Two-Step Adaptive Laboratory Evolution Enhances Osmotolerance in Engineered Escherichia coli for Improved Succinate...

Area of Science:

• Biotechnology
• Metabolic Engineering
• Industrial Microbiology

Background:

• Succinic acid (SA) is a key platform chemical with diverse industrial applications.
• Escherichia coli fermentation for SA is often limited by osmotic stress from alkaline neutralizers.
• Developing osmotically tolerant strains is crucial for efficient microbial SA production.

Purpose of the Study:

• To enhance succinic acid (SA) production in Escherichia coli by improving tolerance to osmotic stress.
• To investigate the metabolic adaptations of an evolved strain for increased SA productivity.
• To demonstrate the efficacy of adaptive laboratory evolution (ALE) for engineering robust microbial cell factories.

Main Methods:

• Employing a two-step adaptive laboratory evolution (ALE) strategy to evolve Escherichia coli strain AFP111.
• Conducting bioreactor fermentation experiments to compare SA production between the evolved strain (ZZT215) and the parent strain (AFP111).
• Utilizing transcriptomic analysis to identify key metabolic changes associated with improved osmotic tolerance.

Main Results:

• The evolved strain ZZT215 exhibited significantly improved Na+ tolerance and SA productivity compared to the parent strain AFP111.
• ZZT215 achieved a peak SA concentration of 87.02 g/L with a productivity of 1.01 g/(L·h) in a 5 L bioreactor.
• Transcriptomic data revealed downregulated TCA cycle genes and upregulated ABC transporters in ZZT215, indicating adaptation to osmotic stress.

Conclusions:

• Multi-step ALE is an effective strategy for developing osmotically robust microbial strains for enhanced succinic acid production.
• The evolved strain ZZT215 represents a promising candidate for industrial-scale microbial synthesis of SA.
• The identified metabolic adaptations provide insights into engineering microbial cell factories for high-value chemical production under stress conditions.