甾体生物碱是 FDA 批准的药物（例如 Zytiga）和有前途的候选药物/先导药物（例如环巴明）；然而，许多 ≥697 种已知的甾体生物碱天然产物仍未充分用作药物，因为在其生产生物体中扩大其生物合成规模可能具有挑战性。 Cyclopamine 是一种由玉米百合 (Veratrum spp.) 植物产生的甾体生物碱，是 Hedgehog (Hh) 信号通路的抑制剂。因此，环巴明是治疗与 Hh 信号传导失调相关的人类疾病的重要候选药物/先导药物，例如基底细胞癌和急性髓性白血病。环巴明及其半合成衍生物已作为基于 Hh 抑制剂的药物在临床（前）试验中进行了研究。然而，扩大环巴明生产规模的挑战减缓了通过（生物）合成衍生化提高其功效和安全性的努力，通常将药物开发限制为环巴明的合成类似物，例如 FDA 批准的药物 Odomzo、Daurismo 和 Erivedge。如果建立了环杷明可规模化和可持续生产的平台，那么它的（生物）合成衍生化、临床开发以及最终的广泛分布就可以加速。为实现这一目标正在进行的努力包括在藜芦植物细胞培养物中生物合成环杷明以及环杷明的半/全化学合成。在此，这项工作推动了一种有前途的未来方法的努力：在工程微生物中生物合成环杷明。我们通过诱导上调天然酵母甲羟戊酸和羊毛甾醇生物合成途径、生物合成的转移，在工程酿酒酵母（S. cerevisiae）中完成了从单糖（即葡萄糖和半乳糖）异源微生物生产维拉嗪（环巴明的生物合成前体）从麦角甾醇（即酿酒酵母中的天然甾醇）到胆固醇（即维拉嗪的生物合成前体）的通量，以及重构的五步维拉嗪生物合成途径的表达。产生维拉嗪的工程酿酒酵母菌株含有来自七个不同物种的八种异源酶。重要的是，通过液相色谱-质谱法，无法将酿酒酵母生产的维拉嗪与商业标准品（藜芦属植物生产的）和本塞姆氏烟草生产的维拉嗪区分开来。据我们所知，这是第一份描述工程酵母异源生产甾体生物碱的报告。通过设计-构建-测试-学习循环，维拉嗪的产量最终提高到最终滴度 83 ± 3 μg/L (4.1 ± 0.1 μg/g DCW)。总之，这项研究为未来环巴明、环巴明（生物）合成衍生物和其他甾体生物碱天然产物的微生物生物合成奠定了基础。版权所有 © 2024 国际代谢工程学会。由爱思唯尔公司出版。保留所有权利。

Steroidal alkaloids are FDA-approved drugs (e.g., Zytiga) and promising drug candidates/leads (e.g., cyclopamine); yet many of the ≥697 known steroidal alkaloid natural products remain underutilized as drugs because it can be challenging to scale their biosynthesis in their producing organisms. Cyclopamine is a steroidal alkaloid produced by corn lily (Veratrum spp.) plants, and it is an inhibitor of the Hedgehog (Hh) signaling pathway. Therefore, cyclopamine is an important drug candidate/lead to treat human diseases that are associated with dysregulated Hh signaling, such as basal cell carcinoma and acute myeloid leukemia. Cyclopamine and its semi-synthetic derivatives have been studied in (pre)clinical trials as Hh inhibitor-based drugs. However, challenges in scaling the production of cyclopamine have slowed efforts to improve its efficacy and safety profile through (bio)synthetic derivatization, often limiting drug development to synthetic analogs of cyclopamine such as the FDA-approved drugs Odomzo, Daurismo, and Erivedge. If a platform for the scalable and sustainable production of cyclopamine were established, then its (bio)synthetic derivatization, clinical development, and, ultimately, widespread distribution could be accelerated. Ongoing efforts to achieve this goal include the biosynthesis of cyclopamine in Veratrum plant cell culture and the semi-/total chemical synthesis of cyclopamine. Herein, this work advances efforts towards a promising future approach: the biosynthesis of cyclopamine in engineered microorganisms. We completed the heterologous microbial production of verazine (biosynthetic precursor to cyclopamine) from simple sugars (i.e., glucose and galactose) in engineered Saccharomyces cerevisiae (S. cerevisiae) through the inducible upregulation of the native yeast mevalonate and lanosterol biosynthetic pathways, diversion of biosynthetic flux from ergosterol (i.e., native sterol in S. cerevisiae) to cholesterol (i.e., biosynthetic precursor to verazine), and expression of a refactored five-step verazine biosynthetic pathway. The engineered S. cerevisiae strain that produced verazine contains eight heterologous enzymes sourced from seven different species. Importantly, S. cerevisiae-produced verazine was indistinguishable via liquid chromatography-mass spectrometry from both a commercial standard (Veratrum spp. plant-produced) and Nicotiana benthamiana-produced verazine. To the best of our knowledge, this is the first report describing the heterologous production of a steroidal alkaloid in an engineered yeast. Verazine production was ultimately increased through design-build-test-learn cycles to a final titer of 83 ± 3 μg/L (4.1 ± 0.1 μg/g DCW). Together, this research lays the groundwork for future microbial biosynthesis of cyclopamine, (bio)synthetic derivatives of cyclopamine, and other steroidal alkaloid natural products.Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.