Control of native metabolic enzyme levels is an important part of engineering strains for overproduction of heterologous compounds. However, for many enzymes involved in central metabolism, static knockdown may result undesired consequences, such as poor growth and poor expression of recombinant proteins. In this work, we have developed a strategy for dynamically modulating the abundance of native enzymes within the host cell and applied this to a model system for the production of myo-inositol, an intermediate in the production of glucaric acid. This system relies on controlled degradation of a key glycolytic enzyme, phosphofructokinase-I (Pfk-I). Through tuning Pfk-I levels, we have been able to develop an E. coli strain with a growth mode close to wild type and a production mode with an increased glucose-6-phosphate pool available for conversion into myo-inositol. The switch to production mode is trigged by inducer addition, allowing yield, titer, and productivity to be managed through induction time. By varying the time of Pfk-I degradation, we were able to achieve a two-fold improvement in yield and titers of myo-inositol.

Dynamic knockdown of E. coli central metabolism for redirecting fluxes of primary metabolites

249th National Meeting (2015)

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