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 previous 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.

Scientific background

Peroxisomal quick facts

Peroxisome biogenesis and inheritance

Cloning strategies and the Yeast Toolbox for Multipart-Assembly

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 Sebastian Schulz (2015) Michael E. Lee (2015) . 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.

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 exampe of your favorite gene 1, YFG1 is explained.

Design of our sub-projects

Protein Import

Scientific background

Peroxisomal matrix proteins are imported post-translationally and in their folded state (Lazarow and Fujiki 1985). The peroxisomal protein import depends on two pathways, both involving a different Peroxisomal targeting signal (PTS) and respective receptor (PEX5 and PEX7). The import cycle can be divided into five conceptual steps: (i) the cytosolic receptors bind their cargo proteins and guide them to a docking site at the peroxisomal membrane, (ii) the receptor-cargo complex translocates to the peroxisomal matrix, (iii) the complex is disassembled, (iv) the receptor is returned to the cytosol (v).

The vast majority of peroxisomal matrix proteins are imported by the PEX5 importer. PEX5 recognizes the C-terminal PTS1 peptide whose evolutionarily conserved Sequence is (S/A/C)-(K/R/H)-(L/M) (Gould et al. 1989). Upon recognition of the PTS1 in the cytosol, PEX5 binds 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). 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.

Some proteins are instead imported by the PEX7 importer, together with the co-receptors PEX18 and PEX21. The targeting signal (PTS2) for this pathway is localized near the N-terminus of the cargo-protein and is comprised of nine different amino acids with a highly variable five amino acid core region and the consensus sequence (R/K)/(L/V/I)X5(H(Q))(L/A). In contrast to the pore formation by PEX5, the pore for import of PTS2 proteins is formed by the co-receptor PEX18 and the docking complex.

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 thailana 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 arabidopsis's PEX5 differ quite a lot, we aligned both sequences to understand where the mutations were set.

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 get 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.

PEX5 variant

In order to achieve an orthogonal import we used a PEX5 knockout strain and transformed it with a plasmid based PEX5 variant that is supposed to detect a non native PTS variant instead of the wild-type one. The construct contains a medium strength promotor, the PEX5 gene and a terminator. The remaining plasmid parts can be seen in the plasmid map below.

mTurquoise−PTS

Our approach for import verification is based on a fluorescent protein tagged with the PTS variants. After several promotor tests with different strength, we decided to express this construct only in low amounts. This is advantageous because 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 the figure below.

Combination of our constructs

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

We then did a co-transformation with the PEX13−mRuby plasmid and the level 2 plasmid to combine everything that is needed into our yeast.

PTS screening

Trusting on our targeted approach alone seemed risky − that is why we planned a PTS screening to find the most favorable PTS for our three receptors. Dueber et al. (2016) used the Violacein assay for a similar purpose. They screened for the best PTS for the wild type receptor 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.

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.

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 and the IPA imine dimer (white color) should be produced in those with functional import.

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. DeLoache, William C., Zachary N. Russ, and John E. Dueber (2016)

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 third one, VioE is targeted to the peroxisome with a PTS1 sequence [1]. 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.

Membrane integration

Scientific background

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.

PEX19-dependent

ER-dependent

Peroxicretion

Scientific background

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 [7]. One step to decrease the costs is to secrete the products into the supernatant [1]. 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.
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.
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.
We overcome this problem by using the "peroxicretion" concept of Sagt and colleagues[9]. They used a v-SNARE (vesicle- synaptosome-associated-Soluble N-ethylmaleimide-sensitive-factor Attachment REceptorprotein) fused to a peroxisomal membrane-protein to secrete the content of peroxisomes. V-SNAREs interact with the t-SNARE (target synaptosome-associated-SNARE) at the cell membrane, which leads to an fusion of the vesicle with the membrane [3]. Sagt and colleagues applied this system in the filamentous fungi Aspergillus niger.

Experimental Design

We will adapt the system of Sagt and colleagues *needs to be change* to secrete the content of our modified compartments [9].
For the application of this system in S. cerevisiae we use a truncated version of the v-SNARE Snc1 to decorate our compartments(Figure 1) [5].

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 1). For PEX26 we use the truncated version published in Halbach et al. (2006). [6]

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 [10]. 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 [10] (Fig 2).

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) [2]. 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.

Membrane permeability and size control

Sensors

Applications

Nootkatone

Our first step was to find a reliable source to prove existence of our precursor FPP in yeast peroxisomes. This task still remains challenging because the availability of this pyrophosphate in yeast is not completely proven yet, however it is expected since it was detected in mammalian and plant peroxisomes. figure of FPP

FPP would then be converted into Valencene by a Valencene synthase. We chose the one from Callitropsis nootkatensis because of its comparably high efficiency in microorganisms Beekwilder et al. (2014). It achieves greater yields in yeast than the citrus Valencene synthase. Furthermore the product specificity is relatively high, while producing less by-products. C.nootkatensis was also chosen because of its robustness towards pH and temperature changes. Our modelling approach revealed that for optimal yields an overexpression of Valencene synthase is necessary because of its slow conversion rate. This is why we chose the strongest promoter of our toolbox for this attempt.

The intermediate Valencene is then converted into Nootkatol by a P450 monooxygenase. The P450-BM3 we chose for this project was taken from the bacterium Bacillus megaterium. Unlike eukaryotic P450’s which are mostly membrane bound, this prokaryotic BM3 is cytosolic which makes it easier to transport it into the peroxisome Girvan et al. (2006). BM3 normally catalyzes the hydroxylation of long chain fatty acids Narhi & Fulco et al. (1986) , which in our case could inhibit the conversion of valencene. This is why we used a mutated version of BM3, AIPLF with a point mutation in the active side for easier accessibility for the rather compact structure Schulz et al. (2015).

Violacein

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.
Synthetic biology offers countless numbers of new opportunities, especially in the field of metabolic engineering. To testify some main parts of our artico 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.
Based on described 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).

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 location 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 GoldenGate 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.

Outlook

Our outlook is characterized by the vision to use our own modeled PTS* import sequence for an real world application. But first we want to further investigate our in vitro strategy including the missing enzymes VioC and VioD and move on towards already developed in vivo tests. It would be great to qualify a statement about the efficiency of different import combinations into the peroxisome. To do so, different level 2 plasmids were planned. On each plasmid the combination of enzymes being peroxisomal or cytosolic is different.

To assure the import of the enzymes and for further analysis it is indispensable to perform peroxisomal purification for an overall quanti- and qualification. Also measurements of the pathway intermediates and their fluxes across the peroxisomal membrane have to further be analyzed. The final step would be the comparison of the differences in the yield level, depending on the localization of the pathway enzymes (cytosolic or peroxisomal). With this the assumed better production in peroxisomes could be shown.