Gc tof ms

Metabolites were analyzed by GC-TOF/MS (Waters Corp., USA). The 1 μL derivatized sample was injected by Agilent 7683 autosampler into GC (Agilent 6890) which was equipped with DB-5MS column (30 m × 0.25 mm × 0.25 μm, J&W Scientific, Folsom, CA). The oven temperature was programmed as 70 °C for 2 min, then increased to 290 °C (5 °C/min), holding for 3 min. The ion source temperature and ionization current were 250 °C and 40 μA, respectively. The mass scan range was 50–800 m/z. Peak detection, deconvolution and peak quantification were performed using Masslynx software 4.1. Metabolites were identified by comparing their mass fragmentation patterns with NIST mass spectral library50 . The area of each acquired peak was normalized against the internal standard and dry cell weight. Multivariate data analysis was preformed by hierarchical cluster analysis (HCA) to view the relative differences in the metabolites concentrations among diverse conditions51 .

Metabolites were analyzed by GC-TOF/MS (Waters Corp., USA) as described previously50 . The 1 μL derivatized sample was injected by Agilent 7683 autosampler into GC (Agilent 6890) which was equipped with DB-5MS column (30 m × 0.25 mm × 0.25 μm, J&W Scientific, Folsom, CA). The oven temperature was programmed as: 70 °C for 2 min, then increased to 290 °C (5 °C/min), holding for 3 min. The ion source temperature and ionization current were 250 °C and 40 μA, respectively. The mass scan range was 50–800 m/z. Peak detection, deconvolution, and peak quantification were performed using Masslynx software 4.151 . Metabolites were identified by comparing their mass fragmentation patterns with NIST mass spectral library52 . The area of each acquired peak was normalized against the internal standard and dry cell weight for calculating the relative abundance. Multivariate data analysis was preformed by principal-components analysis (PCA)53 and hierarchical cluster analysis (HCA)54 to view the relative differences in the metabolites concentrations among diverse conditions.

In order to purify the taxadiene, strain SyBE_001113 were cultured in the 5 L bioreactor with 2 L SD medium. The engineered yeast was prepared in a shake flask at 30°C, 200 rpm till the OD₆₀₀ reached 4.0. The seed was then transferred to the bioreactor with an initial OD₆₀₀ of 0.05, and was cultivated at 350 rpm and 30°C for 66 h with aeration of 1 vvm. The pH was controlled at 5.7. Then 300 ml n-hexane was added to the bioreactor, and stirred for 3 h. The n-hexane phase was pooled and evaporated in vacuum, and the residues were subjected to column chromatography on silica gel eluted with n-hexane for purification. Gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF/MS, Waters) and NMR (Bruker) were used to further identify the purified taxadiene (Fig. S1a and S1b in File S1).
Fermentation cultures (400 µl) of the engineered S. cerevisiae were extracted with the same volume of n-hexane. The mixture was vortexed for 20 min, and 10 µl of the n-hexane phase was analyzed by GC-TOF/MS to quantify the taxadiene. The purified taxadiene was used as standard for qualification by external standard method.