Team:Cologne-Duesseldorf/Design

Project

Project description

Introduction

Compartmentation has been one of nature’s most effective tools for more than a billion years. The tremendous versatility of organisms we see today is only possible because cells have developed the ability of translocating various metabolic processes to subcellular compartments, thereby sequestering them from others. Our project is about harnessing the full potential of this awesome mechanism. What used to have to evolve over millions of years can now be directly controlled and customized through use of our toolbox. Towards this aim we worked on many different sub projects, each targeting a different aspect of compartment customization. Below you will find a description of all of them.

Design and modeling

We have chosen yeast peroxisomes as our chassis for designing synthetic organelles. They are very resistant, have a modifiable import mechanism and are expendable under optimal conditions. We will customize the import machinery of peroxisomes in yeasts in order to regulate the biomolecule import into these compartments. To do so, we modify the TPR-region of the peroxisomal target protein receptor PEX5 bywith modeling, so that it only recognizes a single new designed peroxisomal import signal. The most promising modified PEX5s variants will be implemented into the actual peroxisome.

Real world application

As a proof of concept for our compartimentation strategy we intend to establish the Nootkatone pathway inside the peroxisome. Nootkatone 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 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 peels of millions of grapefruits or synthesized inside of yeast. The problem is that the Nootkatone pathway is toxic for yeast and the efficiency is rather low. Here our compartmentation comes into play: we plan to implement the whole pathway into the modified peroxisome in order to prove, that we have transformed a peroxisome to an independent compartment with all the features required by us

PTS1 Import

The vast majority of peroxisomal matrix proteins is 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 ). 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 Jr. et al 2000). TPR domains are often involved in protein−protein interaction and as it can be seen in the following figure, the TPR regions mediate the binding of the peroxisomal targeting signal.

TPR domain of the human PEX5, with a pentapeptide in its binding pocket (Gatto Jr. et al. , 2000)

The following figure depicts the import mechanism of PTS1 tagged proteins via PEX5.

Import mechanism (Erdmann et al., 2005)
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). 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.

In this subproject we mutated the PEX5 receptor in a way that it recognizes a new signal peptide which does not occur in nature. As PEX5 is responsible for most of the import, we have complete control over its content once we knock out the wild type receptor and replace it with our new mutated one.
Corresponding to the new receptor one needs to design a peroxisomal targeting signal that provides favorable interactions with the residues of the amino acids within the TPR.
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 what Baker et al. did in the moss Physcomitrella patens in 2017. To understand how and where we set the mutations in the PEX5 receptor following this approach, please proceed with the design section.

PTS2 Import

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 only one protein, the 3-Oxoacyl-CoA thiolase Ralf Erdmann(1994), localized in the peroxisome, is instead imported by the receptor Pex7 and some coreceptors Ralf Erdmann (2015).

The targeting signal for this pathway is localized near the N-terminus of each protein. Kunze and colleagues described the PTS2 consensus sequence as the following:

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 till seven are highly variable. Residue number eight consists of Histidine or Glutamine and the ninth is either Leucine or Alanine. Markus Kunze (2015)

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 Ralf Erdmann (2015).

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 tailormade concentrations of different pathway parts in the peroxisome. Besides, 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, 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 Markus Kunze (2015) .

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.

For the application in S. cerevisiae we designed fusion proteins of the v-SNARE Snc1 with different peroxisomal membrane anchors *needs to be change* . We tested the constructs using an GUS Assay. The assays were performed using transformants of the strain BY4742.
Our results *needs to be change* indicate, that it is possible to use our approach for secretion. The best efficiency was achieved using Snc1 fused with a linker to the peroxisomal membrane anchor Pex15. Furthermore the deletion of Pex11 did not increase the amount of active Gus secreted to the supernatant

pH Sensor

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 mechanismen or not. 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 excitation spectrum shifts from 395 to 475 nm Mahon et al. (2011)

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 Mahon et al. (2011) .

roGFP2 Sensor

To maintain thermodynamic driving forces and electron fluxes which are needed at steady state, the intact chemeostasis of the redox machinery is very important (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 dDisulfide 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 488 nm of the reduced form of roGFP exceeds the of the oxidized form and excitation at 405 nm behaves vise verse (Morgan, B. and M. Schwarzländer 2016) .

Nootkatone is an oxidized sesquiterpene, which is highly valuable for industrial and pharmaceutical application. 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 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 Cankar et al. (2010) .

Conversion of valencene to Nootkatol and Nootkatone

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 concentration 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. A s 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. 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.

Yeast viability after 24 h in the presence of (+)-valencene, beta-Nootkatol or nootkatone in different concentrations

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 interest of this sesquiterpene by the industry. 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 different industrial applications in pharmaceuticals and cosmetics.

Violacein is known to have a variety of different biological activities, including an antitumor (Bromberg N et al, 2010), antifungal (Brucker RM et al., 2008) and antiviral (Andrighetti-Fröhner CR et al., 2003) function. 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 gives the prove as a 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 possible 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 pParasite growth was reduced significantly compared to non-treated? animals. Moreover, itIt moreover 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 emergee of resistance to other plant-based malaria drugs becomes more frequently, it is time to look out for other 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 this potential. With the great advantages a peroxisomal import has to offer, we want to develop a solid mechanism to not only proof 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 conditions for the production of violacein to achieve a high yield of the bisindole.

Introduction

In order to get fully controllable artificial compartment, the first step was to design a completely orthogonal import system and the next step was the knockout of endogenous import systems. However, a few proteins are neither imported 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 customizing 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 compartmen

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 or extensive metabolic pathways. Genetic techniques that have historically relied on marker recycling are not able 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. Therefore, 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 assembled together with a Cas9 expression cassette into one vector and then be transformed into yeast. The expression of Cas9 together with gene specific gRNA´s leads to double strand break 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 enable 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.