重组柠檬酸杆菌合成紫色杆菌素的工艺条件优化

以L-色氨酸为前体物,对弗氏柠檬酸杆菌(Citrobacter freundii)基因工程菌株生产紫色杆菌素的工艺条件进行了优化。通过单因素实验研究了培养基种类、培养温度、碳源、溶氧、种子液的状态、接种量、诱导时机、诱导剂的浓度及初始pH对细胞生长和紫色杆菌素产量的影响,通过正交实验研究了这些因素中可能存在交互作用的4个因素(种子液OD660、接种量、诱导时机、诱导剂浓度)的影响主次,确定了这些因素、水平的最佳搭配方案。结果表明,重组柠檬酸杆菌合成紫色杆菌素的最优培养条件为:种子液培养至OD660=3.6时,以3%的接种量接种于以甘油为碳源、初始pH为6.5的磷酸盐发酵培养基E2(25μg.ml-1卡那霉素),先在37℃、200r.min-1下培养至OD660=1.4,然后加入0.5μl.ml-1的诱导剂正辛烷,同时转入20℃,在150r.min-1下诱导培养31h。在此最优条件下,紫色杆菌素粗提物(紫色杆菌素及脱氧紫色杆菌素的混合物)产量可达1.809g.L-1,比优化前(0.514g.L-1)提高了252%,是目前国际上其他研究小组报道的最高摇瓶产量(0.43g.L-1)的4.2倍。质粒稳定性实验结果表明重组柠檬酸杆菌在抗生素选择压力条件下具有良好的遗传稳定性。

Optimization of fermentation conditions for violacein production by recombinant Citrobacter freundii

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.