Synergistic co‐utilization of biomass‐derived sugars enhances aromatic amino acid production by engineered Escherichia coli.

Efficient co‐utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose−xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co‐utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose‐to‐glucose flux ratio. A mutant copy of the xylose‐specific activator (XylR) was then introduced into the phenylalanine‐overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose−xylose co‐utilization. Carbon contribution analysis through 13C‐fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose−xylose mixture; a threefold increase over NST74. Then, using biomass‐derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9‐fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L‐h). This study presents a novel strategy for enhancing phenylalanine production through the co‐utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products. [ABSTRACT FROM AUTHOR]