Previously, we have developed an l-threonine-producing Escherichia coli strain TWF006 in which the regulator-encoding gene iclR was deleted. In this study, further modifications were performed on TWF006 to increase l-threonine yield. Firstly, the regulator-encoding gene fadR was deleted in TWF006, and the resulting strain TWF031 produced 18.86 g l-threonine from 30 g glucose after 24-h cultivation. Secondly, the regulator-encoding genes fabR and lacI in TWF031 were deleted, and the resulting strain TWF033 produced 19.21 g l-threonine from 30 g glucose after 24-h cultivation. Thirdly, additional copies of aceBA and fadBA were inserted into the lacZ locus of TWF033 and the native promoter of acs was replaced by the Ptac-trc; the resulting strain TWF038 produced 20.3 g l-threonine from 30 g glucose after 24-h cultivation. Finally, the genes ppnK, thrA*BC-rhtC, aspC, and ppc were inserted into the chromosome of TWF038; the resulting strain TWF044 produced 21.64 g l-threonine from 30 g glucose, or 28.49 g l-threonine from 40 g glucose after 24-h cultivation. After 48-h fed-batch fermentation, TWF044 produced 103.89 g/l l-threonine. The results suggest that coupling the fatty acid degradation and l-threonine biosynthesis pathway via the glyoxylate shunt could efficiently increase l-threonine production in E. coli.