新型P450scc的挖掘与改造及其在甾体上的应用研究

孕烯醇酮和黄体酮是甾体药物合成中两个核心前体，其传统化学合成极其困难，亟待发展高效、绿色的替代方法。动物来源P450scc可直接将胆固醇转化成孕烯醇酮，为孕烯醇酮合成带来新的契机。然而动物来源P450scc活性低，表达宿主细胞繁殖慢、培养成本高，生产方法尚未确立；同时，动物来源P450scc利用微生物系统表达同样面临蛋白不可溶、活性低等难题，难以实现工业应用。本项目中，我们以首次发现的可将胆固醇和4-胆甾烯-3-酮转化成孕烯醇酮和黄体酮的细菌来源的新型P450scc出发，解析其催化机制和晶体结构，挖掘新的P450scc同源蛋白并拓展底物谱获得新甾体结构，为P450scc活性研究提供基础；运用酶工程、电子传递系统适配和催化条件优化等策略，大幅提高P450scc活性并放大催化反应，建立高效、绿色且具有工业生产前景的合成孕烯醇酮和黄体酮的替代方法，为甾体药物生物合成的工业化带来新突破。

Pregnenolone(PN) and progesterone(PG) are the two core precursors in the synthesis of steroid drugs, their traditional chemical synthesis is extremely difficult, urgently need to develop efficient and green alternatives. The animal-derived P450scc can directly convert cholesterol into pregnenolone, bringing new opportunities for the synthesis of pregnenolone. However, its production method has not been established owing to the low activity of animal-derived P450scc, the slow multiplication and high culturation cost of expression host cell; simultaneously, animal-derived P450scc also faces the conundrum of protein insoluble and low activity when expressed in the microbial system, which is difficult to achieve industrial applications. Here, based on the microorganism-derived P450scc discovered for the first time in our previous studies that can convert cholesterol and 4-cholesten-3-one into pregnenolone and progesterone, we will decipher the catalytic mechanism and determinate.crystal structure, uncover new P450scc homologous proteins and expand the substrate spectrum to obtain new steroid structures, provide a basis for the study of the activity of P450scc; Meanwhile, we will greatly improve the activity of P450scc and amplify the catalytic reaction by combining enzyme engineering、electron transport systems and catalytic condition optimization, ultimately establish an efficient and green alternative method for the synthesis of pregnenolone and progesterone with industrial production prospects, the successful establishment of this method will bring new breakthroughs for the industrialization of steroid drug biosynthesis.