<p>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.</p>
<p>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. </p>
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<h3>Our approach</h3>
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<p>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.</p>
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<p>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.</p>
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<p>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.</p>
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<p>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.</p>
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<h2>Design of our sub-projects</h2>
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<h3>Protein Import</h3>
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<div class="callout"><p>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.</p></div>
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<h4>Scientific background</h4>
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<p>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).</p>
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<p>The vast majority of peroxisomal matrix proteins are imported by the PEX5 importer. PEX5 recognizes the C-terminal PTS1 peptide whose evolutionarily conserved
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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.</p>
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<p>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.</p>
<p>According to our <a href=””>model of the Nootkatone pathway</a> 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 <a href=””>peroxisomal model</a> 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.</p>
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
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.
