Biosynthesis of kratom opioids.

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.

Metabolite and Molecular Characterization of Mitragyna speciosa Identifies Developmental and Genotypic Effects on Monoterpene Indole and Oxindole Alkaloid Composition.

The monoterpene indole alkaloid (MIA) mitragynine has garnered attention as a potential treatment for pain, opioid use disorder, and opioid withdrawal because of its combined pharmacology at opioid and adrenergic receptors in humans. This alkaloid is unique to Mitragyna speciosa (kratom), which accumulates over 50 MIAs and oxindole alkaloids in its leaves. Quantification of 10 targeted alkaloids from several tissue types and cultivars of  M. speciosa revealed that mitragynine accumulation was highest in leaves, followed by stipules and stems, but was absent, along with other alkaloids, in roots. While mitragynine is the predominant alkaloid in mature leaves, juvenile leaves accumulate higher amounts of corynantheidine and speciociliatine. Interestingly, corynantheidine has an inverse relationship with mitragynine accumulation throughout leaf development. Characterization of various cultivars of M. speciosa indicated altered alkaloidal profiles ranging from undetectable to high levels of mitragynine. DNA barcoding and phylogenetic analysis using ribosomal ITS sequences revealed polymorphisms leading M. speciosa cultivars having lower mitragynine content to group with other mitragyna species, suggesting interspecific hybridization events. Root transcriptome analysis of low- and high-mitragynine-producing cultivars indicated significant differences in gene expression and revealed allelic variation, further supporting that hybridization events may have impacted the alkaloid profile of M. speciosa.

Sequence variation of commercially available kratom products at universal DNA barcode regions.

Mitragyna speciosa, commonly known as kratom, is a narcotic plant that is used for its unique mood-enhancing and pain-relieving effects. It is marketed throughout the United States as a 'legal high' and has gained popularity as an alternative to opioids. However, kratom's increasing involvement in accidental overdoses, especially among polydrug users, has prompted warnings from the Drug Enforcement Agency (DEA) and the Food and Drug Administration (FDA). Despite these warnings, kratom remains legal federally, although it is banned in six states. This legal disparity complicates monitoring and enforcement efforts in states where kratom is illegal. Common forensic techniques using morphology or chemical analysis are beneficial in some instances but are not useful in source attribution because most seized kratom is powdered and the alkaloid content of samples can vary within products, making sourcing unreliable. This study focused on developing a DNA barcoding method to access sequence variation in commercial kratom products. It evaluated the utility of one nuclear barcode region (ITS) and three chloroplast barcode regions (matK, rbcL, and trnH-psbA) in assessing sequence variation across commercially available kratom products. Novel polymorphisms were discovered, and the ITS region showed the greatest variation between samples. Among the 15 kratom products tested, only two haplotypes were identified across the four barcoding regions. The findings highlight the potential of DNA barcoding as a forensic tool in the traceability and enforcement against illegal kratom distribution. Nonetheless, the limited haplotypic diversity points to a need for further development and expansion of the M. speciosa DNA sequence database.

Limitation of mitragynine biosynthesis in Mitragyna speciosa (Roxb.) Korth. through tryptamine availability.

Metabolite profiles of Mitragyna speciosa were determined by means of 1H NMR-based and HPLC-based analyses. The results indicated that high contents of secologanin, caffeic acid, gallic acid, epigallocatechin, and mitragynine were accumulated in leaves. In M. speciosa, feedings of tryptamine, tryptophan, phenylalanine or tyrosine significantly increased the mitragynine contents. Feedings of tryptamine and loganin also enhanced the mitragynine accumulation, but feeding of loganin only did not affect the mitragynine level. The mRNA levels of anthranilate synthase alpha subunit (ASA), tryptophan decarboxylase (TDC), and strictosidine synthase (STR) were measured by quantitative real-time polymerase chain reaction (RT-qPCR) in control plants and those exposed to methyl jasmonate (MJ; 10 microM). All genes responded to MJ after a 24-h treatment. The mitragynine contents were also enhanced and corresponded to the transcript levels. From the present results we conclude that a high content of secologanin together with a undetectable level of tryptamine in M. speciosa feature the limitation of mitragynine biosynthesis. Additionally, expression of all the genes limits production of an essential precursor for mitragynine production.

The Mitragyna speciosa (Kratom) Genome: a resource for data-mining potent pharmaceuticals that impact human health.

Mitragyna speciosa (kratom) produces numerous compounds with pharmaceutical properties including the production of bioactive monoterpene indole and oxindole alkaloids. Using a linked-read approach, a 1,122,519,462 bp draft assembly of M. speciosa "Rifat" was generated with an N50 scaffold size of 1,020,971 bp and an N50 contig size of 70,448 bp that encodes 55,746 genes. Chromosome counting revealed that "Rifat" is a tetraploid with a base chromosome number of 11, which was further corroborated by orthology and syntenic analysis of the genome. Analysis of genes and clusters involved in specialized metabolism revealed genes putatively involved in alkaloid biosynthesis. Access to the genome of M. speciosa will facilitate an improved understanding of alkaloid biosynthesis and accelerate the production of bioactive alkaloids in heterologous hosts.

Differentiation of Mitragyna speciosa, a narcotic plant, from allied Mitragyna species using DNA barcoding-high-resolution melting (Bar-HRM) analysis.

Mitragyna speciosa (Korth.) Havil. [MS], or "kratom" in Thai, is the only narcotic species among the four species of Mitragyna in Thailand, which also include Mitragyna diversifolia (Wall. ex G. Don) Havil. [MD], Mitragyna hirsuta Havil. [MH], and Mitragyna rotundifolia (Roxb.) O. Kuntze [MR]. M. speciosa is a tropical tree belonging to the Rubiaceae family and has been prohibited by law in Thailand. However, it has been extensively covered in national and international news, as its abuse has become more popular. M. speciosa is a narcotic plant and has been used as an opium substitute and traditionally used for the treatment of chronic pain and various illnesses. Due to morphological disparities in the genus, the identification of plants in various forms, including fresh leaves, dried leaf powder, and finished products, is difficult. In this study, DNA barcoding combined with high-resolution melting (Bar-HRM) analysis was performed to differentiate M. speciosa from allied Mitragyna and to assess the capability of Bar-HRM assays to identify M. speciosa in suspected kratom or M. speciosa-containing samples. Bar-HRM analysis of PCR amplicons was based on the ITS2, rbcL, trnH-psbA, and matK DNA barcode regions. The melting profiles of ITS2 amplicons were clearly distinct, which enabled the authentication and differentiation of Mitragyna species from allied species. This study reveals that DNA barcoding coupled with HRM is an efficient tool with which to identify M. speciosa and M. speciosa-containing samples and ensure the safety and quality of traditional Thai herbal medicines.

Detection and Identification of Kratom (Mitragyna speciosa) and Marijuana (Cannabis sativa) by a Real-Time Polymerase Chain Reaction High-Resolution Melt Duplex Assay.

Mitragyna speciosa (MS), a plant commonly known as kratom, is a widely used "legal high" opiate alternative for pain relief. DNA extracted from MS and 26 additional plant species was amplified by PCR using primers targeting the strictosidine beta-D-glucosidase (SGD) and secologanin synthase 2 (SLS2) genes and detected by high-resolution melt curves using three intercalating dyes. Amplicon sizes were confirmed using agarose gel electrophoresis. The observed melt temperatures for SGD and SLS2 were 77.08 ± 0.38°C and 77.61 ± 0.46°C, respectively, using SYBR® Green I; 80.18 ± 0.27°C and 80.59 ± 0.08°C, respectively, using Radiant™ Green; and 82.19 ± 0.04°C and 82.62 ± 0.13°C, respectively, using the LCGreen® PLUS dye. The SLS2 primers demonstrated higher specificity and identified MS DNA at 0.05 ng/µL. In a duplex reaction, SLS2 and tetrahydrocannabinoic acid synthase gene primers detected and differentiated MS and Cannabis sativa (CS) by melt peaks at 82.63 ± 0.35°C and 85.58 ± 0.23°C, respectively, using LCGreen® PLUS.

[Discrimination of Kratom Products by an Improved PCR-RFLP Method].

In Japan, mitragynine, 7-hydroxymitragynine and Mitragyna speciosa Korth. (M. speciosa, "Kratom") were controlled as Designated Substances under the Pharmaceutical and Medical Device Act from March 2016. In this study, the origins of 16 Kratom products obtained from the illegal drug market in Japan were investigated by DNA analyses and LC-MS analyses. When the PCR-restriction fragment length polymorphism (RFLP) was performed using the restriction enzyme XmaI (as reported by Sukrong et al. to be able to distinguish M. speciosa), the same DNA fragment patterns were obtained from all 16 products. On the other hand, as a result of the identification of the plant species of each product by nucleotide sequence analyses, the sequences of M. speciosa were detected in only 14 products. Despite the facts that mitragynine and 7-hydroxymitragynine were detected also in the other two products by the LC-MS analyses, M. speciosa DNAs were not amplified from these products by the PCR. Moreover, the DNA amplicons of the other psychotropic plant (Mesembryanthemum sp., e.g. "Kanna") were detected. This plant PCR amplicon has the restriction site for the XmaI at the same position of the M. speciosa PCR amplicon and it is difficult to distinguish "Kratom" and "Kanna" by the conventional PCR-RFLP. When the restriction enzyme XhoI was used simultaneously with the Xmal, the specific DNA fragment was only observed from the M. speciosa amplicon and it was possible to distinguish both species using this improved PCR-RFLP method. This method is useful to identify the origin of Kratom products distributed in the illegal drug market.

Evaluation of Two New Methods for DNA Extraction of "Legal High" Plant Species.

A quick, simple, and high-yield nucleic acid isolation process is crucial for high-quality DNA analysis. The ability of the MicroGEM PDQeX phytoGEM system and Omega Bio-tek E.Z.N.A.® Plant DS Mini kit to extract PCR-ready DNA was evaluated by extracting the forensically relevant "legal high" plant species: Ipomoea purpurea, Artemisia absinthium, Mitragyna speciosa, Datura stramonium, and Papaver somniferum. The plant material was pulverized, processed using the manufacturer's plant protocol for the PDQeX Nucleic Acid Extraction or the manufacturer's protocol for the Omega extraction, quantified using the Invitrogen Qubit 2.0 Fluorometer, and analyzed for amplifiability by PCR using a Qiagen Rotor-Gene Q instrument and published assays. The DNA amplicons for the legal high species produced high-resolution melt curves concordant with the melts observed when DNA was isolated using the Qiagen DNeasy Plant Mini Kit in previous studies.

Mitragyna speciosa: hairy root culture for triterpenoid production and high yield of mitragynine by regenerated plants.

Hairy root cultures of Mitragyna speciosa were established by infection of Agrobacterium rhizogenes ATCC 15834 and maintained in McCown woody plant medium (WPM) supplemented with 0.5 mg/1 naphthaleneacetic acid. The hairy roots were identified for the rooting genes loci of rolA and rolB by polymerase chain reaction. For studying the secondary metabolite production, the n-hexane extract of the hairy roots was prepared and the compounds were isolated by silica gel column chromatography, affording triterpenoids (ursolic acid and oleanolic acid) and phytosterols (beta-sitosterol and stigmasterol). The shoots from the hairy root cultures were regenerated and differentiated to the plantlets. For micropropagation, shoot multiplication was successfully induced from the axillary buds of the regenerated plantlets in WPM supplemented with 0.1 mg/l thidiazuron. The mitragynine contents of 5-month-old regenerated plants and in vitro plantlets (germinated from seeds) were determined using the TLC-densitometric method. The regenerated plants contained (14.25 +/- 0.25) mg/g dry wt mitragynine, whereas the in vitro plantlets contained (4.45 +/- 0.09) mg/g dry wt.