Catching antibiotic factories in action

聚酮生物合成途径产生大量不同的天然产物，并包含许多化学结构，这些结构可用于制药、动物保健和农用化学品产品。聚酮是由模块化聚酮合成酶产生的，这是一种复杂的生物合成多域巨酶，本质上就像装配线一样，可以合理操作以提供功能优化的产品。生产的每个分子的化学结构是由装配线上每一道工序上的酶决定的，这很像一张蓝图。我们了解建造这些工厂的一些规则，并可以重新安排模块的顺序来生产新的化合物，但有时这只会破坏装配线，或者生产出意想不到的化合物。由于生物“工厂”的复杂性，没有一种单一的技术可以提供全景，但这个博士项目将是我们更广泛努力的一部分，我们将把重要的技能和科学专业知识聚集在一起，使不同的“镜头”集中在这个问题上。了解这些系统的工作原理将有助于回答有关其设计原理的重要问题，以便以合理的方式建立新化合物的新途径。为了深入了解分子机制，PHD项目将致力于解决抗MRSA抗生素卡马坦星途径上的聚酮合成酶蛋白质复合体的低温EM和晶体结构(图1A)。这些系统的动态性质使得通过冷冻-EM来研究它们具有挑战性(图1B)，但这将通过使用特定的化学底物模拟探针(即卡马塔星的片段)与冷冻-EM相结合来捕获合成酶的中间构象来解决。您还将与核磁共振光谱仪合作，使用标记的探针(如13C或19F)来实时跟踪中间体的处理。这种实时监测和低温EM结构将一起提供这些抗生素工厂如何工作的独特时空图。重要的是，通过这个合成生物学项目，您将学习包括冷冻-EM、X射线结晶学、核磁共振、微生物学和分子建模/设计的技术，以及与合成化学家合作，提供了广泛的探索途径。

Polyketide biosynthetic pathways generate vast numbers of diverse natural products and encompassing numerous chemical structures that are exploited for applications including pharmaceuticals, animal health and agrochemical products. Polyketides are generated by modular polyketide synthases, sophisticated biosynthetic multi-domain megaenzymes which act essentially like assembly lines and which may be rationally manipulated to deliver functionally optimised products. The chemical structure of each molecule produced is determined by the enzymes present at each tage of the assembly line, rather like a blueprint. We understand some rules for building these factories and can rearrange the order of modules to produce new compounds, but sometimes this just breaks the assembly line, or produces an unexpected compound. Due to the complexity of the biological "factories", no single technique provides the whole picture, but this PhD project will be part of our wider effort to bring together important skills and scientific expertise to focus different "lenses" on the problem. An understanding of how these systems work will help answer important questions about their design principles so new pathways to novel compounds can be built in a rational way. To provide insights into the molecular mechanisms, the PhD project will we aim to solve cryo-EM and crystal structures of polyketide synthase proteins complexes along the pathway of the anti-MRSA antibiotic kalimantacin (Figure 1A). The dynamic nature of these systems makes studying them by cryo-EM challenging (Figure 1B) but this will be solved by using specific chemical substrate-mimetic probes (i.e. fragments of kalimantacin) in combination with Cryo-EM to capture intermediate conformations of the synthase. You will also work with NMR spectroscopists to use labelled probes (eg 13C or 19F) to follow processing of intermediates in real time. Together this real time monitoring and the Cryo-EM structures will provide a unique spatiotemporal picture of how these antibiotic factories work. Crucially, with this synthetic biology project you will learn techniques encompassing Cryo-EM, X-ray crystallography, NMR, microbiology and molecular modelling/design as well as work with synthetic chemists, offering a wide array of avenues to explore.