Illuminating and exploiting programmed O-methylation in trans-AT polyketide synthases

迫切需要生产基于天然产物的药物的新型衍生物，例如抗生素和抗癌剂，以克服耐药性。此外，许多除草剂、杀虫剂和杀真菌剂在保护粮食作物方面发挥着重要作用，它们是天然产品，需要毒性更低、功效更大的新衍生物来养活迅速增长的全球人口。开发天然产物结构修饰的新方法并提高其生产水平是合成生物学的重要应用。许多重要的天然产物是由模块化多酶组装线构建的。这些为合理的生物工程创造新的天然产物衍生物提供了强大的潜力。然而，缺乏对这些固有的移动的蛋白机器的结构、机制和底物耐受性的详细分子见解限制了这一努力的进展。因此，有必要开发新的结构为基础的方法来阐明这种机器的特定组件的机制和底物特异性决定因素。该项目旨在从原子水平上了解装配线机械如何在组装一类重要的天然产物聚酮化合物时将甲基附加到特定的氧原子上。要做到这一点，我们将开发和应用策略，捕捉这些机器在特定的功能状态，这将有助于确定其分子结构使用各种方法，包括核磁共振光谱，卡宾足迹质谱和冷冻电子显微镜的内在动态组件。我们还将使用化学合成的探针分子研究机器关键部件的底物耐受性。所获得的新知识将为未来的生物工程工作提供信息，这些生物工程生产线将用于生产具有改变的药代动力学和药效学特征的新型天然产物衍生物。这有可能被英国的制药，农业化学和生物技术公司积极参与开发基于天然产品的消费品，这将最终造福于更广泛的社会。

There is an urgent need to produce novel derivatives of natural product-based medicines, such as antibiotics and anticancer agents, to overcome drug resistance. Moreover, many herbicides, insecticides, and fungicides, which play an essential role in the protection of food crops, are natural products and new derivatives with lower toxicity and greater efficacy are needed to feed the burgeoning global population. Development of novel methodologies for structural modification of natural products and enhancing their production levels are important applications of synthetic biology. Many important natural products are built by modular multienzyme assembly lines. These offer strong potential for rational bioengineering to create novel natural product derivatives. However, a lack of detailed molecular insight into the structure, mechanism, and substrate tolerance of these inherently mobile protein machines has limited progress in this endeavour. Thus, there is a need to develop novel structure-based approaches to elucidating the mechanism and substrate specificity determinants of specific components of such machinery. This project aims to develop an atomic-level understanding of how assembly line machinery appends methyl groups to specific oxygen atoms during assembly of an important class of natural products known as polyketides. To do this, we will develop and apply strategies for capturing intrinsically dynamic components of these machines in specific functional states, which will facilitate the determination of their molecular structures using a variety of methods, including nuclear magnetic resonance spectroscopy, carbene-footprinting mass spectrometry and cryo-electron microscopy. We will also investigate the substrate tolerance of key parts of the machinery using chemically synthesised probe molecules. The new knowledge gained will inform future efforts to bioengineer these assembly lines to produce novel natural product derivatives with altered pharmacokinetic and pharmacodynamic profiles. This has the potential to exploited by UK-based pharmaceutical, agrochemical and biotechnology companies actively engaged in the development of natural product-based consumer products, which will ultimately benefit to wider society.