A molecular switch that enhances productivity of bioprocesses for heterologous metabolite production

Abstract

Metabolic engineering is the cornerstone of microbial syntheses of value-added, heterologous metabolites, and its toolbox has been extensively employed for maximizing the biosynthetic yields of heterologous molecules. However, there are fewer examples of applying metabolic engineering for improving the productivity of the strains. Productivities of fermentations have hitherto been largely improved through bioprocess engineering. We posited that re-tooling the expression machinery of the host so that it abruptly transitions from biomass accumulation to product generation at a defined time could improve productivity. We verified this hypothesis using a simple mathematical model and subsequently re-engineered the expression machinery in E. coli to switch between these regimes in response to an external stimulus. Specifically, we modified the T7 RNA polymerase that drives expression of the desired metabolic pathway by interrupting its sequence with a temperature-sensitive mutant of the vacuolar membrane ATPase (VMA) intein of S. cerevisiae. This modification temporarily inactivates the T7 RNA polymerase and turns off product formation in favour of biomass accumulation. The polymerase is only activated within the cell when the temperature of the culture is lowered from 37 °C to 18 °C at a defined time, which then coaxes the cells to transition exclusively to product formation. When we tested this molecular control scheme in a strain of E. coli that also expresses the lycopene biosynthetic pathway, we observed that the cells exhibited improved resource allocation, greater stringency of control over expression of the production pathway, and a 15% improvement in productivity. Our results establish a robust and generalizable model for applying metabolic engineering to improve productivity; and, most importantly, our approach seamlessly interfaces metabolic and macroscopic process control.