Amino acid quantification using GC-MS

For metabolomics experiments, cells were pre-cultured in YES overnight, washed and re-suspended in EMM and grown for 7 h till early-exponential phase at the time of harvest. The equivalent of 2 OD_595nm was quenched by direct injection into 100% −80 °C LCMS-grade methanol (Sigma Aldrich). Polar metabolite extraction was adapted from a protocol described in ref. ^84,85 Briefly, samples were extracted twice, for 15 min, on ice in LCMS-grade acetonitrile/methanol/water (2:2:1 v/v) with 1nmol scyllo-inositol internal standard per sample (Sigma Aldrich). Sample debris was then removed via centrifugation and polar metabolite extracts were dried using a SpeedVac Vacuum Concentrator. Dried extracts were phase-separated using −20 °C LCMS-grade chloroform/methanol/water (1:3:3, v/v) (Sigma Aldrich).

240 µl of upper, polar phase was dried into GC-MS glass vial inserts, followed by two 30 µl methanol washes. Derivatisation was performed as previously described⁸⁶ Briefly, samples were incubated overnight in 20 µl of 20 mg ml⁻¹ methoxyamine hydrochloride in pyridine (Sigma Aldrich). The next day, 20 µl of N,O-bis(trimetylsilyl)trifluoroacetamide (BSTFA) and 1% trimethylchlorosilane (TMCS) (Sigma Aldrich) was added and samples were incubated at room temperature for at least 1 h before GC-MS analysis.

Metabolites were detected using Agilent 7890B-MS7000C GC-MS in EI mode as previously described⁸⁶. Samples were injected in a random order alongside metabolite standards and regular hexane washes. Splitless injection was performed at 270 °C in a 30 m + 10 m x 0.25 mm DB-5MS + DG column (Agilent J&W) and helium was used as the carrier gas. The oven temperature was set as follows: 70 °C (2 min), gradient of 70 °C to 295 °C at 12.5 °C per minute, gradient of 295 °C to 350 °C at 25 °C per minute and a 3-min hold at 350 °C. Data was acquired using MassHunter version B.07.02.1938 software (Agilent Technologies).

Samples were analysed using a combination of MassHunter Workstation (Agilent Technologies) and MANIC, an updated version of the software GAVIN⁸⁷ for metabolite identification using retention times and mass spectra and integration of target fragment ion peaks. For abundance quantification, integrals, the known amount of scyllo-inositol internal standard (1 nmol) and the known abundances of a standardised metabolite mix run in parallel to samples (kindly gifted by Dr. James I. MacRae, Francis Crick Institute) were used to calculate an estimated nmol abundance of each metabolite in each sample. The formula used for calculating molar abundances as shown in Eqs. (1) and (2). Abundances were normalised to cell pellet weight.

MRRF molar relative response factor, SI scyllo-inositol (internal standard), met metabolite to be quantified; mm standard metabolite mix, int integrals, s samples.

Raw metabolomics data are shown in Supplementary Data ³.