Enhancing Ergothioneine Production by Combined Protein and Metabolic Engineering Strategies

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Ergothioneine (ERG) is a stable, valuable antioxidant with applications in food, cosmetics, and medicine.
• Microbial production offers a sustainable and efficient alternative to chemical synthesis for ERG.
• Developing microbial cell factories is key for scalable and eco-friendly ERG supply.

Purpose of the Study:

• To establish de novo synthesis of ergothioneine in Escherichia coli.
• To enhance ERG production through protein engineering and strain modification.
• To optimize fermentation conditions for maximizing ERG yield.

Main Methods:

• Introducing Trichoderma reesei genes into E. coli for ERG biosynthesis.
• Employing protein engineering to improve soluble expression of key genes (Tr1, Tr2).
• Implementing strain modifications: knockout of competing pathways and gene copy optimization.
• Optimizing medium composition and fermentation strategies (Erlenmeyer flask and fed-batch).

Main Results:

• Achieved de novo ERG synthesis in engineered E. coli.
• Protein engineering increased ERG production by 198.1%.
• Engineered strain E25 produced 430.9 mg/L ERG in flasks and 2331.58 mg/L in a 5 L bioreactor via fed-batch fermentation.

Conclusions:

• Established a robust platform for efficient and sustainable ergothioneine production using engineered E. coli.
• Demonstrated significant yield improvements through a combination of genetic and process engineering.
• Provided valuable insights for the industrial-scale production of ergothioneine and its derivatives.