新型表观遗传抗肿瘤药物阿扎胞苷的生物合成研究

Azacitidine, a pyrimidine nucleoside alkaloid elaborated by Streptomyces mobaraensis DSM 40578, shows good antitumor activity. As new epigenetic antineoplastic agent, azacitidine is the only natural agent which has been clinically demonstrated to be able to extent a significant overall survival for the patients with higher-risk myelodysplastic syndromes. In addition, azacitidine has been important chemical epigenetic modifier. Chemical structurally, azacytidine, as model compound of simple structure of pyrimidine nucleoside antibiotic molecules with C5 cytidine pyrimidine ring replaced by a nitrogen atom, is very rare in the pyrimidine nucleoside antibiotic, which suggested that azacytidine possesses unusual biosynthetic pathway. Until now, the biosynthesis of azacytidine has been unrevealed. Accordingly, staring with cloning the entire biosynthetic gene cluster, we attempt to establish biosynthetic metabolic pathways of azacitidine through the gene knockout and genetic compensation in vivo and enzyme catalysis in vitro experiment, and focuses on elucidating the enzymology mechanism for pyrimidine ring formation and the introduction of a nitrogen atom at C5. Multiple research technique including molecular biology and chemistry are integrated to applied in this project. Furthermore, we will endeavor to use of rational metabolic engineering, mutation biosynthesis and protein directed evolution method to increase production or the structural diversity. This investigation may fill the blank in the research of azacitidine biosynthesis, supply additional insights into the biosynthesis for nucleoside antibiotic, provide guidance and reference for the biological origin production of azacitidine.

阿扎胞苷是由茂源链霉菌S. mobaraensis DSM 40578产生的嘧啶核苷生物碱，具有良好的抗肿瘤活性，是目前唯一天然来源的且能够延长高风险骨髓增生异常综合征患者总生存期的新型表观遗传抗肿瘤药物，也是重要的化学表观遗传修饰剂。阿扎胞苷是简单结构嘧啶核苷抗生素的代表，其嘧啶环C5位被氮原子替代在核苷类抗生素中非常少见，暗示其生物合成途径较为特殊。时至今日，阿扎胞苷生物合成过程未被揭示。据此，本项目拟运用分子生物学和生物化学等多种研究手段，从克隆生物合成基因簇入手，通过体内基因敲除与回补、体外生化实验揭示其代谢途径，并重点阐明研究嘧啶环形成和C5位氮原子引入的酶学机制。在此基础上本项目将尝试运用代谢工程改造、突变生物合成和蛋白定向进化等手段提高产量或增加结构多样性。这一研究将填补阿扎胞苷生物合成研究的空白，补充人们对核苷类抗生素生物合成的认识，为阿扎胞苷的生物来源生产提供指导和参考。

阿扎胞苷是由茂源链霉菌S. mobaraensis DSM 40578产生的嘧啶核苷生物碱，具有良好的抗肿瘤活性，是目前唯一天然来源的且能够延长高风险骨髓增生异常综合征患者总生存期的新型表观遗传抗肿瘤药物，被美国全国癌症综合网络指南推荐的一线抗癌药物，2016年全球市场销售规模7.38亿美元。 据药智数据分析，2020年国内市场规模保守估算为60亿元人民币。阿扎胞苷是简单结构嘧啶核苷抗生素的代表，其嘧啶环C5位被氮原子替代在核苷类抗生素中非常少见，暗示其生物合成途径较为特殊。本项目通过优化培养基提高了产量，然后运用分子生物学和生物化学等多种研究手段，从克隆生物合成基因簇入手，通过异源表达和基因敲除等实验方法基本揭示其代谢途径。通过比较基因簇研究，找到了抗生素U-44590的生物合成基因簇。这些研究成果将为运用代谢工程改造、突变生物合成和蛋白定向进化等手段提高产量或增加结构多样性提供重要支撑。