Construction of Engineering Bacteria for Degrading Phytosterol to 9α-hydroxy-4-androstene-3, 17-dione and Their Fermentation Process Optimization
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摘要: 9α-羟基雄烯二酮(9α-OH-AD)作为一种不可替代的甾体药物中间体,是合成多种皮质类甾体激素类药物的重要原料。本研究通过在产雄甾-4-烯-3,17-二酮(AD)的菌株Mycobacterium sp.TFZ中表达3-甾酮-9α-羟基化酶(KSH)基因kshA和kshB,获得了一株可直接转化植物甾醇生产9α-OH-AD的工程菌株Mycobacterium sp.TFZ3215,然后以9α-OH-AD产量为评价指标,对油水两相转化体系中添加乳化剂吐温的种类和含量进行优化。研究结果表明:与出发菌株Mycobacterium sp.TFZ相比,工程菌Mycobacterium sp. TFZ3215发酵液中9α-OH-AD的含量由1.7%提高到94.7%,AD的含量由80.1%减少到3.4%。进一步通过在油水两相发酵体系中添加2 g/L的吐温-80后,9α-OH-AD的产量由1.69 g/L增加到7.53 g/L,提高了3.4倍。本研究成功构建了高产9α-OH-AD的工程菌株,并对油水两相发酵体系进行了优化,9α-OH-AD的产量达到7.53 g/L,具有极好的产业化前景。Abstract: 9α-hydroxy-4-androstene-3, 17-dione (9α-OH-AD) was an irreplaceable intermediate of steroid drugs, which was an important raw material for the synthesis of many corticosteroid drugs. In this study, the 3-sterone-9α-hydroxylase (KSH) gene kshA and kshB were successfully expressed in androster-4-ene-3, 17-dione (AD) producing strain Mycobacterium sp.TFZ, and a 9α-OH-AD engineering strain was obtained. The yield of 9α-OH-AD was used as an evaluation index to optimize the type and content of emulsifier Tween in the oil-water two-phase conversion system. The results showed that, compared with the starting strain Mycobacterium sp.TFZ, the content of 9α-OH-AD in the fermentation broth of the Mycobacterium sp.TFZ3215 increased from 1.7% to 94.7%, and the AD contents decreased from 80.1% to 3.4%. After adding 2 g/L of Tween-80 to the oil-water two-phase fermentation system, the yield of 9α-OH-AD increased from 1.69 g/L to 7.53 g/L, which increased by 3.4 times. In this study, a high-yield 9α-OH-AD engineering strain was successfully constructed, and an oil-water two-phase fermentation system was optimized. The yield of 9α-OH-AD reached 7.53 g/L, which had excellent industrialization prospect.
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