Application of a synthetic metabolic toggle switch to isopropanol bio-synthesis

Up-regulation of genes expression level in production pathways and down-regulation of genes in competing pathways are often employed to improve production titer and yield in metabolic engineering. However, the permanent knockout of a pathway responsible for growth and cell maintenance has not previously been employed because of the importance of the cells amount to the production rate and titer, even if its competition with the production pathway is obvious. To avoid the competition between the cell growth and the production, several strategies for optimization of fermentation processes have been developed to date, such as two stage fermentation. In addition to these approaches, a novel methodology to realize a conditional knockout of target endogenous gene is required in order to optimize intracellular metabolism at each fermentation phase for bacterial growth and production. We constructed a metabolic toggle switch as a novel conditional knockout approach in Escherichia coli. We applied it to isopropanol bio-synthesis in E. coli considering the excess metabolic influx to TCA cycle as a target for conditional regulation. The resulting redirection of excess carbon flux caused by interruption of the TCA cycle via switching the expression of gltA encoding citrate synthase OFF improved isopropanol production titer and yield up to 3.7 and 3.1 times, respectively. These improvements were achieved with avoiding significant cell growth inhibition as the results of conditional down-regulation of TCA cycle. This approach is a useful tool to redirect carbon flux responsible for bacterial growth and/or cell maintenance toward a synthetic production pathway.