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 with modeling, so that it only recognizes a single new designed peroxisomal import signal. The most promising modified PEX5s 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 to prove, that we have transformed a peroxisome to an independent compartment with all the features required by us
Protein 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.
The following figure depicts the import mechanism of PTS1 tagged proteins via PEX5.
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.
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:
(R/K) (L/V/I) X1 X2 X3 X4 X5 (H/Q) (L/A) [3]
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.
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 [9].
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
Designing new pathways or transferring pathways into cellular compartments requires a well understanding of the present conditions and content, like cofactors in the peroxisomes. We aim to measure peroxisomal pH, cofactors like NADP+ 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 use ratiometric fluorescent biosensors which we genetically attach to a peroxisomal targeting signal. These measurements give important insights into possible difficulties which may occur if none peroxisomal pathways are transferred into the peroxisome and enable more precise predictions and modelling.
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)
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 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 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) .
Abstract
By transferring the metabolic pathway of nootkatone into the peroxisome we want to overcome the obstacle of intermediate toxicity for the yeast cell. This would pave the way for an efficient, safe and favorable solution of producing and providing an effective insect repellent.
Description
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) .
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.
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.
Abstract
Using the tools of Synthetic Biology in metabolic engineering can unleash the full potential of biofactories. Natural systems use compartmentalization to improve biochemical reactions. Here we are presenting the use of peroxisomal import tags to engineer an artificial compartment in Saccharomyces cerevisiae 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 are aiming 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.
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 Net 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 $1\,mg/kg$) lead 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 Parasite growth was reduced significantly compared to nontreated animals. It 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 emerge 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.
