Systematic Metabolic Engineering of Escherichia coli for High-Level Production of 5-Aminolevulinic Acid

5-Aminolevulinic acid (5-ALA) is a naturally occurring nonproteinogenic amino acid with considerable potential in agricultural and pharmaceutical applications, and its biomanufacturing has attracted growing interest. To meet market demand, this study developed an efficient production process by engineering Escherichia coli. First, we established the C4 biosynthetic pathway for 5-ALA and minimized glycine degradation by targeted deletion of gcvP and kbL. Subsequently, the introduction of a nonoxidative glycolysis (NOG) module combined with enhanced glucose uptake further increased 5-ALA production. To alleviate host toxicity, efflux engineering and antioxidant systems were integrated, further raising the titer to 6.93 ± 0.28 g/L. Dynamic regulation of sucCD expression together with redox rebalancing maintained a balance between precursor supply and cell growth. Finally, attenuation of hemB expression reduced metabolic flux diversion toward heme synthesis, yielding the engineered strain A48, which produced 55.11 g/L of 5-ALA within 41 h in a 5 L bioreactor. The metabolic engineering strategy presented in this study establishes an efficient biosynthetic platform for 5-ALA production.