Mapping Protein-Protein Interactions in Fungal Megasynth(et)ases

真菌负责生产具有工业重要性和结构多样化的天然产品，这些产品在医学(例如治疗传染病、癌症、高胆固醇和移植排斥反应)和农业(例如作物保护和动物保健)中有丰富的应用。这些天然产物的一大类是聚酮类化合物。在真菌中，聚酮是由被称为聚酮合成酶(PKSS)的巨型多域‘巨型合成酶’蛋白合成的，它在结构和催化作用上都与哺乳动物脂肪酸合成酶(FAS)非常相似。然而，与脂肪酸的生物合成不同，真菌PKSS可以通过编程链处理的程度来引入显著的结构复杂性。这最终决定了引入到最终天然产品中的结构元素。自从在真菌中发现PKSS以来，操纵生物合成机器的编程以提供新产品的愿望吸引了大量的研究。然而，到目前为止，重新编制程序的努力在很大程度上没有成功。这主要是由于不相容的结构域组合导致错误的产物和活性和产量的显著降低，限制了PKS的工程潜力，并阻止了进一步的潜在疗法的获得。一个给定的PKS结构域的不同区域调节它如何与其底物和其他结构域相互作用。在PKS内部，这些相互作用对于正确的反应顺序和有效的聚酮结构至关重要。通过在分子水平上深入了解PKS系统来实现对结构域功能的控制，对于实现和加强这些生物工程策略是至关重要的。本项目将使用最先进的生化和生物物理技术来研究真菌PKS中蛋白质-蛋白质相互作用(PPI)的分子细节。这些包括：设计和合成捕捉蛋白质复合体的新型机制交联剂；丙氨酸扫描突变和卡宾足迹相互作用位点；完整蛋白质质谱学，以可视化生物合成过程；以及X射线结晶学，以获得PKS内各个结构域的原子级结构。这些数据将指导PKS中的突变/结构域切换，以允许对PKS机制进行有效的“重新编程”，并产生新的产品。

Fungi are responsible for the production of industrially-important and structurally-diverse natural products that have a wealth of applications in medicine (e.g. the treatment of infectious diseases, cancer, high cholesterol and transplant rejection) and agriculture (e.g. crop protection and animal health). A major class of these natural products are polyketides. In fungi, polyketides are synthesised by giant multi-domain 'megasynthase' proteins known as polyketide synthases (PKSs), which closely resemble the mammalian fatty acid synthase (FAS) in both domain architecture and catalysis. However, unlike fatty acid biosynthesis, fungal PKSs can introduce significant structural complexity by programming the degree of chain processing. This ultimately dictates the structural elements introduced into the final natural product.Since the discovery of PKSs in fungi, the desire to manipulate the programming of the biosynthetic machinery to provide novel products has attracted intensive research. However, to date, reprogramming efforts have been largely unsuccessful. This is mostly due to incompatible domain combinations resulting in erroneous products and dramatically reduced activity and yields, limiting the engineering potential of PKSs, and preventing access to further potential therapeutics. Different regions of a given PKS domain regulate how it interacts with both its substrate and with other domains. Within the PKS itself, these interactions are critical for the correct ordering of reactions and efficient polyketide construction. Achieving control over domain function via an in-depth understanding of PKS systems, at the molecular level, is essential for achieving and enhancing these bioengineering strategies.This project will examine the molecular details of protein-protein interactions (PPIs) in fungal PKSs using state-of-the-art biochemical and biophysical techniques. These include: design and synthesis of novel mechanism-based crosslinkers to trap protein complexes; alanine scanning mutagenesis and carbene footprinting to pin-point interaction sites; intact protein mass spectrometry to visualise the biosynthetic process; and X-ray crystallography to obtain atomic-level structures of individual domains within the PKS. These data will guide mutations/domain switches in the PKS to allow effective 'reprogramming' of the PKS machinery and yield novel products.