Pathway Refactoring for Efficient 7-Dehydrocholesterol Production in Saccharomyces cerevisiae

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• 7-Dehydrocholesterol (7-DHC) is a crucial sterol and the primary precursor to vitamin D3.
• Efficient and sustainable production of 7-DHC is vital for various applications.
• Current methods for 7-DHC production may face limitations in scalability and sustainability.

Purpose of the Study:

• To construct a Saccharomyces cerevisiae strain capable of de novo biosynthesis of 7-dehydrocholesterol.
• To optimize the engineered yeast strain for enhanced 7-DHC production through metabolic engineering strategies.
• To achieve high-titer, scalable, and sustainable production of 7-DHC.

Main Methods:

• Construction of a de novo 7-DHC biosynthetic pathway in Saccharomyces cerevisiae.
• Introduction of heterologous DHCR24 and overexpression of key enzymes.
• Optimization of ergosterol pathway, use of organic solvents, surfactants, and ε-polylysine.
• Subcellular localization of the pathway in peroxisomes and redox level rebalancing.
• Scale-up fermentation in a 5 L bioreactor.

Main Results:

• Initial 7-DHC titer of 109.0 mg L⁻¹ achieved through pathway construction and enzyme modification.
• An 86.3% increase in 7-DHC titer by dynamic regulation of the ergosterol pathway and DHCR24 multicopy expression.
• A 99.1% increase in 7-DHC titer with the addition of ε-polylysine.
• Final 7-DHC titer of 517.4 mg L⁻¹ in shake flasks after pathway refactoring.
• Successful scale-up to 3.26 g L⁻¹ in a 5 L bioreactor.

Conclusions:

• The engineered Saccharomyces cerevisiae strain efficiently produces 7-dehydrocholesterol via a de novo biosynthetic pathway.
• Metabolic engineering strategies, including pathway optimization and cofactor balancing, significantly enhance 7-DHC yield.
• The developed pathway refactoring strategy offers a sustainable and scalable method for 7-DHC production.