Plug-in repressor library for precise regulation of metabolic flux in Escherichia coli

In metabolic engineering, enhanced production of value-added chemicals requires precise flux control between growth-essential competing and production pathways. Although advances in synthetic biology have facilitated the exploitation of a number of genetic elements for precise flux control, their use requires expensive inducers, or more importantly, needs complex and time-consuming processes to design and optimize appropriate regulator components, case-by-case. To overcome this issue, we devised the plug-in repressor libraries for target-specific flux control, in which expression levels of the repressors were diversified using degenerate 5′ untranslated region (5’ UTR) sequences employing the UTR Library Designer. After we validated a wide expression range of the repressor libraries, they were applied to improve the production of lycopene from glucose and 3-hydroxypropionic acid (3-HP) from acetate in Escherichia coli via precise flux rebalancing to enlarge precursor pools. Consequently, we successfully achieved optimal carbon fluxes around the precursor nodes for efficient production. The most optimized strains were observed to produce 2.59 g/L of 3-HP and 11.66 mg/L of lycopene, which were improved 16.5-fold and 2.82-fold, respectively, compared to those produced by the parental strains. These results indicate that carbon flux rebalancing using the plug-in library is a powerful strategy for efficient production of value-added chemicals in E. coli . ● Plug-in repressor libraries with diverse expression levels were devised for precise flux control in Escherichia coli. ● The expression levels of the repressors were varied using degenerate 5′ untranslated region sequences employing the UTR Library Designer. ● The repressor libraries enabled precise flux control without expensive inducers and laborious process to find & optimize suitable regulation elements to each individual case. ● As examples, the improved production of 3-hydroxypropionic acid from acetate and lycopene from glucose in E. coli were demonstrated.