Systematic engineering to enhance 3-carene production in yeast

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• 3-Carene, a bicyclic monoterpene with pine aroma, has applications as a flavoring agent, food additive, and insecticide.
• Current microbial synthesis of 3-carene is limited by low 3-carene synthase (CarTS) activity and insufficient precursor supply in Saccharomyces cerevisiae.

Purpose of the Study:

• To establish an efficient microbial cell factory for 3-carene production.
• To overcome limitations in 3-carene synthesis by enhancing precursor supply and enzyme activity.

Main Methods:

• Engineered Saccharomyces cerevisiae using an orthogonal pathway (NPP pathway) and a truncated 3-carene synthase (SsCarTS).
• Optimized enzyme performance through subcellular localization and molecular chaperone screening.
• Enhanced 3-carene production via medium optimization, including metal ions and vitamins, in shake flask and bioreactor cultures.

Main Results:

• Introduced the NPP pathway and SsCarTS, increasing 3-carene yield by 32.1-fold.
• Further improved yield by 39.0% to 50.6 mg/L in shake flasks using enzyme optimization.
• Achieved a record titer of 506.7 mg/L in a 5-L bioreactor through medium optimization.

Conclusions:

• Successfully developed a microbial cell factory for efficient 3-carene biosynthesis.
• The optimized pathway and conditions represent a significant advancement in producing bicyclic monoterpenes.
• Provides a valuable platform for the biosynthesis of other bicyclic monoterpenes.