Microbial fatty acid-derived fuels have emerged as promising alternatives to petroleum-based transportation fuels. Here we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titre improvements in a multi-gene fatty acid metabolic pathway. On the basis of central pathway architecture, E. coli fatty acid biosynthesis was re-cast into three modules: the upstream acetyl coenzyme A formation module; the intermediary acetyl-CoA activation module; and the downstream fatty acid synthase module. Combinatorial optimization of transcriptional levels of these three modules led to the identification of conditions that balance the supply of acetyl-CoA and consumption of malonyl-CoA/ACP. Refining protein translation efficiency by customizing ribosome binding sites for both the upstream acetyl coenzyme A formation and fatty acid synthase modules enabled further production improvement. Fed-batch cultivation of the engineered strain resulted in a final fatty acid production of 8.6 g l(-1). The modular engineering strategies demonstrate a generalized approach to engineering cell factories for valuable metabolites production.