New tools for elucidating natural product biosynthesis in-situ at atomic resolution

天然产物及其衍生物已经并将继续是高价值化合物的重要来源，具有广泛的应用，包括杀虫剂、除草剂、抗癌剂和抗生素。在过去的50年里，科学家们已经开始发现这些具有生物重要性的化合物在自然界中合成的极其复杂和多样化的方式。聚酮类化合物是一类重要的天然产物，广泛存在于细菌和真菌等生物体中。虽然这些生物可能被视为简单的有机体，但就清洁有效地将简单的原料转化为复杂分子的能力而言，它们的表现可以说超过了世界上最好的合成化学家。我们希望利用的正是这种非凡的力量。通过充分了解自然界的生物合成机制(即分子是如何产生的)，我们可以设计生物合成途径，使用环境友好的方法生产新的化合物。过去几年的另一个兴趣是这些新化合物的更广泛的潜在应用，例如，微生物生物合成生物燃料或复杂的分子支架，可以作为合成化学家在制造、健康和农业中使用其他重要分子的起始单元出售。但是，我们仍然有很长的路要走，才能使我们自己设计的系统足够正常和高效地工作，以实现可靠的大规模生产。这是为什么？事实证明，聚酮是由巨型蛋白质组件催化的一系列化学反应产生的，这些组件在微生物内部充当纳米工厂。简单的有机分子在一端被激活和装载，连接在一起，然后在另一端作为完整的(通常是精细的)产品释放。纳米工厂将简单的积木连接到单个模块的装配线上，类似于一组在汽车制造中执行操作的机器人。因此，每个分子的化学结构是由装配线上每个阶段的酶决定的，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。更重要的是，许多被称为反式AT途径的途径以受控的方式招募额外的酶，其中一些途径建立了新的化学分支，称为β分支，从导致重要化学和生物特性(抗菌活性、抗癌特性和毒素)的分子中分离出来。我们目前没有合适的工具来理解这一切是如何工作的，以及一切是如何在正确的时间发生作用的。我们已经设计了一种新的工具，将核磁共振和化学合成结合在一起，来查看重建的TRAN-AT工厂是如何工作的。化学合成使我们基本上可以在分子中引入一个微型天线(碳13标记)，而核磁共振则让我们观察或“调谐”这个信号。因此，一个特定分子的命运可以被实时跟踪，而它仍然在“工厂”中。这通常需要隔离它，到那时，我们已经丢失了关于它正在做什么以及如何做的所有信息！我们的目标是研究几个非常不同的反式-AT“工厂”的结构和功能，这些工厂生产带有这些β-支链的分子。通过观察所有东西是如何组装在一起的，我们将更好地交换部件，从而使这些分支多样化，这可能会给它们带来宝贵的新特性。由于生物“工厂”的复杂性，没有单一的技术能提供全部情况，但我们的团队汇集了重要的技能和科学专业知识，将“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。

Natural products and their derivatives have, and will continue to be, an important source of high value compounds with a wide range of applications including pesticides, herbicides, anti-cancer agents and antibiotics. Over the last fifty years scientists have begun to uncover the remarkably complex and diverse ways in which these biologically important compounds are synthesised in nature. Polyketides are an important class of these natural products which are found in a wide range of organisms including bacteria and fungi. Whilst these might be viewed as simple organisms, they arguably outperform the world's best synthetic chemists in terms of their ability to convert simple feedstocks to complex molecules cleanly and efficiently. It is this remarkable power we wish to harness. By fully understanding nature's biosynthetic machinery (i.e. how molecules are created) we can engineer biosynthetic pathways to deliver new compounds using environmentally friendly methods for their production. A further interest in the last few years is the wider potential application of these new compounds so, for example, the microorganisms biosynthesise biofuels or complex molecular scaffolds that can be sold as starter units for synthetic chemists to use en-route to other molecules of importance in manufacture, health and agriculture.However, we are still a long way off making our own designed systems work properly and efficiently enough for reliable large-scale production. Why is this? It turns out that polyketides are made by a series of chemical reactions catalysed by mega-protein assemblies that act as nano-scale factories inside the microbe. Simple organic molecules are activated and loaded at one end, joined together and then released as completed (usually elaborate) products at the other end. The nano-factories join the simple building blocks on an assembly line of individual modules, akin to a group of robots performing operations in vehicle manufacture. The chemical structure of each molecule is thus determined by the enzymes present at each stage 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. What is more, many pathways, known as trans-AT pathways, recruit additional enzymes in a controlled manner, some of which build new chemical branches, called beta branches, off the molecules leading to important chemical and biological properties (antibacterial activity, anti-cancer properties and toxins). We don't currently have the right tools to be able to understand how this all works and how everything acts at just the right time.We have designed a new tool that combines NMR and chemical synthesis, to view how reconstructed trans-AT factories work. Chemical synthesis allows us to essentially introduce a micro-antenna (a carbon-13 label) within a molecule and NMR lets us look at or "tune into" this signal. Hence the fate of a particular molecule can be followed, in real time whilst it is still in "the factory". This would normally require it to be isolated, by which time we have lost all the information about what it was doing and how! Our aim is to investigate the structure and function of several very different trans-AT "factories" that produce molecules with these beta-branches. By seeing how everything fits together, we will be better placed to swap parts and therefore diversify these branches, which could give them valuable new properties.Due to the complexity of the biological "factories", no single technique provides the whole picture, but our team brings 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.