Engineering metabolic biosynthetic pathways has enabled the microbial production of many useful chemicals. However, pathway productivities and yields are often limited by metabolic imbalances. Synthetic regulatory circuits have been shown to be able to balance engineered pathways, improving titers and productivities. Here we developed a negative feedback regulatory circuit based on a malonyl-CoA-based sensor-actuator. Malonyl-CoA is biosynthesized from acetyl-CoA by the acetyl-CoA carboxylase, which is the rate-limiting step for fatty acid biosynthesis. Overexpression of acetyl-CoA carboxylase improves fatty acid production, but slows down cell growth. We have devised a malonyl-CoA sensor-actuator that controls gene expression levels based on intracellular malonyl-CoA concentrations. This sensor-actuator is used to construct a negative feedback circuit to regulate the expression of acetyl-CoA carboxylase. The negative feedback circuit is able to up-regulate acetyl-CoA carboxylase expression when the malonyl-CoA concentration is low and down-regulate acetyl-CoA carboxylase expression when excess amounts of malonyl-CoA have accumulated. We show that the regulatory circuit effectively alleviates the toxicity associated with acetyl-CoA carboxylase overexpression. When used to regulate the fatty acid pathway, the feedback circuit increases fatty acid titer and productivity by 34% and 33%, respectively.