Engineering Yarrowia lipolytica for Campesterol Overproduction.

Area of Science:

• Biotechnology
• Synthetic Biology
• Microbial Engineering

Background:

• Campesterol is a vital precursor for synthesizing sterol-based pharmaceuticals like progesterone and hydrocortisone.
• Developing microbial production platforms for sterols is crucial for sustainable drug manufacturing.

Purpose of the Study:

• To engineer the yeast Yarrowia lipolytica for efficient microbial production of campesterol.
• To optimize fermentation conditions and identify the best carbon source for maximizing campesterol yield.

Main Methods:

• Disruption of the ERG5 gene and constitutive expression of heterologous 7-dehydrocholesterol reductase (DHCR7) in Yarrowia lipolytica.
• Screening of codon-optimized DHCR7 genes from different species, with Xenopus laevis DHCR7 showing superior performance.
• Optimization of fermentation strategies, including high cell density fed-batch fermentation with carbon source restriction.

Main Results:

• The Yarrowia lipolytica strain expressing Xenopus laevis DHCR7 achieved the highest campesterol titer, attributed to a specific amino acid substitution (D409) in the enzyme's substrate-binding site.
• Sunflower seed oil supported a higher cell growth rate compared to glucose or glycerol.
• A record microbial campesterol titer of 453±24.7 mg/L was achieved using sunflower seed oil as the carbon source under optimized fed-batch fermentation.

Conclusions:

• Engineering Yarrowia lipolytica with specific genetic modifications and optimized fermentation conditions can lead to high-level microbial production of campesterol.
• This work provides valuable insights into the microbial biosynthesis of complex sterols and enhances the potential for producing valuable molecules in yeast.