Aas18 analytical standard

Mycelium for amino acid analysis was harvested during steady-state according to the method described by de Jonge et al.^{65 (link)}. In brief, samples of 10 ml or less were quenched directly into −20 °C 40% methanol (20 ml), weighted and filtered using a vacuum pump followed by a single washing 1x with the same volume of ice-cold 40% methanol before freezing in liquid nitrogen and storage at −80 °C until extraction. For extraction, filter papers with frozen sample were directly placed in 50 ml falcon tubes containing 20 ml 73 °C hot 75% ethanol, shaken vigorously, boiled for 3 min at 95 °C, chilled on ice for 5 min, centrifuged for 5 min at 4000 × g⁻¹ and filtered over a 0.2 µm cellulose acetate filter (VWR). 1 ml aliquots were concentrated in a speed-vac (Eppendorf) for 45 min at 30 °C, centrifuged for 10 min at 10.000 × g⁻¹. Supernatant was stored at −80 °C if not used immediately for LC-MS analysis. All extractions were performed in quadruplicate per bioreactor run and analyzed in technical duplicate on LC-MS. Amino acid retention times were verified by a standard mixture (AAS18 Analytical standard; Sigma Aldrich) or dilutions of pure amino acids in 10 mM HCl (for Asn, Gln, Trp). Peak areas were corrected for extracted biomass and concentrations were calculated using a calibration curve.

This analysis was done to characterize the compounds presence in the EBN extract. Approximately 2 mg of EBN extract was dissolved in 1 ml of HPLC grade methanol. The solution was sonicated for 15 min and filtered through a membrane filter with pore size 0.22 μm before analysis. About 10 μL of the sample was injected into the AccelaTM UHPLC System (Thermo Scientific, San Jose, United States) equipped with quaternary pump, a build degasser, a PDA detector and an auto-sampler. Separation of the EBN sample was carried out by using A Luna Kinetex RP C18 column (2.6 μm, 2.1 mm I.D. x 150 mm) at a flow rate of 200 μL/min over 40 min by using acetonitrile: 0.1% formic acid in water as eluent. The step gradient and isocratic solvent composition was depicted in Supplementary Table S1.
The sample was analyzed by LTQ mass spectrometer (Thermo Scientific, San Jose, United States) with m/z spectrum ranged from 50 to 1000 was recorded in negative ionization mode. The setting of electrospray ionization modes was as follows: source accelerating voltage, 4.0 kV; capillary temperature, 350°C; sheath gas flow, 40 arb and auxiliary gas, 20 arb.
A reference standard of 18 types of amino acid (AAS18, analytical standard) was obtained from Sigma-Aldrich and used in this analysis. This reference standard was separated and analyzed under the same setting explained above.