Combinatorial biosynthesis of fungal benzenediol lactone polyketides

 性状（由申请方提供）：苯并吡喃内酯（BDL）是具有多种生物活性的真菌聚酮化合物天然产物。不同的BDL作为热应激反应系统或多种促分裂原活化蛋白激酶的高度特异性和有效的抑制剂，而其他的是各种受体的配体。MAP激酶的抑制转化为对依赖于这些调节剂的突变形式的癌细胞系的有效抗增殖活性。类似地，进化上保守的分子伴侣Hsp 90是癌症化疗的经验证的靶标，其抑制导致多种致癌途径的组合阻断。BDL是由成对的协同迭代聚酮酶（iPKS）生物合成的，代表了向模块化聚酮酶的概念性步骤。BDL生物合成还涉及由特定剪裁酶催化的聚酮化合物支架的PKS后修饰。对于当前的应用程序，我们提出了一个四管齐下的方法，将：1。为BDL生物合成组装前所未有的遗传工具箱; 2.使用此工具箱开发组合生物合成方法，以生产各种各样的非天然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.在各种生物测定中评估所产生的BDL，以发现用于药物发现的先导化合物。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，并监测新的和已知的BDL的生物合成。我们将通过混合和匹配各种iPKS来研究组合生物合成;通过结构域交换来创建混合酶;以及使用后PKS酶进行组合剪裁。我们还将使用结构域交换，酶的结构数据，同源性建模和定点诱变来定义酶的结构特征，确定第一个环的环化几何形状和链终止库。最后，新的BDL同源物将被分离，结构鉴定，并在各种基于细胞的生物活性和体外生物化学测定评价。这些经验教训将促进我们对BDL生物合成和真菌聚酮生物合成过程中反复酶催化的理解。改进的组合生物合成方法将允许生产新的BDL和其他聚酮化合物。在这些实验中获得的BDL类似物可以提供用于开发癌症、免疫系统疾病、炎症或神经退行性病症和真菌感染性疾病的治疗的先导化合物。

 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.