The optimal conditions for violacein production by the genetically engineered strains of Citrobacter freundii using L-tryptophan as a precursor were systematically studied.The effects of different cultivation parameters (culture medium

temperature

carbon source

dissolved oxygen

seed state

inoculums size

inducer concentration

initial pH and etc.) on cell growth and violacein production were studied by design of single-factor experiments.By orthogonal experiments study

the primary-secondary order and their optimal mix of four factors ［the optical density (OD660) of the seeding culture

inoculums size

induction timing

inducer concentration］ that were likely to occur interaction were determined.The results showed that the optimal conditions for violacein production by the recombinant C.freundii were as follows: 3 % (vol) of seed culture (OD660=3.6) was inoculated in phosphate medium E2 (25 μg·ml-1 kanamycin) with glycerol as carbon source (initial pH6.5)

after fermentation with shaking at 37℃

200 r·min-1 for 5 h until the OD660 reached 1.4

0.5 ml·L-1 n-octane was added as inducer

then the cultures were transferred to 20℃

150 r·min-1 to induce the synthesis of violaceins for 31 h.Under the optimal conditions

the produced crude violaceins (the mixture of violacein and deoxyviolacein) reached 1.809 g·L-1

which was 252% higher than that before the optimization (0.514 g·L-1)

and was 4.2 folds of the highest yield by flask culture previously reported by other research groups (0.43 g·L-1).Moreover

plasmid stability experiment showed that recombinant C.freundii has a good genetic stability under selective pressure of antibiotics.