Bioproduction of 3-Hydroxypropionic Acid by Enhancing the Precursor Supply with a Hybrid Pathway and Cofactor Regeneration

3-Hydroxypropionic acid (3-HP) is one of the 12 valuable platform chemicals with versatile applications in the chemical, food, and cosmetic industries. However, the biosynthesis of 3-HP faces challenges due to the lack of robust chassis and the high costs associated with the fermentation process. To address these challenges, we made efforts to augment the robustness of 3-HP-producing chassis by exploiting metabolic regulation, controlling carbon flux, balancing cofactor generation, and optimizing fermentation conditions. First, the malonyl-CoA (MCA) pathway was recruited and rebalanced in <i>Escherichia coli</i>. Subsequently, a hybrid pathway integrating the Embden-Meyerhof-Parnas pathway with the nonoxidative glycolysis pathway was systematically modulated to enhance carbon flux to the MCA pathway, followed by fine-tuning NADPH regeneration. Then, by optimizing the fermentation conditions, 3-HP production was significantly improved, reaching 6.8 g/L. Finally, in a fed-batch experiment, the final chassis produced 42.8 g/L 3-HP, corresponding to a 0.4 mol/mol yield and 0.6 g/(L·h) productivity.