Metabolic engineering of <i>Corynebacterium glutamicum</i> for enhanced 5-aminolevulinic acid production via precise porphobilinogen synthase activity modulation

5-Aminolevulinic acid (5-ALA) is a valuable precursor for pharmaceuticals and agriculture, but its microbial production is limited by tight coupling to essential heme biosynthesis. Here, we introduce a systematic, activity-graded tuning strategy for porphobilinogen synthase (PBGS, encoded by <i>hemB</i>) to decouple 5-ALA synthesis from heme metabolism in <i>Corynebacterium glutamicum</i>. Guided by structural and functional analyses, PBGS variants with progressively reduced activities were constructed to investigate the quantitative relationship between enzyme activity, cell growth, and 5-ALA accumulation. Controlled attenuation of PBGS activity maintained essential metabolism while markedly enhancing 5-ALA accumulation and minimizing porphyrin by-products. The engineered strain FA3 [<i>hemB</i>(D128E), <i>hemA</i> overexpression] achieved an optimal balance of growth and productivity. Metabolomic profiling confirmed that PBGS downregulation primarily suppressed porphyrin biosynthesis with minimal impact on central carbon metabolism. Subsequent metabolic and process optimizations, including <i>gdhA</i> and <i>aceA</i> deletion, dynamic <i>rhtA</i> expression, and cultivation control, further boosted production to 14.44 g/L 5-ALA in shake-flask culture, representing the highest shake-flask titer reported to our knowledge for <i>C. glutamicum</i> under similar conditions. This work provides the first systematic dissection of PBGS activity-dependent metabolic regulation and demonstrates that graded control of an essential enzyme enables rational metabolic decoupling, offering a broadly applicable framework for robust, high-yield microbial production of valuable compounds.IMPORTANCE5-Aminolevulinic acid (5-ALA) is an important precursor with pharmaceutical and agricultural applications, but microbial production is often constrained by its tight linkage to essential heme metabolism. Here, we systematically tuned porphobilinogen synthase activity to decouple 5-ALA accumulation from excessive porphyrin flux while maintaining cell growth. This strategy not only enabled the highest reported 5-ALA titer in <i>Corynebacterium glutamicum</i> but also highlights a broadly applicable framework for rationally engineering essential metabolic enzymes to achieve robust, high-yield microbial production of valuable compounds.