α-Ketoglutarate is accumulated as the main byproduct during the aerobic succinate production from glycerol by Escherichia coli BL21(DE3) in minimal medium. To address this issue, here a strategy of directed pathway evolution was developed to enhance the alternative succinate production route-the glyoxylate shunt. Via the directed pathway evolution, the glyoxylate shunt was recruited as the primary anaplerotic pathway in a ppc mutant, which restored its viability in glycerol minimal medium. Subsequently, the operon sdhCDAB was deleted and the gene ppc was reverted in the evolved strain for succinate production. The resulting strain E2-Δsdh-ppc produced 30 % more succinate and 46 % less α-ketoglutarate than the control strain. A G583T mutation in gene icdA, which significantly decreased the activity of isocitrate dehydrogenase, was identified in the evolved strain as the main mutation responsible for the observed phenotype. Overexpression of α-ketoglutarate dehydrogenase complex in E2-Δsdh-ppc further reduced the amount of byproduct and improved succinate production. The final strain E2-Δsdh-ppc-sucAB produced 366 mM succinate from 1.3 M glycerol in minimal medium in fed-batch fermentation. The maximum and average succinate volumetric productivities were 19.2 and 6.55 mM h(-1), respectively, exhibiting potential industrial production capacity from the low-priced substrate.