Engineering of multiple modular pathways for high-yield production of 5-aminolevulinic acid in Escherichia coli

•Balancing the expression of hemA and hemL by mutating RBS improved ALA synthesis.•ALA catabolism was weakened by substituting the promoter of hemB with fliCp.•Strengthening the native synthesis pathway of PLP improved GST-AT activity.•ALA production increased to 5.25 g/L with a pH two-stage strategy. 5-aminolevulinic acid (ALA), an important precursor of tetrapyrroles, has various applications in medicine and agriculture fields. Several methods have been adopted to enhance ALA synthesis in our previous studies. In this study, systematic metabolic engineering strategies were implemented to further improve ALA production in Escherichia coli. Firstly, hemA and hemL with different strength of RBS from the artificially constructed mutation libraries were randomly assembled to balance metabolic flux. Then the expression of ALA dehydratase was rationally regulated by replacing promoter with fliCp to weaken ALA catabolism. Besides, the activity of glutamate-1-semialdehyde aminotransferase was increased through strengthening the native biosynthesis pathway of cofactor pyridoxal 5′-phosphate. Moreover, plasmid stability was improved by 21.4% by deleting recA and endA in the recombinant. Finally, stepwise improvements in ALA production were increased to 5.25 g/L with a pH two-stage strategy in a 3-L fermenter. This study proved the importance of metabolic balance in the pathway.