Engineering Escherichia coli for robust Co-utilization of glucose and xylose enables high-titer succinate production from lignocellulosic hydrolysates

Succinate is a biobased platform chemical with wide applications in food, pharmaceuticals, and biodegradable polymers such as polybutylene succinate. Despite advances in microbial fermentation, cost-effective production remains limited by inefficient utilization of lignocellulosic hydrolysates, where glucose and xylose are the predominant sugars. In this study, we systematically engineered <i>Escherichia coli</i> C600 to enhance succinate biosynthesis from mixed sugars and hydrolysates. Competing by-product pathways were eliminated, the phosphotransferase system was modified to relieve carbon catabolite repression, and the <i>pck</i> gene from <i>Bacillus subtilis</i> was introduced to alleviate the ATP burden in xylose metabolism. To further improve xylose utilization, heterologous oxidative pathways (Weimberg and Dahms) from <i>Caulobacter crescentus</i> were integrated and fine-tuned using ribosome binding site libraries. The optimized strain exhibited flexible glucose-xylose co-utilization across varying sugar ratios, maintaining high succinate yields. A global transcriptional regulator library was then applied, and a <i>crp</i> mutant ESC6_crp-W68⁺ was identified and enabled efficient growth and succinate production using inorganic nitrogen as the sole nitrogen source. Scale-up fermentation in a 5-L bioreactor confirmed the industrial relevance of the engineered strain: ESC6_crp-W68⁺ produced 87.7 g/L succinate from synthetic mixed sugars with a yield of 1.15 mol/mol, and 77.3 g/L from corn stover hydrolysate with a yield of 1.02 mol/mol. This multi-layered engineering framework established a metabolically robust and cost-efficient <i>E</i>. <i>coli</i> platform, enabling high-titer succinate production directly from lignocellulosic hydrolysates.