Product-Responsive Regulation of NADPH and Malonyl-CoA Allocation Enhances 3-Hydroxypropionic Acid Production in...

Efficient microbial production of platform chemicals requires precise regulation of metabolic fluxes. However, static control strategies─including constitutive overexpression and multi-inducer systems requiring manual optimization─cannot adapt in real time to changing intracellular states and therefore hinder fine-tuning of cofactor and precursor pools. Here, we constructed a product-responsive regulatory circuit in Escherichia coli that coordinates two key biosynthetic determinants─NADPH regeneration and malonyl-CoA allocation─in response to intracellular 3-hydroxypropionic acid (3-HP) accumulation. Following initial IPTG induction of the 3-HP biosynthesis module, intracellular 3-HP accumulation activates the downstream product-responsive regulatory circuit, enabling coordinated modulation of NADPH regeneration and malonyl-CoA allocation without additional external intervention. The final engineered strain, SBH40, achieved a 3-HP titer of 3.0 g/L and a yield of 0.19 g/g glucose, representing an approximately 5-fold improvement in both metrics relative to a nonregulated control strain (0.64 g/L; 0.04 g/g glucose). These results demonstrate that a single-input product-responsive circuit can autonomously coordinate multiple metabolic nodes after pathway initiation, offering a practical and modular framework for dynamic regulation of cofactor- and precursor-intensive biosynthesis.