Agilent 1290 infinity 6460

10 mL of cultured supernatant were directly dried down to 0.5 mL and then were filtered using Nylon Syringe Filters (0.22 μm, 13 mm). Neutral loss experiments were carried out on an Agilent LC-QQQ-MS system (Agilent 1290 Infinity-6460, Agilent Technologies, Santa Clara, CA, USA) in both positive and negative modes. The mobile phase and gradient were as the same as LC-QTOF analysis. Each neutral loss spectrum was scanned from m/z 150 to 1000 in centroid mode with a scan rate of 1.5 spectra/s.

The culture medium containing 3-HP was collected by centrifuging at 10,000×g for 10 min and the supernatant was used for product analysis. The concentration of 3-HP was determined by LC–MS carried out on an Agilent LC-QQQ-MS system (Agilent 1290 Infinity-6460, Agilent Corp, SantaClara, CA, USA) in the negative-ion mode. 3-HP standard was purchased from Tokyo Chemical Industry (Tokyo, Japan). The sample was separated on an Agilent SB C18 column (100 × 2.1 mm, 1.8 μm). Mobile phase A consisted of 10% (v/v) buffer in water, while mobile phase B was 10% (v/v) buffer in acetonitrile. The buffer consisted of 200 mM formic acid adjusted to pH 4.0 with ammonium hydroxide solution. The linear gradient was as following: 0–3 min, 5–10% B; 3–5 min, 100% B; 5–7 min, 5% B. The flow rate was 0.2 ml/min and the column was set at 35 °C. The injection volume was 5 μl. The 3-HP titer was measured in biological triplicate. The significance of differences between the different engineered strains was determined by t tests (MS excel) with a p value less than 0.05 considered to be statistically significant.