Fig. 8

Strain improvement by varying isobutanol pathway gene copy number. a Shake-flask fermentation of PP310 strain, which has one additional copy of the PpIlv6_T2A_PpIlv2 expression cassette integrated into the yeast chromosome. b Shakeflask fermentation of PP302 strain, which contains additional copies of isobutanol pathway genes LlkivD and ScADH7 on an episomal plasmid. c Shake-flask fermentation of PP303 strain, which contains additional copies of isobutanol pathway genes PpIlv5 and PpIlv3 on an episomal plasmid. d Shake-flask fermentation of PP304 strain, which contains additional copies of isobutanol pathway genes PpIlv6 and PpIlv2 on an episomal plasmid. The engineered strains were pre-cultured in 5-mL aliquots in MGY (2% glycerol) minimal medium overnight and used to inoculate 50 mL fresh MGY_glu (10% glucose) in 250-mL Erlenmeyer flasks to achieve an initial optical density of 0.05 at 600 nm (OD600). The cultures were grown at 30 °C and 250 rpm in an orbital shaking incubator. Samples were taken at several time points and the supernatants were analyzed on HPLC to quantify the levels of isobutanol, glucose and other metabolites. Values are the mean of three biological replicates ± standard deviation (n = 3)