Two‐Step Adaptive Laboratory Evolution Enhances Osmotolerance in Engineered <i>Escherichia coli</i> for Improved Succinate Biosynthesis

Succinic acid (SA) is a promising platform chemical with broad applications in agricultural, food, and pharmaceutical industries. Microbial production of SA using Escherichia coli typically requires alkaline neutralizers to maintain pH during fermentation, leading to elevated osmotic pressure that severely inhibits SA production. The strain ZZT215, evolved from AFP111 using two-step adaptive laboratory evolution (ALE) strategy, exhibited the improved Na⁺ tolerance and SA productivity without further genetic modification. In 5 L bioreactor fermentation, ZZT215 accumulated 87.02 g/L of SA with a productivity at 1.01 g/(L·h), representing 24.9% and 21.7% increases compared to the parent strain AFP111, respectively. Transcriptomic analysis revealed downregulated TCA cycle genes and upregulated ABC transporters, indicating metabolic adaptation to osmotic stress. These findings highlight the potential of multiple-step ALE for engineering robust microbial cell factories for SA and other high-value chemicals.