Characterization and Engineering of Fungal Megasynthases

描述(由申请人提供)：真菌来源的聚酮合成酶(PKS)是已知的微生物次级代谢中最神秘的酶机制之一。这些酶与非常重要的真菌天然产物的生物合成有关，既有益，如来自土曲霉的降胆固醇重磅炸弹药物洛伐他汀；也有害，如来自不同曲霉的致癌真菌毒素黄曲霉毒素。高度还原真菌PKS(HRPKS)在结构和力学上不同于已知的细菌PKS，因此产生不同结构多样性和生物活性的化合物。与细菌I型PKS或结构相关的哺乳动物FAS相比，我们对真菌HRPKS的编程规则的了解仍然非常有限，特别是关于如何在最终产物的合成中迭代使用和精确编排一组结构域，通常超过30个催化步骤。因此，了解真菌的HRPKS功能将增强我们对多结构域、过程系统的天然产物生物合成和酶学的认识。在这个方案中，我们将使用几个模型系统和化合物来全面研究HRPKSS的机制。我们的首要目标是了解并能够预测HRPKS序列与产品结构之间的关系。在建议期间，将并行实现四个目标：1)根据功能和产物结构对真菌HRPKS进行分类和挖掘；2)对HRPKS剪裁结构域的特异性进行指纹分析；3)了解HRPKS卸载机制中的蛋白质-蛋白质相互作用；以及4)阐明PKS后分子内环化反应的酶。每个目标都针对HRPKS功能的一个独特方面，这对结构多样性的产生至关重要。基本的酶学问题也将在解决这些目标时得到回答。我们的工作解决了聚酮生物合成中最不被理解、最困难，也可能是最后一个前沿的问题。

DESCRIPTION (provided by applicant): Polyketide synthases (PKSs) of fungal origin are among the most enigmatic enzyme machineries known in microbial secondary metabolism. These enzymes are associated with the biosynthesis of very important fungal natural products, both beneficial, such as the cholesterol lowering blockbuster drug lovastatin from Aspergillus terreus; and deleterious, such as the carcinogenic mycotoxin aflatoxin from different aspergilli. The highly reducing fungal PKSs (HRPKS) are architecturally and mechanistically distinct from the well-characterized bacterial PKSs, and therefore produce compounds of different structural diversity and biological activities. In contrast to bacterial Type I PKSs or the structurally relatd mammalian FASs, our knowledge of the programming rules of fungal HRPKSs remains very limited, especially with regard to how a single set of domains are iteratively used and precisely orchestrated in the synthesis of the final product, often exceeding a combined 30 catalytic steps. Therefore, understanding fungal HRPKS function will enhance our knowledge of natural product biosynthesis and enzymology of multidomain, processive systems. In this proposal, we will comprehensively investigate the mechanisms of HRPKSs using several model systems and compounds. Our overarching goal is to understand, and to be able to predict, the relationship between HRPKS sequence and product structure. Four aims will be pursued in parallel during the proposal period: 1) Classifying and mining fungal HRPKS based on function and product structure; 2) Fingerprinting the specificity of HRPKS tailoring domains; 3) Understanding protein-protein interactions in HRPKS off-loading mechanisms; and 4) Elucidating the enzymes responsible for post- PKS intramolecular cyclization reactions. Each aim is targeted at one unique aspect of HRPKS function that is vital to structural diversity generation. Fundamental enzymology questions will also be answered in addressing these aims. Our work tackles the least understood, the most difficult and perhaps the last frontier of polyketide biosynthesis.