Synergistic effect of chaperone-guided folding and energy allocation for enhancing 5-aminolevulinic acid in engineered Escherichia coli

5-Aminolevulinic acid (5-ALA), the universal precursor of tetrapyrroles, has widely applications such as photodynamic therapy of skin cancer, agriculture booster and cosmetics. 5-ALA production relies on the efficiency of 5-aminolevulinate synthase (ALAS) and energy balance for cell growth. In this study, the folding capacity of ALAS was enhanced by chromosomal integration of molecular chaperone systems, including Trigger Factor (TF), GroEL/GroES (GroELS), and DnaK/DnaJ (DnaKJ), in engineered E. coli expressing Rhodobacter capsulatus ALAS (RcALAS). Among all systems, the DnaKJ-integrated strain markedly improved solubility, further plasmid-based and co-expression of DnaKJ increased 5-ALA production to 7.15 g/L, representing a 2.23-fold increase over the control. By refining carbon source utilization and feeding strategies to encounter the ATP demand, 5-ALA titer was further increased to 9.36 g/L within 36 h in shake flask cultivation. To validate the process performance, bench-scale cultivation in a 2.5 L Ultra-Yield flask demonstrated its scalability, achieving a final 5-ALA titer of 12.1 g/L. The successful 5-ALA biosynthesis assisted by DnaKJ chaperone, coupled with carbon flux redirection, showed a synergistic effect that improved heterologous enzyme expression and evaluated overall efficiency of 5-ALA production.