Real world application
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 scent. 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.
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) (Gould et al. 1989). 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 (Gatto et al. 2000). TPR domains are often involved in protein−protein interactions. As can be seen in the following figure, the TPR regions mediate the binding of the peroxisomal targeting signal.
The following figure depicts the import mechanism of PTS1 tagged proteins via Pex5.
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.
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.
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 Molecular Dynamics. In the model section we explain the molecular dynamics approach in more detail.
Our second approach relies on recently published literature. We designed a receptor similar to the one designed by Cross et al. in the moss Physcomitrella patens in 2017. To understand how and where we set the mutations in the Pex5 receptor in this approach, please proceed with the design section.
The peroxisomal import depends on two pathways. A vast majority of the proteins normally found in the peroxisome are imported via the Pex5 importer. In S. cerevisiae there is only one protein, the 3-Oxoacyl-CoA thiolase (Erdmann 1994), localized in the peroxisome, is imported by the receptor Pex7 and some coreceptors instead (Erdmannet al. 2007).
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)
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 (Erdmann et al. 2007).
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 (PTS1 import).
Kunze et al. 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, tryptophane 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 (Kunze et al. 2011) .
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.
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-Pex3 dependent and an ER-dependent one (Sparkes et al. 2005) .
They rely on a so called mPTS sequence, that is used to mark the proteins for transport to and integration into the peroxisomal membrane ( Tabak et al. 2003). We will try to take advantage 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.
As a proof of concept, we will incorporate three proteins through three different approaches into the peroxisomal membrane: (i) mRuby2-Pex26 as proof for the Pex19-dependent mechanism, (ii) Pex3-mRuby2 itself to showcase the ER-dependent mechanism and (iii) bacteriorhodopsin, a unidirectional proton pump, fused to the N-terminal anchor of Pex3.
The exact mechanisms of mPTS binding, Pex3/Pex19 disassembly, mPTS-PMP binding, and release from the Pex3/Pex19 mediated mPTS-PMP docking to the full integration into the membrane are still unknown (Schueller et al. 2010) . However, general principles of the integration of a new peroxisomal membrane protein (PMP) through Pex19 and Pex3 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 more 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 Pex3p, 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 (Liu et al. 2016) .
Downstream processing is a very important part of industrial biological compound production. For most biotechnologically produced compounds, it is the most expensive part of the production
(Keller et al, 2001)
. One step to decrease the costs is to secrete the products into the supernatant
(Berlec et al, 2013)
. 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 is of great economic value as well.
In regards to the whole project, this is an important part of making the compartment more applicable by enabling immediate extraction of metabolites 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 provide the basis for our compartment do not possess a known natural secretion mechanism.
We overcome this problem by using the "peroxicretion" concept of Sagt and colleagues (Sagt et al, 2009) . 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 (Chen et al, 2001) .
Peroxisome Biogenesis and Proliferation
Peroxisomes can be generated in different ways and their size and abundance is controlled by a number of pathways (Yan et al. 2005). 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 (Smith and Aitchison 2013). Both pathways are still being investigated and to date haven’t been fully understood.
Peroxisomes are extremely sensitive to environmental cues and are able to proliferate or be degraded accordingly (Yan et al. 2005). Depending on the growth medium and their extracellular environment, peroxisomes are able to divide and multiply separately from cell division (Yan et al. 2005). 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 (Zhou et al. 2016). .
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 (Zhou et al. 2016). Since S. cerevisiae 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.
Due to its unique ability to promote peroxisome division and its role in peroxisome biogenesis (Li and Gould 2002)
, the first peroxin we chose for our purpose is Pex11, which is located in the inner surface of the peroxisomal membrane (Gurvitz et al. 2001). 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 (Zhou et al. 2016). 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 (Smith and Aitchison 2013). Other media, like oleate-containing ones, cause an induction of peroxisomal proliferation, which is due to an oleate responsive element of the Pex11 promoter (Zhou et al. 2016).
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.
According to Zhou et al., the genes of the Pex30 – 32 family have been shown to influence peroxisome proliferation (Zhou et al. 2016). 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 (Zhou et al. 2016). Due to these side effects we decided to discard working with a Pex31,32 knockout for now.
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 (Zhou et al. 2016, Tower et al. 2011) . 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 (Zhou et al. 2016). 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 (Zhou et al. 2016).
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.
How does it integrate into the overall project?
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. 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 (Zhou et al. 2016, Tower et al. 2011) . 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 (Smith and Aitchison 2013) and increase the production of target molecules while decreasing byproduct formation (Zhou et al. 2016). 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 (Smith and Aitchison 2013), 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.
Overall goal of this subproject
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.
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 neither 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 Mahon et al. (2011).
To maintain thermodynamic driving forces and electron fluxes which are needed at steady state, the intact chemeostasis of the redox machinery is of great importance (2016, Schwarzländer). Glutathione is considered to be inside the peroxisomal lumen (Elbaz-Alon, Y., et al. 2014). 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 (Morgan, B. and M. Schwarzländer 2016).
Nootkatone is an oxidized sesquiterpene, which is highly valuable for industrial and pharmaceutical applications. We will focus on its repellent effect towards insects Zhu et al. (2001). Also, therapeutic activities of nootkatone have been reported, such as anti-platelet effects in rats Seo et al. (2011), anti-proliferative activity towards cancer cell lines Gliszczyńska et al. (2011) and enhancement of energy metabolism through AMP-activated protein kinase activation in skeletal muscle and liver Murase et al. (2010).
Nootkatone can be extracted from grapefruit, 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 Cankar et al. (2010).
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 Schulz et al. (2015).
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 Saccharomyces cerevisiae at concentrations higher than 100 mg/L Gavira et al. (2013). 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 Gavira et al. (2013). 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 Cooper et al. (2000), Block et al. (1991), we assume that beta-nootkatol does not affect the peroxisomal membrane either. However, in order to validate this hypothesis, we have to collect and evaluate our own data on how beta-nootkatol affects the peroxisome membrane and thus the yield of nootkatone.
Our goal is the successful integration of the nootkatone pathway into our compartment in order to overcome 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.
Violacein (C20H13N3O3), 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 Chromobacterium violaceum. Due to its wide range of biological properties, violacein is useful for various industrial applications in pharmaceuticals and cosmetics.
Violacein is known to have a variety of different biological activities, including antitumor (Bromberg N et al, 2010), antifungal (Brucker RM et al., 2008) and antiviral (Andrighetti-Fröhner CR et al., 2003) effects. Furthermore, it has been shown that violacein enhances the effect of most commercial antibiotics by working synergistically with them (Subramaniam S et al., 2014). This is especially of high interest in the fight against recent antibiotic-resistant strains of pathogenic bacteria such as MRSA (multi resistant Staphylococcus aureus). Violacein’s antibacterial action against S. aureus has been proven by Cazoto LL et al. (2011) .
It is of high medical interest that toxic effects of Violacein on cultured cancer cells were shown within in vitro tests. Furthermore, the Ehrlich ascites tumor (EAT) mouse model provides prove as an in vivo 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 (Seong Yeol Choi et al., 2015) . The ability to weaken cancer growth draws more attention to violacein as a potential cancer therapeutic. de Carvalho DD et al. (2006) showed that violacein is capable to induce apoptosis in various cancer cells by inducing the production of oxygen radicals.
A main focus also lies in violacein’s antimalarial activity, which was tested in vitro and in vivo on human and murine blood stage forms of Plasmodium parasites (Stefanie C. P. Lopes et al., 2009) . P. falciparum is known to be the deadliest Plasmodium species that causes malaria in humans (Stephen M. Rich et al., 2009) . Violacein acted effectively against diseases caused by both, young and mature parasite strains, of P. falciparum, and parasite growth was reduced significantly compared to non-treated animals. Moreover, it has a protective effect as mice infected with a lethal strain (P. chabaudi chabaudi) died within 10 days, whereas the majority (80 %) treated with violacein survived the infection (Stefanie C. P. Lopes et al., 2009) . 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 (Peplow M, 2016) .
As the commercial production of violacein is rather difficult and limited for low productivity
(Hongnian Sun et al., 2016)
, researchers are working on improving the fermentative titers by metabolic engineering.
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 microenvironment with optimized working conditions for the production of violacein to achieve a high yield of the bisindole.
Our first idea was to optogenetically control peroxisomal protein import via PTS1. For this we used the protein LOV2, fused to sfGFP which is further described in (Smith and Aitchison 2013). The idea behind this was to use fluorescence microscopy to check whether GFP was mainly found inside peroxisomes after an illumination experiment.
Peroxisomes possess two protein import pathways: via Pex5 and via Pex7. The targeting sequence for Pex7 is N-terminal, so LOV2 cannot be used for controlling the Pex7-import. Therefore we used a different optogenetic switch which does work, namely phytochrome B and its binding partner PIF6 (Zhou et al., 2016). Our plan involved blocking the protein of interests PTS2 with a fluorescent protein and using Phytochrome B and PIF6 to reassemble a split TEV-protease. This would lead to cleavage of the fluorescent protein blocking the PTS2-sequence of the protein of interest and subsequent import via Pex7.
Optogenetic gene expression
Our optogenetic approach was about controlling the expression of genes of interest. For this we used a system which was already used by last year’s iGEM team optoptosis and also involves Phytochrome B and PIF6.