New insight into the codon usage and medium optimization toward stable and high-level 5-aminolevulinic acid production in Escherichia coli

5–Aminolevulinic acid (5-ALA) is recently being used as a critical prodrug used for the photodynamic therapy of cancers. In this study, we first demonstrated via codon shuffling that codon adaptation index (CAI) and tRNA adaptation index (tAI) influenced the translational level and folding of the recombinant ALA synthase from Rhodobacter capsulatus (Rc*) for 5-ALA production in E. coli. A genome-based chassis Rc*GI was achieved by genomic integration and co-expression of chaperone (GroELS) with Rc*, which increased the cell growth of E. coli and 5-ALA production (up to 5.9 g/L) in antibiotic-free culture. However, the homocysteine methyltransferase (metE) and ferrienterobactin receptor (fepA), both ferric dependent and heme related proteins were stimulated after continuous culture for 7 batches, thus the addition of ferric ion into the culture boosted the cell growth and 5-ALA production. Finally, the glucose-glycerol mixed carbon source was employed to extend cell growth and the highest 5-ALA titer of 15.6 g/L was achieved via fed-batch fermentation. We provide new insights into the economical and efficient bioprocessing of 5-ALA production with a robust chassis.