Culture conditions of S. erythraea
XM solid medium was used for the growth of S. erythraea spores
which were incubated at 34 °C for 6 days. About
1 cm2 XM solid
medium covered with dense spores was picked into 300 mL flasks with 30
mL preculture medium. The component of XM solid medium and preculture
medium were described previously (C. Chen et al., 2017). After a
preculture of 48 h, 3 mL seed culture was collected, resuspended in 1 mL
PBS buffer (pH=7.4) and inoculated into flasks with 27 mL modified
minimal liquid medium for phenotype characterization. The modified
minimal liquid medium (MMLM) contained the following per 800 mL: 2 g
(NH4)2SO4, 5 g casamino
acids (DifcoTM, BD), 0.6 g MgSO4∙7H2O,
0.001 g ZnSO4∙7H2O, 0.001 g
FeSO4∙7H2O, 0.001 g
MnCl2∙4H2O, 0.001 g
CaCl2. 800 mL MMLM was then dispensed in 80 ml aliquots.
15 mL
NaH2PO4/K2HPO4buffer (0.1 M, pH=6.8) was added in 80 mL liquid medium for pH
buffering. After autoclaving, 25% (w/v) glucose as sole carbon source
was injected in the medium to a final concentration 20 g/L. Liquid
culture in flasks was performed in shaking incubator with an agitation
speed of 220 rpm at 34
°C.
The fermentations of E3 and E3::sucBA were carried out in a 5-L
fermenter (Shanghai Guoqiang Bioengineering Equipment Co., Ltd., China).
Samples were taken every 12 or 24 h for monitoring cell growth and
erythromycin production. CER were determined by process mass
spectrometer.