Engineering genetic circuits for dynamic control of central metabolism.

Genetic regulation tools have been examined for their ability to enable sophisticated dynamic control of biosynthesis in microbial cell factories, enhancing the production performance of valuable compounds. However, most genetic tools are pathway- or intermediate-specific, hindering their broad applicability in synthetic biology. Moreover, their potential to balance metabolic fluxes in central metabolism between cell growth and product formation remains under-explored, raising the question of whether they can facilitate efficient biosynthesis. To answer this, we established the PdhR biosensor system that responds to pyruvate to dynamically regulate metabolic flux distribution in central metabolism. In this study, we first characterized the dose response of PdhR biosensor system by screening multiple PdhR homologs derived from various microorganisms. Computational analysis further guided the identification of key factors contributing to their functional differences, enabling the optimization of biosensor properties through site-directed mutagenesis. As proof of concept, we employed our biosensor system to improve the biosynthesis of trehalose and 4-hydroxycoumarin (4HC), respectively. Specifically, trehalose titer increased to 3.72 g/L, which is 2.33-fold higher than the control group. In addition, we improved the 4HC titer to 491.5 mg/L, which possessed a 1.63-fold increase over the static strategy. In summary, the established central metabolism-responsive biosensor system underlined the necessity of metabolic flux distribution and validated its broad applicability in the biosynthesis of central metabolism-derived compounds.