Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways

DeLoache, William C.; Russ, Zachary N.; Dueber, John E.

doi:10.1038/ncomms11152

Cited by 152 publications

(183 citation statements)

References 45 publications

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“…TCA cycle, β-oxidation), exposing an enzyme to different conditions such as concentrations of substrates (or inhibitors), pH or redox cofactors. Localization tags from native genes have been used to target heterologous (or native) enzymes to specific organelles as a way to increase local enzyme concentration and improve titers (34,35). However, fusion tags can be laborious to design and assemble since they require understanding of protein trafficking and are therefore not used to their fullest extent in synthetic biology.…”

Section: Resultsmentioning

confidence: 99%

A Cas9-based toolkit to program gene expression inSaccharomyces cerevisiae

et al. 2016

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Despite the extensive use of Saccharomyces cerevisiae as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation and solubility tags. We facilitated the use of these parts via a web-based tool, that automates the generation of DNA fragments for integration. Our system builds upon existing gene editing methods in the thoroughness with which the parts are standardized and characterized, the types and number of parts available and the ease with which our methodology can be used to perform genetic edits in yeast. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled us to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production.

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Section: Resultsmentioning

confidence: 99%

A Cas9-based toolkit to program gene expression inSaccharomyces cerevisiae

et al. 2016

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“…Recent reports have explored the localization of engineered proteins to eukaryotic compartments such as the peroxisome 21,22 , mitochondria 19 , and vacuoles 30 . While peer-reviewed) is the author/funder.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to approaches that leverage existing organelles [19][20][21][22] , encapsulins have the advantage of being completely orthogonal to endogenous eukaryotic compartments. There is also ample choice of different encapsulin protein variants derived from different families of bacteria and archaea.…”

Section: Introductionmentioning

confidence: 99%

Prokaryotic nanocompartments form synthetic organelles in a eukaryote

Lau

Giessen

Altenburg

et al. 2018

Preprint

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Compartmentalization of proteins into organelles is a promising strategy for enhancing the productivity of engineered eukaryotic organisms. However, approaches that co-opt endogenous organelles may be limited by the potential for unwanted crosstalk and disruption of native metabolic functions. Here, we present the construction of synthetic non-endogenous organelles in the eukaryotic yeast Saccharomyces cerevisiae, based on the prokaryotic family of self-assembling proteins known as encapsulins. We establish that encapsulins self-assemble to form nanoscale compartments in yeast, and that heterologous proteins can be selectively targeted for compartmentalization. Housing destabilized proteins within encapsulin compartments affords protection against proteolytic degradation in vivo, while the interaction between split protein components is enhanced upon co-localization within the compartment interior. Furthermore, encapsulin compartments can support enzymatic catalysis, with substrate turnover observed for an encapsulated yeast enzyme. Encapsulin compartments therefore represent a modular platform, orthogonal to existing organelles, for programming synthetic compartmentalization in eukaryotes.

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“…Furthermore, additional work is needed to determine whether an increased peroxisomal compartmentalization of metabolic pathways is a mechanism exploited by cancer cells to improve the efficiency of biomass production. Lending support to this notion, compartmentalization of heterologous metabolic pathways in yeast peroxisomes dramatically improves product titer, presumably due to isolation of pathways in a compact and suitable environment [87,88]. …”

Section: Concluding Remarks and Future Perspectivesmentioning

confidence: 99%

Peroxisomal Dysfunction in Age-Related Diseases

Cipolla

Lodhi

2017

Trends in Endocrinology & Metabolism

141

119

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Peroxisomes carry out many key functions related to lipid and reactive oxygen species (ROS) metabolism. The fundamental importance of peroxisomes for health in humans is underscored by the existence of devastating genetic disorders caused by impaired peroxisomal function or lack of peroxisomes. Emerging studies suggest that peroxisomal function may also be altered with aging and contribute to the pathogenesis of a variety of diseases, including diabetes and its related complications, neurodegenerative disorders, and cancer. With increasing evidence connecting peroxisomal dysfunction to the pathogenesis of these acquired diseases, the possibility of targeting peroxisomal function in disease prevention or treatment becomes intriguing. Here, we review recent developments in understanding the pathophysiological implications of peroxisomal dysfunctions outside the context of inherited peroxisomal disorders.

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Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways

Cited by 152 publications

References 45 publications

A Cas9-based toolkit to program gene expression inSaccharomyces cerevisiae

A Cas9-based toolkit to program gene expression inSaccharomyces cerevisiae

Prokaryotic nanocompartments form synthetic organelles in a eukaryote

Peroxisomal Dysfunction in Age-Related Diseases

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