Harnessing yeast organelles for metabolic engineering

Hammer, Sarah K.; Avalos, José L.

doi:10.1038/nchembio.2429

Cited by 170 publications

(126 citation statements)

References 99 publications

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“…Many natural product biosyntheses evolved in the context of highly specialized cells that offer pathway compartmentalization and differential gene expression. Production of MIAs and other natural products in yeast will therefore rely on the continued development of tools for coordinated pathway induction (Zalatan et al, 2015) and localized pathway expression (Hammer and Avalos, 2017). Stepwise analysis around two critical steps in iridoid biosynthesis permitted a more complete understanding of the biochemical details surrounding shunt product formation, enabling rational metabolic engineering.…”

Section: Discussionmentioning

confidence: 99%

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

Billingsley

DeNicola

Barber

et al. 2017

Metabolic Engineering

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Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous ‘ene’-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,β-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs.

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Section: Discussionmentioning

confidence: 99%

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

Billingsley

DeNicola

Barber

et al. 2017

Metabolic Engineering

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“…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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“…Subcellular compartmentalization is an evolved strategy for coping with unproductive or harmful crosstalk [5][6][7][8] . In eukaryotes, lipid bilayer membrane-enclosed organelles are used to direct enzymatic activity toward specific substrates.…”

Section: Introductionmentioning

confidence: 99%

“…Engineered compartmentalization has garnered considerable attention in recent years 7,[14][15][16][17][18] .…”

Section: Introductionmentioning

confidence: 99%

Repurposing the yeast peroxisome to compartmentalize a toxic enzyme enables improved (S)-reticuline production

Grewal¹,

Samson²,

Baker

et al. 2020

Preprint

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Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with other factors in the cytosol. Increasingly, engineers are researching ways in which synthetic compartmentalization could be used to address challenges in metabolic engineering. Here, we identified that norcoclaurine synthase (NCS), the enzyme which catalyzes the first committed reaction in benzylisoquinoline alkaloid (BIA) biosynthesis, is toxic when expressed cytosolically in Saccharomyces cerevisiae and, consequently, restricts (S)-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased (S)-reticuline titer, achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and product across the peroxisome membrane. We identified that peroxisome protein capacity in S. cerevisiae becomes a limiting factor for further improvement of BIA production and demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing BIA titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering.

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Harnessing yeast organelles for metabolic engineering

Cited by 170 publications

References 99 publications

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

Prokaryotic nanocompartments form synthetic organelles in a eukaryote

Repurposing the yeast peroxisome to compartmentalize a toxic enzyme enables improved (S)-reticuline production

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