2.2 Culture conditions
The E. coli strains was grown in Luria Bertani (LB) or Terrific Broth (TB) medium at 37 ℃, supplemented where necessary with 100 μg/mL kanamycin (Kan). Flask fermentation was performed as follows: E. coli BL21 (DE3) harboring pET24a-GDH was grown in 3 mL of LB liquid medium overnight at 37℃. Then, 300 uL of the resulting culture were used to inoculate 30 mL of Terrific Broth (TB) medium in a 250ml shake flask. Cells were cultured at 37 ℃ to an OD600 of 2-4, at which point IPTG was added to a final concentration of 0.3 mM, and the fermentation was allowed to continue at 28℃ for an additional 40 h.
The GDH expression strains were first pre-cultured at 37 ℃, 220 rpm for 16 h seeded into a 500 mL shake flask containing 100 mL of TB medium, and cultured at 37 oC, 220 rpm for 5 h. In the scale-up fermentation experiments, the resulting 100 mL seed culture was used to inoculate a 7.5-L fermenter containing 4 L of TY medium (Tao et al., 2014) at 37 ℃ at an aeration rate of 2.5 vvm. Agitation was steadily increased to a maximum of 800 rpm to maintain 20% dissolved oxygen (DO). When the DO further increased to more than 60%, 400 g/L glycerol, 50 g/L yeast extract, and 25 g/L tryptone were added into the medium, and the fermentation was continued at 800 rpm at an aeration rate of 2.5 vvm. When the OD600 reached 20, 0.2 mM IPTG was added to the medium and the cells were allowed to express the target protein at 28 ℃ for 43 h.