Yeast extract

Fermentation was
performed as follows: to test the riboflavin biosynthesis
ability of the constructed strains, 20 μL of a cryopreservation
stock stored at −80 °C was mixed with 100 μL of
sterile ddH₂O, spread on an agar plate, and incubated at
37 °C for 24 h. After that, the colonies on the plate were scraped
and suspended in 1 mL of the fermentation medium to acquire the seed
suspension, which was used to inoculate 500 mL baffled shake flasks
containing 80 mL of the fermentation medium, adjusting the initial
OD₆₀₀ to 0.1. The fermentation medium contained 40 g/L
sucrose, 16.5 g/L corn steep powder (Solarbio, Beijing, China), 5
g/L (NH₄)₂SO₄, 5 g/L yeast extract
(Solarbio, Beijing, China), 0.5 g/L MgSO₄, 3 g/L K₂HPO₄, 1 g/L KH₂PO₄, and corresponding
antibiotics. The fermentation was conducted at 37 °C and 180
rpm for 48 h, during which samples were drawn at 12, 18, 24, 36, 42,
and 48 h to measure the cell growth and the titer of riboflavin. Cell
growth was monitored by measuring the optical density at 600 nm, and
the riboflavin concentration was determined as follows: culture samples
were diluted with 0.01 M NaOH to the linear range of the spectrophotometer.
After dissolving for 20 min, 1 mL of samples was centrifuged at 10,000g for 2 min to remove the cell debris, and the absorption
at 444 nm (A₄₄₄) was measured immediately.^{7 (link)}