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

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.

Bacterial Type 1 trans-AT polyketide synthases are complex yet well-suited for engineering to produce new bioactive compounds by virtue of their non-linear processing and chemical diversity via the recruitment of many different in-trans enzymes. The underlying mechanistic control exerted by this interplay of factors is complex and understanding is limited, in part due to technical challenges of in-situ investigations. To advance the field, we propose to optimise our recently pioneered selective labelling/NMR assay to monitor [13C]-labelled protein bound intermediates in vitro which will enable mechanistic details of polyketide processing to be probed at atomic resolution.

This interdisciplinary project will combine new NMR methodology, isotopic labelling, chemical synthesis and purified enzymes to study beta-branching biosynthetic transformations that are determined by the balance between in-cis versus in-trans processing of an ACP bound polyketide intermediate. Their widespread occurrence in natural products with potent biological activity ensures these structural and mechanistic findings will have general and future applicability.

We will advance our assay with real-time 2D NMR methods and synthetic 13C labelled substrate analogues to capture the kinetics of how ACP bound intermediates are processed. This will elucidate how dehydratase (ECH1), in-trans decarboxylase (ECH2) and an in-cis modular ECH2 (mECH) domains compete to produce a precise beta-branch as exemplified in the biosynthesis of the antibiotic kalimantacin.

We will determine how in-trans ECH2 domains are specifically excluded or linked to specific off-loading mechanisms to curtail beta-branch processing applied to the biosynthesis of bongkreckic acid.

We will investigate the biosynthesis of the anti-tumour antibiotic leinomycin and establish how curtailed beta-branch intermediates may be trapped via a conjugate addition of cysteine to provide sulfur incorporation.