Pathway remodeling and adaptive evolution enable efficient co-utilization of glucose and xylose in Escherichia coli

Efficient co-utilization of glucose and xylose is critical for microbial bioconversion of lignocellulosic hydrolysates. However, carbon catabolite repression prevents simultaneous sugar consumption in conventional industrial strains such as Escherichia coli . Here, we blocked the Embden−Meyerhof−Parnas and pentose phosphate pathways in E. coli MG1655 by deleting pgi and gnd , generating strain E. coli MD0 that metabolizes glucose exclusively via the Entner−Doudoroff pathway. Adaptive laboratory evolution yielded mutant E. coli MDE with superior glucose-xylose co-utilization, outperforming E. coli MG1655Δ ptsG . Genomic analysis identified gntR and xylR mutations as key contributors to this phenotype. Similar engineering in Klebsiella oxytoca also enhanced glucose and xylose co-utilization. Seven byproduct genes were deleted in E. coli MDE, and efficient production of pyruvate from straw hydrolysate was achieved by using the constructed strain E. coli MDE-6. Further introducing the budRABC operon in E. coli MDE-6 resulted in 2,3-butanediol generation from straw hydrolysate. This study establishes E. coli MDE as a robust chassis for lignocellulose biorefinery. IMPORTANCE The inability of industrial microbes to co-utilize glucose and xylose severely limits lignocellulosic biomass valorization in bioproduction. Here, we combined pathway separation and adaptive laboratory evolution to develop an Escherichia coli mutant MDE capable of simultaneous glucose and xylose utilization. Strain E. coli MDE was engineered to efficiently produce pyruvate and 2,3-butanediol from straw hydrolysate and showed robust sugar co-utilization at various ratios. We identified two key beneficial mutations enabling this phenotype. When introduced into Klebsiella oxytoca alongside the pathway separation strategy, these mutations also conferred efficient glucose-xylose co-utilization. This study provides a generalizable strategy for engineering simultaneous sugar utilization in diverse microbial chassis.