Search

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.)

The isonitrile moiety is an electron-rich functionality that decorates various bioactive natural products isolated from diverse kingdoms of life. Isonitrile biosynthesis was restricted for over a decade to isonitrile synthases, a family of enzymes catalyzing a condensation reaction between l-Trp/l-Tyr and ribulose-5-phosphate. The discovery of ScoE, a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase, demonstrated an alternative pathway employed by nature for isonitrile installation. Biochemical, crystallographic, and computational investigations of ScoE have previously been reported, yet the isonitrile formation mechanism remains obscure. In the present work, we employed in vitro biochemistry, chemical synthesis, spectroscopy techniques, and computational simulations that enabled us to propose a plausible molecular mechanism for isonitrile formation. Our findings demonstrate that the ScoE reaction initiates with C5 hydroxylation of ( R )-3-((carboxymethyl)amino)butanoic acid to generate 1 , which undergoes dehydration, presumably mediated by Tyr96 to synthesize 2 in a trans configuration. ( R )-3-isocyanobutanoic acid is finally generated through radical-based decarboxylation of 2 , instead of the common hydroxylation pathway employed by this enzyme superfamily.