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. Intriguingly, mutants of FabF (a ketoacyl synthase involved in acyl-ACP elongation) that exhibit both (i) adjusted substrate specificities and (ii) reduced bindinging affinities for ACP enable additional control over profile width. This work provides a detailed framework for using coordinated shifts in the concentrations of native and nonnative enzymes to exert tight control over the outputs of fatty acid pathways. It concludes by discussing applications in oleochemical biosynthesis.

Comprehensive kinetic framework for controlling fatty acid biosynthesis in E. coli

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.