A Gram-Scale Limonene Production Process with Engineered Escherichia coli

Area of Science:

• Biotechnology
• Metabolic Engineering
• Industrial Microbiology

Background:

• Monoterpenes like limonene are valuable natural products with diverse applications.
• Traditional plant extraction faces limitations; microbial production offers a sustainable alternative.
• High titers and space-time yields are crucial for economically viable bioprocesses.

Purpose of the Study:

• To develop a stable, high-titer limonene production process using metabolically engineered Escherichia coli (E. coli).
• To evaluate glycerol as a sole carbon source for E. coli growth and limonene synthesis.
• To achieve industrially relevant yields for limonene bioproduction.

Main Methods:

• Metabolic engineering of E. coli BL21 (DE3) with optimized mevalonate pathway and limonene synthase genes on a single plasmid.
• Fed-batch fermentation in minimal medium with a non-toxic organic phase at the bioreactor scale.
• Optimization of fermentation conditions using glycerol as the sole carbon source.

Main Results:

• Achieved limonene titers of up to 7.3 g·L_org^-1 (3.6 g·L^-1 in aqueous phase).
• Obtained industrially viable space-time yields of 0.15 g·L^-1·h^-1.
• Demonstrated the highest monoterpene concentrations reported to date using a microbial system.

Conclusions:

• The developed E. coli bioprocess is stable and achieves unprecedented limonene titers and yields.
• Glycerol is a feasible carbon source for efficient limonene production in E. coli.
• This study provides a foundation for an economically viable and industrially relevant limonene bioprocess.