HPLC Analysis of Metabolites

The concentrations of L-phenylalanine and 2-phenylethanol were measured by HPLC (HP 1200, Agilent, USA) equipped with a reversed phase column of Poroshell 120 SB-C18 (4.6 × 150 mm, 2.7 micron, Agilent, USA) and a UV absorbance detector (Agilent, USA) at 258 nm with the mobile phase of 70% water and 30% acetonitrile containing 0.1% trifluoroacetic acid at the flow rate of 0.6 mL/min. Organic acids and sugars were measured by HPLC (HP 1200, Agilent, USA) equipped with a cation-exchange column of Aminex HPX-87H (7.8 × 300 mm, BioRad, USA), a refractive index (RI) detector (Agilent, USA), and a UV absorbance detector (Agilent, USA) with the mobile phase of 3 mM sulfuric acid solution at the flow rate of 0.5 mL/min. Acetate, lactate, and shikimate were detected by the UV detector at 210 nm, while the RI detector was used to measure glucose and xylose. Cell growth was monitored by measuring the optical density of cultures at 600 nm (OD₆₀₀), and one unit of optical density corresponded to 0.31 dry cell weight (DCW) (g/L) [27 (link)]. The determination of reductive activity of AdhA was performed as described previously [16 (link)].