?-caryophyllene is a common sesquiterpene compound currently being studied as a promising precursor for the production of high-density fuels. Acute demand for high-density fuels has provided the impetus to pursue biosynthetic methods to produce ?-caryophyllene from reproducible sources. In this study, we produced ?-caryophyllene by assembling a biosynthetic pathway in an engineered Escherichia coli strain of which phosphoglucose isomerase gene has been deleted. The 1- deoxy-d-xylulose 5-phosphate (DXP) or heterologous mevalonate (MVA) pathways were employed. Meanwhile, geranyl diphosphate synthase, glucose-6-phosphate dehydrogenase and ?-caryophyllene synthase genes were co-overexpressed in the above strain. The final genetically modified strain, YJM59, produced 220 ? 6 mg/L of ?-caryophyllene in flask culture. We also evaluated the use of fed-batch fermentation for the production of ?-caryophyllene. After induction for 60 h, the YJM59 strain produced ?-caryophyllene at a concentration of 1520 mg/L. The volumetric production fermented in the aerobic fed-batch was 0.34 mg/(L?h?OD600) and the conversion efficiency of glucose to ?-caryophyllene (gram to gram) was 1.69%. Our results are the first successful attempt to produce ?-caryophyllene using E. coli BL21(DE3), and provide a new strategy that is green and sustainable for the production of ?-caryophyllene.