Biosynthesis of Kratom Opioids

Abstract

Mitragyna speciosa (kratom) derived monoterpenoid indole alkaloids (MIAs) such as mitragynine and 7-hydroxymitragynine are a new class of opioids with a corynanthe MIA pharmacophore that is responsible for their significantly reduced side effects and superior safety profiles. While botanical kratom has been historically used for stimulation and pain management in Southeast Asia, the biosynthesis of kratom MIAs is not known. In this study, we identified and characterized 9 reductases bearing various degrees of demethyldihydrocoryanthine/demethylcorynantheidine synthase activity and a new SABATH type methyltransferase that catalyzes highly unusual non-aromatic enol methylation from kratom and several other species, which are required in kratom opioids biosynthesis. With unnatural substrate 4-hydroxytryptamine, we further showed the biosynthesis of mitragynine and its epimer speciogynine using these characterized enzymes. The promiscuity of kratom opioid biosynthetic enzymes suggests that derivatives and analogs of kratom opioids may be manufactured in heterologous systems with appropriate enzymes and substrates.

Metabolic engineering of yeast forde novoproduction of kratom monoterpene indole alkaloids

“…This approach was also recently reported in a N. benthamiana leaf transient expression platform, but 9-OH-corynantheidine (3a/b) was not observed in tobacco leaves although 9-OH-strictosidine was detected (Schotte et al, 2023). To validate the substrate promiscuity observed by Kim et al (2023) and demonstrate de novo production of 9-OH-corynantheidine (3a/b) in yeast, we expressed the fungal tryptamine 4-monooxygenase PcPsiH from P. cubensis together with its reductase PcCPR in MIA-CZ-1, generating MIA-KM-1. Here, we employed a CYP/CPR design previously engineered in yeast to produce psylocibin (Milne et al, 2020).…”

“…Recent work established that MG and SG (its C20 epimer) are produced in M. speciosa in five enzymatic steps from the common MIA precursor strictosidine (Fig. 1, "Canonical pathway") (K. Kim et al, 2023;Schotte et al, 2023). Strictosidine first undergoes de-glycosylation (SGD, strictosidine-O-β-D-glucosidase), followed by action of a medium chain alcohol dehydrogenase (DCS, dihydrocorynantheine synthase), enol O-methyltransferase (OMT, EnolMT), and finally oxidation and methylation at C9 position.…”

“…The stereoselectivity of the different DCS variants is responsible for the production of either mitragynine (20S) or speciogynine (20R). While the last two enzymes responsible for the formation of the methoxy group in C9 in M. speciosa are still unknown, Kim et al, (2023) reported an enzyme named Hp9OMT from firebush Hamelia patens capable of methylating 9-OHcorynantheidine to form MG and SG, and further reported the refactoring of a 6-enzyme bioconversion of supplemented MIA precursors tryptamine and secologanin to MG and SG at µg/L-scale in yeast (Kim et al, 2023). Over the course of this study, the core kratom pathway, comprising SGD, MsDCS1 and EnolMT, was refactored in a strictosidine-producer yeast strain leading to 0.9 mg/L de novo corynantheidine production (Dror et al, 2024).…”

Total Synthesis and Structural Plasticity of Kratom Pseudoindoxyl Metabolites**

“…[27] Finally, we believed that our secologanin analogue could further broaden the synthetic landscape, as it could provide access to both diastereomeric kratom pseudoindoxyls of the corynanthe type, allo (mitragynine) and normal (speciogynine) isomers, by selective hydrogenation of its olefinic bond. [28,29] As such, this synthetic development was inspired by the manner in which nature assembles mitragynine pseudoindoxyl (3), including rapid complexity generation through cascade reactions of tryptamine and secologanin analogues, step and redox economy, divergency and protecting-group-free assembly. [30] Herein, we report the first total synthesis of mitragynine pseudoindoxyl (3) and its diastereomer, speciogynine pseudoindoxyl (4).…”

Syntheses and structural plasticity of kratom pseudoindoxyl metabolites

“…24 Finally, we believed that our secologanin analog could further broaden the synthetic landscape, as it could provide access to both diastereomeric kratom pseudoindoxyls of the corynanthe-type, allo (mitragynine) and normal (speciogynine) isomers, by selective hydrogenation of its olefinic bond. 25,26 As such, this synthetic study was designed to mimic the manner through which nature assembles mitragynine pseudoindoxyl (3), including rapid complexity generation via cascade reactions of tryptamine and secologanin analogs, step-and redox economy, divergency and protecting group-free assembly. 27 Herein, we report the first total synthesis of mitragynine pseudoindoxyl (3) and its diastereomer, speciogynine pseudoindoxyl (4).…”