Engineering Escherichia coli for Glutarate Production as the C₅ Platform Backbone

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Industrial Biotechnology

Background:

• Glutarate, a C5 dicarboxylic acid, is crucial for producing polyesters and polyamides like nylon-4,5 and nylon-5,5.
• Current glutarate production relies on petroleum-derived feedstocks via chemical synthesis, leading to significant pollution and greenhouse gas emissions.
• Previous biological glutarate production methods, often from lysine, suffered from low yields, titers, and extended fermentation times.

Purpose of the Study:

• To develop a more efficient and sustainable bio-based method for glutarate production in Escherichia coli.
• To engineer a novel synthetic pathway for glutarate synthesis, overcoming limitations of previous biological approaches.
• To achieve high glutarate titers in E. coli, making bio-based production economically feasible.

Main Methods:

• A five-step reverse adipate degradation pathway (RADP) from Thermobifida fusca was introduced into Escherichia coli.
• Strain optimization involved fermentation parameter adjustments and genetic modifications using CRISPR/Cas9 to eliminate competing metabolic pathways (ΔarcA, ΔldhA, ΔatoB, ΔpflB).
• Glutarate production was evaluated in shaken flasks and optimized via fed-batch fermentation.

Main Results:

• The engineered E. coli strain (Bgl146) initially produced detectable glutarate in shaken flasks.
• Fermentation optimization increased glutarate titer to 4.7 ± 0.2 mM in shaken flasks.
• The final optimized strain (Bgl4146), with deleted competing pathways, achieved a record titer of 36.5 ± 0.3 mM glutarate via fed-batch fermentation.

Conclusions:

• The study successfully established a novel, efficient bio-production route for glutarate in E. coli.
• The achieved glutarate titer represents the highest reported for E. coli, demonstrating the potential of the engineered RADP.
• This work provides a feasible and sustainable alternative to chemical glutarate synthesis, reducing environmental impact and reliance on fossil fuels.