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性的天然产物，这些天然产物在医学（例如治疗传染病，癌症，高胆固醇和移植排斥）和农业（例如作物保护和动物健康）中具有丰富的应用。这些天然产物的主要类别是聚酮化合物。在真菌中，聚酮化合物由称为聚酮化合物合成酶（PKS）的巨型多结构域“大合成酶”蛋白合成，其在结构域结构和催化作用方面与哺乳动物脂肪酸合成酶（FAS）非常相似。然而，与脂肪酸生物合成不同，真菌PKS可以通过编程链加工的程度来引入显著的结构复杂性。这最终决定了引入最终天然产物的结构元件。自从在真菌中发现PKS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深入的研究。然而，迄今为止，重编程的努力基本上是不成功的。这主要是由于不相容的结构域组合导致错误的产物和显著降低的活性和产率，限制了PKS的工程潜力，并阻止获得进一步的潜在治疗剂。给定PKS结构域的不同区域调节其如何与其底物和与其他结构域相互作用。在PKS本身内，这些相互作用对于反应的正确排序和有效的聚酮化合物构建是至关重要的。通过对PKS系统的深入了解，在分子水平上实现对结构域功能的控制，对于实现和增强这些生物工程策略至关重要。本项目将使用最先进的生物化学和生物物理技术研究真菌PKS中蛋白质-蛋白质相互作用（PPI）的分子细节。其中包括：设计和合成新的机制为基础的交联剂，以陷阱蛋白质复合物;丙氨酸扫描诱变和卡宾足迹针点相互作用的网站;完整的蛋白质质谱，以可视化的生物合成过程;和X射线晶体学，以获得原子水平的结构内的PKS的个别域。这些数据将指导PKS中的突变/结构域转换，以允许PKS机制的有效“重编程”并产生新的产物。

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.