Mycelium Amino Acid Analysis Protocol

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.