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.

中文翻译：

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从工程酿酒酵母中的单糖合成维拉津


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（例如环巴胺）;然而，许多已知的 ≥697 甾体生物碱天然产物仍未作为药物得到充分利用，因为在其生产生物体中扩大它们的生物合成规模可能具有挑战性。环巴胺是玉米百合 （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，环巴胺是治疗与 Hh 信号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。环帕胺及其半合成衍生物已在（前）临床试验中作为基于 Hh 抑制剂的药物进行研究。然而，扩大环帕胺生产规模的挑战减缓了通过（生物）合成衍生化提高其疗效和安全性的努力，通常将药物开发限制在环帕胺的合成类似物上，例如 FDA 批准的药物 Odomzo、Daurismo 和 Erivedge。如果建立了一个可扩展和可持续生产环巴胺的平台，那么其（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。为实现这一目标而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帕胺和环帕胺的半/全化学合成。在此，这项工作朝着一种有前途的未来方法迈进：在工程微生物中生物合成环帕胺。我们在工程酿酒酵母 （S. cerevisiae），通过天然酵母甲羟戊酸酯和羊毛甾醇生物合成途径的诱导上调，将生物合成通量从麦角甾醇（即酿酒酵母中的天然甾醇）转移到胆固醇（即维拉嗪的生物合成前体），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包含来自 7 个不同物种的 8 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（植物生产的藜芦属）和本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中甾体生物碱异源产生的报告。Verazine 的产量最终通过设计-构建-测试-学习循环增加，最终滴度为 83 ± 3 μg/L （4.1 ± 0.1 μg/g DCW）。总之，这项研究为未来环巴胺的微生物生物合成、环巴胺的（生物）合成衍生物和其他甾体生物碱天然产物奠定了基础。