Improving CsOAC Activity in Saccharomyces cerevisiae for Directed Production of Olivetolic Acid through Rational...

Area of Science:

• Biotechnology
• Synthetic Biology
• Enzyme Engineering

Background:

• Olivetolic acid (OA) is a key precursor in cannabinoid biosynthesis.
• Current methods in Saccharomyces cerevisiae produce olivetol (OL) as a significant byproduct during OA production.
• Optimizing OA production requires enhancing the activity and specificity of olivetolic acid cyclase (CsOAC).

Purpose of the Study:

• To engineer olivetolic acid cyclase (CsOAC) for increased olivetolic acid (OA) production in Saccharomyces cerevisiae.
• To reduce the formation of olivetol (OL) as a side product during OA biosynthesis.
• To identify and characterize improved CsOAC variants through rational design and in vivo screening.

Main Methods:

• In silico protein-ligand docking to identify potential CsOAC variants.
• Rational design of CsOAC based on computational predictions.
• In vivo screening of engineered CsOAC variants in Saccharomyces cerevisiae.
• Evaluation of OA and OL production levels for different CsOAC variants.

Main Results:

• Four engineered CsOAC variants demonstrated improved properties for OA production.
• The best variant, G82A/L92Y, achieved a 1.7-fold increase in OA production.
• A significant shift in product ratio favoring OA over OL was observed with the optimized CsOAC variant.

Conclusions:

• Rational design and in vivo screening are effective strategies for engineering enzyme specificity.
• The engineered CsOAC variant G82A/L92Y significantly enhances OA production in yeast.
• This work provides a foundation for improving cannabinoid precursor biosynthesis in microbial systems.