Ethylene glycol (EG) is an important chemical used for several industrial applications including poly(ethylene terephthalate) synthesis. In this study, Escherichia coli was metabolically engineered to efficiently produce EG from xylose. To biosynthesize EG, the Dahms pathway was introduced by expressing xylBC genes from Caulobacter crescentus (xylBC(ccs)). Various E. coli strains and glycolaldehyde reductases were screened to find E. coli W3110 strain and glycolaldehyde reductase (yqhD) as optimal combination for EG production. In silico genome-scale metabolic simulation suggested that increasing the native xylose pathway flux, in the presence of the overexpressed Dahms pathway, is beneficial for EG production. This was achieved by reducing the Dahms pathway flux by employing a synthetic small regulatory RNA targeting xylB(ccs). Fed-batch culture of the final engineered E. coli strain produced 108.2 g/L of EG in a xylose minimal medium. The yield on xylose and EG productivity were 0.36 g/g (0.87 mol/mol) and 2.25 g/L/h, respectively. (c) 2018 American Institute of Chemical Engineers