Metabolic engineering was formally defined more than two decades ago (Bailey, 1991) and it is now an established discipline. Metabolic engineering is generally defined as the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or the introduction of new ones with the use of recombinant DNA technology (Bailey, 1991; Stephanopoulos, 1998). Therefore, the analysis and engineering/synthesis of metabolic pathways is of central importance to metabolic engineering. The analytical part uses a number of experimental and modeling techniques for the systematic study of cellular responses (in terms of RNA, protein and metabolite levels, metabolic fluxes, etc.) to genetic and environmental perturbations. This facilitates a rational design of metabolic modifications, which are implemented using recombinant DNA technology. Both, the analysis and the synthesis of metabolic pathways will be covered in this review. Recent efforts on the engineering of fermentative and biosynthetic pathways for biofuel production in Escherichia coli, as well as those enabling the utilization of novel feedstocks, will be highlighted.