Agilent 1260 infinity series instrument

HPLC analysis of reactions was generally carried out on an Agilent 1260 Infinity series instrument (Agilent Technologies Inc., USA) using a reversed phase C18 column (Kinetex® 5 µm C18 100 Å, LC Column 150 × 4.6 mm, Phenomenex, USA) or a polar column (Comixsep®, P/N FMG-BPF5-EONU, Polar BiPFP 5 u, 250 × 4.6 mm, China) with UV detection at 256 or 304 nm under the following program: solvent A, 10% acetonitrile (MeCN) in water supplementing with 0.1% formic acid; solvent B, 90% MeCN in water; 5% B to 80% B (0–20 min), 80% B to 100% B (20–21 min), 100% B (21–24 min), 100% B to 5% B (24–25 min), 5% B (25–30 min); flow rate at 1 mL min⁻¹.

The fermentation of the recombinant strains was carried out using 4^# (fish peptone 8 g·L⁻¹, soluble starch 15 g·L⁻¹, cornmeal 8 g·L⁻¹, tryptone 5 g·L⁻¹, glycerol 5 g·L⁻¹, CaCO₃ 2 g·L⁻¹, KBr 0.2 g·L⁻¹, artificial sea salt 30 g·L⁻¹, PH 7.2-7.4) medium in 250 mL flask by shaking at 200 rpm and 28 °C for 4 days. A 5 mL fermentation broth was extracted with 5 mL butanone, and the extract was then concentrated under vacuum. The residues were dissolved into 100 μL of methanol. For analyzing the metabolites of recombinant strains, the HPLC was used by Agilent 1260 infinity series instrument (Agilent Technologies Inc. Santa Clara, USA) and a reversed phase column (Phenomenex kinetex, C18, 5 μm, 250 mm × 4.6 mm) with UV detection at 265 nm under the following program: solvent system (solvent A, 10% CH₃CN in water supplemented with 0.08% formic acid; solvent B, 90% CH₃CN in water), 5% B to 100% B (0–20 min), 100% B (21–25 min), 100% B to 5%B (25–26 min), 5% B (26–30 min), flow rate at 1 mL min⁻¹.

A typical in vitro enzyme reaction of FlsO1 (or its homologous enzymes) was conducted in 100 μL phosphate buffer (50 mM, pH 7.0) comprising of 200 μM PJM (8), 2 mM NADPH, 10 μM FlsO1 (or FlsO2, FlsO3, FlsO4, FlsO5, AlpK, and Nes26). The reactions were incubated for 30 min at 30 °C and then were quenched by mixing with 100 μL of ice-cold MeOH. When using NEN C (14) as the mimic substrate, the reaction was conducted in a 100 μL reaction mixture consisting of 200 μM 14, 10 μM FlsO1 (or AlpK, Nes26), 2 mM NADPH with or without 4 mM l-cysteine in 50 mM phosphate buffer (pH 7.0). The reaction mixtures were incubated for 30 min at 30 °C and were stopped by adding 100 μL ice-cold MeOH. HPLC analysis of the enzyme reactions was carried out on the Agilent 1260 Infinity series instrument (Agilent Technologies Inc., USA) using a reversed phase column Luna C18 (5 μm, 150 × 4.6 mm, Phenomenex) with UV detection at 304 nm under the following program: solvent system (solvent A, 10% MeCN in water supplemented with 0.1% formic acid; solvent B, 90% MeCN in water); 5% B to 80 % B (0–20 min), 100% B (21–24 min), 100% B to 5% B (24–25 min), 5% B (25–30 min); flow rate at 1 mL/min. For the analysis of reaction with NEN C (14), 0.1% formic acid in solvent A was replaced by 0.1% TFA (trifluoroacetic acid).

Tissues used in this study were collected from 1-year-old (leaves, stems, bark, and roots) and mature elite (flowers and hulls) black walnut trees located at Martell Forest (West Lafayette, IN, USA). Naphthoquinone standards of juglone, phylloquinone, menaquinone-4 (MK-4), and plumbagin were from Sigma-Aldrich. Unless otherwise mentioned, all other reagents were from Fisher Scientific. Ultrapure water and high-performance liquid chromatographic (HPLC)- or gas chromatographic (GC)-grade solvents were used for all metabolite extractions. HPLC analyses were carried out on an Agilent 1260 Infinity series instrument (Agilent Technologies) equipped with diode array and fluorometer detection modules employing Chemstation software. An Agilent 7890B GC coupled with a 5977A mass spectrometer (MS) employing Chemstation software was used to perform GC-MS analyses.