Quenching and extraction of intracellular metabolites
Approximately 5 mL sample was rapidly taken from fermenter and injected into 25 mL precooled (–40°C) 60% (v/v) methanol solution to interrupt the cell metabolism as soon as possible, resulting in a final concentration of 50% (v/v) methanol. The quenching process lasted 3 min. Cell pellets were then separated using a fast vacuum dependent filtration (0.45 μm nominal pore size) and washed with 5 mL precooled (–40°C) 50% (v/v) methanol solution to remove the residual medium component on the cell surface. Next the filter adhered with cell pellet was frozen by liquid nitrogen, and rapidly transferred into 9.5 mL precooled (–40°C) extraction solution composed of 2.5 mL methanol, 5 mL chloroform, and 2 mL fresh Tricine-EDTA buffer solution (3 mmol/L Tricine and 3 mmol/L EDTA, pH 7.0, stored at 4°C). The mixture was vigorously vibrated for 60 min at –40±5°C (dry ice-ethanol bath). The first aqueous phase was collected by a centrifugation step (12,000 rpm, 5 min, –19°C), more methanol and Tricine-EDTA buffer solution was added in for a second extraction, and the second aqueous phase was collected and pooled with the first one. The aqueous phase was stored at −80°C and evaporated to dryness before use. The dry residue was reconstituted in 100 μL of Mill-Q water for LC-MS/MS analysis.