| Summary: | Abstract Background 5-Aminolevulinic acid (5-ALA) is a promising biostimulant, feed nutrient, and photodynamic drug with wide applications in modern agriculture and therapy. Although microbial production of 5-ALA has been improved realized by using metabolic engineering strategies during the past few years, there is still a gap between the present production level and the requirement of industrialization. Results In this study, pathway, protein, and cellular engineering strategies were systematically employed to construct an industrially competitive 5-ALA producing Escherichia coli. Pathways involved in precursor supply and product degradation were regulated by gene overexpression and synthetic sRNA-based repression to channel metabolic flux to 5-ALA biosynthesis. 5-ALA synthase was rationally engineered to release the inhibition of heme and improve the catalytic activity. 5-ALA transport and antioxidant defense systems were targeted to enhance cellular tolerance to intra- and extra-cellular 5-ALA. The final engineered strain produced 30.7 g/L of 5-ALA in bioreactors with a productivity of 1.02 g/L/h and a yield of 0.532 mol/mol glucose, represent a new record of 5-ALA bioproduction. Conclusions An industrially competitive 5-ALA producing E. coli strain was constructed with the metabolic engineering strategies at multiple layers (protein, pathway, and cellular engineering), and the strategies here can be useful for developing industrial-strength strains for biomanufacturing.
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