2.1 Strains and culture conditions
Engineering Y. lipolytica stain YL-C11 (matA, leucine+, uracil+, xpr2-322, axpl-, Δku70, Δsnf:: tHMG-carB-carRA-ggs1, Δgut2:: did2-ura3) was used in this study.
The rejuvenation of strain was carried out on YPD agar medium. Single colony was pre-cultured in YPD medium at 30℃, 180 rpm for 24h. Then draw 1 ml the pre-cultured solution to the 250 ml shake-flask containing 50 ml medium, which containing (100 ml) 3 g glucose, 1 g casein peptone, 1 g yeast, 0.3 g (NH4)2SO4, 0.25 g KH2PO4, and 0.05 g MgSO4. The seed was incubated at 30℃, 180 rpm for 48 h.
2.2 Fed-Batch fermentationandDO-stat Fed-batch fermentation
Fed-batch and DO-stat fed-batch fermentation were performed in a 5 L bioreactor (Bioflo 110, New Brunswick, USA) with working volume at 4 L. The medium was identical to the shake flask medium apart from glucose concentration. An aeration rate of 0.25-1.25 vvm, agitation speed was at 400-900 rpm, 30℃, and pH 5.5 was controlled with 15% ammonia, separately.
In fed-batch fermentation, the glucose was maintained at around 5 g/L. In the early stages of fermentation, when the value of O2 was less than 15%, the aeration rate and agitation speed increased alternately until the aeration rate and agitation speed were reached a maximum of 1.25 vvm and 900 rpm.
In DO-stat fed-batch fermentation, when the aeration rate and agitation speed reached max, the pure glucose solution was pumped into the fermenter to maintained DO at 10-20%. (NH4)2SO4 (5%) was added at 24th, 48th, and 72nd hour to avoid pH rise.