Development of bifunctional biosensors for sensing and dynamic control of glycolysis flux in metabolic engineering

Glycolysis is the primary metabolic pathway in all living organisms. Maintaining the balance of glycolysis flux and biosynthetic pathways is the crucial matter involved in the microbial cell factory. Few regulation systems can address the issue of metabolic flux imbalance in glycolysis. Here, we designed and constructed a bifunctional glycolysis flux biosensor that can dynamically regulate glycolysis flux for overproduction of desired biochemicals. A series of positive-and negative-response biosensors were created and modified for varied thresholds and dynamic ranges. These engineered glycolysis flux biosensors were verified to be able to characterize in vivo fructose-1,6-diphosphate concentration. Subsequently, the biosensors were applied for fine-tuning glycolysis flux to effectively balance the biosynthesis of two chemicals: mevalonate and N-acetylglucosamine. A glycolysis flux-dynamically controlled Escherichia coli strain achieved a 111.3 g/L mevalonate titer in a 1L fermenter.