Dissecting and Harnessing Carrier Protein Interactions in Fungal Megasynth(et)ases

真菌聚酮合成酶(PKS)是一种巨型多结构域蛋白质，负责合成大量具有生物活性的天然产物。虽然许多是作为医药或农用化学品的重要用途，但有些是对人类健康和农业都有害的毒素。这些复杂化合物的精确组装依赖于生物合成中间体共价连接到的小载体蛋白(CP)结构域与PKS中的单个催化结构域之间的高度程序化的蛋白质-蛋白质相互作用(PPI)，从而确保整个过程高效并保持产品的保真度。尽管这些相互作用很重要，但人们对它们的了解仍然很少，主要是因为它们的短暂性质，需要有效地研究各种技术的组合。揭示控制编程的分子因素是我们理解真菌PKS的最大遗留问题，并代表着将这些酶重新连接到用户设计的分子的巨大障碍。该项目旨在结合前沿的质谱学、结构生物学和生化技术来阐明支持真菌PKS机制的依赖于CP的相互作用的分子细节。这项研究最初将集中在两个类似的PKS系统中的PPI的特征；一个参与环孢菌素(临床使用的免疫抑制剂)的生物合成，另一个负责构建洛伐他汀(临床使用的降胆固醇药物)。这些PKS在单个催化结构域的定位上有所不同，因此代表了建立依赖CP相互作用的共同原理的优秀模型系统。综上所述，这些工作将大大加深我们对PPI在真菌PKS中所起作用的理解。它还将形成一个知识库，开始利用这种相互作用来构建能够生产新型天然产品类似物的工程系统。

Fungal polyketide synthases (PKS) are giant multi-domain proteins responsible for the biosynthesis of a vast number of biologically active natural products. Whilst many find important applications as medicinal or agrochemical agents, some are toxins that are harmful to both human health and agriculture. The precise assembly of these complex compounds relies on the highly programmed protein-protein interactions (PPIs) between a small carrier protein (CP) domain, to which biosynthetic intermediates are covalently tethered, and the individual catalytic domains within the PKSs, ensuring that the overall process is efficient and maintains product fidelity. Despite the importance of these interactions, they remain poorly understood, primarily due to their ephemeral nature, requiring a combination of techniques to be studied effectively. Uncovering the molecular factors governing programming is the greatest remaining problem in our understanding of fungal PKSs, and represents a huge obstacle to rewiring these enzymes towards user-designed molecules. This project aims to combine cutting-edge mass spectrometry, structural biology and biochemical techniques to elucidate the molecular details of CP-dependent interactions underpinning fungal PKS machinery. The research will initially focus on characterising PPIs in two similar PKS systems; one involved in the biosynthesis of cyclosporin (clinically used immunosuppressant), the other responsible for the construction of lovastatin (clinically used cholesterol-lowering agent). These PKSs differ by the positioning of a single catalytic domain, and therefore represent excellent model systems to establish the common principles underlying CP-dependent interactions. Taken together, this body of work will significantly deepen our understanding of the roles played by PPIs in fungal PKSs. It will also form a knowledge-base to begin exploiting such interactions to construct engineered systems capable of producing novel natural product analogues.