Stable Isotope Labeling Method

¹³C-labeling analysis was performed by the protocol reported before with a slight modification [59 (link)]. 15 ml cells of the later exponential phase at the OD₆₀₀ of 1.3 ± 0.1 were pre-cultured in minimal medium with ¹²C-methanol and 5 g/l ¹²C-labeled β-alanine, and then were rapidly passed through the membrane filter (0.22 μm, 47 mm, Sartorius). The filter was immediately removed and placed on an agar plate with ¹²C-methanol and ¹²C-labeled β-alanine for 30 min at 30 °C, then transferred to another agar plate with the same concentration of ¹²C methanol and ¹³C-labeled β-alanine for different times (i.e. 2, 5 and 12 h). Then the filter was immediately transferred to a 50 ml tube with liquid nitrogen for quenching. The sample was stored at − 80 °C freezer until it was ready for subsequent extractions. The metabolites were extracted and analyzed as the described above.