Growth and culture conditions

Escherichia coli strains were grown at 37 °C in Luria-Bertani medium supplemented with kanamycin sulfate (50 mg.L⁻¹). P. pastoris and Y. lipolytica strains were grown at 30 °C in YPD medium or in buffered rich medium supplemented with appropriate carbon sources for each yeast. The buffered rich medium consisted in 6.7 g.L⁻¹ YNB without amino acids, potassium phosphate buffer 100 mM pH 6, 10 g.L⁻¹ yeast extract, 20 g.L⁻¹ soytone, 0.4 mg.L⁻¹ biotin, and 1.2% PTM1 solution. For Y. lipolytica, the buffered rich medium was supplemented with 20 g.L⁻¹ glycerol and 10 g.L⁻¹ erythritol as carbon source and inducer, respectively (YSPGE). For P. pastoris, the buffered rich medium was supplemented with 9.44 g.L⁻¹ sorbitol and 10 g.L⁻¹ methanol as carbon source and carbon source / inducer, respectively (YSPSM). Yeast cultures were inoculated at an initial optical density at 600 nm (OD₆₀₀) of 0.5. MG-132 was used at a final concentration of 500 μM (stock solution at 50 mM in DMSO). Shake-flask cultures were performed for 48 h in triplicate in 100- or 250-mL flasks, with incubation at 30 °C and 150 rpm. Bioreactor cultures were performed for 72 h in duplicate in DASGIP DASbox Mini Bioreactors SR0250ODLS (Eppendorf, Hamburg, Germany). Temperature was set at 30 °C, agitation at 800 rpm and aeration at 1 vvm. pH was automatically adjusted to 6 by addition of 42.5% H₃PO₄ (8 M) or NaOH (12.5 M).