Monoterpene biosynthesis pathway construction in Escherichia coli

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Microbial Biotechnology

Background:

• (-)-Carvone is a valuable spearmint monoterpene ketone with applications in flavor and fragrance industries.
• Microbial production offers a sustainable alternative to plant extraction.
• Efficient biosynthesis pathways in microbial hosts remain a challenge.

Purpose of the Study:

• To engineer Escherichia coli for the biosynthesis of (-)-carvone.
• To investigate the limiting factors in the microbial production of carvone and its intermediates.
• To assess the functionality of downstream enzymes in the carvone pathway.

Main Methods:

• Constructed a synthetic pathway in E. coli by introducing four genes for (-)-carvone biosynthesis.
• Utilized inducible gene overexpression for pathway component production.
• Analyzed pathway intermediates and enzyme activity.
• Performed feeding studies with (-)-limonene to evaluate downstream conversion.

Main Results:

• Achieved production of ~5 mg/l of (-)-limonene, which was excreted into the medium.
• Downstream products (-)-carveol and (-)-carvone were not detected under initial overexpression conditions.
• Enzyme assays revealed limited flux due to C(5) isoprenoid precursor availability.
• Feeding studies confirmed enzyme functionality but showed compromised efficiency due to uptake/trafficking issues.

Conclusions:

• The engineered E. coli can produce (-)-limonene, but precursor supply is a major bottleneck.
• Downstream conversion to (-)-carvone is hindered by both precursor limitations and cellular transport restrictions.
• Further optimization requires addressing isoprenoid precursor pool enhancement and improving substrate uptake/trafficking mechanisms.