Living systems have evolved remarkable molecular functions that can be redesigned for in vivo chemical synthesis as we gain a deeper understanding of the underlying biochemical principles for de novo construction of synthetic pathways. We have focused on developing pathways for next-generation biofuels as they require carbon to be channeled to product at quantitative yields. However, these fatty acid-inspired pathways must manage the highly reversible nature of the enzyme components. For targets in the biodiesel range, the equilibrium can be driven to completion by physical sequestration of an insoluble product, which is a mechanism unavailable to soluble gasoline-sized products. In this work, we report the construction of a chimeric pathway assembled from three different organisms for the high-level production of n-butanol (4,650 ± 720 mg l⁻¹) that uses an enzymatic chemical reaction mechanism in place of a physical step as a kinetic control element to achieve high yields from glucose (28%).