2.3 Culture and fermentation conditions
In 24-well plate fermentation and flask fermentation, all E. colistrains were cultivated and fermented in LB medium at 37 ℃. The strains were all shaken at 220 rpm/min while growing in liquid medium.
24-well plates and flask fermentation were performed as follows: BL21 (DE3) strains containing pEBI and various IUP pathway plasmids of pRhaIUPnIDIsc were selected and transferred into 48-well plates/ tubes with 500 μL/3 mL LB liquid medium adding according antibiotics (chloramphenicol (CmR) 25 μg/mL and ampicillin (AmpR) 100 μg/mL) for 12 h at 37 ℃. Then, bacterial culture was transferred into 24-well plates/flask with 2 mL diluted to OD600=0.1 and then cultivated in liquid LB medium at 37 ℃. When OD600 reached 0.6-0.8, L-arabinose (Ara), L-rhamnose (Rha) and anhydrotetracycline (aTc) were added to a final concentration of 10mMol/L, 10mMol/L and 100 ng/mL respectively. When OD600 reached 2-4, 0.005~ 0.1 mMol/L IPTG and 1~3 g/L prenol/isoprenol was added and the fermentation continued for 24 h at 37 ℃.
The methods for the fed-batch fermentation are referred to this study with modifications as follows: the fed-batch fermentation for lycopene production was performed in a 5 L bioreactor with a working volume of 2.4 L at 37 ℃ for 40 h. 10 mM/L Rha was added after 3 hours’ cultivation and 2 g/L substrates (prenol: isoprenol=1:3) were added to the bioreactor at 6 h.