Dynamic Metabolic Control Improves the Biosynthesis of Chemical Molecules in Engineered E. coli

Abstract

Metabolic engineering is an effective strategy to optimize the biosynthesis of chemical molecules in genetically modified microbes. However, many current metabolic engineering strategies are limited by the requirements for cellular growth. To further optimize cell factories and overcome this limitation, we have applied 2-stage dynamic metabolic control strategies to optimize the biosynthesis of several compounds in E. coli. Using this strategy, cells grow without being impacted by product biosynthesis. We use phosphate depletion as a trigger to both force cells into a stationary production stage and initiate product synthesis. Phosphate depletion also dynamically removes enzymes involved in competitive or inhibitory metabolic pathways. This is accomplished by both inhibiting new transcription with CRISPR interference and degrading existing proteins via DAS+4 degron mediated proteolysis. Through my work, we demonstrate that the implementation of 2-stage dynamic metabolic control can indeed improve the biosynthesis of several small molecule chemicals in engineered E. coli, including: xylitol, citramalate and ethylene glycol. Rates, titers and yields were improved significantly. In addition, my work explored the mechanisms underlying improvements in performance. Specifically, we conclude that dynamic dysregulation of feedback control over central metabolism can lead to greatly improved stationary phase sugar uptake rates and pathway fluxes.