Engineered production of 5-aminolevulinic acid in recombinant <i>Escherichia coli</i> BL21

5-aminolevulinic acid (ALA) is a non-protein amino acid that has been widely used in the fields of medicine and agriculture. This study aims to engineer the C5 pathway of the ALA biosynthesis in <i>Escherichia coli</i> BL21 to enhance ALA production. The ALA synthase genes <i>gltX</i>, <i>hemA,</i> and <i>hemL</i> were overexpressed in <i>E. coli</i> BL21 to lead to the increase in the production of ALA. The sRNA RyhB was also overexpressed to downregulate the expression of ALA dehydratase to reduce the downstream bioconversion of ALA to porphobilinogen. Next, the gene <i>arcA</i> was knocked out by CRISPR-Cas9 technology to open the TCA cycle to promote the respiratory metabolism of the strain to reduce the feedback inhibition of heme to ALA. The fermentation conditions of the engineered strain were optimized by response surface experiments. The time-course analysis of the ALA production was carried out in a 1 L shake flask. Through these efforts, the production of ALA in engineered strain reached 2953 mg/L in a 1 L shake flask. This study contributes to the industrial production of ALA by the engineered <i>E. coli</i> in the future.