Itaconic acid is an excellent polymeric precursor with wide range of industrial applications. Here, efficient production of itaconate from various renewable substrates was demonstrated by engineered Escherichia coli. Limitation in the itaconate precursor supply was revealed by feeding of the key intermediate citrate to the culture medium. Efforts of enhancing the cis-aconitate flux and preserving the isocitrate pool increased itaconate productivity by nearly 100-fold. Elimination of the isocitrate lyase lowered the itaconate production by 10-30%, suggesting the potential positive role of glyoxylate shunt. High aeration was essential for efficient synthesis of itaconate due to its inability to serve as a fermentation product. Using the best strain, we achieved by far the highest itaconate titer from xylose and glycerol individually, reaching 20-22g/L in 72h with an average yield of 0.5g/g using bench-scale flasks. Compare to the use of phosphoenolpyruvate (PEP) carboxylase, overexpression of pyruvate carboxylase consistently led to higher production of itaconate from substrates such as glucose and glycerol whose dissimilation involves PEP-dependent phosphorylation. With high density fermentation in the fed-batch bioreactor, the titer of itaconate was further pushed to 43g/L in 32h with a final yield around 0.6g/g of glycerol.