Fig. 3

Fine-tuning of ALAS expression for 5-ALA production. a Effects of regulating ALAS expression via RBS engineering on 5-ALA production. b Correlation between ALAS activity in crude extract and 5-ALA production level. The x-axis shows mean of ALAS activities in crude extract measured by ALAS formation rate of three parallel experiments. Strains were cultivated in shake flasks with modified M9 medium. OD_600nm value, 5-ALA titer and yield at 36 h are shown. Cells were collected at the same time and lysed for ALAS activity assay. The reaction mixture contained 100 mM Tris–HCl (pH 7.5), 200 mM glycine, 0.2 mM succinyl-CoA, 0.1 mM pyridoxal phosphate (PLP) and 20 µg crude extract. After proceeding at 37 ℃ for 10 min, the reaction was terminated by the addition of 10% (v/v) trichloroacetic acid. Concentration of 5-ALA in the supernatant was determined. Error bars indicate standard deviations from three parallel experiments