Abstract
The development of ‘designer’ organelles could be a key strategy to enable foreign pathways to be efficiently controlled within eukaryotic biotechnology. A fundamental component of any such system will be the implementation of a bespoke protein import pathway that can selectively deliver constituent proteins to the new compartment in the presence of existing endogenous trafficking systems. Here we show that the protein–protein interactions that control the peroxisomal protein import pathway can be manipulated to create a pair of interacting partners that still support protein import in moss cells, but are orthogonal to the naturally occurring pathways. In addition to providing a valuable experimental tool to give new insights into peroxisomal protein import, the variant receptor-signal sequence pair forms the basis of a system in which normal peroxisomal function is downregulated and replaced with an alternative pathway, an essential first step in the creation of a designer organelle.
Highlights
The development of ‘designer’ organelles could be a key strategy to enable foreign pathways to be efficiently controlled within eukaryotic biotechnology
The chemical space that can be covered by all possible variants of the receptor and signal sequence is vast, which makes the development of an assay for a peroxin 5 (PEX5)*-peroxisomal targeting signal 1 (PTS1)* pair problematic
We reduced the dimensionality of this search by designing modifications to the PEX5 component and rapidly surveying which peptide sequences could be bound by each mutated receptor (Fig. 1a)
Summary
The development of ‘designer’ organelles could be a key strategy to enable foreign pathways to be efficiently controlled within eukaryotic biotechnology. The creation of a bespoke intracellular compartment would be a highly desirable synthetic biology tool, as it would enable non-natural pathways to be isolated from other cellular processes Such isolation could lead to increased yields of synthetic proteins in biotechnology applications and prevention of adverse effects on existing metabolic pathways, as well as alteration of post-translational modifications of high-value protein products. We show that it is possible to remodel the protein–protein interactions that control peroxisomal protein import to create an orthogonal signal-receptor pair that is functional in vivo. This allows switching of the selectivity of protein import to effectively ‘hijack’ the function of the pre-existing peroxisome, simultaneously downregulating import of native peroxisomal proteins — an essential first step towards creating a designer organelle that could exist in parallel with ‘normal’ peroxisomes. Concomitant expression of PEX5* with a cargo protein bearing PTS1* results in a
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