2 oxoglutarate

Determination of metabolites in the extracellular medium was performed as in Díaz-Troya et al. (2020) (link) with minor modifications. A 6 ml aliquot of culture samples was centrifuged (15 000 g for 10 min at 4 °C) and supernatants were quick-frozen in liquid nitrogen, lyophilized (VirTis BenchTop Pro Freeze dryer, SP Scientific), and stored at –20 °C until analyzed. Samples were resuspended in 600 µl of H₂O, and 10–100 µl aliquots were analyzed by enzyme-coupled assays in 375 mM Tris–HCl (pH 7.5) with 0.11 mM NADH (and 50 mM NH₄Cl for 2-oxoglutarate quantification). The reaction was triggered by the addition of 5 mU of lactate dehydrogenase (Sigma) or 12 mU of glutamate dehydrogenase (Sigma) for the determination of pyruvate or 2-oxoglutarate, respectively. When NADH oxidation was completed, the remaining NADH was measured spectrophotometrically at 340 nm to calculate the concentration of the metabolite compared with standard curves of known amounts of pyruvate (Sigma) and 2-oxoglutarate (Sigma).

Nicotine amide adenine dinucleotide (NAD) disodium salt, 3-phosphoglycerate [D-(–)-3-phosphoglyceric acid disodium salt], l-homoserine, d-homoserine, l-homocysteine, 2-oxoglutarate (α-ketoglutaric acid sodium salt), oxaloacetate, cis-aconitate, molecular weight standards (Gel Filtration Markers Kit for Protein Molecular Weights) for size exclusion chromatography analyses, and Murashige–Skoog (MS) vitamin solution were purchased from Sigma–Aldrich Co., Ltd. (St. Louis, MO, USA). The other amino acids, the salt mixture of MS medium, Good’s buffers, NADH (β-diphosphopyridine nucleotide disodium salt, reduced form), fumarate, malate (l-malate), citrate (citric acid monohydrate), and isocitrate [trisodium ( ± )-isocitrate n-hydrate] were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).