Improved Biosynthesis of Ethylene Glycol From Xylose in Engineered E. coli Utilizing Two‐Stage Dynamic Control

ABSTRACT We engineered Escherichia coli for improved ethylene glycol (EG) biosynthesis using two‐stage dynamic metabolic control coupled with pathway optimization. An engineered Dahms pathway ( xylB, xylC, xylD, yqhD ) was induced upon phosphate depletion to enable EG biosynthesis during stationary phase. The performance of our initial strain, expressing a baseline pathway, was improved by the addition of “stoichiometric” valves enabling proteolytic degradation of xylose isomerase and soluble transhydrogenase, upon phosphate depletion. Reduction of these enzymes, which compete for substrates with EG biosynthesis, led to a 3‐fold increase in specific productivity. In contrast, enabling “regulatory” dynamic metabolic valves targeting enoyl‐ACP reductase and glucose‐6‐phosphate dehydrogenase, designed to enhance NADPH flux, did not improve production in this control. Following pathway optimization, regulatory strains exhibited enhanced production rates exceeding 0.5 g gCDW − 1 h − 1 , a 14‐fold improvement over the control, while stoichiometric valves conferred no benefit. Scale‐up in fed‐batch bioreactors achieved 140 g L − 1 EG in 70 h with a yield of 0.38 g g − 1 xylose (92% of theoretical), representing one of the highest reported titers in E. coli . These results demonstrate that optimal engineering strategies shift with pathway capacity. While elimination of competing fluxes benefits low‐flux systems, high‐flux pathways require enhanced cofactor regeneration.