Abstract
5-Aminolevulinic acid (5-ALA), a versatile precursor for tetrapyrrole derivatives (such as heme, chlorophyll, and cobalamin), drives advancing microbial cell factories to meet growing biomedical and industrial demands. However, there remain two challenges that limit yield and scalability: the limitations of conventional plasmid-based gene expression systems and the lack of fine regulation of succinyl-CoA. In this study, to address these limitations, we integrated multiple copies of hemA (C132A) of the heterologous C4 pathway on the genome. For fine regulating the supply of succinyl-CoA, the genes related to the tricarboxylic acid cycle (TCA cycle) oxidation branch pathway were combinatorially screened. The optimal combination of icd and lpd was confirmed by ribosome binding site (RBS) engineering, which was integrated on the genome with optimized expression intensity. Succinyl-CoA supply was further increased by genome integration and expression optimization of key CoA biosynthetic gene coaA, pantothenic acid synthesis-related gene panB-panC, and β-alanine synthesis-related gene panD. The optimized genomically stable chassis achieved a high 5-ALA production of 6.38 ± 0.16 g/L, which was 8.63-fold higher than the single hemA (C132A) copy strain A1 (0.74 ± 0.07 g/L). From these findings, a stable and high-yield 5-ALA synthetic strain was successfully constructed, providing a new strategy for production of biochemicals derived from succinyl-CoA in C. glutamicum.