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 Escherichia coli 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 pck gene from Bacillus subtilis was introduced to alleviate the ATP burden in xylose metabolism. To further improve xylose utilization, heterologous oxidative pathways (Weimberg and Dahms) from Caulobacter crescentus 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 crp 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 E . coli platform, enabling high-titer succinate production directly from lignocellulosic hydrolysates.