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.