The strategic design of this study aimed at producing succinate and polyhydroxyalkanoate (PHA) from substrate mixture of glycerol/glucose and fatty acid in Escherichia coil. To accomplish this, an E. coli KNSP1 strain derived from E. coil LR1110 was constructed by deletions of ptsG, sdhA and pta genes and overexpression of phaC1 from Pseudomonas aeruginosa. Cultivation of E. coil KNSP1 showed that this strain was able to produce 21.07 g/L succinate and 0.54 g/L PHA (5.62 wt.% of cell dry weight) from glycerol and fatty acid mixture. The generated PHA composed of 58.7 mol% 3-hydroxyoctanoate (3HO) and 41.3 mol% 3-hydroxydecanoate (3HD). This strain would be useful for complete utilization of byproducts glycerol and fatty acid of biodiesel production process. (C) 2011 Elsevier Ltd. All rights reserved.