<jats:title>ABSTRACT</jats:title> <jats:p> 5-Aminolevulinic acid (ALA), a nonprotein amino acid involved in tetrapyrrole synthesis, has been widely applied in agriculture, medicine, and food production. Many engineered metabolic pathways have been constructed; however, the production yields are still low. In this study, several 5-aminolevulinic acid synthases (ALASs) from different sources were evaluated and compared with respect to their ALA production capacities in an engineered <jats:named-content content-type="genus-species">Corynebacterium glutamicum</jats:named-content> CgS1 strain that can accumulate succinyl-coenzyme A (CoA). A codon-optimized ALAS from <jats:named-content content-type="genus-species">Rhodobacter capsulatus</jats:named-content> SB1003 displayed the best potential. Recombinant strain CgS1/pEC-SB produced 7.6 g/liter ALA using a mineral salt medium in a fed-batch fermentation mode. Employing two-stage fermentation, 12.46 g/liter ALA was produced within 17 h, with a productivity of 0.73 g/liter/h, in recombinant <jats:named-content content-type="genus-species">C. glutamicum</jats:named-content> . Through overexpression of the heterologous nonspecific ALA exporter RhtA from <jats:named-content content-type="genus-species">Escherichia coli</jats:named-content> , the titer was further increased to 14.7 g/liter. This indicated that strain CgS1/pEC-SB-rhtA holds attractive industrial application potential for the future. <jats:p> <jats:bold>IMPORTANCE</jats:bold> In this study, a two-stage fermentation strategy was used for production of the value-added nonprotein amino acid 5-aminolevulinic acid from glucose and glycine in a generally recognized as safe (GRAS) host, <jats:named-content content-type="genus-species">Corynebacterium glutamicum</jats:named-content> . The ALA titer represented the highest in the literature, to our knowledge. This high production capacity, combined with the potential easy downstream processes, made the recombinant strain an attractive candidate for industrial use in the future.