Kinetically guided, ratiometric tuning of fatty acid biosynthesis

Cellular metabolism is a nonlinear reaction network in which dynamic shifts in enzyme concentration help regulate the flux of carbon to different products. Despite the apparent simplicity of these biochemical adjustments, their influence on metabolite biosynthesis tends to be context-dependent, difficult to predict, and challenging to exploit in metabolic engineering. This study combines a detailed kinetic model with a systematic set of in vitro and in vivo analyses to explore the use of enzyme concentration as a control parameter in fatty acid synthesis, an essential metabolic process with important applications in oleochemical production. Compositional analyses of a modeled and experimentally reconstituted fatty acid synthase (FAS) from Escherichia coli indicate that the concentration ratio of two native enzymes-a promiscuous thioesterase and a ketoacyl synthase-can tune the average length of fatty acids, an important design objective of engineered pathways. The influence of this ratio is sensitive to the concentrations of other FAS components, which can narrow or expand the range of accessible chain lengths. Inside the cell, simple changes in enzyme concentration can enhance product-specific titers by as much as 125-fold and elicit shifts in overall product profiles that rival those of thioesterase mutants. This work develops a kinetically guided approach for using ratiometric adjustments in enzyme concentration to control the product profiles of FAS systems and, broadly, provides a detailed framework for understanding how coordinated shifts in enzyme concentration can afford tight control over the outputs of nonlinear metabolic pathways.