Peroxisome compartmentalization of a toxic enzyme improves alkaloid production

Grewal, Parbir S.; Samson, Jennifer A.; Baker, Jordan J.; Choi, Brian; Dueber, John E.

doi:10.1038/s41589-020-00668-4

Cited by 96 publications

(67 citation statements)

References 48 publications

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“…In contrast, the growth profiles of strains with encapsulated MIOX were similar to that of a non-MIOX-expressing control strain (Figure S7), suggesting that overexpression of the MIOX protein may be inherently toxic to yeast. This is similar to a recent yeast study where targeting of norcoclaurine synthase into peroxisomes was found to alleviate cellular toxicity associated with the enzyme 47 . GFP signal detected by flow cytometry (Figure 7b) and western blot (Figure 7c) was used as a proxy for intracellular MIOX levels in the three GFP-tagged strains.…”

Section: Resultssupporting

confidence: 90%

An artificial self-assembling nanocompartment for organising metabolic pathways in yeast

Cheah

Stark

Adamson

et al. 2021

Preprint

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Metabolic pathways are commonly organised by sequestration into discrete cellular compartments. Compartments prevent unfavourable interactions with other pathways and provide local environments conducive to the activity of encapsulated enzymes. Such compartments are also useful synthetic biology tools for examining enzyme/pathway behaviour and for metabolic engineering. Here, we expand the intracellular compartmentalisation toolbox for budding yeast (Saccharomyces cerevisiae) with engineered Murine polyomavirus virus-like particles (MPyV VLPs). The MPyV system has two components: VP1 which self-assembles into the compartment shell; and a short anchor, VP2C, which mediates cargo protein encapsulation via binding to the inner surface of the VP1 shell. Destabilised GFP fused to VP2C was specifically sorted into VLPs and thereby protected from host-mediated degradation. In order to access metabolites of native and engineered yeast metabolism, VLP-based nanocompartments were directed to assemble in the cytosol by removal of the VP1 nuclear localisation signal. To demonstrate their ability to function as a metabolic compartment, MPyV VLPs were used to encapsulate myo-inositol oxygenase (MIOX), an unstable and rate-limiting enzyme in D-glucaric acid biosynthesis. Strains with encapsulated MIOX produced ~20% more D-glucaric acid compared to controls expressing ‘free’ MIOX - despite accumulating dramatically less expressed protein - and also grew to higher cell densities. These effects were linked to enzyme stabilisation and mitigation of cellular toxicity by the engineered compartment. This is the first demonstration in yeast of an artificial biocatalytic compartment that can participate in a metabolic pathway and establishes the MPyV platform as a promising synthetic biology tool for yeast engineering.

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Section: Resultssupporting

confidence: 90%

An artificial self-assembling nanocompartment for organising metabolic pathways in yeast

Cheah

Stark

Adamson

et al. 2021

Preprint

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“…This clearly illustrates the importance of the control of the subcellular distribution of actors from a heterologous metabolic pathway as also strengthened by the artificial targeting of the plant norcoclaurine synthase to yeast peroxisomes. [10] Finally, the implementation of the last module (module IV) dedicated to expression of final step enzymes for hyoscyamine and scopolamine biosynthesis required an additional step of pathway elucidation. By using emerging omics-based strategies that facilitate the discovery of missing enzymes from complex metabolic pathways, they identified the hyoscyamine aldehyde to hyoscyamine converting enzyme in A. belladonna (hyoscyamine dehydrogenase, HDH), through a "guilty by association" study using known genes of TA synthesis as baits.…”

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confidence: 99%

Engineered Microbes for Producing Anticholinergics

2020

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The tropane alkaloids (TAs) hyoscyamine and scopolamine function as acetylcholine receptor antagonists and are used clinically as parasympatholytics to treat neuromuscular disorders in humans. While TAs are synthesized in a small subset of plant families, these specialized metabolites are only accumulated in limited quantities in plant organs. The complex chemical structures of these compounds make their industrial production by chemical synthesis very challenging, Therefore, the supply of these TAs still relies on intensive farming of Duboisia shrubs in tropical countries. Many adverse factors such as climate fluctuations and pandemics can thus influence annual world production. Based on the landmark microbial production of the antimalarial semi‐synthetic artemisinin, the Smolke group recently developed a yeast cell factory capable of de novo synthesizing hyoscyamine and scopolamine, thus paving the way for an alternative production of these compounds.

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“…Together with the increase of the peroxisome density triggered by engineered transcription factors, such an approach substantially improved BIA production. 33 Similarly, in plants, rerouting the diterpene biosynthesis from the chloroplasts (synthesis through the methylerythritol pathway) to the cytosol (mevalonate pathway) dramatically increased synthesis titers and allowed production of the allopathic momilactone B in N. benthamiana. 34…”

Section: A Booming Discovery Of Pnp Biosynthetic Pathwaysmentioning

confidence: 99%