title: Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways.
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.
authors.affiliation: UC Berkeley and UCSF Graduate Program in Bioengineering, University of California, Berkeley (UC Berkeley), Berkeley, California 94720, USA.|Department of Bioengineering, UC Berkeley, Berkeley, California 94720, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: DeLoache
authors.affiliation: UC Berkeley and UCSF Graduate Program in Bioengineering, University of California, Berkeley (UC Berkeley), Berkeley, California 94720, USA.|Department of Bioengineering, UC Berkeley, Berkeley, California 94720, USA.
authors.forename: Zachary N
authors.identifier: http://orcid.org/0000-0002-0571-8252
authors.initials: ZN
authors.lastname: Russ
authors.affiliation: Department of Bioengineering, UC Berkeley, Berkeley, California 94720, USA.
authors.forename: John E
authors.initials: JE
authors.lastname: Dueber
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001681:Biological Assay; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D053858:Metabolic Networks and Pathways; D055442:Metabolome; D009844:Oligosaccharides / Q000378:metabolism; D010539:Permeability; D020675:Peroxisomes / Q000378:metabolism*; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: D000596:Amino Acids; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins