Combinatorial biosynthesis of fungal benzenediol lactone polyketides

 说明书(申请人提供)：苯二醇内酯(BDLS)是真菌聚酮类天然产物，具有多种生物活性。不同的BDL是热应激反应系统或不同的丝裂原激活的蛋白激酶的高度特异和有效的抑制物，而另一些则是各种受体的配体。对MAP激酶的抑制可以转化为对依赖于这些调节因子突变形式的癌细胞株的强大的抗增殖活性。同样，进化上保守的伴侣蛋白Hsp90是癌症化疗的有效靶点，其抑制会导致多条致癌途径的联合阻断。BDL是由一对相互协作的迭代聚酮合成酶(IPKS)生物合成的，代表着朝着模块化聚酮合成酶迈出的概念性一步。BDL的生物合成还包括在特定剪裁酶的催化下，对聚酮支架进行PKS后修饰。对于目前的应用，我们提出了一个四管齐下的方法：1.组装一个史无前例的BDL生物合成工具箱；2.利用这个工具箱开发组合生物合成方法，产生大量非天然的BDL类似物和同系物；3.破译酶结构对IPKS生物合成规则的贡献；以及4.在各种生物测试中对产生的BDL进行评估，以发现用于药物开发的先导化合物。我们将使用传统的克隆管道以及基因组挖掘和合成生物学从各种真菌中克隆大量的BDL生物合成簇。我们将在酵母异源宿主中表达生物合成基因，并将监测新的和已知的BDL的生物合成。我们将通过混合和匹配不同的IPKS来研究组合生物合成；通过结构域交换来创建混合合成酶；以及使用后PKS酶进行组合剪裁。我们还将使用结构域交换、酶结构数据、同源建模和定点突变来定义决定第一环环化几何形状和链终止谱系的酶结构特征。最后，将分离、结构鉴定新的BDL同系物，并在各种基于细胞的和体外生化分析中评估其生物活性。这些经验教训将促进我们对迭代酶催化BDL生物合成特别是真菌聚酮生物合成的理解。改进的组合生物合成方法将允许生产新的BDL和其他聚酮类化合物。在这些实验中获得的BDL类似物可能为开发癌症、免疫系统紊乱、炎症或神经退行性疾病以及真菌感染性疾病的治疗方法提供先导化合物。

 DESCRIPTION (provided by applicant): Benzenediol lactones (BDLs) are fungal polyketide natural products with diverse biological activities. Different BDLs act as highly specific and potent inhibitors of the heat stress response system or diverse mitogen-activated protein kinases, while others are ligands for various receptors. Inhibition of MAP kinases translates to a potent antiproliferative activity against cancer cell lines that depend on mutant forms of these regulators. Similarly, the evolutionarily conserved chaperone Hsp90 is a validated target for cancer chemotherapy whose inhibition leads to a combinatorial blockade of multiple cancer-causing pathways. BDLs are biosynthesized by pairs of collaborating iterative polyketide synthases (iPKSs), representing a conceptual step towards the modular polyketide synthases. BDL biosynthesis also involves post-PKS modification of the polyketide scaffolds, catalyzed by specific tailoring enzymes. For the current application, we propose a four-pronged approach that will: 1. assemble an unprecedented genetic toolbox towards BDL biosynthesis; 2. use this toolbox to develop combinatorial biosynthetic methodologies to produce a large variety of unnatural BDL analogues and congeners; 3. decipher enzyme structural contributions to the biosynthetic rules of iPKSs; and 4. evaluate the produced BDLs in various biological assays to discover lead compounds for drug discovery. We will clone a large variety of BDL biosynthetic clusters from various fungi using traditional cloning pipelines as well as genome mining and synthetic biology. We will express the biosynthetic genes in a yeast heterologous host, and we will monitor the biosynthesis of new and known BDLs. We will investigate combinatorial biosynthesis by mixing and matching various iPKSs; creating hybrid synthases by domain swaps; and using post-PKS enzymes for combinatorial tailoring. We will also use domain exchanges, enzyme structural data, homology modeling and site-directed mutagenesis to define enzyme structural features that determine first ring cyclization geometry and chain termination repertoire. Finally, novel BDL congeners will be isolated, structurally identified, and evaluated for biological activities in various cell-based and in vitro biochemical assays. The lessons learned will advance our understanding of iterative enzymatic catalysis during BDL biosynthesis in particular and fungal polyketide biosynthesis in general. Improved methodologies for combinatorial biosynthesis will allow the production of novel BDLs and other polyketides. BDL analogs obtained in these experiments may provide lead compounds for developing treatments for cancer, immune system disorders, inflammatory or neurodegenerative conditions, and fungal infectious diseases.