Conversion of biomass feedstock to chemicals and fuels has attracted increasing attention recently. Soybean meal, containing significant quantities of carbohydrates, is an inexpensive renewable feedstock. Glucose, galactose, and fructose can be obtained by enzymatic hydrolysis of soluble carbohydrates of soybean meal. Free fatty acids (FFAs) are valuable molecules that can be used as precursors for the production of fuels and other value-added chemicals. In this study, free fatty acids were produced by mutant Escherichia coli strains with plasmid pXZ18Z (carrying acyl-ACP thioesterase (TE) and (3R)-hydroxyacyl-ACP dehydratase) using individual sugars, sugar mixtures, and enzymatic hydrolyzed soybean meal extract. For individual sugar fermentations, strain ML211 (MG1655 fadD(-)fabR(-))/pXZ18Z showed the best performance, which produced 4.22, 3.79, 3.49g/L free fatty acids on glucose, fructose, and galactose, respectively. While the strain ML211/pXZ18Z performed the best with individual sugars, however, for sugar mixture fermentation, the triple mutant strain XZK211 (MG1655 fadD(-)fabR(-)ptsG(-))/pXZ18Z with an additional deletion of ptsG encoding the glucose-specific transporter, functioned the best due to relieved catabolite repression. This strain produced approximately 3.18g/L of fatty acids with a yield of 0.22g fatty acids/g total sugar. Maximum free fatty acids production of 2.78g/L with a high yield of 0.21g/g was achieved using soybean meal extract hydrolysate. The results suggested that soybean meal carbohydrates after enzymatic treatment could serve as an inexpensive feedstock for the efficient production of free fatty acids. Biotechnol. Bioeng. 2015;112: ???-???. (c) 2015 Wiley Periodicals, Inc.