Design
We designed a novel toolbox for complete control over all major functions of the peroxisome. The toolbox is our solution to improve 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 our constructs 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 either naturally occurring pathways in eukaryotes or in 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 projects 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.
Design of our sub-projects
Protein Import
The peroxisome has two pathways for importing proteins with the main transport proteins being PEX5 and PEX7. We created an orthogonal PEX5 binding pocket and corresponding recognition peptide (PTS1) by structural modeling. We also created a library of PEX7 recognition sequences for import of proteins incompatible with the PTS1 peptide.
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 developed at late point in our project − as it turned out the research group of Alison Baker et al. did similar work on the PEX5 receptor and published a paper in September 2017. They designed a receptor which is a combination of the PEX5 from Physcomitrella patens and Arabidopsis thaliana that did interact with a new PTS. Understandably, we were curious if we could use their results to get an orthogonal import pathway in yeast and did sequence alignments to figure out which amino acids have to be changed.
We then set point mutations at the corresponding positions in the yeasts PEX5 and started molecular dynamics simulations with a couple of PTS variants − one of them was actually the variant they used in the paper (YQSYY). The details and results of our strucutral modeling can be found in the modeling section. Fortuneatly evaluation revealed that this receptor variant could be a promising candidate an thus we synthesized it. Together with the 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, one of the toolbox fluorescent proteins, with our PTS variants. Additionally we wanted to mark the peroxisomal membrane, so that we could be absolutely sure about the localization within the peroxisome − we chose the transmembrane domain of PEX13 tagged with mRuby.
Once we figured out our experimental design, we thought about our constructs.
Pex13−mRuby
We wanted to use the peroxisomal membrane protein PEX13 as a fluorescent marker, but instead of using the whole protein which could severly influence the peroxisomal properties we just used the transmembrane domain with a short linker. Literature research revealed that such constructs have been tested before − Erdmann et al. (2004) described a construct containing only PEX13200-310 with a C-terminal GFP. Instead of GFP we use mRuby to get a higher differentiation from mTurquoise which we use for another construct decribed later.
Geneious version 10.2 created by Biomatters. Available from https://www.geneious.com
PEX5 variant
In order to achieve an orthogonal import we use a PEX5 knockout strain and transform it with a plasmid based PEX5 variant that is supposed to detect a non native PTS variant instead of the wildtype one. Regarding the constructs design we tested two variants.
The first construct contains a medium strength Promotor, the PEX5 gene and a terminator. Everything else can be seen in the plasmid map below.
The second design differs only in a C-terminal 3XFLAG-6XHis-tag − with that we are able to do a verification via western blot.
We designed two constructs because there was no literature if a C-terminal fusion to the PEX5 protein would inhibit the import process. With our design we are able to test this and if it does we still have the undisturbed PEX5 variant without 3XFLAG-6XHis-tag. The disadvantage of this construct is, that its presence can only be shown by a successful import.
mTurqouise−PTS
Our approach for import verification is based on a fluorescent protein tagged with the PTS variants. After a couple of tests of promotors 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 distrubuted in the whole cytosole and 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 mTurqouise constructs into a level 2 plasmid portrayed below.
We than did a co-transformation with the PEX13−mRuby plasmid and the level 2 plasmid to get everything thats 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.
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 production of Prodeoxyviolacein.
Our rests upon the follwing two plasmids which are co-tranformed into yeast.
As shown above, we created one plasmid containing VioA, VioB and one of our PEX5 variants while the other plasmid only contains VioE. We then designed primers which bind to the VioE plasmid and 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 Escherischia 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
Membrane integration
PEX19-dependent
ER-dependent
Peroxicretion
Membrane permeability and size control
Sensors
Applications
Nootkatone
Model influence on Nootkatone expression
According to our model of the Nootkatone pathway we found that overexpression of Valencene Synthase is necessary to maximize the Nootkatone yield, while both alcohol dehydrogenase and p450-BM3 have only minor effects on the yield. When comparing the cytosolic model to our peroxisomal model we found that if our assumption that neither Nootkatone nor Nootkatol are able to pass the peroxisomal membrane holds up we can greatly increase Nootkatone production.
