NAD⁺-driven modular multi-enzyme cascade system enables the in vitro synthesis of 3-hydroxypropionic acid from glucose.

3-Hydroxypropionic acid (3-HP) is an important bio-based platform chemical with broad industrial potential. Most reported in vitro biosynthetic systems rely on costly NADPH, which limits scalability. Here, a modular in vitro multi-enzyme cascade pathway was designed using D-glucose as the sole carbon source, driven exclusively by the low-cost NAD⁺/NADH redox pair. Three functional modules were constructed: a pyruvate accumulation module built on thermophilic enzymes, an alanine cycling module proposed as a conceptual strategy for β-alanine regeneration through a CO₂-mediated carboxylation-decarboxylation cycle, and a 3-HP production module centered on an NAD⁺-dependent 3-HP dehydrogenase (HPD). Through systematic enzyme screening, CaHPD from Candida albicans was identified as a highly efficient biocatalyst, achieving a productivity of 0.83 g/L/h. Following reaction optimization, the 3-HP titer increased 3.2-fold from 2.3 mM (23% molar conversion) to 7.26 mM (72.6% molar conversion). Under high-glucose conditions (50 mM), the 3-HP titer increased to 26.47 mM. Analysis of the alanine cycling module revealed that insufficient thermodynamic driving force in the reverse carboxylation step is the key bottleneck for in situ β-alanine regeneration. This study establishes a novel NADH-dependent modular pathway for multi-enzyme-catalyzed 3-HP biosynthesis and offers a framework for developing cost-effective, scalable cell-free biomanufacturing platforms.