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Abstract
Compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk and improving pathway efficiency, but improved tools and design rules are needed to make this strategy available to more engineered pathways. Here we focus on the Saccharomyces cerevisiae peroxisome and develop a sensitive high-throughput assay for peroxisomal cargo import. We identify an enhanced peroxisomal targeting signal type 1 (PTS1) for rapidly sequestering non-native cargo proteins. Additionally, we perform the first systematic in vivo measurements of nonspecific metabolite permeability across the peroxisomal membrane using a polymer exclusion assay. Finally, we apply these new insights to compartmentalize a two-enzyme pathway in the peroxisome and characterize the expression regimes where compartmentalization leads to improved product titre. This work builds a foundation for using the peroxisome as a synthetic organelle, highlighting both promise and future challenges on the way to realizing this goal.
Highlights
Compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk and improving pathway efficiency, but improved tools and design rules are needed to make this strategy available to more engineered pathways
Metabolic engineers have begun to harness the subcellular organelles of Saccharomyces cerevisiae to take advantage of these properties: the mitochondrion was used to enhance the production of isobutanol[7], and the vacuole was used as a site for methyl halide synthesis[8]
Further enhancements should be achievable by overexpressing peroxisome biogenesis genes, removing endogenous peroxisomal cargo, or using hosts with naturally voluminous peroxisomes, such as Pichia pastoris[10]
Summary
Compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk and improving pathway efficiency, but improved tools and design rules are needed to make this strategy available to more engineered pathways. Metabolic engineers have begun to harness the subcellular organelles of Saccharomyces cerevisiae to take advantage of these properties: the mitochondrion was used to enhance the production of isobutanol[7], and the vacuole was used as a site for methyl halide synthesis[8] These successes relied on substrates that naturally accumulate in the mitochondria and vacuole, limiting the applicability to new pathways. Peroxisomes have already attracted some interest from metabolic engineers, who have placed lycopene and polyhydroxyalkanoate biosynthetic pathways in the peroxisomal lumen[14,15] These efforts left many open challenges, including validation and optimization of protein import, measurements of peroxisomal permeability and verification that intermediates are trapped within the peroxisome. A systematic experimental analysis of their effect on import has yet to be performed
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