Monoterpene indole alkaloids (MIAs) from Mitragyna speciosa (“kratom”), such as mitragynine and speciogynine, are promising novel scaffolds for opioid receptor ligands for treatment of pain, addiction, and depression. While kratom leaves have been used for centuries in South-East Asia as stimulant and pain management substance, the biosynthetic pathway of these psychoactives have only recently been partially elucidated. Here, we demonstrate the de novo production of mitragynine and speciogynine in Saccharomyces cerevisiae through the reconstruction of a five-step synthetic pathway from common MIA precursor strictosidine comprising fungal tryptamine 4-monooxygenase to bypass an unknown kratom hydroxylase. Upon optimizing cultivation conditions, a titer of ∼290 μg/L kratom MIAs from glucose was achieved. Untargeted metabolomics analysis of lead production strains led to the identification of numerous shunt products derived from the activity of strictosidine synthase (STR) and dihydrocorynantheine synthase (DCS), highlighting them as candidates for enzyme engineering to further improve kratom MIAs production in yeast. Finally, by feeding fluorinated tryptamine and expressing a human tailoring enzyme, we further demonstrate production of fluorinated and hydroxylated mitragynine derivatives with potential applications in drug discovery campaigns. Altogether, this study introduces a yeast cell factory platform for the biomanufacturing of complex natural and new-to-nature kratom MIAs derivatives with therapeutic potential.

中文翻译：

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酵母的代谢工程用于从头生产卡痛叶单萜吲哚生物碱


来自 Mitragyna speciosa（“kratom”）的单萜吲哚生物碱 （MIA），例如 mitragynine 和 speciogynine，是有前途的阿片受体配体治疗疼痛、成瘾和抑郁的新型支架。虽然卡痛叶在东南亚被用作兴奋剂和疼痛管理物质已有几个世纪的历史，但这些精神活性物质的生物合成途径直到最近才被部分阐明。在这里，我们通过重建由真菌色胺 4-单加氧酶组成的常见 MIA 前体 strictosidine 绕过未知的卡痛叶羟化酶的五步合成途径，展示了酿酒酵母中 mitragynine 和 speciogynine 的从头生产。优化培养条件后，葡萄糖中的滴度达到 ∼290 μg/L kratom MIA。对先导生产菌株的非靶向代谢组学分析导致鉴定出许多源自狭素合酶 （STR） 和二氢鸡棒腺合酶 （DCS） 活性的分流产物，突出了它们是酶工程的候选者，以进一步改善酵母中 kratom MIA 的产生。最后，通过喂养含氟色胺和表达人类定制酶，我们进一步证明了氟化和羟基化 mitragynine 衍生物的生产，在药物发现活动中具有潜在应用。总而言之，本研究介绍了一个酵母细胞工厂平台，用于生物制造具有治疗潜力的复杂天然和新自然 kratom MIAs 衍生物。