De novo design of the global transcriptional factor Cra‐regulated promoters enables highly sensitive glycolysis flux biosensor for dynamic metabolic control

Abstract Glycolytic flux is a fundamental index in microbial cell factories. A glycolytic flux biosensor that can monitor glucose metabolism efficiency is a promising strategy in rewiring metabolic flux to balance growth and biosynthesis. A key design feature of the glycolytic flux biosensors is the interaction between the global transcriptional factor Cra and its regulated promoters. However, overexpression and mutation of Cra has unpredictable effects on global metabolism in Escherichia coli. Therefore, new orthogonal biosensor design strategies should be developed to circumvent metabolic issues. In this report, the promoters in glycolytic flux biosensor were replaced with synthetic promoters of varying strengths or phage‐derived promoters, and the Cra DNA‐binding sites were deployed into promoters at different positions and distances to yield biosensors. The de nova biosensors that depended on Cra could sense Fructose‐1,6‐diphosphate (FBP) with broad dynamic ranges and low basal leakage. Then the negative‐response biosensors were applied to fine‐tune the target ATP synthesis gene, leading to the desired increase in pyruvate production (the highest 9.66 g/L) and cell growth. Moreover, the membrane synthesis gene plsC was also dynamically activated by the positive‐response biosensor, leading to effective accumulation of lycopene in the cell membrane and a 50‐fold increase in lycopene titre (100.3 mg/L) when compared with the control strain, demonstrating the effective and broader usages of our biosensors.