Mes monohydrate

Ultrapure water was obtained from a MilliQ purification system from Millipore (Bedford, MA, USA). Acetonitrile (ACN) and methanol (MeOH) were LC-MS grade (Optima^®) and obtained from Fisher Chemical (Reinach, Switzerland). Trifluoroacetic acid (TFA, ≥99.0%) was obtained from Biosolve BV (Valkenswaard, the Netherlands). Thorium-232 (Th) pure standard at 1.0 mg mL⁻¹ in 2% HNO₃ was purchased from PerkinElmer (Schwerzenbach, Switzerland). Ammonium hydroxide (28.0–30.0%), citric acid (≥99.5%), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA, 99.0–101.0%), formic acid (≥98.0%), MES monohydrate (≥99.5%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, ≥98.0%), N-hydroxysuccinimide (≥99.0%), N,N-dimethylacetamide (DMA, 99.8%), p-aminobenzoic acid (pABA, 99.0%), and succinimide (≥98.0%) were obtained from Sigma-Aldrich (Buchs, Switzerland). The 3,2-HOPO chelator and TOP-succinimide, as well as the ACCs, were provided by Bayer AG (Wuppertal, Germany).

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC), N-hydroxysulfo- succinimide (Sulfo-NHS) and 1-step PNPP solution were obtained from Thermo Scientific Inc. (Rockford, IL, USA). 1,2-Distearoyl-sn-glycero-3-phosphocholine, rosmarinic acid, gallic acid, sodium hydroxide, potassium hydroxide, MES monohydrate, Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma (St Louis, MO, USA). Magnesium hydroxide was purchased from Showa Chemical Co. (Tokyo, Japan). Sesame oil was purchased from a local market. Lithospermic acid B (LSB) was purified and provided by the Ko Da Pharmaceutical Co. (Taoyuan, Taiwan). Sodium lithospermate (Na-LSB), potassium lithospermate (K-LSB) and magnesium lithospermate (Mg-LSB) were prepared as described previously [27 (link)]. Triton X-100 and Tris base were purchases form Amersco (Solon, OH, USA). Polyethylene glycol 8000 was purchased from Bio Basic Inc. (Markharn, Ontario, Canada). 96-well flat-bottomed Immuno Maxisorp plates were purchased from Thermo (Nunc, Roskilde, Denmark). Alkaline phosphatase conjugated affinipure Rabbit Anti-Chicken IgY was purchased from Jackson Immuno-Research (West Grove, PA, USA).

To measure the production of melatonin, bacterial cells were cultivated in deep-well plates in glucose slow-release (GSR) medium mimicking fed-batch fermentation at 30°C. 1 L of GSR medium contains 15 g of Maltodextrin (dextrose equivalent 4.0–7.0, Sigma-Aldrich), 200U of Amyloglucosidase (Sigma-Aldrich, ∼70 U/mg) for glucose release, 40 g of MES monohydrate (Sigma-Aldrich), 1.2 g of K₂HPO₄, 7 g of Ammonium sulfate, 120 mg of Sodium citrate, 8 mg of ZnCl₂, 12 mg of FeSO₄⋅7H₂O, 9 uM of CaCl₂, 12.5 mM of MgSO₄⋅7H₂O, as well as trace elements and vitamins. The media pH was adjusted to 6.4. The medium was supplemented with 500 mg/L tryptophan, 50 mg/L kanamycin, and 100 mg/L ampicillin. The bacterial cells were cultivated at 30°C in M9 medium supplemented with 0.5% glucose for 24 h. The precultures were diluted 100 times into the GSR medium and cultured at 30°C. After 24 h, the supernatant was collected by filtering the broth with 0.2 μm filters (Pall, New York, United States). The concentration of melatonin in the supernatant was determined by HPLC as previously described (Luo et al., 2019 (link)).