Targeted proteomics for metabolic pathway optimization: application to terpene production.

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Proteomics

Background:

• Metabolic engineering requires robust methods for pathway assembly and optimization.
• Identifying production bottlenecks beyond simple product titers is crucial for microbial strain improvement.
• Analytical techniques are essential for pinpointing limiting factors in engineered biological systems.

Purpose of the Study:

• To apply targeted proteomics for identifying rate-limiting steps in microbial production of amorpha-4,11-diene.
• To investigate the impact of specific protein levels on sesquiterpene biosynthesis.
• To improve amorpha-4,11-diene production in Escherichia coli.

Main Methods:

• Utilized selected-reaction monitoring (SRM) mass spectrometry for targeted proteomics.
• Quantified protein levels of mevalonate pathway enzymes in engineered Escherichia coli.
• Implemented gene codon optimization and stronger promoter expression for bottleneck enzymes.

Main Results:

• Identified mevalonate kinase (MK) and phosphomevalonate kinase (PMK) as potential bottlenecks in amorpha-4,11-diene production.
• Demonstrated significant increases in MK and PMK protein abundance after genetic modifications.
• Achieved over a three-fold increase in amorpha-4,11-diene titer, exceeding 500 mg/L.

Conclusions:

• Targeted proteomics is effective for identifying metabolic pathway bottlenecks.
• Optimizing expression of identified bottleneck proteins (MK and PMK) substantially enhances sesquiterpene production.
• This study provides a refined strategy for improving microbial biosynthesis of valuable compounds.