Targeted Omics Informed Engineering to Improve C5 Alcohol Production in E. coli

C5 alcohols are attractive targets for microbial production due to their importance as “platform” chemicals and potential use as advanced biofuels.  Here we leverage targeted omics data and standardized parts to construct a modified mevalonate pathway in E. coli for the improved production of three C5 alcohols: 3-methyl-3-butenol, 3-methyl-2-butenol, and 3-methyl-1-butanol.  By correlating pathway proteomics with diagnostic metabolites, we first assessed pathway behavior and revealed the primary determinants of efficient precursor production and carbon utilization.  Informed by this methodology, we increased 3-methyl-3-butenol titer to 1.5 g/L, identified toxic metabolites, and prioritized future engineering targets.  Further improvement of 3-methyl-3-butenol titer to ~2 g/L (70% theoretical) was achieved through selective RBS engineering of NudB—a promiscuous phosphatase—and targeted gene replacements.  Expression of a fusion protein and reductase resulted in the production of all three C5 alcohols including over 250 mg/L of 3-methyl-1-butanol, a >20-fold improvement over a previous engineering effort.  This targeted approach to engineering should prove useful for the analysis and optimization of increasingly complex metabolic pathways.