figure>
+     <p>As shown above, we created one plasmid containing VioA, VioB and one of our Pex5 variants while the other plasmid only contained VioE. We then designed primers which bind to the VioE plasmid to amplify the whole plasmid except the terminator − random PTS1 variants were attached to VioE with the help of a random primer library. Following up, we did the ligation with the corresponding terminator and obtained a mix of several different VioE-PTS1 plasmids.
-<p>
+     <br>
-In this subproject we mutated the PEX5 receptor in a way that enables it to recognize a new signal peptide which does not occur in nature. As PEX5 is responsible for the import of most proteins , we have complete control over the peroxisomal content once we knock out the wild type receptor and replace it with our newly mutated one.
+     After plasmid amplification in <i>Escherichia coli</i> we then co-transformed yeast with the two constructs and waited for the colonies to grow. With the yeast growing, prodeoxyviolacein should be produced in yeast cells with absent import (green color) and the IPA imine dimer (white color) should be produced in those with functional import.
-Corresponding to the new receptor, a peroxisomal targeting signal that provides favorable interactions with the residues of the amino acids within the TPR needs to be designed.
-<br>
-Our first approach for the mutation deals with the introduction of site-directed mutagenesis in the TPR of PEX5 followed by computational simulation of the binding affinity between our new designed PEX5 receptor and several peptide variants via <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655909/">Molecular Dynamics</a>. In the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model">model section </a>we explain the molecular dynamics approach in more detail.
 <br>
-Our second approach relies on recently published literature. We designed a receptor similar to what <a href="https://www.nature.com/articles/s41467-017-00487-7">Baker <i>et al.</i> </a> did in the moss <i>Physcomitrella patens</i> in 2017. To understand how and where we set the mutations in the PEX5 receptor following this approach, please proceed with the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design">design section</a>.
+Plasmid preparation of those with white color and subsequent sequencing leads to the identification of functional PTS1 variants. Afterwards, we repeat the cloning steps described before to obtain a mTurquoise&minus;PTS1* construct and co-transform it with the corresponding Pex5 variant. Eventually, the correct localization of mTurquoise tagged with these PTS1 variants provides proof for its function.</p>
-</p>
-<h3>PTS2 Import</h3>
+<h3>Mutagenesis of PTS2</h4>
-<p>The peroxisomal import depends on two pathways. A vast majority of the proteins normally found in the peroxisome are imported via the <a href="#PTS1">Pex5 importer</a>.
-In <i>S. cerevisiae</i> only one protein, the 3-Oxoacyl-CoA thiolase <a href="http://onlinelibrary.wiley.com/doi/10.1002/yea.320100708/full"><abbr title="Erdmann, R. (1994). The peroxisomal targeting signal of 3‐oxoacyl‐CoA thiolase from Saccharomyces cerevisiae. Yeast, 10(7), 935-944."> (Erdmann)</abbr></a>, localized in the peroxisome, is imported by the receptor Pex7 and some coreceptors instead <a href="https://www.ncbi.nlm.nih.gov/pubmed/17445803"><abbr title="Platta, H. W., & Erdmann, R. (2007). The peroxisomal protein import machinery. FEBS letters, 581(15), 2811-2819.
-"> (Erdmann)</abbr></a>.</p>
-<p>The targeting signal for this pathway is localized near the N-terminus of each protein. Kunze and colleagues described the PTS2 consensus sequence (see figure 2.1)</p>
-<figure>
+<p>To characterize the import efficiency for the site-directed PTS2 firefly luciferase was used. Luciferase is a luminescent protein which can be split in a C- and a N-terminal part. Only when combined, luminescence can be detected. To measure the import efficiency the two parts will be expressed and imported into the peroxisome in a separated way. The smaller part (Split2) of the split luciferase will be brought into the peroxisome first via the PTS1 dependent pathway. The other part is imported via the respective modified PTS2 sequence. The better this sequence is recognized by Pex7, the stronger the luminescence of the assembled luciferase can be detected in the peroxisome. There is a chance of split parts of the luciferase assembling in the cytosol if the import is too slow. To avoid wrong conclusions of the luciferase localisation, we designed a negative control experiment. It includes a split luciferase similar to the one used in the initial experiment, but without the peroxisomal targeting sequence. Consequently there will be no import into the peroxisome. If we subtract the luminescence of the negative control experiment from the luminescence of the main experiment we can define the degree of import.</p>
-    <img src="https://static.igem.org/mediawiki/2017/c/c3/T--cologne-duesseldorf--PTS_richtig.png  ">
+<p>In addition to a directed approach according to Kunze and colleagues we also want to perform a random  mutagenesis experiment to alter the five variable amino acids of the core region of the PTS2 sequence in an unbiased manner. The aim is to generate a library of different peroxisomal PTS2. The 15 nucleotides are assembled by chance. In the DNA synthesis this sequence will either be described as [NNN]<sub>5</sub> or [DNK]<sub>5</sub>. N stands for all four nucleotides mixed, K for either G or T and D for A,G or T. The “DNK” composition prohibits two out of three termination codons. Additionally with this library the amino acid frequency is improved towards a balanced ratio in between the different kinds <a href="https://www.ncbi.nlm.nih.gov/pubmed/27025684 "><abbr title="DeLoache, W. C., Russ, Z. N., & Dueber, J. E. (2016). Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways. Nature communications, 7.">(Dueber)</abbr></a>.</p>
-    <figcaption>Figure 2.1: The peroxisomal targeting signal type two consists of nine amino acids. Residue one contains Arginine or Lysine, residue two Leucine, Valine or Isoleucine. The amino acids three to seven are highly variable. Residue number eight consists of Histidine or Glutamine and the ninth is either Leucine or Alanine.
+<p>Each approach could generate up to 415 DNA sequences, which is roughly 1,07 billion. On the level of the amino acid sequence there are 3,2 million possibilities, since each residue can be taken by 20 different amino acids.  For the assay we therefore need a high throughput method. </p>
-<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3247985/ ">
+<p>We adapted work of DeLoache, Russ and Dueber using the violacein pathway to measure the import effectiveness of tripeptides. The pathway consists of Violacein A (VioA), Violacein B (VioB) and Violacein E (VioE). It converts tryptophan into the green product prodeoxyviolacein (PDV). The first two enzymes, VioA and VioB, are expressed in the cytosol, and the third one, VioE, is targeted to the peroxisome with a PTS1 sequence. The degree of import can be measured by the intensity of green colour of the colonies. An efficient import signal leads to a strong import of the VioE into the peroxisome and subsequently to white colonies, because the intermediates cannot diffuse into the peroxisome to its respective enzyme. DeLoache <i>et al.</i> showed that there is a proportional correlation between the concentration of the green product PDV and a red fluorescent substance. The concentration of this product displays the import efficiency of the respective sequence.</p>
-<abbr title="2011, Kunze, M., Neuberger, G., Maurer-Stroh, S., Ma, J., Eck, T., Braverman, N., Schmid, J., Eisenhaber, F. & Berger, J. - Structural requirements for interaction of peroxisomal targeting signal 2 and its receptor PEX7."> (Kunze)</abbr></a>
+<p>This assay has been used for the evaluation of the generated PTS2 sequences. The VioE-PTS2 plasmids are harvested and cotransformed with a VioA-VioB plasmid. Each plasmid contains a specific auxotrophy marker. Consequently every growing colony contains both plasmids. To evaluate the respective sequence the concentration of the red fluorescence is measured. The more fluorescence is detected the more VioE is in the cytosol. Therefore the respective PTS2 is not that efficient. The other way around a low concentration of the fluorescent substance correlates with an efficient import via the respective PTS2. </p>
-</figcaption>
+</div>
-</figure>
-<p>The five amino acids in the center are not conserved and highly variable. In yeast, among other organisms, the protein Pex7 works as a soluble chaperone, which recognizes PTS2 and directs the protein to the import pore at the peroxisomal membrane
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/17445803 "> <abbr title="2007, Platta, Harald W., and Ralf Erdmann - The peroxisomal protein import machinery">(Erdmann)</abbr></a>.</p>
-<p>Towards the aim of implementing a valuable import device for our toolbox we created a library of different PTS2 versions showing variable import efficiencies. Subsequently, one can ensure customizable concentrations of different pathway parts in the peroxisome. Moreover, proteins which require an unmodified C-terminus can be imported via PTS2 since this sequence is located on the N-terminus of the protein (<a href="#PTS1">PTS1 import</a>).</p>
-<p>Kunze <i>et al.</i> performed a mutational analysis for the PTS2 containing human thiolase, specifically for the five variable residues in the core region. The wild type sequence of those residues was defined as glutamine, valine, valine, leucine and glycine. These amino acids were substituted by specific amino acids to be able to evaluate the effect of distinct types in the above stated positions within the sequence. The selected amino acids represent different groups to investigate the biochemical effects of different side chains or other factors: aspartate as a negatively charged, tryptophan as an aromatic, arginine as a basic, leucine as a bulky and lysine as a positively charged amino acid. The thiolase import was subsequently measured with immunofluorescence microscopy. The recognition and import of the PTS2 harboring protein of interest by Pex7 worked out with aspartate at position X1, but not on X2 or X3. Lysine on residue X3 lead to a strong decrease of import activity. Kunze et al. concluded that the import of a given protein relies highly on the amino acid groups in the core region of the PTS2 <a href="https://www.ncbi.nlm.nih.gov/pubmed/22057399 ">       <abbr title="2011, Kunze, M., Neuberger, G., Maurer-Stroh, S., Ma, J., Eck, T., Braverman, N., Schmid, J., Eisenhaber, F. & Berger, J. - Structural requirements for interaction of peroxisomal targeting signal 2 and its receptor PEX7.">(Kunze) </abbr></a>.</p>
-<p>Besides a biased approach, which relies on substitution of single residues in the amino acid sequence of the PTS2, in a second approach we aim to randomly change the sequence to characterize a huge library of different sequence compositions.</p>
-</div>
 <button class="accordion">
-<h2  id="MembraneIntegration">Membrane Integration</h2>
+  <h2 id="Membrane_Integration">Membrane integration</h2>
-<p>To optimize the luminal conditions of our compartment we focused on integrating new proteins to its membrane. This way we can alter specific properties or supply reactions inside with necessary co-factors. To test the import and integration mechanism, we fused our designed membrane anchors to fluorescent marker proteins and finally integrated the protein pump bacteriorhodopsin into the membrane to acidify our compartment</p>
+  <p>Abstract</p>
-</button>
+  </button>
 <div class="panel">
-<h3>Introduction</h3>
+<!-- <h3>Scientific background</h3>
+<p>Peroxisomal membrane proteins are synthesized on free polysomes in the cytosol and afterwards integrated into the membrane via two major pathways: one dependent on the endoplasmatic reticulum and one dependent on Pex19 and Pex3.
+<h3>Pex19-dependent</h3>
+<h3>ER-dependent</h3> -->
+<h3>Experimental Work/Design</h3>
+<p>In order to test our hypothesis we fused the last 59 amino acids of the C-terminus of human <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> (AA 246-305) to a red fluorescent protein, to further elucidate the  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>/Pex19-dependent import. mRuby is generally used as a marker in combination with a fluorescent microscope to visualize the localization of the fusion protein. The C-terminus of <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> contains a helical signal-anchor, which serves as both, a mPTS and transmembrane domain. We designed our construct with mRuby2 fused to the N-terminal side of the <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a>-C-terminus, this way the mRuby should face the cytosolic side of the peroxisomal membrane.
+Quite similar to our mRuby-<a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> approach, we designed a construct for the ER-dependent import. Therefore, we fused the mRuby2 fluorescent protein to the N-terminus of  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> (AA 1-39). This construct should be N-terminally anchored in the peroxisomal membrane, with mRuby2 again facing the cellular lumen.</p>
+<img src="https://static.igem.org/mediawiki/2017/4/4e/PMP_figures_Pex3_and_PEX26.png">
+<p>Our main goal is to introduce a rather complex membrane protein to the peroxisome that can alter specific traits. For that we fused the  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> N-terminus (AA 1-39) to a <i>Halobacterium salinarum</i> <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> protein (AA 16-262), replacing the first 16 amino acids (<a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>-BacR). The original archaeal <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> acts as a proton pump by capturing light energy to move protons across the membrane out of the cell. The resulting proton gradient is subsequently converted into chemical energy.
+Our assumption is that the first transmembrane segment determines the orientation of the following protein and that therefore due to the N-terminal anchoring signal the <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> will be inserted in reverse orientation, pumping the protons into the peroxisome. This way the pH of the peroxisomal lumen could actively be controlled and adjusted.</p>
-<p>Many reactions rely on optimal conditions like pH and co-factors. Thus, this subproject aims at the optimization of those circumstances through the integration of new membrane proteins, which alter specific properties of the peroxisomal lumen. Such an approach promises to be very useful for metabolic engineering projects as it can help to adjust the pH, provide cofactors to enzymes or increase/decrease the concentrations of metabolites inside to peroxisome. In nature two distinct mechanisms exist, which are used for the integration of membrane proteins into the peroxisomal membrane – a Pex19-<a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> dependent and an ER-dependent one  <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1255988"> <abbr title="I.A. Sparkes, C. Hawes, A. Baker, AtPEX2 and AtPEX10 are targeted to peroxisomes independently of known endoplasmic reticulum trafficking routes, Plant Physiol. 139 (2005) 690–700"> (2005, Sparkes <i>et al.</i>) </abbr> </a>.</p>
+<p>Finally, our project involved combining the work of other subteams to verify the localization of our constructs in the peroxisome and analyze the effects they have on the import. Therefore, we are using the superfolded-GFP protein, another fluorescent marker, which is in our case fused to the peroxisomal import sequence PT1, and a version of Pex11 that is fused to the fluorescent marker Venus. Both markers emit light in the green light spectrum, were as mRuby2 emits light in the red part of the spectrum, giving us a strong contrast and an easy way of differentiating between the two under the fluorescent microscope.  </p>
+<p>To physically create our constructs, we researched the DNA sequences of <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a>,  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> and <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> via UniProt and pre-designed our fusion constructs with the software tool „Geneious“. We ordered the synthesis of three separate parts ( <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>, <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> and  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>-BacR) from IDT. To ease out the process of assembling our plasmids, we used the „<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-2">Dueber Toolbox</a>", containing various parts such as promoters and terminators, to tailor the plasmids specific to your needs. Finally, to combine all the selected parts, we used the „Golden Gate” assembly method.</p>
-<img src="https://static.igem.org/mediawiki/2017/8/8c/PMP_pH_dependent_enzymes.png">
-<p>They rely on a so called mPTS sequence, that is used to mark the proteins for transport to and integration in the peroxisomal membrane  <a href="https://www.ncbi.nlm.nih.gov/pubmed/12839494"> <abbr title="H.F. Tabak, J.L. Murk, I. Braakman, H.J. Geuze, Peroxisomes start their life in the endoplasmic reticulum, Traffic 4 (2003) 512–518"> (2003, H.F. Tabak <i>et al.</i>)</abbr> </a>. We will try to utilize the capability of both mechanisms to incorporate new proteins into the peroxisomal membrane.
-However, to test whether yeast can integrate and use the foreign proteins in its peroxisomal membrane, we will design three different constructs, which will hopefully give us insights into the mechanisms and its efficiency to incorporate new proteins into the peroxisomal membrane.</p>
+</div>
-<img src="https://static.igem.org/mediawiki/2017/7/7b/PMP_Import_ways.png">
+<button class="accordion">
+  <h2 id="Secretion">Secretion</h2>
+  <p>For the peroxisome secretion in <i>S. cerevisiae</i> we designed fusion proteins of the v-SNARE <a href="http://parts.igem.org/Part:BBa_K2271060">  Snc1 </a> with different peroxisomal membrane anchors.
+We tested the constructs using a GUS Assay. The assays were performed using transformants of the strain BY4742.</p>
+  </button>
+<div class="panel">
+<h3>Experimental Design </h3>
+<p>We will adapt the system of  Sagt and colleagues  to secrete the content of our modified compartments <a href=" https://www.ncbi.nlm.nih.gov/pubmed/19457257">
+  <abbr title=" Sagt, C. M., ten Haaft, P. J., Minneboo, I. M., Hartog, M. P., Damveld, R. A., van der Laan, J. M., ... & van der Klei, I. (2009). Peroxicretion: a novel secretion pathway in the eukaryotic cell. BMC biotechnology, 9(1), 48.
+">
+      (Sagt <i> et al</i>, 2009)
+  </abbr>
+</a>. <br>
+For the application of this system in <i>S. cerevisiae</i> we use a truncated version of the v-SNARE Snc1 to decorate our compartments(Figure 3.1) <a href=" http://www.jbc.org/content/272/26/16591.short">
+  <abbr title=" Gerst, J. E. (1997). Conserved α-helical segments on yeast homologs of the synaptobrevin/VAMP family of v-SNAREs mediate exocytic function. Journal of Biological Chemistry, 272(26), 16591-16598.
+">
+      (Gerst <i> et al</i>, 1997)
+  </abbr>
+</a>. <br>
-<p>As a proof of concept, we will incorporate three proteins through three different approaches into the peroxisomal membrane: (i) mRuby2-<a href="http://www.uniprot.org/uniprot/Q7Z412"><a href="http://www.uniprot.org/uniprot/Q7Z412" style="color:#DB8321">PEX26</a></a> as a proof for the Pex19-dependent mechanism, (ii)  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>-mRuby2 itself to showcase the ER-dependent mechanism and (iii) <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a>, a unidirectional proton pump, fused to the N-terminal anchor of  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>. </p>
-<h4>Pex19-dependent Mechanism</h4>
+ <figure>
-<p>The exact mechanisms of mPTS binding,  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>/Pex19 disassembly, mPTS-PMP binding, and release from the  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>/Pex19 mediated mPTS-PMP docking to the full integration into the membrane are yet unknown <a href="https://www.ncbi.nlm.nih.gov/pubmed/20531392"> <abbr title="2010, Schueller - The peroxisomal receptor Pex19p forms a helical mPTS recognition domain"> (2010, Schueller <i>et al.</i>)</abbr> </a>. However, general principles of the integration of a new peroxisomal membrane protein (PMP) through Pex19 and  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> are studied. Most PMPs feature a membrane targeting signal (mPTS), multiple binding sites for Pex19p, and at least one transmembrane domain (TMD). The mPTS can appear in two different ways, either located in the middle of the primary amino acid sequence, which is the rather complex form, or it can be found at the N-terminal part of the PMP as in Pex25. Pex19p is a cytosolic protein, which recognizes the mPTS of the PMP to be incorporated. In the first step Pex19p attaches to the PMP by binding to the mPTS and acts like a chaperone, guiding it to the peroxisome. Next, Pex19p binds N-terminally to the peroxisomal membrane protein  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>p, which is attached to the peroxisomal membrane through an N-terminal membrane anchor. This will bring the PMP in close proximity to the peroxisomal membrane. Last, Pex19p initiates the membrane integration of the PMP.  <a href="https://www.ncbi.nlm.nih.gov/pubmed/26777132">
+   <img src="https://static.igem.org/mediawiki/2017/f/f9/T--Cologne-Duesseldorf--Snc1_gerst.png">
-   <abbr title="(2016, Liu et al)">.</a></p>
-<h4>Additional Sources/References</h4>
+   <figcaption> <b>Figure 3.1 A diagram of the general domain structure of Snc1. </b> V is a variable domain which is not important for the binding to the t-SNARE. TM is the transmembrane domain. H1 and H2 are the a-helical segments, forming the SNAREpin with the t-SNARE  <a href=" http://www.jbc.org/content/272/26/16591.short">
+  <abbr title=" Gerst, J. E. (1997). Conserved α-helical segments on yeast homologs of the synaptobrevin/VAMP family of v-SNAREs mediate exocytic function. Journal of Biological Chemistry, 272(26), 16591-16598.
+">
+      (Gerst <i> et al</i>, 1997)
+  </abbr>
+</a></figcaption>
-<p>2001, Jones - Multiple Distinct Targeting Signals in Integral Peroxisomal Membrane Proteins</p>
+</figure>
-<p>2004, Jones - PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins</p>
-<p>2004, Rottensteiner - Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals</p>
-<p>2016, Mayerhofer - Targeting and insertion of peroxisomal membrane proteins ER trafficking versus direct delivery to peroxisomes</p>
-<p>2016, Hua - Multiple paths to peroxisomes Mechanism of peroxisome maintenance in mammals</p>
-<p>2016, Giannopoulou - Towards the molecular mechanism of the integration of peroxisomal membrane proteins</p>
-</div>
+<p>To decorate the compartments with the SNARE we use a <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#MembraneIntegration"> peroxisomal transmembrane protein </a>  . In our case we use the proteins Pex15 or PEX26, which were further investigated in another sub project, and fuse Snc1 to the N-terminus. We expressed these constructs of membrane anchor and Snc1 constitutively under control of the RPL18B promotor. In case of Pex15 we used a truncated version, lacking a large part of the N-terminus, only consisting of the transmembrane domain (315-383) (Figure 3.1). For PEX26 we use the truncated version published in Halbach et al. <a href=" http://jcs.biologists.org/content/119/12/2508">
+  <abbr title=" Halbach, A., Landgraf, C., Lorenzen, S., Rosenkranz, K., Volkmer-Engert, R., Erdmann, R., & Rottensteiner, H. (2006). Targeting of the tail-anchored peroxisomal membrane proteins PEX26 and PEX15 occurs through C-terminal PEX19-binding sites. Journal of cell science, 119(12), 2508-2517.
+">
+      (Halbach <i> et al</i>, 2006)
+  </abbr>
+</a>.
+</p>
+<figure>
+        <img src="https://static.igem.org/mediawiki/2017/6/61/T--Cologne-Duesseldorf--Peroxicretion_concept_Pex26.png">
-<button class="accordion">
-<h2 id="Secretion">Secretion</h2>
+        <figcaption><b>Figure 3.2 Concept of secreting peroxisomal contents to the supernatant. </b>For the secretion, the membrane anchor Pex15 or Pex26 is used. This anchor is used to decorate peroxisomes or our modified compartments with the v-SNARE Snc1. For the secretion Snc1 interacts with the t-SNAREs in the cell membrane. Induced from this interaction the vesicle and cell membrane fuse and the content of the compartment is secreted to the supernatant.</figcaption>
-<p>Downstream processing is not only time consuming but also cost and energy intensive. Therefore, we aim to simplify the purification of compounds produced in our artificial compartment. We used a concept based on the peroxicretion described by Sagt and colleagues <a href=" https://www.ncbi.nlm.nih.gov/pubmed/19457257">
-  <abbr title=" Sagt, C. M., ten Haaft, P. J., Minneboo, I. M., Hartog, M. P., Damveld, R. A., van der Laan, J. M., ... & van der Klei, I. (2009). Peroxicretion: a novel secretion pathway in the eukaryotic cell. BMC biotechnology, 9(1), 48.">
-      (Sagt <i> et al</i>, 2009)
+</figure>
-  </abbr>
+<p>We verified our secretion using Beta-glucuronidase (GUS) as a reporter protein. In 2012 Stock and colleagues described the GUS reporter assay for unconventional secretion <a href=" https://www.ncbi.nlm.nih.gov/pubmed/22446315 ">
-</a>. </p>
+   <abbr title=" Stock, J., Sarkari, P., Kreibich, S., Brefort, T., Feldbrügge, M., & Schipper, K. (2012). Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis. Journal of biotechnology, 161(2), 80-91.
-</button>
-<div class="panel">
+">
-<h3>Introduction </h3>
+       (Stock <i> et al</i>, 2012)
-<p>Downstream processing is a very important part of industrial biological compound production. For most biotechnological produced compounds, it is the most expensive part of the production <a href=" https://www.ncbi.nlm.nih.gov/pubmed/11602307 ">
-   <abbr title=" Keller, K., Friedmann, T., & Boxman, A. (2001). The bioseparation needs for tomorrow. TRENDS in Biotechnology, 19(11), 438-441.">
-       (Keller <i> et al</i>, 2001)
    </abbr>
 </a>
-. One step to decrease the costs is to secrete the products into the supernatant <a href=" https://www.ncbi.nlm.nih.gov/pubmed/23385853">
+. With it, it is possible to determine whether a protein is secreted conventional and is <i>N</i>-glycosylated or secreted unconventional and not <i>N</i>-glycosylated. GUS is a bacterial protein with an <i>N</i>-glycosylation-site, which is active only if the protein is not <i>N</i>-glycosylated. The GUS-activity can be measured with different reagents in plate or liquid assays. Liquid assays can be applied qualitatively as well as quantitatively to measure differences in activity. If GUS is secreted by the conventional pathway the <i>N</i>-glycosylation leads to inactivation of the enzyme <a href=" https://www.ncbi.nlm.nih.gov/pubmed/22446315 ">
-   <abbr title=" Berlec, A., & Štrukelj, B. (2013). Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells. Journal of industrial microbiology & biotechnology, 40(3-4), 257-274.
+   <abbr title=" Stock, J., Sarkari, P., Kreibich, S., Brefort, T., Feldbrügge, M., & Schipper, K. (2012). Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis. Journal of biotechnology, 161(2), 80-91.
 ">
-       (Berlec <i> et al</i>, 2013)
+       (Stock <i> et al</i>, 2012)
    </abbr>
-</a>. After secretion, it is possible to remove most cellular compounds from valuable products with one simple centrifugation step. Due to this, secretion is not only a great tool for a compartment toolbox but also has an economic value. <br>
+</a>
-In regards to the whole project, this is an important part for making the compartment more applicable. Through it we go a step further by thinking about the extraction of products after production.<br>
+ (Fig 3.2).</p>
-At the end of this sub project it should be possible to secrete every compound produced in the modified compartment to the supernatant. This is not trivial because peroxisomes, which are the basis of our compartment do not possess a known natural secretion mechanism. <br>
-We overcome this problem by using the "peroxicretion" concept of Sagt and colleagues <a href=" https://www.ncbi.nlm.nih.gov/pubmed/19457257">
-  <abbr title=" Sagt, C. M., ten Haaft, P. J., Minneboo, I. M., Hartog, M. P., Damveld, R. A., van der Laan, J. M., ... & van der Klei, I. (2009). Peroxicretion: a novel secretion pathway in the eukaryotic cell. BMC biotechnology, 9(1), 48.">
-      (Sagt <i> et al</i>, 2009)
-  </abbr>
-</a>. They used a v-SNARE (<b>v</b>esicle- synaptosome-associated-<b>S</b>oluble <b>N</b>-ethylmaleimide-sensitive-factor <b>A</b>ttachment <b>RE</b>ceptorprotein) fused to a peroxisomal membrane-protein to secrete the content of peroxisomes. V-SNAREs interact with the t-SNARE (<b>t</b>arget synaptosome-associated-SNARE) at the cell membrane, which leads to an fusion of the vesicle with the membrane <a href=" https://www.nature.com/nrm/journal/v2/n2/full/nrm0201_098a.html">
-  <abbr title=" Chen, Y. A., & Scheller, R. H. (2001). SNARE-mediated membrane fusion. Nature reviews Molecular cell biology, 2(2), 98-106.">
-      (Chen <i> et al</i>, 2001)
-  </abbr>
-</a>.
-</p>
-</div>
+<figure>
+        <img src="https://static.igem.org/mediawiki/2017/0/0e/T--Cologne-Duesseldorf--Gus_Erkl%C3%A4rung.png">
+        <figcaption><b>Figure 3.3 The GUS Assay. </b> GUS secreted with an unconventional secreted protein like Cts1 from Ustilago maydis active in the supernatant. GUS secreted with a conventional Signal peptide (Sp) inactive in the supernatant. If GUS is in the cytoplasm there is also no activity (Lysis control) <a href=" https://www.ncbi.nlm.nih.gov/pubmed/23455565">
+  <abbr title=" Feldbrügge, M., Kellner, R., & Schipper, K. (2013). The biotechnological use and potential of plant pathogenic smut fungi. Applied microbiology and biotechnology, 97(8), 3253-3265.
+">
+      (Feldbrügge <i> et al</i>, 2013)
+  </abbr>
+</a>. </figcaption>
-<button class="accordion" >
-<h2 id="SizeAndNumber">Size and Number</h2>
-<p>
-Peroxisomes are eukaryotic organelles that are involved in a vast variety of metabolic processes, most notably the metabolism of lipids as well as various oxidative reactions [1, 6]. They are remarkably versatile when it comes to adapting to both, intra- and extracellular cues and changes, responding to environmental variation by changing their size, shape and content accordingly. These exceptional abilities of peroxisomes do not only provide cells with a microenvironment for metabolic reactions but also enable them to respond to metabolic or environmental stress as well as cope with needs of cell division [6]. Controlling the size and shape of peroxisomes is therefore not only crucial to understanding peroxisome dynamics and utility but also provides a promising opportunity to influence bioengineering pathways within cells. Many studies that have been conducted in order to investigate the complexity of the shape, size and number of peroxisomes have led to the recognition of several PEX genes involved in peroxisome biogenesis and proliferation [3]. In order to control the size and number of peroxisomes as part of our toolbox, we further investigated the Pex11, Pex31,32, and Pex34 genes.
-</p>
-</button>
-<div class="panel">
-<h3>Introduction</h3>
-<h4>Peroxisome Biogenesis and Proliferation</h4>
-<p>
-Peroxisomes can be generated in different ways and their size and abundance is controlled by a number of pathways [8]. In yeast, peroxisomes can be generated de novo by budding from the endoplasmatic reticulum (ER) or through division from pre-existing peroxisomes using new proteins and lipids supplied from the ER in the form of vesicles [1]. Both pathways are still being investigated and to date haven’t been fully understood.
-</p>
-<figure>
-    <img class="half-width" src="https://static.igem.org/mediawiki/2017/4/44/T--Cologne-Duesseldorf--Peroxisomes-can-form-through-two-pathways.jpg">
-    <figcaption>Fig.1: Peroxisomes can form through two pathways Nat Rev Mol Cell Biol. 2013 Dec; 14(12): 803–817.</figcaption>
 </figure>
+<p>GUS will be imported to the peroxisome with the PTS1 sequence and measured quantitatively in the supernatant. We will use a coexpression of GUS-PTS1 and Snc1-Pex15 or Snc1-PEX26 to identify the secretion of the compounds. Furthermore, we will use GUS-PTS1 expressed in <i>S. cerevisiae</i> without Snc1 fused to a membrane anchor for a control. We will measure the active GUS in the supernatant with a liquid assay based on the turnover of  4-methylumbelliferyl-beta-D-glucuronide to 4-methyl umbelliferone (4-MU)<a href=" http://cshprotocols.cshlp.org/content/2007/2/pdb.prot4688.abstract">
+  <abbr title="Blázquez, M. (2007). Quantitative GUS activity assay of plant extracts. Cold Spring Harbor Protocols, 2007(2), pdb-prot4690.
+">
+      (Blázquez <i> et al</i>, 2007)
+  </abbr>
+</a>. Here we expect a higher activity of GUS in the supernatant of cultures with Snc1 decorated peroxisomes. <br>
+To increase the variability of our constructs we also designed vectors with and without a GS-Linker connecting the Snc1 with the Pex15. Additionally we tested our constructs in strains with a <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#SizeAndNumber"> deletion of Pex11 </a>. This deletion leads to formation of <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#SizeAndNumber"> larger peroxisomes</a> and may increase the efficiency of our secretion mechanism.</p>
+</div>
-<p>
+<button class="accordion">
-Peroxisomes are extremely sensitive to environmental cues and are able to proliferate or be degraded accordingly [3]. Depending on the growth medium and their extracellular environment, peroxisomes are able to divide and multiply separately from cell division [3]. Their size and number is directly influenced by the presence of e.g. fatty acids, which lead to an increase in both size and number. Furthermore, peroxisome population is regulated by different peroxisomal integral membrane proteins, so called peroxins [2].
+  <h2 id="SizeAndNumber">Size and Number</h2>
-</p>
+  <p>Abstract</p>
-<h4>Peroxins</h4>
+  </button>
-<p>
+<div class="panel">
-The formation of peroxisomes, both by de novo generation as well as growth and fission, is a highly controlled mechanism. Multiple studies have shown that the growth and division of peroxisomes are regulated by protein families specific to peroxisomes, so called peroxins [2]. Since <I>S. cerevisiae</I> naturally contains a very small amount of peroxisomes when growing under glucose-rich conditions, biosynthesis and target yield can be increased by altering peroxisome size and number. A short introduction to the peroxins used in this part of the project is given hereafter.
+  <h3>Heading</h3>
-</p>
+    <p>
-<h5>PEX11</h5>
-<p>
-Due to its unique ability to promote peroxisome division and its role in peroxisome biogenesis [5], the first peroxin we chose for our purpose is Pex11, which is located in the inner surface of the peroxisomal membrane [9]. Erdmann and Blobel have shown that the deletion of the Pex11 gene in S. cerevisiae results in cells with fewer, larger peroxisomes, whereas overexpression results in cells with a higher quantity of smaller peroxisomes [2]. Studies conducted by Smith and Aitchison confirmed that Pex11p-deficient cells growing on fatty acids failed to increase the amount of peroxisomes and instead the accumulation of a few giant peroxisomes was observed [1]. Other media, like oleate-containing ones, cause an induction of peroxisomal proliferation, which is due to an oleate responsive element of the Pex11 promoter [2].
+    </p>
+</div>
+<button class="accordion">
+  <h2 id="Sensors"><i>In Vivo</i> Sensors</h2>
+  <p>Surveillance of key physiology factors is indispensable for a good understanding and optimization of metabolic processes in compartments. In comparison to <i>in vitro</i> measurements, <i>in vivo</i> sensors have major advantages in terms of measure relevant physiological properties in living systems. Former methods required destructive assays, expensive and time consuming preprocessing of cells or compartments to determining fluxes inside cells <a href="https://www.ncbi.nlm.nih.gov/pubmed/20854260"> <abbr title="2010, Frommer et al. -Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors."> Frommer<i>et al.</i> (2010)</abbr></a>. This leads to inexact measurements with poor spacial and temporal resolution. The usage of <i>in vivo</i> sensors tackles all of these problems at once. They allow detecting fluxes inside the cell and compartments in real time and supersede difficult and time consuming cell and organelle extraction. </p>
+  </button>
+<div class="panel">
+<h3>Sensors</h3>
+<p>
+To enrich our toolbox we decided to measure four essential physiological factors: ATP, NADPH, Glutathione and the pH.
+<br>ATP/ADP conversion is used as an energy currency in many cellular processes like translocation of proteins and metabolites or anabolic and catabolic turnovers. It is assumed that ANT1P an ATP/AMP antiporter is located in the peroxisomal membrane and that the re shuttle mechanism of the peroxisomal protein import machinery is ATP dependent
+<a
+href="https://www.ncbi.nlm.nih.gov/pubmed/11566870">
+<abbr title="Identification and functional reconstitution of the yeast peroxisomal adenine nucleotide transporter"> (Palmieri L. <i>et al.</i>, 2001)</abbr></a>. NADPH plays an important role in anabolic pathways and is also indispensable to our desired pathways of <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-5"> nootkatone </a>
+and <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#Violacein">violacein.
+</a>Glutathione is an antioxidant and redox buffer which is also found in yeast peroxisomes. It is used as cofactor by at least two types of peroxisomal proteins the glutathione peroxidases and glutathione transferases, which reduce lipid- and hydrogen peroxides or transfer glutathione to lipid peroxides for the purpose of detoxification
+<a href="http://www.jbc.org/content/276/17/14279.full.pdf">
+<abbr title="2001, Horiguchi H. et al.- Antioxidant System within Yeast Peroxisome">
+(Horiguchi H. <i>et al.</i>, 2001)</abbr></a>.
+Furthermore, it partly represents the redox state of the peroxisome. Knowing the pH of a compartment is important to predict the activities of almost all enzymatic processes inside of it and to follow up acidification and basification upon conversion of metabolites.
 <br>
-In order to find out more about the complexity of peroxisome biogenesis and proliferation and also get constructive feedback on our work, we consulted with Florian David from Biopetrolia in Sweden. Their company specializes in yeast engineering in order to improve production titers, yields and rates for the production of biofuels, pharmaceuticals and other products. He suggested to expand our project to working not only with Pex11, but Pex31,32 and Pex34 as well.
+<br>
+We finally chose two ratiometric sensors to perform measurements with. The pH sensitive and glutathione redox state reporting green fluorescent proteins pHLuorin2
+<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152828">
+<abbr title="2011, Mahon M. J. - pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein">
+(Mahon M. J. <i>et al.</i>, 2011)
+</abbr>
+</a>
+and roGFP2
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539">
+<abbr title="2016, Schwarzländer et al.- Dissecting Redox Biology Using Fluorescent Protein Sensors">
+(Schwarzländer M. <i>et al.</i>, 2016)</abbr></a>.
+We aim to target them either in the peroxisomal lumen or the cytosol. To achieve peroxisomal targeting we attach the peroxisomal targeting signal 1 with
+ <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-2"> Golden Gate cloning</a>.
 </p>
+<div class="flex-row-2">
+ 	<div>
+   	<img src="https://static.igem.org/mediawiki/2017/c/cb/RoGFP2_lvl1_cytosolic_medium_promotor.png">
+ 	</div>
+ 	<div>
+   	<img src="https://static.igem.org/mediawiki/2017/8/8e/RoGFP2_lvl1_peroxisomal_medium_promotor.png">
+ 	</div>
+	</div>
-<h5>PEX30-32</h5>
-<p>
-According to Zhou et al., the genes of the Pex30 – 32 family have been shown to influence peroxisome proliferation [2]. Their deletion resulted in the production of a higher quantity of large peroxisomes. Zhou et al. further investigated the effect of a Pex31,32 knockout, showing both number and size increase, also leading to a higher metabolic yield. However, a Pex31,32 knockout has been proven to attribute to a change in the membrane structure, resulting in higher permeability of the peroxisome membrane for fatty aldehydes and other intermediates and byproducts [2]. Due to these side effects we decided to discard working with a Pex31,32 knockout for now.
-<p/>
+<div class="flex-row-2">
+ 	<div>
+   	<img src="https://static.igem.org/mediawiki/2017/e/ed/PHlourin2_lvl1_cytosolic_medium_promotor2.png
+">
+ 	</div>
+ 	<div>
+   	<img src="https://static.igem.org/mediawiki/2017/3/3a/PHlourin2_lvl1_peroxisomal_medium_promotor.png">
+ 	</div>
+	</div>
-<h5>PEX34</h5>
+<figcaption><font size="2">
-<p>
+<strong> Figure 5.1 </strong>Level1 plasmids with  promoters of medium strength and uracil auxotrophy. Top left roGFP2 cytosolic. Top right roGFP2 peroxisomal. Bottom left pHLourin2 cytosolic. Bottom right pHLourin2 peroxisomal.</font>
-Similar to Pex11, Pex34p is another peroxisomal integral membrane protein that can act both, independently and in combination with Pex11p, Pex25p, and Pex27p to control the peroxisome morphology and population. Pex34p is suggested to directly influence peroxisome proliferation as well as constitutive peroxisome division. Specifically, Pex34p overexpression positively affects peroxisome numbers in wild type and pex34 cells, whereas Pex34 deletion results in cells with fewer peroxisomes [6, 2]. In their studies Zhou et al. targeted synthetic pathways to peroxisomes in order to increase the production of fatty-acid-derived fatty alcohols, alkanes and olefins. By harnessing peroxisomes to produce fatty-acid-derived chemicals and biofuels they were able to show that peroxisome increases the production of target molecules while decreasing byproduct formation. Additionally, analyzing the effect of peroxin knockouts and overexpression, their research revealed that Pex34 overexpression significantly increased their yield [2].  The main advantage of working with Pex34p over Pex31,32 is the effect on the peroxisomal membrane. While Pex31,32 significantly increases membrane permeability, Pex34 has less effects on the membrane structure [2].
-</p>
-<figure>
-    <img src="https://static.igem.org/mediawiki/2017/5/52/T--Cologne-Duesseldorf--peroxisome-quantity-and-morphology.png">
-    <figcaption>Fig. 2: Regulation of peroxisome quantity and morphology by different peroxins
 </figcaption>
-</figure>
-<h4>Our Project</h4>
 <p>
-One step towards achieving the creation of a fully controllable artificial compartment is the regulation of and control over its morphology. In our case we are aiming at achieving the exact regulation of the size and number of the peroxisome. As a first approach we have chosen to control the Pex11 concentration in the cell. Furthermore Pex11 is to be designed as a 3b toolbox part so it can be combined and its effects tested with different promoters. For that purpose we are working with two constitutive promoters of varying strength as well as two inducible promoters which increase gene expression when grown in varying concentrations of galactose or copper sulfate. To control the range from a few giant peroxisomes to a high quantity of small ones we are working in a pex11D knockout strain. Secondly, following the advice of Florian David from Biopetrolia, we intend to increase both, the size and quantity of peroxisomes in the cell via a Pex34 overexpression. By working with Pex34 we will not only be able to control the peroxisome morphology, but also positively influence production yields.
+Using promoters with different expression strength in order to find optimal measurement conditions is of high interest. There is a trade off between a high signal-to-noise ratio and self induced effects which are both dependent on expression levels
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539">
+<abbr title="2016, Schwarzländer et al.- Dissecting Redox Biology Using Fluorescent Protein Sensors">
+(Schwarzländer M. <i>et al.</i>, 2016)
+</abbr></a>.
+This cannot be generalized for each sensor. For example, pHlourin2 has sparse influence on the existing pH because of the buffer effect of proteins.
+<br>
+Validation of the peroxisomal localization can be achieved via fluorescence overlap of the sensor and a peroxisomal marker in our case pex13-mRuby (<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-4">import mechanism</a>).
 </p>
-<h4>How does it integrate into the overall project?</h4>
+<div class="flex-row-2">
-<p>
+ 	<div>
-Controlling the size and number of peroxisomes is one of the multiple functions we plan to integrate into our artificial compartment toolbox so that it can be utilized for various projects. However, even though the exact control of proliferation can help understand the complex matter of peroxisome dynamics, the advantages of these findings exceed mere foundational research.
+   	<img src="https://static.igem.org/mediawiki/2017/4/45/RoGFP2_lvl2_peroxisomal_strong_promotor_Peroxin13_mRuby.png">
-Integrating synthetic pathways into cells is often impeded by competing pathways and accruing intermediates or undesired byproducts that negatively influence biosynthesis. In order to achieve feasible results from microbial production, respective pathways need to be isolated into a suitable environment. Compartmentation provides microenvironments for metabolic functions of cells shielding them from the interference of simultaneously occurring reactions and therefore favoring biosynthesis. Going one step further, establishing synthetic pathways into a fully controlled compartment has the potential to increase the efficiency and productivity of these pathways resulting in higher yields of target products [2,6]. In our case, we change the peroxisome’s morphology by knocking out or overexpressing Pex11 and Pex34 to obtain either a large amount of smaller peroxisomes or a high amount of enlarged ones. Especially the overexpression of Pex34 which results in a high quantity of large peroxisomes has been shown to actively regulate metabolic processes [1] and increase the production of target molecules while decreasing byproduct formation [2]. Furthermore, evidence indicates that changing the morphology of a compartment, including both, its shape and size, influences the amount of chemical reactions embedded in that compartment [1], a trait that can be used to increase the yield of otherwise inefficient reactions.  Ultimately, even though we decided to discard our work on a Pex31,32 knockout, the effects this knockout has on membrane permeability and structure could potentially be used for further pathways within the peroxisome. </p>
+ 	</div>
+ 	<div>
+   	<img src="https://static.igem.org/mediawiki/2017/9/99/PHlourin2_lvl2_peroxisomal_strong_promotor_peroxin13_mRuby.png">
+ 	</div>
+	</div>
+<figcaption><font size="2">
+<strong>Figure 5.2 </strong>Level2 plasmids with peroxisomal marker Peroxin13-mRuby and uracil auxotrophy for colocalization. Left roGFP2 with strong promoter. Right pHLourin2 with strong promotor.</font>
+</figcaption>
-<h4>Overall goal of this subproject</h4>
 <p>
-In our subproject we want to achieve full control over peroxin concentrations in the yeast cell, in order to establish a simple method to regulate the peroxisome morphology and quantity.</p>
+It can also be validated by transforming the sensors attached to the PTS1 sequence into Pex5 knockout yeast strain. The sensor is expected to show no specific localisation, because of the missing import sequence. We calibrate the sensors in living yeast cells and physiological ranges so that we can not only perform relative but also quantitative measurements. We aim to confirm our hypothesis of an more oxidized redox state of roGFP2 in peroxisomes with <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#Violacein">violacein pathway
+</a> activity and want to measure differences in pH within yeast strains with peroxisomal membrane anchored pex3-bacteriorhodopsin protein
+<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#MembraneIntegration"> (membrane proteins) </a>.
+<br>
+<br>
+Once expression and localization of the sensor is proven by microscopy, measurements with a plate reader or a fluorometer are acceptable. This allows a high number of replicates to be measured accurately in reasonable time. Microscopy is performed with a filter based Nikon Eclipse TI fluorescence microscope at 100-fold magnification and plate reader measurements are performed with a Tecan infinite plate reader
+<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152828">
+<abbr title="2011, Mahon et al.- pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein">
+(Mahon M. J. <i>et al.</i>, 2011)</abbr>
+</a>
+. For pHLuorin2 emission intensity is measured at 535 nm upon excitation at 405 nm and 485 nm. Same settings were used for roGFP2
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539">
+<abbr title="2016, Schwarzländer et al.- Dissecting Redox Biology Using Fluorescent Protein Sensors">(Schwarzländer M. <i>et al.</i>, 2016)</abbr>
+</a>
+. Evaluation of the reported signals is done by the excitation ratio of the the corresponding excitation wavelength.
+</p>
 </div>
 <button class="accordion">
-<h2 id="Sensors"><i>In Vivo</i> Sensors</h2>
+  <h2>Nootkatone</h2>
-<p>Designing new pathways or transferring pathways into cellular compartments requires a sound understanding of the present conditions and content, like cofactors in the peroxisomes.
+ <p>In order to have a proof of concept for our compartmentation strategy we intend to establish the nootkatone pathway inside the peroxisome. Therefore we aim to target the three enzymes for the nootkatone production to compartment. Before starting the actual lab work for the ambitious plan  to establish a completely new pathway in the peroxisome there are several aspects that have to be considered first.</p>
-We aimed  measuring the peroxisomal pH, cofactors like NADP<sup>+</sup> and ATP in wild type yeast and our designed mutants over different time periods as well as in response to changing physiological conditions. Therefore, we used ratiometric fluorescent biosensors which we genetically attached to a peroxisomal targeting signal.
+  </button>
-These measurements provide important insights into possible issues which may occur if non-peroxisomal pathways are transferred into the peroxisome and furthermore enable more precise predictions and modelling.</p>
-</button>
 <div class="panel">
-<h4> pH Sensor </h4>
-<p>The activity of enzymatic Proteins is mostly pH-dependent. Therefore, it is of high interest to understand the pH-regulating mechanism of the peroxisome and the effects on the imported pathways. Literature has not agreed whether there is a common peroxisomal pH nor whether there is a regulating mechanism. For our measurements, we use pH Lourin2, a GFP variant with a bimodal excitation spectrum with peaks at 395 and 475 nm and an emission maximum at 509 nm. Upon acidification, the excitation spectrum shifts from 395 to 475 nm <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152828/"> <abbr title="2011, Mahon et al.- pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein"> Mahon <i>et al.</i> (2011)</abbr>.</a>
-</p>
+<p>The initial step is to find a reliable source to prove the abundance of our precursor <i>Farnesyl pyrophosphate</i> (FPP) in yeast peroxisomes. At this time there is no proof of existence of FPP inside yeast peroxisomes yet. However it is predicted to be present, as it was detected in mammalian and plant peroxisomes
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/11108725">
+<abbr title="2000, Olivier et al. - Identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes. AA-CoA thiolase, hmg-coa synthase, MPPD, and FPP synthase.">
+   	Olivier <i>et al.</i> (2000)
+	</abbr>
+</a>.</p>
 <figure>
-	<img src="https://static.igem.org/mediawiki/2017/9/93/Artico_pHLuorin2_Verlauf.png">
+     	<img src="https://static.igem.org/mediawiki/2017/b/b9/Farnesylpyrophosphat.png">
-	<figcaption><font size="3"> <strong>Figure5.1</strong>
+      	<figcaption><strong>Figure 7.1</strong> structure FPP </figcaption>
-pHLuorin2 emission at 509 nm, excited at wavelengths between 350 nm and 500 nm . Five different pH values, ranging from 5.8 to 7.8 are shown <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152828/"><abbr title="2011, Mahon et al.- pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein"><font size="3"> Mahon <i>et al.</i> (2011)</font></abbr></a>.</font> </figcaption>
+  	</figure>
-</figure>
-<h4> roGFP2 Sensor </h4>
-<p>To maintain thermodynamic driving forces and electron fluxes which are needed at steady state, the intact chemeostasis of the redox machinery is of high importance<a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539"> <abbr title="2016, Schwarzländer, et al. - Dissecting Redox Biology Using Fluorescent Protein Sensors"> (2016, Schwarzländer)</abbr></a>. Glutathione is considered to be inside the peroxisomal lumen <a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539"> <abbr title="2014, Elbaz-Alon, Y., et al. -The Yeast Oligopeptide Transporter Opt2 Is Localized to Peroxisomes and Affects Glutathione Redox Homeostasis">(Elbaz-Alon, Y., et al. 2014)</abbr></a>. We therefore wanted to monitor glutathione redox potentials inside the peroxisomal lumen using the  GFP variant roGFP2, which is able to precisely detect redox changes of glutathione. Two cysteines in the beta barrel structure can either form two thiols or one disulfide bondage dependent on whether they are reduced or oxidized. This influences the proton transfer of the chromophore and ultimately leads to a ratiometric shift in excitation. Excitation at 485 nm of the reduced roGFP2 exceeds the excitation of oxidized roGFP2 at 485 whereas excitation at 405 nm  of oxidized roGFP2 exceeds excitation of reduced roGFP2?????? Was meinen dieses? <a href="https://link.springer.com/article/10.1007/s12268-016-0683-2"> <abbr title="Morgan, B. and M. Schwarzländer 2016 et al.- The Yeast Oligopeptide Transporter Opt2 Is Localized to Peroxisomes and Affects Glutathione Redox Homeostasis">(Morgan, B. and M. Schwarzländer 2016)</abbr></a>. </p>
-</div>
-<button class="accordion">
+<p>The precursor FPP is converted into valencene by a valencene synthase (ValS). We chose the one from <i>Callitropsis nootkatensis</i> because of its comparably high efficiency in microorganisms. It achieves greater yields in yeast than the citrus valencene synthase. Furthermore, the product specificity is relatively high, while production of byproducts is low
-<h2 id="Nootkatone">Nootkatone</h2>
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/24112147"><abbr title="Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene."> Beekwilder et al. (2014)</abbr></a>.
-<p>As a proof of concept for our compartment toolbox we decided to shift the metabolic pathway of nootkatone into the peroxisome. With this transfer we want to overcome the obstacle of intermediate toxicity for the yeast cell. A working metabolism will pave the way for an efficient, safe and favorable solution of producing and providing an effective insect repellent. </p>
-</button>
-<div class="panel">
-<p>Nootkatone is an oxidized sesquiterpene, which is highly valuable for industrial and pharmaceutical application. We will focus on its repellent effect towards insects
+The valencene synthase was also chosen because of its robustness towards pH and temperature changes
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/11441443">
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/24112147">
-<abbr title="2001, Zhu et al. - Nootkatone is a repellent for Formosan subterranean termite (Coptotermes formosanus)">
+<abbr title="2014, Beekwilder et al. - Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.">
-    	Zhu <i>et al.</i> (2001)
+   	Beekwilder <i>et al.</i> (2014)
-	</abbr>
+</abbr>
 </a>.
-Also, therapeutic activities of nootkatone have been reported, such as anti-platelet effects in rats
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/21354294">
-<abbr title="2011, Seo et al. - Antiplatelet effects of Cyperus rotundus and its component (+)-Nootkatone">
-    	Seo <i>et al.</i> (2011)</abbr>
-</a>,
- anti-proliferative activity towards cancer cell lines
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/21377882">
-<abbr title="2011, Gliszczyńska et al. - Microbial Transformation of (+)-Nootkatone and the Antiproliferative Activity of Its Metabolites">
-   	Gliszczyńska <i>et al.</i> (2011)
-	</abbr>
-</a>
- and enhancement of energy metabolism through AMP-activated protein kinase activation in skeletal muscle and liver
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/24624065">
-<abbr title="2010, Murase et al. - Habituation of the responsiveness of mesolimbic and mesocortical dopamine transmission to taste stimuli
-">
-   	Murase <i>et al.</i> (2010)
-	</abbr>
-</a>.</p>
-<p>Nootkatone can be extracted from grapefruits, but the organic material is limited and the yield is very low. So far, industrial production of nootkatone requires toxic substances such as heavy metals and strong oxidants like tert-butyl hydroperoxide which is known to be carcinogenic
+Our modelling approach revealed that for optimal yields an overexpression of valencene synthase is necessary because of its slow conversion rate <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#MetabolicModel">(Model)</a>. This is why we chose the strongest promoter of the yeast toolbox
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/21115006">
+<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#yeast-toolbox">(Dueber Toolbox)</a> for this attempt.
-<abbr title="2010, Cankar et al. -  A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene
+</p>
-">
-   	Cankar <i>et al.</i> (2010)
-	</abbr>
-</a>.</p>
 <figure>
-     	<img src="https://static.igem.org/mediawiki/2017/d/d9/Valencene_Nootkatol_Nootkatone.jpeg">
+         <img src="https://static.igem.org/mediawiki/parts/a/a8/ValS_lvl1_PTS1.png">
-       	<figcaption><strong>Figure 7.1</strong> Conversion of valencene  to Nootkatol and Nootkatone </figcaption>
+          <figcaption> <strong>Figure 7.2</strong> ValS lvl.1 PTS1 plasmid </figcaption>
+      </figure>
+<img src="https://static.igem.org/mediawiki/2017/a/a7/Valencene.png">
+       	<figcaption><strong>Figure 7.3</strong> structure Valencene</figcaption>
    	</figure>
+<p>The intermediate valencene is then converted into nootkatol by a P450 monooxygenase as well as into small amounts of our desired product nootkatone.  The P450 monooxygenase we chose for this project was taken from the bacterium <i>Bacillus megaterium</i>. In this  case it is not only a simple P450 monooxygenase, but an entire P450 system, consisting of a soluble P450 fused to a cytochrome P450 reductase (CPR) enzyme, making an additional reductase obsolete
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/12419614">
+<abbr title="2002, De Mot et al. - A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes.">
+   	De Mot <i>et al.</i>  (2002)
+</abbr>
+</a>.
+Unlike eukaryotic P450s, which are mostly membrane bound, this prokaryotic BM3-P450 is located in the cytosol facilitating an easier transport into the peroxisome, as membrane integration of proteins is a more difficult task to achieve than import of cytosolic proteins
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/17073779">
+<abbr title="2006, Girvan et al. - Flavocytochrome P450 BM3 and the origin of CYP102 fusion species.">
+   	Girvan <i>et al.</i> (2006)
+</abbr>
+</a>.
+BM3 normally catalyzes the hydroxylation of long chain fatty acids
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/3086309">
+<abbr title="1986, Narhi et al. - Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium.">
+   	Narhi  <i>et al.</i> (1986)
+</abbr>
+</a>, which in our case could inhibit the conversion of valencene. For that reason we used a mutated version of BM3, which is called AIPLF. This variant is an enhanced version of the BM3 AIP version, which has a ten times better substrate oxidation rate for valencene than the wildtype BM3 and produces less byproduct when valencene concentration is saturated. Additionally to previous named benefits the AIPLF variant with
+<abbr title="R47L/Y51F/F87A/A328I/I401P">
+point mutations
+	</abbr>
+in the active side has a significantly lower binding affinity towards long chain fatty acids and therefore increase the transposition rate of valencene.
-<p>The synthesis of nootkatone starts from the precursor farnesyl pyrophosphate (FPP) and requires at least two enzymes. The initial step is the formation of valencene from FPP by a valencene synthase (ValS) followed by the production of nootkatol, nootkatone and other by-products by a P450 BM3 monooxygenase (BM3). The co-expression of an alcohol dehydrogenase (ADH) with ValS improves nootkatone production by favoring the conversion from nootkatol into nootkatone
+<a href="http://onlinelibrary.wiley.com/doi/10.1002/cctc.201402952/full">
- <a href="http://onlinelibrary.wiley.com/doi/10.1002/cctc.201402952/full">
 <abbr title="2015, Schulz et al. - Selective Enzymatic Synthesis of the Grapefruit Flavor (+)-Nootkatone">
     	Schulz <i>et al.</i> (2015)
+</abbr>
+</a>
+<a href="https://docserv.uni-duesseldorf.de/servlets/DerivateServlet/Derivate-43193/Dissertation_SvenCarstenLehmann.pdf">
+<abbr title="2016, Lehmann - Entwicklung eines P450-basierten Ganzzellkatalysators
+für die selektive Oxyfunktionalisierung von α-Pinen">
+   	Lehmann (2016)
 	</abbr>
-</a>.</p>
+</a>
+</p>
+<figure>
+     	<img src="https://static.igem.org/mediawiki/parts/f/fe/BM3_lvl1_PTS1.png">
+      	<figcaption><strong>Figure 7.4</strong> BM3 lvl.1 PTS1 plasmid </figcaption>
+  	</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2017/7/72/Nootkatol.png">
+      	<figcaption><strong>Figure 7.5</strong> structure Nootkatol </figcaption>
+  	</figure>
+<p>The alcohol dehydrogenase from Pichia pastoris subsequently converts nootkatol into nootkatone by oxidation. It uses NAD+ as a cofactor which is reduced in the reaction. The regeneration of this cofactor is facilitated by the BM3, which oxidizes NADH
+<a href="http://onlinelibrary.wiley.com/doi/10.1002/cctc.201402952/full">
+<abbr title="2015, Schulz et al. - Selective Enzymatic Synthesis of the Grapefruit Flavor (+)-Nootkatone">
+   	Schulz <i>et al.</i> (2015)
+</abbr>
+</a>.</p>
+<figure>
+     	<img src="https://static.igem.org/mediawiki/2017/e/e2/ADH_lvl1_PTS1.png">
+      	<figcaption><strong>Figure 7.6</strong> ADH lvl.1 PTS1 plasmid </figcaption>
+  	</figure>
-<p>Previous approaches of nootkatone synthesis in yeast often failed due to toxic intermediates. A specific problem is the toxicity of beta-nootkatol and nootkatone itself for <i>Saccharomyces cerevisiae</i> at concentration higher than 100 mg/L
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/23518241">
-<abbr title="2013, Gavira et al. - Challenges and pitfalls of P450-dependent (þ)-valencene bioconversion by Saccharomyces cerevisiae">
-   	Gavira <i>et al.</i> (2013)
-	</abbr>
-</a>.
-For an efficient industrial production, concentrations need to be in the range of g/L, which is lethal for yeast cells. Beta-nootkatol seems to accumulate in membranes because of its hydrophobic characteristics, resulting in changes of the membrane permeability, integrity and the function of membrane proteins
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/23518241">
-<abbr title="2013, Gavira et al. - Challenges and pitfalls of P450-dependent (þ)-valencene bioconversion by Saccharomyces cerevisiae">
-   	Gavira <i>et al.</i> (2013)
-	</abbr>
-</a>.
-It is presumed that the toxicity is partly caused by this effect. As one of the original purposes of the peroxisome is to reduce hydrogen peroxide, which is harmful to the cell and also alters the membrane composition
-<a href="https:/https://www.ncbi.nlm.nih.gov/books/NBK9930/">
-<abbr title="2000, Cooper et al. - The Cell: A Molecular Approach. 2nd edition">
-   	Cooper <i>et al.</i> (2000)
-	</abbr>
-</a>
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/1902481">
-<abbr title="1991, Block et al. - Hydrogen peroxide alters the physical state and function of the plasma membrane of pulmonary artery endothelial cells">
-   	Block <i>et al.</i> (1991)
-	</abbr>
-</a>
-, we assume that beta-nootkatol does not affect the peroxisomal membrane either. But to be fully sure if this hypothesis is true, we have to collect and evaluate our own data on how beta-nootkatol affects the peroxisome membrane and thus the yield of nootkatone.</p>
-<div class="half-width">
 <figure>
-     	<img src="https://static.igem.org/mediawiki/2017/3/3c/Graph1.png">
+<img class="half-width" src="https://static.igem.org/mediawiki/2017/5/5f/Nootkatone.png">
-       	<figcaption><strong>Figure 7.2</strong> Yeast viability after 24 h in the presence of (+)-valencene, beta-Nootkatol or nootkatone in different concentrations <a href="https://www.ncbi.nlm.nih.gov/pubmed/23518241">
+       	<figcaption><strong>Figure 7.7</strong> structure Nootkatone </figcaption>
-<abbr title="2013, Gavira et al. - Challenges and pitfalls of P450-dependent (þ)-valencene bioconversion by Saccharomyces cerevisiae">
-   	Gavira <i>et al.</i> (2013)
-	</abbr>
-</a> </figcaption>
    	</figure>
-</div>
+<p>The first milestone to achieve our goal is the separate integration of each of our three enzymes ValS, BM3 and ADH into level 1 vectors <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#yeast-toolbox">(Dueber Toolbox)</a>  in yeast and to verify their expression by Western Blot analysis. Therefore, a 3xFlag/6xHis-tag was added to the N-terminus of each of the proteins.  It enables us to use an anti-His antibody followed by an anti-mouse-antibody to make protein abundances visible. Subsequently, the two enzymes ADH and ValS were combined in a  level 2 cassette plasmid.  BM3 is designed as a level 1 plasmid and will be co-expressed with the level 2 plasmid to achieve a nootkatone production. The expression of the enzymes in the cytoplasm is again verified by Western Blot analysis.
+The further approach aims to provide the enzymes with C-terminal peroxisomal targeting signals type one, which finally converts the nootkatone pathway into our artificial compartment. </p>
+<p>Since the production of nootkatone does not lead to a change of colour we need to apply different methods for verifying substrates. Once we succeed with the qualitative validation via Western Blot analysis, we can verify the presence of nootkatone by using high performance liquid chromatography and mass spectrometry, respectively.</p>
-<p>Our goal is the successful integration of the nootkatone pathway into our compartment and to bypass the problem of high concentration toxicity of beta-nootkatol and nootkatone for the yeast cell. This would not only be a more efficient but also a more environmentally friendly method to satisfy the great industrial demand of this sesquiterpene. It would also facilitate the access to a high performing insect repellent in less developed regions of the world and therefore decrease the spread of diseases like malaria, dengue or the Zika virus.</p>
-</div>
+<h3>Model influence on Nootkatone expression</h3>
+<p>We modeled the nootkatone biosynthesis pathway using ordinary differential equations in order to optimize nootkatone production. We found two hard and an easy problem, all of which we could find a solution for. The easy problem is optimization of the enzyme concentrations of the biosynthesis pathway. According to our <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#MetabolicModel">model of the Nootkatone pathway</a> we found that overexpression of Valencene Synthase is necessary to maximize the Nootkatone yield, while both alcohol dehydrogenase and p450-BM3 have only minor effects on the yield. </p>
+<p>One of the hard problems, as shown in our <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#Penalty-Model">penalty model</a> is the toxicity of nootkatone and nootkatol. Since the toxicity most likely stems from both nootkatone and nootkatol clogging up the cell wall we present our peroxisome as a solution for this problem. When comparing the cytosolic model to our <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#Peroxisome-model">peroxisomal model</a> we found that, if our assumption that neither Nootkatone nor Nootkatol are able to pass the peroxisomal membrane holds up, we can greatly increase Nootkatone production.</p>
+<p>The last hard problem is the influx of the pathway precursor farnesyl pyrophosphate (FPP). We used <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#OptKnock">OptKnock analysis</a> to design yeast strains with optimized FPP production. With this analysis we got hints that growing the yeast cells on a fatty acid medium might be a simple alternative to
+knocking out the desired genes.</p>
+</div>
 <button class="accordion">
-<h2 id="Violacein">Violacein</h2>
+  <h2 id="Violacein">Violacein</h2>
-<p> Using the tools of synthetic biology in metabolic engineering unleashes the full potential of biofactories. Natural systems use compartmentalization to improve biochemical reactions. Here we present the use of peroxisomal import tags to engineer an artificial compartment in <i>Saccharomyces cerevisiae</i> cells to be further used in metabolic engineering approaches. As an application and proof of concept we are using the well studied biosynthetic pathway of violacein. By designing an import library for the different enzymes we aim to understand basic design principles that can guide future design of compartmentalization for metabolic engineering. We chose violacein, not only because of its wide range of biological benefits but also as a solid foundation to proof a sophisticated import machinery. </p>
+<p>Working on a project is a process of well planned steps. It starts with an idea and theoretical research to create a design. When finally demonstrating the mechanism of the project it is important to point out the benefits with a suitable application.
-</button>
+<br>Synthetic biology offers countless numbers of new opportunities, especially in the field of metabolic engineering. To testify some main parts of our <strong>artico</strong> project, we decided to relocate a metabolic pathway into yeast peroxisomes. There are several reasons why this approach fits perfectly as a proof for our concept.
-<div class="panel">
+</p>
-<figure class="floatleft">
-<img class="half-width" src="https://static.igem.org/mediawiki/parts/1/17/T--Cologne-Duesseldorf--Violacein_Struktur.png">
+ </button>
+<div class="panel">
+<p> Based on described of<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#Violacein"> advantages </a> of violacein this pathway was chosen. Violacein is naturally produced in numerous bacterial strains, most popular in the gram-negative <i> Chromobacterium violaceum </i>. It is related to biofilm production and shows typical activities of a secondary metabolite <a href="https://www.hindawi.com/journals/bmri/2015/465056/">
+<abbr title="Violacein: Properties and Production of a Versatile Bacterial Pigment">
+(Seong Yeol Choi <i>et al.</i>, 2015)
+</abbr>
+.
+</a>
+</p>
+<figure>
+<img src="https://static.igem.org/mediawiki/2017/0/09/T--Cologne-Duesseldorf--Violacein_Pathway_komplett.png">
 <figcaption>
-<strong>Figure 8.1</strong> Structural formula of violacein.
+<strong>Figure 8.1</strong> The synthesis of Violacein requires five enzymes encoded by the VioABCDE operon. VioA, a flavin-dependent L-tryptophan oxidase and VioB, a heme protein, work in combination to oxidize and dimerize L-tryptophan to an IPA imine dimer. Hydrogen peroxide is released as a by-product of the VioA reaction. Next step by VioE is the rearrangement of the IPA imine dimer to protodeoxyviolaceinic acid, which can non-enzymatically oxidize to prodeoxyviolacein or, by VioC via deoxyviolaceinic acid, oxidize to pink deoxyviolacein. The flavin-dependent oxygenases VioC and VioD require interaction with the oxidized form of flavin-adenine dinucleotide (FAD)
-</figcaption>
+<a href="http://www.uniprot.org/uniprot/Q9S3U9">
-</figure>
+<abbr title="UniProtKB - Q9S3U9 (VIOC_CHRVO)">
-<p>
+(uniprot,
-Violacein (C<sub>20</sub>H<sub>13</sub>N<sub>3</sub>O<sub>3</sub>), a bisindole, is a violet pigment, formed by condensation of two tryptophan molecules. It can naturally be found in numerous bacterial strains, for example in the gram-negative <i> Chromobacterium violaceum</i>. Due to its wide range of biological properties, violacein is useful for various industrial applications in pharmaceuticals and cosmetics.
-<br>
-Violacein is known to have a variety of different biological activities, including an antitumor
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/20416285">
-<abbr title="Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor">
-(Bromberg N<i> et al</i>, 2010)
 </abbr>
-</a>, antifungal
+</a>
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/18949519">
+<a href="http://www.uniprot.org/uniprot/Q9S3U8">
-<abbr title="Amphibian chemical defense: antifungal metabolites of the microsymbiont Janthinobacterium lividum on the salamander Plethodon cinereus">
+<abbr title="UniProtKB - Q9S3U8 (VIOD_CHRVO)">
-(Brucker RM <i>et al.</i>, 2008)
+uniprot)
 </abbr>
 </a>
-and antiviral
+. The two enzymes act sequentially: first, VioD hydroxylates protodeoxyviolaceinic acid, leading to protoviolaceinic acid. Second, VioC creates the oxindole at the 2-position of one indole ring, leading to violet violacein
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/14595466">
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/17176066">
-<abbr title="Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum">
+<abbr title="In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum">
-(Andrighetti-Fröhner CR <i> et al.</i>, 2003)
+(Balibar CJ <i> et al.</i>, 2006)
 </abbr>
 </a>
-function. Furthermore, it has been shown that violacein enhances the effect of most commercial antibiotics by working synergistically with them
+<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025692/">
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/24073823">
+<abbr title="Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum">
-<abbr title="Synergistic antimicrobial profiling of violacein with commercial antibiotics against pathogenic micro-organisms">
+(Janis J. Füller <i> et al.</i>, 2016)
-(Subramaniam S <i> et al.</i>, 2014)
 </abbr>
-</a>. This is especially of high interest in the fight against recent antibiotic-resistant strains of pathogenic bacteria such as MRSA (multi resistant <i>Staphylococcus aureus</i>). Violacein’s antibacterial action against <i>S. aureus</i> has been proven by
+</a>
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/21364597">
+.
-<abbr title="Antibacterial activity of violacein against Staphylococcus aureus isolated from bovine mastitis">
+</figcaption>
-Cazoto LL <i>et al.</i> (2011)
+</figure>
+<p> Relocating the pathway into the peroxisome enables proximity of the enzymes and substrates. Furthermore the yeast cell is protected from the toxic substance hydrogen peroxide. Yeast peroxisomes have no problem with this as their main function is the beta-oxidation of fatty acids and the detoxification of the thereby produced H<sub>2</sub>O<sub>2</sub>
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/17445803">
+<abbr title="The peroxisomal protein import machinery">
+(Erdmann R. <i>et al.</i>, 2007)
 </abbr>
-</a>.
+</a>. Because VioC and VioD are FAD-dependent, it is additionally an evidence for FAD availability inside of the peroxisome, if the synthesis of Violacein works. Otherwise the two enzymes would not be able to catalyze the reaction.
-<br>It is of high medical interest that toxic effects of Violacein on cultured cancer cells were shown within <i>in vitro</i> tests. Furthermore, the Ehrlich ascites tumor (EAT) mouse model gives the prove as an <i>in vivo</i> test: daily injection of violacein ($0.1\,\mu g/kg$ up to $1\,mg/kg$) led to a significant increased survival rate of the mice
+<br>
-<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538413/">
+The genes for VioA, VioB and VioE were amplificated via PCR with Golden Gate compatible overhangs from the biobrick VioABCE
-<abbr title="Violacein: Properties and Production of a Versatile Bacterial Pigment">
+<a href="http://parts.igem.org/Part:BBa_K274004">
-(Seong Yeol Choi <i>et al.</i>, 2015)
+<abbr title="Part:BBa_K274004">
+(Part: BBa_K274004)
 </abbr>
-</a>. The ability to weaken cancer growth draws more attention to violacein as a possible cancer therapeutic.
+</a>
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/16889929">
+.
-<abbr title="Cytotoxic activity of violacein in human colon cancer cells">
-de Carvalho DD <i>et al.</i> (2006)
-</abbr>
-</a>showed that violacein is capable to induce apoptosis in various cancer cells by inducing the production of oxygen radicals.
-<br> A main focus also lies in violacein’s antimalarial activity, which was tested <i>in vitro</i>  and <i>in vivo</i> on human and murine blood stage forms of <i>Plasmodium</i> parasites
-<a href="http://aac.asm.org/content/53/5/2149.full">
-<abbr title="Violacein Extracted from Chromobacterium violaceum Inhibits Plasmodium Growth In Vitro and In Vivo">
-(Stefanie C. P. Lopes <i> et al.</i>, 2009)
-</abbr>
-</a>. <i>P. falciparum</i> is known to be the deadliest <i>Plasmodium</i> species that causes malaria in humans
-<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2720412/">
-<abbr title="The origin of malignant malaria">
-(Stephen M. Rich <i>et al.</i>, 2009)
-</abbr>
-</a>. Violacein acted effectively against diseases caused by both, young and mature parasite strains, of <i>P. falciparum</i>, and parasite growth was reduced significantly compared to non-treated animals. Moreover, it has a protective effect as mice infected with a lethal strain (<i>P. chabaudi chabaudi</i>) died within 10 days, whereas the majority (80 %) treated with violacein survived the infection
-<a href="http://aac.asm.org/content/53/5/2149.full">
-<abbr title="Violacein Extracted from Chromobacterium violaceum Inhibits Plasmodium Growth In Vitro and In Vivo">
-(Stefanie C. P. Lopes <i> et al.</i>, 2009)
-</abbr>
-</a>. Not at least because the emerge resistance to plant-based malaria drugs becomes more frequent, it is time to look out for further possibilities in the worldwide battle against malaria <a href="https://www.ncbi.nlm.nih.gov/pubmed/26911755">
-<abbr title="Synthetic biology's first malaria drug meets market resistance">
-(Peplow M, 2016)
-</abbr>
-</a>.
 </p>
-<p> As the commercial production of violacein is rather difficult and limited for low productivity
-<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4997675/">
+<div class="quarter-width">
-<abbr title="Engineering Corynebacterium glutamicum for violacein hyper production">
+<figure>
-(Hongnian Sun <i>et al.</i>, 2016)
+<img class="half-width" src="https://static.igem.org/mediawiki/parts/0/00/T--Cologne-Duesseldorf--BioBrickViolaceinPlatte.png">
-</abbr>
+</figure>
-</a>, researchers are working on improving the fermentative titers by metabolic engineering.
+</div>
-<br>Here we want to make use of the existing potential violacein has and even try to promote it. With the great advantages a peroxisomal import has to offer, we want to develop a solid mechanism to not only prove the concept of our project, but also take advantage of violacein’s biological opportunities. By relocalization of the violacein pathway into yeast peroxisomes we want to create a space with optimized working conditions for the production of violacein to achieve a high yield of the bisindole.
+<p>
+By Golden Gate cloning the peroxisomal targeting sequence (PTS1) was attached to the C-terminus of every pathway protein. Combined with the other necessary parts of the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-2"> toolbox </a> they represent the level 1 plasmids.
 </p>
+<div class="flex-row-2">
+    <div>
+    <img src="https://static.igem.org/mediawiki/2017/b/b2/T--Cologne-Duesseldorf--VioA_pts1_plasmid.jpg">
+    </div>
+    <div>
+    <img src="https://static.igem.org/mediawiki/2017/1/1b/T--Cologne-Duesseldorf--VioB_lvl1_pts1_plasmid.jpg">
+    </div>
+</div>
+<div class="flex-row-2">
+    <div>
+ <img src="https://static.igem.org/mediawiki/2017/6/63/VioC_lvl1_pts1_plasmid.jpg">
+    </div>
+    <div>
+    <img src="https://static.igem.org/mediawiki/2017/4/43/T--Cologne-Duesseldorf--VioE_lvl1_pts1_plasmid.jpg">
+    </div>
 </div>
+<p>
+The PTS-tag marks the proteins for the import into peroxisomes. This should first of all point out the functionality of the yeast’s natural import mechanism and also be the basis for demonstrating our own modeled PTS*, proving our designed <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#Pts1Import">orthogonal import mechanism</a>.
+Furthermore we also aim to optimize the working conditions for the enzymes inside of the reaction room - the peroxisomes. For example to vary the pH with new <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#MembraneIntegration"> membrane proteins </a> such as bacteriorhodopsin. To secure this change, we can also check the current conditions by our designed <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#Sensors"> sensors</a>.
+</p>
+<p>
+There are several methods to verify the pathway’s enzymes. First of all, violacein and several intermediates (prodeoxyviolacein, deoxyviolacein, proviolacein) are colorful and the production in yeast can be visualized easily. Furthermore we added a His-/Flag-tag to the N-terminus of every protein (see geneious plasmid cards) to confirm their expression via SDS page and western blot. After verifying the presence of the enzymes the next step is to test their functionality. Before performing <i>in vivo</i> experiments in yeast an <i>in vitro</i> assay was implemented. For this the three enzyme pathway leading to PDV was reconstructed, testing VioA, VioB and VioE. To enable the best conditions for the enzymes, the pathway was studied intensively and all needed cofactors were calculated and added to the <i>in vitro</i> reaction (see protocol <a href="https://static.igem.org/mediawiki/2017/a/aa/T--Cologne-Duesseldorf--prodeoxyviolacein_assay.pdf"> prodeoxyviolacein <i>in vitro </i>assay</a>). This included FAD, MgCl<sub>2</sub>, catalase for decomposition of hydrogen peroxide, and the substrate L-tryptophan. The <i>in vitro</i> reaction was followed by qualitative analysis via HPLC and mass spectrometry.
+</p>
+<button class="accordion">
+ <h2 id="designrules">Design Rules For Genome Engineering Regarding Customization of Peroxisome Properties</h2>
+ <p>In order to reach the ultimate goal of creating a fully controllable artificial compartment, genome engineering can be utilized for customizing the compartment's properties,.These include aspects such as membrane permeability, size/number, or decoupling of peroxisomes from the cytoskeleton, the peroxisomal proteome or metabolome. In our project we used the Crispr Cas9 system for knocking out several genes (Pex9, Pex31&Pex32, INP1, POT1) at the same time in order to engineer the previously mentioned properties. Furthermore, we designed a yeast strain  with a completely replaced protein-import machinery for controlling the entire peroxisomal lumen.</p>
+<p>For the future one could think of much more radical strategies for peroxisomal engineering with a final goal of a “minimal peroxisome” by redirecting metabolic pathways through changing the protein-localization-signal in the yeast genome. Additionally, endogenous metabolic pathways could be redirected to our novel artificial compartment for establishing a customized metabolism specifically tailored for the user's application.</p>
+  </button>
+<div class="panel">
+  <h3>Introduction</h3>
+<p>In order to achieve a fully controllable artificial compartment, the first step was to design a completely orthogonal import system. Next was the knockout of endogenous import systems. However, a few proteins are imported neither by the Pex5 nor the Pex7 import machinery. Therefore, specific genome engineering designs, such as knockouts, deleting or redirecting the protein localization could be utilized for the ultimate goal of creating a synthetic organelle.</p>
+<p>Additionally, knockouts or genome integrations enable customization of the peroxisomal properties, such as membrane permeability, size/number, decoupling of peroxisomes from cytoskeleton and the peroxisomal metabolism.</p>
+<p>All these strategies allow a rational design of an artificial compartment, which is fully engineerable regarding  the proteome, metabolome and the entire peroxisomal environment.</p>
+<h3>Design of yeast multi -knockout strains</h3>
+<h4>The Crispr Cas9 System</h4>
+<p>The demands on yeast engineering have significantly increased with the design of more complex systems and extensive metabolic pathways. Genetic techniques that have historically relied on marker recycling are unable to keep up with the ambitions of synthetic biologists. In recent years the Crispr Cas9 system has been used for several strain-engineering purposes, including:</p>
+<ul>
+  <li>Markerless integration of multiple genetic cassettes into selected genomic loci</li>
+  <li>Multiplexed and iterative gene knockouts without the need to recycle a marker</li>
+  <li>Precise genome editing – nucleotide substitutions, etc.</li>
+</ul>
+<p>We utilized the Cas9 system as a tool for peroxisomal engineering and have adopted the existing toolbox from <abbr title="Lee, Michael E.; DeLoache, William C.; Cervantes, Bernardo; Dueber, John E. (2015): A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. In: ACS synthetic biology 4 (9), S. 975–986. DOI: 10.1021/sb500366v.">(Lee et al. 2015)</abbr> and the complete cloning system which also provides the possibilities for genome integration and gene editing by Cas9. For this, two oligonucleotides have to be designed for targeting the Cas protein to the gene of interest.</p>
-</article>
+<figure>
+  <img src="https://static.igem.org/mediawiki/2017/3/3b/--T--cologne-duesseldorf--Cas9_1.PNG">
+  <figcaption>Figure 1: Plasmid construction for the gRNA expression plasmid<br>Two oligos, containing the targeting sequence of the gRNA, have to be annealed and can then be integrated in the gRNA entry Vector by a Golden Gate reaction. Adapted from (<abbr title="Lee, Michael E.; DeLoache, William C.; Cervantes, Bernardo; Dueber, John E. (2015): A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. In: ACS synthetic biology 4 (9), S. 975–986. DOI: 10.1021/sb500366v.">Lee et al. 2015</abbr>)</figcaption>
+</figure>
+<p>Several gRNA vectors can subsequently be assembled into one vector with a Cas9 expression cassette and then be transformed into yeast. The expression of Cas9 together with gene specific gRNA´s leads to double strand breakage followed by non-homologous end joining repair or homologous recombination, in case of added repair DNA (figure 3).</p>
+<figure>
+  <img src="https://static.igem.org/mediawiki/2017/6/69/--T--cologne-duesseldorf--Cas9_2.PNG">
+  <figcaption>Figure 2: Plasmid construction for the expression plasmid containing Cas9 and gRNA´s<br>Vector for Cas9 and gRNA expression, assembled by a Golden Gate reaction, containing a URA marker, Cen6 yeast origin and a kanamycin resistance. Adapted from (<abbr title="Lee, Michael E.; DeLoache, William C.; Cervantes, Bernardo; Dueber, John E. (2015): A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. In: ACS synthetic biology 4 (9), S. 975–986. DOI: 10.1021/sb500366v.">Lee et al. 2015</abbr>)</figcaption>
+</figure>
+<p>The combination of the Cas9 system with DNA repair sequences enables not only knockouts of peroxisomal proteins, but also allows redirecting protein localization by changing protein targeting signals or integration of linear DNA into yeast chromosomes. Genome engineering facilitates yeast strain development for customized peroxisomes.</p>
+<figure>
+  <img src="https://static.igem.org/mediawiki/2017/9/9b/--T--cologne-duesseldorf--Cas9_3.PNG">
+  <figcaption>Figure 3: Design of repair DNA sequences for homologous recombination after inducing double strand break by Cas9<br>
+  Repair DNA sequences can be used to increase the efficiency for Cas9 guided knocking out of specific genes, but would also allow genomic integration of targeting signals or complete genes.  Adapted from (<abbr title="Lee, Michael E.; DeLoache, William C.; Cervantes, Bernardo; Dueber, John E. (2015): A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. In: ACS synthetic biology 4 (9), S. 975–986. DOI: 10.1021/sb500366v.">Lee et al. 2015</abbr>)</figcaption>
+</figure>
+<h3>The peroxisomal proteome of yeast (saccharomyces cerevisiae)</h3>
+<p>The peroxisomal proteome is studied extensively for saccharomyces cerevisiae and contains exactly 67 proteins (<abbr title="Kohlwein, Sepp D.; Veenhuis, Marten; van der Klei, Ida J. (2013): Lipid droplets and peroxisomes: key players in cellular lipid homeostasis or a matter of fat--store 'em up or burn 'em down. In: Genetics 193 (1), S. 1–50. DOI: 10.1534/genetics.112.143362.">Kohlwein et al. 2013</abbr>). The function is characterized for the most of those proteins and it is known, that yeast peroxisomes are expendable under optimal growth conditions. Nevertheless, some knockouts are lethal under oleate or stress conditions.</p>
+<table>
+  <thead>
+    <tr>
+      <th>Gene</th>
+      <th>Required for growth on oleate</th>
+      <th>Expression induced by oleate</th>
+      <th>Enzyme/activity</th>
+      <th>Molecular mass (kDa)</th>
+      <th>Isoelectric point</th>
+      <th>Molecules per cell </th>
+      <th>Localization</th>
+      <th>Function</th>
+    </tr>
+  </thead>
+  <tbody>
+    <tr>
+      <td colspan=”9”>ß-Oxidation enzymes</td>
+    </tr>
+    <tr>
+      <td>PCS60 (FAT2)</td>
+      <td>No</td>
+      <td>Yes</td>
+      <td>Medium chain fatty acyl-CoA synthetase</td>
+      <td>60.5</td>
+      <td>9.98</td>
+      <td>8.770</td>
+      <td>Peripheral peroxisomal membrane and matrix</td>
+      <td>Activates fatty acids with a preference for medium chain lengths, C9-C13</td>
+    </tr>
+    <tr>
+      <td>FAT1</td>
+      <td>No</td>
+      <td>-</td>
+      <td> Very long chain fatty acyl-CoA synthetase and long chain fatty acid transporter</td>
+      <td>77.1</td>
+      <td>8.47</td>
+      <td>16,900</td>
+      <td>Lipid droplet, ER, peroxisome Three predicted TM</td>
+      <td>Activates fatty acids with a preference for very long chain lengths, C20–C26</td>
+    </tr>
+    <tr>
+      <td>POX1</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Acyl-CoA- oxidase</td>
+      <td>84.0</td>
+      <td>8.73</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Oxidation of acyl-CoA</td>
+    </tr>
+    <tr>
+      <td>CTA1</td>
+      <td>No</td>
+      <td>Yes</td>
+      <td>Catalase</td>
+      <td>58.6</td>
+      <td>7.46</td>
+      <td>623</td>
+      <td>Peroxisomal matrix</td>
+      <td>Degrades hydrogen peroxide produced by Pox1</td>
+    </tr>
+    <tr>
+      <td>FOX2 (POX2)</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Multifunctional enzyme; 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase</td>
+      <td>98.7</td>
+      <td>9.75</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>-</td>
+    </tr>
+    <tr>
+      <td>POT1 (FOX3, POX3)</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>3-Ketoacyl-CoA thiolase</td>
+      <td>44.7</td>
+      <td>7.56</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Cleaves 3-ketoacyl-CoA into acyl-CoA and acetyl-CoA</td>
+    </tr>
+    <tr>
+      <td>DCI1 (ECI2)</td>
+      <td>-</td>
+      <td>-</td>
+      <td> Δ(3,5)-Δ(2,4)-dienoyl-CoA isomerase (putative)</td>
+      <td>30.1</td>
+      <td>8.83</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Auxiliary enzyme of fatty acid β-oxidation; role in β-oxidation debated</td>
+    </tr>
+    <tr>
+      <td>SPS19 (SPX1)</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>2,4-Dienoyl-CoA reductase</td>
+      <td>31.1</td>
+      <td>9.67</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Auxiliary enzyme of fatty acid β-oxidation</td>
+    </tr>
+    <tr>
+      <td>ECI1</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Δ3, Δ2-enoyl-CoA isomerase</td>
+      <td>31.7</td>
+      <td>8.21</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Auxiliary enzyme of fatty acid β-oxidation</td>
+    </tr>
+    <tr>
+      <td>TES1 (PTE1)</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Acyl-CoA thioesterase</td>
+      <td>40.3</td>
+      <td>9.58</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Auxiliary enzyme of fatty acid β-oxidation</td>
+    </tr>
+    <tr>
+      <td>MDH3</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Malate dehydrogenase</td>
+      <td>37.3</td>
+      <td>10.00</td>
+      <td>3,300</td>
+      <td>Peroxisomal matrix</td>
+      <td>Required for the malate-oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+, part of the glyoxylate cycle
+      </td>
+    </tr>
+    <tr>
+      <td>IDP3</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>NADP+ dependent isocitrate dehydrogenase</td>
+      <td>47.91</td>
+      <td>10.02</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Required for the 2-ketoglutarate/isocitrate shuttle, exchanging peroxisomal NADP+ for cytosolic NADPH</td>
+    </tr>
+    <tr>
+      <td>CAT2</td>
+      <td>No</td>
+      <td>No</td>
+      <td>Carnitine acetyl-CoA transferase</td>
+      <td>77.2</td>
+      <td>8.34</td>
+      <td>470</td>
+      <td>Peroxisome mitochondria</td>
+      <td>Transfers activated acetyl groups to carnitine to form acetylcarnitine which can be shuttled across membranes</td>
+    </tr>
+    <tr>
+      <td colspan=”9”>Glyoxylate cycle</td>
+    </tr>
+    <tr>
+      <td>CIT2</td>
+      <td>No</td>
+      <td>-</td>
+      <td>Citrate synthase</td>
+      <td>51.4</td>
+      <td>6.34</td>
+      <td>2,310</td>
+      <td>Peroxisomal matrix</td>
+      <td>Condensation of acetyl CoA and oxaloacetate to form citrate</td>
+    </tr>
+    <tr>
+      <td>MDH3</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Malate dehydrogenase</td>
+      <td>37.3</td>
+      <td>10.00</td>
+      <td>3,300</td>
+      <td>Peroxisomal matrix</td>
+      <td>Required for the malate–oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+</td>
+    </tr>
+    <tr>
+      <td>MLS1</td>
+      <td>Yes</td>
+      <td>-</td>
+      <td>Malate synthase</td>
+      <td>62.8</td>
+      <td>7.18</td>
+      <td>ND</td>
+      <td>Peroxisomal protein</td>
+      <td>Required for utilization of nonfermentable carbon sources</td>
+    </tr>
+    <tr>
+      <td colspan=”9”> Other peroxisome-associated enzyme activities</td>
+    </tr>
+    <tr>
+      <td> GPD1 (DAR1, HOR1, OSG1, OSR5</td>
+      <td>-</td>
+      <td>-</td>
+      <td> NAD-dependent glycerol-3-phosphate dehydrogenase</td>
+      <td>42.9</td>
+      <td>5.26</td>
+      <td>807</td>
+      <td>Peroxisome, cytosol, nucleus</td>
+      <td> Key enzyme of glycerol synthesis, essential for growth under osmotic stress</td>
+    </tr>
+    <tr>
+      <td>PNC1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Nicotinamidase</td>
+      <td>25.0</td>
+      <td>6.23</td>
+      <td>7,720</td>
+      <td>Peroxisome, cytosol</td>
+      <td>Converts nicotinamide to nicotinic acid as part of the NAD(+) salvage pathway</td>
+    </tr>
+    <tr>
+      <td>NPY1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>NADH diphosphatase</td>
+      <td>43.5</td>
+      <td>6.26</td>
+      <td>846</td>
+      <td>Peroxisome cytosol</td>
+      <td>Hydrolyzes the pyrophosphate linkage in NADH and related nucleotides</td>
+    </tr>
+    <tr>
+      <td>STR3</td>
+      <td>-</td>
+      <td>-</td>
+      <td> Cystathionine β-lyase</td>
+      <td>51.8</td>
+      <td>7.96</td>
+      <td>ND</td>
+      <td>Peroxisome</td>
+      <td>Converts cystathionine into homocysteine</td>
+    </tr>
+    <tr>
+      <td>STR3</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Cystathionine ß-lyase</td>
+      <td>51.8</td>
+      <td>7.96</td>
+      <td>ND</td>
+      <td>Peroxisome</td>
+      <td>Converts cystathionine into homocysteine</td>
+    </tr>
+    <tr>
+      <td>GTO1</td>
+      <td>-</td>
+      <td>-</td>
+      <td> ω-Class glutathione transferase </td>
+      <td>41.3</td>
+      <td>9.53</td>
+      <td>-</td>
+      <td>Peroxisome</td>
+      <td>Induced under oxidative stress</td>
+    </tr>
+    <tr>
+      <td>AAT2(ASP5)</td>
+      <td>-</td>
+      <td>Yes</td>
+      <td>Aspartate aminotransferase</td>
+      <td>46.1</td>
+      <td>8.50</td>
+      <td>7,700</td>
+      <td>Cytosol, peroxisome</td>
+      <td>Involved in nitrogen metabolism</td>
+    </tr>
+    <tr>
+      <td>PCD1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Nudix pyrophosphatase with specificity for coenzyme A and CoA derivatives</td>
+      <td>39.8</td>
+      <td>6.59</td>
+      <td>238</td>
+      <td>Peroxisome</td>
+      <td>May function to remove potentially toxic oxidized CoA disulfide from peroxisomes</td>
+    </tr>
+    <tr>
+      <td>LPX1</td>
+      <td>-</td>
+      <td>Yes</td>
+      <td>Triacylglycerol lipase</td>
+      <td>43.7</td>
+      <td>8.16</td>
+      <td>2,350</td>
+      <td>Peroxisomal matrix</td>
+      <td>-</td>
+    </tr>
+    <tr>
+      <td colspan=”9”>Peroxisomal transporters</td>
+    </tr>
+    <tr>
+      <td>PXA1 (LPI1, PAL1, PAT2, SSH2</td>
+      <td>-</td>
+      <td>-</td>
+      <td> Subunit of a heterodimeric ATP-binding cassette transporter complex</td>
+      <td>100.0</td>
+      <td>10.34</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>Import of long-chain fatty acids into peroxisomes</td>
+    </tr>
+    <tr>
+      <td>PXA2 (PAT1)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Subunit of a heterodimeric ATP-binding cassette transporter complex</td>
+      <td>97.1</td>
+      <td>9.47</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>Import of long-chain fatty acids into peroxisomes</td>
+    </tr>
+    <tr>
+      <td>ANT1(YPR118C)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Adenine nucleotide transporter</td>
+      <td>36.4</td>
+      <td>10.6</td>
+      <td>2,250</td>
+      <td>Peroxisomal membrane</td>
+      <td>Involved in β-oxidation of medium-chain fatty acids</td>
+    </tr>
+    <tr>
+      <td colspan=”9”>Peroxins</td>
+    </tr>
+    <tr>
+      <td>Pex1 (PAS1)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>AAA ATPase</td>
+      <td>117.3</td>
+      <td>6.93</td>
+      <td>2,100</td>
+      <td>Peroxisomal membrane</td>
+      <td>Involved in recycling of Pex5, forms heterodimer with Pex6</td>
+    </tr>
+    <tr>
+      <td>Pex2 (RT1, PAS5)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>E3 ubiquitin ligase</td>
+      <td>30.8</td>
+      <td>9.02</td>
+      <td>339</td>
+      <td>Peroxisomal membrane</td>
+      <td>RING finger protein, forms complex with Pex10 and Pex12. Involved in matrix protein import</td>
+    </tr>
+    <tr>
+      <td>Pex3 (PAS3)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>50.7</td>
+      <td>6.29</td>
+      <td>1,400</td>
+      <td>Peroxisomal membrane</td>
+      <td>Required for proper localization of PMPs</td>
+    </tr>
+    <tr>
+      <td>Pex4 (PAS2, UBC10)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Ubiquitin conjugating enzyme</td>
+      <td>21.1</td>
+      <td>5.36</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>Involved in matrix protein import</td>
+    </tr>
+    <tr>
+      <td>Pex5 (PAS10)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Soluble PTS1 receptor</td>
+      <td>69.3</td>
+      <td>4.79</td>
+      <td>2,070</td>
+      <td>Cytosol and peroxisomal matrix</td>
+      <td>Required for import of PTS1-containing peroxisomal proteins, contains TPR domains</td>
+    </tr>
+    <tr>
+      <td>Pex6 (PAS8)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>AAA ATPase</td>
+      <td>115.6</td>
+      <td>5.44</td>
+      <td>1,630</td>
+      <td>Peroxisomal membrane</td>
+      <td>Involved in recycling of Pex5, forms heterodimer with Pex1</td>
+    </tr>
+    <tr>
+      <td>Pex7 (PAS7, PEB1)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Soluble PTS2 receptor</td>
+      <td>42.3</td>
+      <td>8.34</td>
+      <td>589</td>
+      <td>Cytosol and peroxisomal matrix</td>
+      <td>Requires Pex18 and Pex21 for association to the receptor docking site, contains WD40 repeat </td>
+    </tr>
+    <tr>
+      <td>PEX8 (PAS6)</td>
+      <td>-</td>
+      <td>-</td>
+      <td> Intra peroxisomal organizer of the peroxisomal import machinery </td>
+      <td>68.2</td>
+      <td>7.62</td>
+      <td>538</td>
+      <td>Peroxisomal matrix and luminal membrane face</td>
+      <td>Pex5-cargo dissociation</td>
+    </tr>
+    <tr>
+      <td>Pex9</td>
+      <td>-</td>
+      <td>-</td>
+      <td>PTS-receptor</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+    </tr>
+    <tr>
+      <td>Pex10</td>
+      <td>-</td>
+      <td>-</td>
+      <td>E3 ubiquitin ligase</td>
+      <td>39.1</td>
+      <td>9.88</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>RING finger protein involved in Ubc4-dependent Pex5 ubiquitination. Forms complex with Pex2 and Pex12 </td>
+    </tr>
+    <tr>
+      <td>PEX11 (PMP24, PMP 27)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>26.9</td>
+      <td>10.65</td>
+      <td>1,630</td>
+      <td>Peroxisomal membrane</td>
+      <td>Involved in peroxisome fission, required for medium-chain fatty acid oxidation </td>
+    </tr>
+    <tr>
+      <td>Pex12 (PAS11)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>E3 ubiquitin ligase</td>
+      <td>46.0</td>
+      <td>9.86</td>
+      <td>907</td>
+      <td>-</td>
+      <td>RING finger protein, forms complex with Pex2 and Pex10</td>
+    </tr>
+    <tr>
+      <td>Pex13 (PAS20)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Component of docking complex for Pex5 and Pex7</td>
+      <td>42.7</td>
+      <td>9.83</td>
+      <td>7,900</td>
+      <td>Peroxisomal membrane</td>
+      <td>Forms complex with Pex14 and Pex17</td>
+    </tr>
+    <tr>
+      <td>Pex14</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Central component of the receptor docking complex</td>
+      <td>38.4</td>
+      <td>4.61</td>
+      <td>2,570</td>
+      <td>Peroxisomal membrane</td>
+      <td>Interacts with Pex13</td>
+    </tr>
+    <tr>
+      <td>Pex15 (PAS21)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>43.7</td>
+      <td>8.42</td>
+      <td>1,070</td>
+      <td>Peroxisomal membrane</td>
+      <td>Recruitment of Pex6 to the peroxisomal membrane, tail anchored PMP</td>
+    </tr>
+    <tr>
+      <td>Pex17 (PAS9)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Component of docking complex for Pex5 and Pex7</td>
+      <td>23.2</td>
+      <td>10.24</td>
+      <td>656</td>
+      <td>Peroxisomal membrane</td>
+      <td>Forms complex with Pex13 and Pex14</td>
+    </tr>
+    <tr>
+      <td>Pex18</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Required for PTS2 import</td>
+      <td>32.0</td>
+      <td>4.78</td>
+      <td>ND</td>
+      <td>Interacts with Pex7 partially redundant with Pex21</td>
+    </tr>
+    <tr>
+      <td>Pex19 (PAS12)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Chaperone and import receptor for newly synthesized PMP</td>
+      <td>38.7</td>
+      <td>4.08</td>
+      <td>5,350</td>
+      <td>Cytosol, peroxisome, farnesylated</td>
+      <td>Interacts with PMPs, involved in PMP sorting. Also interacts with Myo2 and contributes to peroxisome partitioning</td>
+    </tr>
+    <tr>
+      <td>Pex21</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Required for PTS2 protein import</td>
+      <td>33.0</td>
+      <td>6.67</td>
+      <td>ND</td>
+      <td>Cytosol</td>
+      <td>Interacts with Pex7, partially redundant with Pex18</td>
+    </tr>
+    <tr>
+      <td>Pex22(YAF5)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Required for import of peroxisomal proteins</td>
+      <td>19.9</td>
+      <td>8.33</td>
+      <td>259</td>
+      <td>Peroxisomal membrane</td>
+      <td>Recruits Pex4 to the peroxisomal membrane</td>
+    </tr>
+    <tr>
+      <td>Pex25</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome size and maintenance, required for re-introduction of peroxisomes in peroxisome deficient cells</td>
+      <td>44.9</td>
+      <td>9.77</td>
+      <td>2,420</td>
+      <td>Peripheral peroxisomal membrane</td>
+      <td>Recruits GTPase RhoI to peroxisomes, interacts with homologous protein Pex27</td>
+    </tr>
+    <tr>
+      <td>Pex27</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome size and number</td>
+      <td>44.1</td>
+      <td>10.49</td>
+      <td>382</td>
+      <td>Peripheral peroxisomal membrane</td>
+      <td>Interacts with homologous protein Pex25</td>
+    </tr>
+    <tr>
+      <td>Pex28</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome size, number and distribution</td>
+      <td>66.1</td>
+      <td>7.09</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>May act upstream of Pex30, Pex31 and Pex 32</td>
+    </tr>
+    <tr>
+      <td>Pex29</td>
+      <td>-</td>
+      <td>-</td>
+      <td>63.5</td>
+      <td>6.8</td>
+      <td>5,040</td>
+      <td>Peroxisomal membrane</td>
+      <td>May act upstream of Pex30, Pex31 and Pex32</td>
+    </tr>
+    <tr>
+      <td>Pex30</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome number</td>
+      <td>59.5</td>
+      <td>5.59</td>
+      <td>4,570</td>
+      <td>Peroxisomal membrane</td>
+      <td>Negative regulator, partially functionally redundant with Pex31 and Pex32</td>
+    </tr>
+    <tr>
+      <td>Pex31</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome number</td>
+      <td>52.9</td>
+      <td>10.15</td>
+      <td>238</td>
+      <td>Peroxisomal membrane</td>
+      <td>Negative regulator, partially functionally redundant with Pex30 and Pex32 </td>
+    </tr>
+    <tr>
+      <td>Pex32</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome number</td>
+      <td>48.6</td>
+      <td>9.14</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>Negative regulator partially functionally redundant with Pex30 and Pex31</td>
+    </tr>
+    <tr>
+      <td>PEX34</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome number</td>
+      <td>16.6</td>
+      <td>10.30</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>-</td>
+    </tr>
+    <tr>
+      <td colspan=”9”>Peroxisome fission and inheritance</td>
+    </tr>
+    <tr>
+      <td>DYN2 (SLC1)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Light chain dynein</td>
+      <td>10.4</td>
+      <td>9.03</td>
+      <td>1,310</td>
+      <td>Cytosol</td>
+      <td>Microtubule motor protein</td>
+    </tr>
+    <tr>
+      <td>SEC20</td>
+      <td>-</td>
+      <td>-</td>
+      <td>v-SNARE</td>
+      <td>43.9</td>
+      <td>5.92</td>
+      <td>4,910</td>
+      <td>Golgi, ER</td>
+      <td>Involved in retrograde transport from the Golgi to the ER, interacts with the Dsl1 complex through Tip20</td>
+    </tr>
+    <tr>
+      <td>SEC39(DSL3)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Component of the Ds11p-tethering complex</td>
+      <td>82.4</td>
+      <td>4.65</td>
+      <td>1,840</td>
+      <td>ER, nuclear envelope</td>
+      <td>Proposed to be involved in protein secretion</td>
+    </tr>
+    <tr>
+      <td>DSL1 (RNS1)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Component of the ER target site that interacts with coatomer</td>
+      <td>88.1</td>
+      <td>4.69</td>
+      <td>8,970</td>
+      <td>Peripheral ER, Golgi membrane</td>
+      <td>Forms a complex with Sec39 and Tip20 that interacts with ER SNAREs, Sec20 and Use1</td>
+    </tr>
+    <tr>
+      <td>FIS1 (MDV2)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Required for peroxisome fission</td>
+      <td>17.7</td>
+      <td>9.87</td>
+      <td>2,410</td>
+      <td>Peroxisomal membrane mitochondria</td>
+      <td>Tail anchored protein recruits Dnm1 via Mdv1/Caf4; also involved in mitochondrial fission</td>
+    </tr>
+    <tr>
+      <td>DNM1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>GTPase, dynamin like protein involved in peroxisome fission</td>
+      <td>85.0</td>
+      <td>5.25</td>
+      <td>9,620</td>
+      <td>-</td>
+      <td>Also involved in mitochondrial fission</td>
+    </tr>
+    <tr>
+      <td> VPS1 (GRD1, LAM1, SPO15, VPL1, VPT26)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>GTPase, dynamin like protein involved in peroxisome fission</td>
+      <td>78.7</td>
+      <td>8.15</td>
+      <td>5,960</td>
+      <td>-</td>
+      <td>Also involved in vacuolar protein sorting</td>
+    </tr>
+    <tr>
+      <td> VPS34 (END12, PEP15, VPL7, VPT29, STT8, VPS7)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Phosphatidylinositol 3-kinase</td>
+      <td>100.9</td>
+      <td>7.79</td>
+      <td>1,080</td>
+      <td>-</td>
+      <td>Forms complex with Vps15</td>
+    </tr>
+    <tr>
+      <td>INP1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in retention of peroxisomes in mother cells</td>
+      <td>47.3</td>
+      <td>8.34</td>
+      <td>639</td>
+      <td>Peroxisomal membrane</td>
+      <td>Recruited to the peroxisome by binding to Pex3</td>
+    </tr>
+    <tr>
+      <td>INP2</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Myo2 receptor, involved in peroxisome inheritance</td>
+      <td>81.5</td>
+      <td>9.41</td>
+      <td>736</td>
+      <td>Peroxisomal membrane</td>
+      <td>-</td>
+    </tr>
+    <tr>
+      <td>RHO1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>GTP binding protein of the Rho subfamily of Ras like proteins, involved in actin assembly at the peroxisome</td>
+      <td>23.2</td>
+      <td>6.07</td>
+      <td>ND</td>
+      <td>-</td>
+      <td>Involved in <em>de novo</em> peroxisome formation recruited to peroxisomes by Pex25</td>
+    </tr>
+  </tbody>
+</table>
+<h3>Knockout designs in our project</h3>
+<h4>Pex 9</h4>
+<p>Pex9 is a recently discovered import receptor for PTS1 proteins, which is induced by oleate
+and is an import receptor for both malate synthase isoenzymes Mls1p and Mls2p. In order to get a completely empty reaction room, a Pex9 knockout was designed to prevent unintended protein import.</p>
+<h4>Pex 31 & Pex 32</h4>
+<p>It has been shown that knockouts of Pex31 and Pex32 leads to an increased Peroxisomal size, but additionally the membrane permeability was affected (<abbr title="Zhou, Yongjin J.; Buijs, Nicolaas A.; Zhu, Zhiwei; Gómez, Diego Orol; Boonsombuti, Akarin; Siewers, Verena; Nielsen, Jens (2016): Harnessing Yeast Peroxisomes for Biosynthesis of Fatty-Acid-Derived Biofuels and Chemicals with Relieved Side-Pathway Competition. In: Journal of the American Chemical Society 138 (47), S. 15368–15377. DOI: 10.1021/jacs.6b07394.">Zhou et al. 2016</abbr>). This effect can be used as a tool for engineering membrane permeability by knocking out or overexpress both genes. A knockout would lead to an increased permeability and one could think of an opposite effect in case of overexpression, but this has not been shown yet.</p>
+<h4>INP1</h4>
+<p>The INP1 knockout was designed after the skype call of Prof Dueber, who recommended us, decoupling of peroxisomes from cytoskeleton in order to improve the secretion efficiency. INP1 is responsible for the tethering of peroxisome, which would inhibit the secretion of peroxisomes.</p>
+<h4>POT1</h4>
+<p>The only protein, which is imported by the Pex7 import machinery in saccharomyces is the 3-ketoacyl-CoA thiolase (POT1). A knockout of POT1 would enable utilizing the Pex7 import for proteins of interest, which cannot be tagged at the C-terminus with pts1, without having unintended import of other enzymes.
+<h3>Genomic integration of our novel Pex5 import receptor</h3>
+<p>After testing our new Pex5 import systems, which is completely orthogonal to the natural import, the next step would be to replace the endogenous system with our artificial import system. Therefore, an integration plasmid was designed with help of the previously described yeast toolbox, containing HO locus homologies and a hygromycin resistance (Figure 4). Afterwards the plasmid was transformed into the yeast strain which was created by our collaboration partner Aachen (double knockout strain Pex5 & Pex7).</p>
+<figure>
+  <img src="https://static.igem.org/mediawiki/2017/2/27/--T--cologne-duesseldorf--Cas9_4.PNG">
+  <figcaption>Figure 4: Design of integration plasmid for integrating our orthogonal Pex5 import receptor.<br>Therefore, an integration plasmid was designed with help of the previously described yeast toolbox, containing HO locus homologies and a hygromycin resistance</figcaption>
+</figure>
+<p>The resulting yeast strain allows full control over the peroxisomal matrix proteome, by replacing the whole protein import machinery, which is the first step for creating our artificial compartment.</p>
+<h3>Outlook</h3>
+<p>Besides the genome engineering approaches, which were performed in our project one could think of more radical strategies for peroxisomal engineering. A final goal could be a “minimal peroxisome”, which contains only the proteins that are required for the biogenesis of the peroxisome and import of proteins and metabolites. On the one hand peroxisomal pathways could be redirected to cytosol or other organelles and one the other hand endogenous metabolic pathways could be redirected to our novel artificial compartment by changing the protein localization signal in the yeast genome with help of the Cas9 system. All these strategies would allow tremendous improvements for metabolic engineering applications by creating an artificial compartment, which can be rational designed and customized for specific metabolic pathways.</p>
+</div>
+  </article>
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