高产莫能菌素肉桂地链霉菌的透明颤菌vgb基因异源表达

Heterologous integrated expression of Vitreoscilla vgb in highly monensin producing Streptomyces cinnamonensis

肉桂地链霉菌(Streptomyces cinnamonensis)发酵产生的聚醚类抗生素莫能菌素(Monensin)因其环保且高效的特性而被广泛应用在医药、农业等领域,但莫能菌素实际生产中普遍的低氧条件严格限制了肉桂地链霉菌发酵莫能菌素水平。该文在前期整合表达莫能菌素生物合成基因簇中途径特异性正向调控基因(monH、monRⅡ)基础上,尝试通过异源整合表达透明颤菌血红蛋白基因vgb,以期通过改善菌株摄取溶氧能力来提高莫能菌素产量。结果显示,在40 mmol/L Ca^(2+)、供受体菌比例100∶1和培养18 h覆盖抗生素条件下,接合转移效率提高3倍;成功将基因vgb与莫能菌素生物合成途径特异性调控基因monH/monRⅡ进行串联整合,得到工程菌2110-monH-vgb和2110-monH-monRⅡ-vgb,其摇瓶生物量在高限氧条件下稍有提高(6%),但发酵效价分别较异源表达前提高18.0%和21.3%;后者在5 L罐水平生物量较异源表达前略有提高,发酵效价高达11.5 kU/mL,较出发菌提高47.3%。通过优化遗传操作系统并用于肉桂地链霉菌发酵产莫能菌素基因工程改造,成功获得有效提高莫能菌素发酵效价的vgb异源串联整合表达菌,为其后续的工业应用奠定基础。

Monensin is a kind of polyether antibiotic produced by Streptomyces cinnamonensis,which is widely used in fields including medicine and agriculture due to its environmentally friendly and efficient properties.However,the low oxygen condition commonly existing in the monensin production process strictly limited the monensin fermentation level.Based on the previous integrated expression of the monensin biosynthesis pathway specific regulatory genes monH and monRII,this study attempted to integrating Vitreoscilla hemoglobin gene vgb by heterologous expression to increase the monensin fermentation titer by improving the oxygen uptake capacity of strains.Results showed that the optimized conditions(40 mmol/L Ca^(2+),donor-recipient ratio 100∶1,antibiotics adding time 18 h)significantly improved the conjugation efficiency by 3 times.With successful integration of vgb with monH and monRII,two engineering strains,2110-monH-vgb and 2110-monH-monRII-vgb were obtained.They produced 18.0%and 21.3%of the monensin higher than that without heterologous expression,respectively,under the extremly weak oxygen shake-flask condition.The biomass of 2110-monH-monRII-vgb in a 5 L bio-fermentor was slightly higher than that of the starting strain,but its monensin titer reached 13.4 kU/mL,which was 55.7%higher than that of the starting strain.In conclusion,by optimizing the conjugation efficiency of S.cinnamonensis and applying it to the genetic engineering of S.cinnamonensis in monensin fermentation,this study successfully obtained a vgb integrated strain by heterologous expression that effectively increased the monensin fermentation level,which would supply a substantial foundation for its subsequent industrial application.