LC-HRESIMS analysis

For LC-HRESIMS analyses, separation was performed in a Luna C18 column (100 × 3 mm, 3 µm, 100 Å, Phenomenex). Mixtures of MeOH/H₂O 1:1 (v/v) with 0.1% formic acid (eluent A) and IPA with 0.1% formic acid (eluent B) were used as mobile phase, with a flow rate of 0.4 mL min⁻¹. Depending on the sample to be analyzed, different elution programs and injection volumes/concentrations were used, as detailed below.

Crude extracts and flash chromatography fractions obtained from the initial feeding experiments were separated (10 μL of a 1 mg mL⁻¹ solution were injected) using a gradient from 9:1 to 3:7 eluent A/eluent B in 10 min and held for 7 min before returning to the initial conditions.

Crude extracts obtained from feeding with 7-bromoheptanoic, caprylic, butyric, lauric and palmitic acids were separated using a gradient from 9:1 to 3:7 eluent A/eluent B in 10 min and held for 12 min before returning to the initial conditions.

Crude extracts of biomass from cultures that were not supplemented with fatty acids were analyzed by LC-HRESIMS using a gradient from at 9:1 to 1:4 eluent A/eluent B in 3 min and held for 28 min before returning to initial conditions. Fractions obtained from semipreparative HPLC during purification of natural bartoloside esters were analyzed (10 μL at 1 mg mL⁻¹ per injection) using a similar program, but with an isocratic step of only 24 min. Semipreparative HPLC subfractions that were analyzed by LC-HRESIMS/MS were separated (10 μL at 0.5 mg mL⁻¹ injected) using a gradient from 9:1 to 7:13 eluent A/eluent B in 5 min, increasing to 3:7 eluent A/eluent B over 15 min and held for 5 min before returning to the initial conditions.

Analysis of enzymatic reaction samples was carried out by injecting 10 μL of supernatant from the methanol/acetonitrile-quenched reaction mixture (see Enzymatic assays). Separation involved an isocratic step of 9:1 eluent A/eluent B over 2 min, followed by a linear gradient to 7:13 eluent A/eluent B over 3 min and held for 10 min, followed by a linear gradient to 3:17 eluent A/eluent B over 3 min and held for 8 min before returning to the initial conditions. For the activity dependence on temperature, esterification in D₂O assay and assays for the determination of kinetic parameters, separation employed an isocratic step of 9:1 eluent A/eluent B over 2 min, followed by a linear gradient to 7:13 eluent A/eluent and held for 7 min before returning to the initial conditions. A longer program that allowed for the separation of monoesters 7a/7b was used to analyze the assays designed to study the selectivity of the esterification of 1 and palmitic acid. It consisted of a flow rate of 0.6 mL min⁻¹ and an isocratic step of 9:1 eluent A/eluent B over 2 min, followed by a linear gradient to 9:11 eluent A/eluent B over 2 min, held for 38 min, then by a linear gradient to 3:17 eluent A/eluent B over 5 min and was held for 8 min before returning to the initial conditions.