Fig. 4

Fine-tuning of PPC expression for 5-ALA production. a Effects of regulating PPC expression via RBS engineering on 5-ALA production. b Correlation between PPC activity in crude extract and 5-ALA production level. The x-axis shows mean of PPC activities in crude extract measured by NADH oxidation 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 PPC activity assay. The PPC activity was determined by a coupling reaction catalyzed by malate dehydrogenase at 30 ℃. The reaction mixture contained 100 mM Tris–HCl (pH 7.5), 2 mM phosphoenolpyruvate, 10 mM NaHCO₃, 10 mM MnSO₄, 0.1 mM NADH, 20% (v/v) glycerol, 1.6 U malate dehydrogenase, and 20 µg crude extract. The activity was assayed spectrophotometrically by monitoring the decrease in absorbance of NADH at 340 nm. Error bars indicate standard deviations from three parallel experiments