Structure of the <i>Cannabis sativa</i> olivetol‐producing enzyme reveals cyclization plasticity in type III polyketide synthases

Kearsey, Lewis J.; Prandi, Nicole; Karuppiah, V.; Yan, Cunyu; Leys, D.; Toogood, Helen S.; Takano, Eriko; Scrutton, Nigel S.

doi:10.1111/febs.15089

Cited by 28 publications

(31 citation statements)

References 33 publications

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“…2a) (Gagne et al 2012;Tan et al 2018;Kearsey et al 2019). Kearsey et al (2019) also confirmed that, in the absence of OAC, a nonenzymatic C2 → C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol instead of OLA ( Fig. 2a).…”

Section: Cannabinoids Are Synthesized Through a Common Pathway In Trisupporting

confidence: 61%

“…This conundrum, OLA cannot be prepared in vitro, but the in vitro product, olivetol, is not observed in vivo, has since been resolved by evidence that the process requires olivetolic acid cyclase (OAC), to conduct the intramolecular C2 → C7 aldol condensation without decarboxylation ( Fig. 2a) (Gagne et al 2012;Tan et al 2018;Kearsey et al 2019). Kearsey et al (2019) also confirmed that, in the absence of OAC, a nonenzymatic C2 → C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol instead of OLA ( Fig.…”

Section: Cannabinoids Are Synthesized Through a Common Pathway In Trimentioning

confidence: 95%

“…Kearsey et al also investigated the crystal structure of TKS in the presence of CoA (Fig. 3a) and also performed a structure-guided mutagenesis study to investigate why the tetraketide intermediate is released prior to OAC- free cyclization (Kearsey et al 2019). Noel and coworkers had suggested that an 'aldol switch' is necessary to trigger tetraketide release, thereby enabling subsequent olivetolic acid production catalyzed by OAC (Austin et al 2004).…”

Section: Cannabinoids Are Synthesized Through a Common Pathway In Trimentioning

confidence: 99%

“…During the formation of OLA, small quantities of pentyl diacetic lactone (PDAL) and hexanoyl triacetic acid lactone (HTAL) are also formed from non-enzymatic hydrolysis of the mono-and di-malonylated intermediates respectively ( Fig. 2a) (Taura et al 2007b;Gagne et al 2012;Kearsey et al 2019).…”

Section: Cannabinoids Are Synthesized Through a Common Pathway In Trimentioning

confidence: 99%

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The biosynthesis of the cannabinoids

et al. 2021

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Cannabis has been integral to Eurasian civilization for millennia, but a century of prohibition has limited investigation. With spreading legalization, science is pivoting to study the pharmacopeia of the cannabinoids, and a thorough understanding of their biosynthesis is required to engineer strains with specific cannabinoid profiles. This review surveys the biosynthesis and biochemistry of cannabinoids. The pathways and the enzymes’ mechanisms of action are discussed as is the non-enzymatic decarboxylation of the cannabinoic acids. There are still many gaps in our knowledge about the biosynthesis of the cannabinoids, especially for the minor components, and this review highlights the tools and approaches that will be applied to generate an improved understanding and consequent access to these potentially biomedically-relevant materials. Graphical abstract

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Section: Cannabinoids Are Synthesized Through a Common Pathway In Trisupporting

confidence: 61%

Section: Cannabinoids Are Synthesized Through a Common Pathway In Trimentioning

confidence: 95%

Section: Cannabinoids Are Synthesized Through a Common Pathway In Trimentioning

confidence: 99%

Section: Cannabinoids Are Synthesized Through a Common Pathway In Trimentioning

confidence: 99%

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The biosynthesis of the cannabinoids

et al. 2021

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“…For MALDI TOF/TOF, the peptides were eluted with 2-10 µl of 0.1% TFA in 95% ACN and then coated onto MALDI plate with matrix. For LC-MS/MS, peptides were eluted with 2-10 µl of 0.1% formic acid in 95% ACN and then dispensed into auto-sampler vial 18 . MS/MS spectra were searched against NCBInr database using the parameters as mentioned in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Purification and biochemical characterization of Hel a 6, a cross-reactive pectate lyase allergen from Sunflower (Helianthus annuus L.) pollen

Ghosh

Sircar

Asam

et al. 2020

Sci Rep

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Sunflower pollen was reported to contain respiratory allergens responsible for occupational allergy and pollinosis. The present study describes the comprehensive characterization of a major sunflower allergen Hel a 6. Natural Hel a 6 was purified from sunflower pollen by anion exchange and gel filtration chromatography. Hel a 6 reacted with IgE-antibodies from 57% of 39 sunflower-sensitized patient sera suggesting it to be a major allergen. The patients were of Indian origin and suffering from pollinosis and allergic rhinitis. Hel a 6 exhibited allergenic activity by stimulating mediator release from basophils. Monomeric Hel a 6 displayed pectate lyase activity. The effect of various physicochemical parameters such as temperature, pH, and calcium ion on the functional activity of Hel a 6 revealed a stable nature of the protein. Hel a 6 was folded, and its melting curve showed reversible denaturation in which it refolded back to its native conformation from a denatured state. Hel a 6 displayed a high degree of sequence conservation with the pectate lyase allergens from related taxonomic families such as Amb a 1 (67%) and Art v 6 (57%). The IgE-cross reactivity was observed between Hel a 6 and its ragweed and mugwort homologs. The cross-reactivity was further substantiated by the mediator release when Hel a 6-sensitized effector cells were cross-stimulated with Art v 6 and Amb a 1. Several putative B cell epitopes were predicted and mapped on these 3 allergens. Two antigenic regions were found to be commonly shared by these 3 allergens, which could be crucial for cross-reactivity. In conclusion, Hel a 6 serves as a candidate molecule for diagnosis and immunotherapy for weed allergy.

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Engineering cannabinoid production in Saccharomyces cerevisiae

Schmidt,

Aras,

Kayser

2024

Biotechnology Journal

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Phytocannabinoids are natural products with highly interesting pharmacological properties mainly produced by plants. The production of cannabinoids in a heterologous host system has gained interest in recent years as a promising alternative to production from plant material. However, the systems reported so far do not achieve industrially relevant titers, highlighting the need for alternative systems. Here, we show the production of the cannabinoids cannabigerolic acid and cannabigerol from glucose and hexanoic acid in a heterologous yeast system using the aromatic prenyltransferase NphB from Streptomyces sp. strain CL190. The production was significantly increased by introducing a fusion protein consisting of ERG20WW and NphB. Furthermore, we improved the production of the precursor olivetolic acid to a titer of 56 mg L−1. The implementation of the cannabinoid synthase genes enabled the production of Δ9‐tetrahydrocannabinolic acid, cannabidiolic acid as well as cannabichromenic acid, where the heterologous biosynthesis of cannabichromenic acid in a yeast system was demonstrated for the first time. In addition, we found that the product spectrum of the cannabinoid synthases localized to the vacuoles of the yeast cells was highly dependent on extracellular pH, allowing for easy manipulation. Finally, using a fed‐batch approach, we showed cannabigerolic acid and olivetolic acid titers of up to 18.2 mg L−1 and 117 mg L−1, respectively.

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Structure of the Cannabis sativa olivetol‐producing enzyme reveals cyclization plasticity in type III polyketide synthases

Cited by 28 publications

References 33 publications

The biosynthesis of the cannabinoids

The biosynthesis of the cannabinoids

Purification and biochemical characterization of Hel a 6, a cross-reactive pectate lyase allergen from Sunflower (Helianthus annuus L.) pollen

Engineering cannabinoid production in Saccharomyces cerevisiae

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