Verazine biosynthesis from simple sugars in engineered Saccharomyces cerevisiae

Area of Science:

• Biotechnology
• Metabolic Engineering
• Natural Product Biosynthesis

Background:

• Steroidal alkaloids are valuable pharmaceuticals, but their natural production is difficult to scale.
• Cyclopamine, a steroidal alkaloid, inhibits the Hedgehog signaling pathway and is a promising cancer drug candidate.
• Current limitations in cyclopamine production hinder its therapeutic development.

Purpose of the Study:

• To establish a scalable and sustainable method for producing steroidal alkaloids.
• To engineer microorganisms for the biosynthesis of cyclopamine precursors.
• To lay the groundwork for microbial production of cyclopamine and its derivatives.

Main Methods:

• Engineered Saccharomyces cerevisiae (yeast) for heterologous production of verazine.
• Upregulated native yeast pathways (mevalonate, lanosterol) and diverted flux from ergosterol to cholesterol.
• Expressed a refactored five-step verazine biosynthetic pathway using eight heterologous enzymes from seven species.

Main Results:

• Achieved microbial production of verazine from simple sugars (glucose, galactose).
• Confirmed verazine identity and purity using liquid chromatography-mass spectrometry, matching plant-produced standards.
• Reached a final verazine titer of 83 ± 3 μg/L (4.1 ± 0.1 μg/g DCW) after optimization.

Conclusions:

• Demonstrated the first heterologous production of a steroidal alkaloid in engineered yeast.
• Established a foundational platform for microbial biosynthesis of cyclopamine and related compounds.
• Paved the way for scalable, sustainable production of steroidal alkaloid natural products for pharmaceutical applications.