Fig. 9

Production of isobutyl acetate (a) and isoamyl acetate (b) in engineered P. pastoris. Engineered strains were pre-cultured in 5-mL aliquots in MGYH minimal medium overnight and used to inoculate 5 mL fresh MGYH (with 10% glucose as the main carbon source) to achieve an initial optical density of 0.05 at 600 nm (OD600). The cultures were overlayed with 5 mL hexadecane and grown at 30 °C and 250 rpm in an orbital shaking incubator. The hexadecane layer from each sample was taken at two different time points (48 and 96 h) and the samples were analyzed on GC–MS to quantify the isobutyl acetate ester and isopentyl acetate ester content