Reprogramming Escherichia coli metabolism for butyrolactam production by the temperature-powered multi-genes control circuit

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Butyrolactam is a crucial platform chemical for nylon production.
• Current butyrolactam biosynthesis methods face challenges with static control, limiting efficiency.
• Limited research exists on optimizing butyrolactam production pathways.

Purpose of the Study:

• To engineer Escherichia coli for enhanced butyrolactam synthesis.
• To overcome limitations in static control for improved biomanufacturing efficiency.
• To develop a novel bioswitch for precise gene regulation in microbial systems.

Main Methods:

• Systematic engineering of Escherichia coli chassis.
• CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) for gene regulation.
• Development of a temperature-powered bioswitch (AITB circuit) for simultaneous gene up- and down-regulation.
• Fed-batch culture and optimization of α-ketoglutarate metabolic pathways.

Main Results:

• Identified and addressed the inadequate α-ketoglutarate pool as a key bottleneck.
• Achieved a highest titer of 59.87 g/L butyrolactam in fed-batch culture.
• Demonstrated production of 14.96 g/L butyrolactam using cassava starch as a renewable feedstock.

Conclusions:

• The engineered Escherichia coli strain shows high efficiency in butyrolactam production.
• The developed AITB circuit provides an effective tool for dynamic gene control in biomanufacturing.
• This study paves the way for sustainable production of bio-based chemicals from renewable resources.