Catching antibiotic factories in action

聚酮化合物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，其被开发用于包括药物、动物健康和农用化学产品在内的应用。聚酮化合物由模块化聚酮化合物脱氢酶产生，模块化聚酮化合物脱氢酶是复杂的生物合成多结构域巨酶，其作用基本上像装配线，并且可以被合理地操纵以递送功能优化的产物。生产的每个分子的化学结构是由装配线上每个阶段的酶决定的，就像一张蓝图。我们了解建造这些工厂的一些规则，可以重新排列模块的顺序来生产新的化合物，但有时这只是打破了装配线，或者生产出意想不到的化合物。由于生物“工厂”的复杂性，没有单一的技术提供了全貌，但这个博士项目将是我们更广泛努力的一部分，汇集重要的技能和科学专业知识，集中不同的“镜头”的问题。了解这些系统如何工作将有助于回答有关其设计原理的重要问题，以便以合理的方式构建新化合物的新途径。为了深入了解分子机制，博士项目将致力于解决沿着抗MRSA抗生素kalimantacin途径的聚酮合酶蛋白复合物的cryo-EM和晶体结构（图1A）。这些系统的动态性质使得通过cryo-EM对其进行研究具有挑战性（图1B），但这将通过使用特定的化学底物模拟探针（即卡利曼霉素片段）与Cryo-EM结合捕获合酶的中间构象来解决。您还将与NMR光谱学家一起使用标记探针（例如13 C或19 F）来跟踪中间体的真实的处理。这种真实的时间监测和冷冻EM结构将共同提供这些抗生素工厂如何工作的独特时空图片。至关重要的是，通过这个合成生物学项目，您将学习包括Cryo-EM，X射线晶体学，NMR，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.