Quantifying Intracellular Coenzyme Levels

The intracellular concentration of CoA, acetyl-CoA, and butanoyl-CoA was determined using ¹³C-isotope labelling experiment following the protocol used in the previous study with minor modifications [18 (link)]. Fully ¹³C-labeled cell extracts of BUOHSE and DC7 strains were prepared. Cells were cultivated as described previously except that NaH¹³CO₃ (> 98 atom % ¹³C, Cambridge Isotope Laboratories, Inc., USA) was used instead of NaHCO₃. Sampling was performed 3 days after IPTG induction in the same way as described above except that pre-cooled deionized water was used as washing solvent instead of NH₄CO₃. Cells were extracted as described above except without the freeze-drying step. Extraction was repeatedly conducted for four times. After samples were concentrated for 2 h using centrifugal concentration, all samples were combined in a 15 mL centrifuge tube. This ¹³C-labelled cell extract was used as an internal standard. Six point calibration curve was constructed for each metabolite using peak area ratios of U-¹²C to U-¹³C, as described previously [18 (link)]. The detailed description of calibration curve for each metabolite is listed in Additional file 1: Table S1. Analysis mode was multiple reaction monitoring (MRM) mode in IP-RP-LC/QqQ-MS system (Additional file 1: Table S4).