Improved protein glycosylation enabled heterologous biosynthesis of monoterpenoid indole alkaloids and their unnatural derivatives in yeast

Shahsavarani, Mohammadamin; Utomo, Joseph Christian; Kumar, Rahul; Paz-Galeano, Melina; Garza-García, Jorge Jonathan Oswaldo; Mai, Zhan; Ro, Dae‐Kyun; Qu, Yang

doi:10.1016/j.mec.2022.e00215

Cited by 10 publications

(7 citation statements)

References 33 publications

(48 reference statements)

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“…For pathway assembly in yeast, codon‐optimized dCrSTR (Shahsavarani et al ., 2022) was subcloned from pUG19 vector into pESC‐His vector within NotI/ClaI site. MsDCS1 was subcloned from the respective pET30b+ vector into pESC‐His‐dCrSTR within the BamHI/SalI sites.…”

Section: Methodsmentioning

confidence: 99%

“…The expression cassette of codon‐optimized CrSGD was subcloned from pESC‐His‐CrSGD vector (Gao et al ., 2022) by the primer set (31/32), and ligated into pESC‐His‐dCrSTR‐MsDCS1 vector within the DraIII site. The PcuT4H open reading frame was synthesized (Twist Biosciences, South San Francisco, CA, USA) and subcloned into pESC‐Leu‐CrCPR vector (Shahsavarani et al ., 2022) within the BamHI/SalI sites. MsOMT1 was subcloned from the respective pET30b+ vector into pESC‐Ura vector within BamHI/SalI sites.…”

Section: Methodsmentioning

confidence: 99%

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Biosynthesis of kratom opioids

et al. 2023

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Summary Mitragynine, an analgesic alkaloid from the plant Mitragyna speciosa (kratom), offers a safer alternative to clinical opioids such as morphine, owing to its more favorable side effect profile. Although kratom has been traditionally used for stimulation and pain management in Southeast Asia, the mitragynine biosynthesis pathway has remained elusive. We embarked on a search for mitragynine biosynthetic genes from the transcriptomes of kratom and other members of the Rubiaceae family. We studied their functions in vitro and in vivo. Our investigations led to the identification of several reductases and an enol methyltransferase that forms a new clade within the SABATH methyltransferase family. Furthermore, we discovered a methyltransferase from Hamelia patens (firebush), which catalyzes the final step. With the tryptamine 4‐hydroxylase from the psychedelic mushroom Psilocybe cubensis, we accomplished the four‐step biosynthesis for mitragynine and its stereoisomer, speciogynine in both yeast and Escherichia coli when supplied with tryptamine and secologanin. Although we have yet to pinpoint the authentic hydroxylase and methyltransferase in kratom, our discovery completes the mitragynine biosynthesis. Through these breakthroughs, we achieved the microbial biosynthesis of kratom opioids for the first time. The remarkable enzyme promiscuity suggests the possibility of generating derivatives and analogs of kratom opioids in heterologous systems.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Biosynthesis of kratom opioids

et al. 2023

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“…While chemical synthesis methods for producing mescaline exist (Cassels & Sáez‐Briones, 2018), researchers are increasingly turning to synthetic biology approaches to produce a range of plant‐derived compounds in a sustainable and scalable manner. Identification of biosynthetic genes will facilitate the development of synthetic biosystems for the industrial production of mescaline, as has been the case for other psychoactive plant‐derived products including opiates (Dastmalchi, Chang, et al., 2019a; Pyne et al., 2020), tropane alkaloids (Chavez et al., 2022; Srinivasan & Smolke, 2021) and monoterpene indole alkaloids (Dusséaux et al., 2020; Shahsavarani et al., 2023). Additionally, several enzymes identified here, such as LwOMT10 and LwNMT, displayed broad phenethylamine substrate ranges (Tables S2 and S3).…”

Section: Discussionmentioning

confidence: 99%

Elucidation of the mescaline biosynthetic pathway in peyote (Lophophora williamsii)

Watkins,

Li,

Yeaman

et al. 2023

The Plant Journal

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SUMMARYPeyote (Lophophora williamsii) is an entheogenic and medicinal cactus native to the Chihuahuan desert. The psychoactive and hallucinogenic properties of peyote are principally attributed to the phenethylamine derivative mescaline. Despite the isolation of mescaline from peyote over 120 years ago, the biosynthetic pathway in the plant has remained undiscovered. Here, we use a transcriptomics and homology‐guided gene discovery strategy to elucidate a near‐complete biosynthetic pathway from l‐tyrosine to mescaline. We identified a cytochrome P450 that catalyzes the 3‐hydroxylation of l‐tyrosine to l‐DOPA, a tyrosine/DOPA decarboxylase yielding dopamine, and four substrate‐specific and regiospecific substituted phenethylamine O‐methyltransferases. Biochemical assays with recombinant enzymes or functional analyses performed by feeding putative precursors to engineered yeast (Saccharomyces cerevisiae) strains expressing candidate peyote biosynthetic genes were used to determine substrate specificity, which served as the basis for pathway elucidation. Additionally, an N‐methyltransferase displaying broad substrate specificity and leading to the production of N‐methylated phenethylamine derivatives was identified, which could also function as an early step in the biosynthesis of tetrahydroisoquinoline alkaloids in peyote.

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“…Other examples of genetic manipulation include expressing constitutive biosynthetic enzymes, transcription factors, or transporters known to regulate MIA accumulation [ 43 , 45 , 46 ]. While most engineering efforts of the MIA pathways into microbial and plant systems are intended to increase the yield of the desired MIAs, a notable advantage of reconstitution of natural product pathways includes the expansion of chemical diversity, potentially enabling the production of other bioactive MIAs that are not readily available in nature [ 47 , 48 , 49 ].…”

Section: The Complexities Of Mia Biosynthetic Pathwaymentioning

confidence: 99%

Advances in Metabolic Engineering of Plant Monoterpene Indole Alkaloids

Salim,

Jarecki,

Vick

et al. 2023

Biology

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Monoterpene indole alkaloids (MIAs) encompass a diverse family of over 3000 plant natural products with a wide range of medical applications. Further utilizations of these compounds, however, are hampered due to low levels of abundance in their natural sources, causing difficult isolation and complex multi-steps in uneconomical chemical syntheses. Metabolic engineering of MIA biosynthesis in heterologous hosts is attractive, particularly for increasing the yield of natural products of interest and expanding their chemical diversity. Here, we review recent advances and strategies which have been adopted to engineer microbial and plant systems for the purpose of generating MIAs and discuss the current issues and future developments of manufacturing MIAs by synthetic biology approaches.

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Improved protein glycosylation enabled heterologous biosynthesis of monoterpenoid indole alkaloids and their unnatural derivatives in yeast

Cited by 10 publications

References 33 publications

Biosynthesis of kratom opioids

Biosynthesis of kratom opioids

Elucidation of the mescaline biosynthetic pathway in peyote (Lophophora williamsii)

Advances in Metabolic Engineering of Plant Monoterpene Indole Alkaloids

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