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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 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. • DnaKJ-mediated folding mitigated the bottleneck by enhancing ALAS solubility. • Synergistic chaperone engineering and carbon allocation boosted 5-ALA titer by 127%. • An ATP-based index quantified energy utilization efficiency for 5-ALA synthesis. • Bench-scale cultivation yielded 12.1 g/L 5-ALA with optimal energy and ATP balance.
Hou et al. (Sun,) studied this question.