二硫吡咯酮生物合成途径中N4甲基转移酶的表征及组合生物合成应用

Dithiolopyrrolone antibiotics have broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, and even possess anti-cancer property. It has been also reported that a series of chemical derivatives of dithiolopyrrolone compounds are high-effective for increasing white blood cells. In chemical structure, the differences among the members of dithiolopyrrolone family are the methylation on N4 and acylation on N7, which implies the modification on N4 and N7 during the biosynthesis of this kind of compound could be manipulated. The cloning of biosynthesis gene cluster and the biosynthesis pathway of representative members of dithiolopyrrolone like holomycin, thiolutin and thiomarinol were reported previously and the metabolic engineering application of those pathway is starting up. However, the enzyme responsible for the N4 methylation of this kind of compound has never been characterized before and the combinatorial biosynthesis application system of its biosynthesis pathway hasn’t been built yet. Our project is planning to use the genome sequencing and high-thoughput conjugation to position the N4 methylation which is located outside of the gene cluster, using in vivo and in vitro function characterization to elucidate the molecular machnism of its catalysis. By constructing chassis cell and expression vector of each functional component, the combinatorial biosynthesis system could been built and synthesis more than one types of dithiolopyrrolone compound could be realized, which could provide theoretical basis and Technical guidance for drug design and development of this types of compound.

二硫吡咯酮类抗生素具有广谱的抗革兰氏阳性和阴性菌活性，并能有效抑制肿瘤细胞的生长，其一系列结构衍生物还具有显著升高外周血白细胞的功效，但是其生物合成途径的组合生物合成应用系统一直未被建立。该家族化合物结构上的差异在于N4位的甲基化和N7位的酰基化，意味着N4、N7位具有潜在的可工程操作性。前期研究报道了二硫吡咯酮代表性成员全霉素、硫磺藤菌素和Thiomarinol的生物合成基因簇的克隆及其生物合成途径，但负责该类化合物N4位甲基化的酶一直未被表征，一定程度上影响了这些途径的代谢工程应用。因此，本项目拟以基因组测序和高通量接合转移的方法，定位基因簇以外的N4位甲基转移酶，采用体内体外功能表征阐明其催化的分子机制，并通过构建底盘细胞和各个功能元件的表达载体，建立二硫吡咯酮的组合生物合成体系，为该类化合物的设计与开发提供理论依据和技术指导。

微生物天然产物是新药发现和发展的重要源泉。然而近年来新结构、新活性的天然产物发现率愈来愈低，组合生物合成与合成生物学是突破这一瓶颈的最佳选择之一。本项目从新分离的一株土壤链霉菌Streptomyces sp. MG17入手，通过全基因组测序、生物信息学和分子生物学手段，克隆了一个新的二硫吡咯酮类化合物生物合成基因簇（tan）。深入的序列分析找出了该类化合物生物合成的8个必需酶。通过高通量接合转移和转座子插入随机失活定位了N4甲基转移酶（Tan15）并进一步对其进行表征和代谢工程应用，在体内实现了holomycin向thiolutin的转化，填补二硫吡咯酮生物合成途径中的空白。.为建立二硫吡咯酮类化合物的生物组合生物合成体系，本项目通过收集菌种资源建立二硫吡咯酮类抗生素的菌种与分子信息资源库、构建二硫吡咯酮类化合物产生的底盘细胞。利用N7位酰基转移酶的种类多样性和底物选择宽泛性的特点，在底盘细胞里添加其它来源的酰基转移酶或者添加长链脂肪酸活化相关的酶RkA，实现了holomycin及其同系物的合成，从而证实二硫吡咯酮类化合物的生物合成途径可以进行组合生物合成。为进一步通过组合生物合成和代谢工程的方法获得更多的结构新颖、高效低毒的“非天然”二硫吡咯酮类化合物提供理论借鉴。