Systems Metabolic Engineering of Dual Pathways with Stage-Specific Activation for High-Level 5-Aminolevulinic Acid Production in Escherichia coli

5-Aminolevulinic acid (5-ALA) is an important non-proteinogenic amino acid with wide applications in agriculture and medicine. To achieve high-level microbial production, this study established a dual-pathway reconstruction strategy in Escherichia coli by integrating the endogenous C5 pathway with an inducible exogenous C4 pathway. Multi-copy overexpression of gltX , hemA , and hemL , combined with enhanced glutamate supply and the introduction of a non-oxidative glycolysis pathway, effectively increased the C5 pathway flux and carbon efficiency, while product toxicity was significantly alleviated by strengthening efflux mechanisms and oxidative stress tolerance. A quorum sensing–based regulatory system was employed to dynamically regulate hemB expression, thereby balancing cell growth and 5-ALA biosynthesis, and a controlled glycine feeding strategy specifically activated the C4 pathway during the later fermentation stage, forming a stage-specific dynamic activation mechanism. Promoter regulation of sucC/sucD expression and enhanced endogenous PLP biosynthesis further stabilized the C4 flux. Fed-batch fermentation in a 5 L bioreactor resulted in a final 5-ALA titer of 37.34 g/L, demonstrating the industrial potential of this systems metabolic engineering strategy combining dual pathways and stage-specific activation control.