Summary Biological systems build complex molecules for many different purposes - building blocks for cells, and supracellular structures, the catalytic and energy storage systems that drive living cells, the messenger molecules that allow communication and information storage within and between organisms and finally the molecules that allow defence or aggression against other organisms. Mankind has learnt to exploit many of these natural compounds, not least those that act as antibiotics. One class of compounds, the polyketides, are of great importance because they include many molecules with a great diversity of structures which cover a whole range of useful activities - not just antibacterials, but also antifungals, anticancer and anticholesterol agents, to name just a few. Typically, the molecular 'backbones' of these compounds are made by joining simple building blocks on an assembly line, each building block being added by a separate 'module' that also processes the new segment to one of a number (normally three) of modifications (known as the Type I PKS pathway). The resulting molecular chain can be of different lengths and combinations of modifications and can then be decorated with different side chains to produce a unique product ('tailoring'). However, an increasing number of atypical pathways are being uncovered that appear to use additional mechanisms not yet defined. These provide ways of producing new structures in a controlled way. One such pathway, found in Pseudomonas fluorescens, synthesises the clinically important antibiotic mupirocin. It is most active against Gram positive bacteria and is particularly used against MRSA (Methicillin Resistant Staphylococcus aureus), one of the most dangerous 'superbugs'. Biosynthesis of mupirocin involves an atypical Type I PKS along with a large number of 'tailoring' enzymes some of which we have discovered act in tandem with the PKS in building the backbone of mupirocin. This is in contrast to typical type I PKS modules which within themselves contain all the information needed to build the backbone. This project integrates microbial molecular genetics, biochemistry and chemistry to study the biosynthetic machinery both in living cells and with purified enzymes to understand the role of the different PKS and 'tailoring' components in building the final active product. It will explore the reactions carried out by different parts of the pathway and their flexibility to produce new compounds. These will be made available for screening for new biological activities that may be of use as prophylactic or therapeutic agents.

Technical Abstract:
Mupirocin is composed of a polyketide-derived acid, monic acid (MA) esterified by 9-hydroxy-nonanoic acid (9-HN). MA is formed via an 'AT-less' modular Type 1 PKS, with many features common to this increasing generic group. To understand how the tetrahydropyran ring and other features of MA are formed we will extend ongoing analysis of mutants defective in the pathway to study intermediates involved and create double mutants to establish the order in which particular gene products work. We will purify MmpD encoding the first four Type I PKS modules to explore how trans-acting tailoring genes mupC and mupD carry out reductions on module 3/4 intermediates. We will test MupC and MupF on synthetic substrates to establish their activity/specificity. We will determine whether MmpD processes altered substrates if module 1 is inactivated and alternative substrates are fed. We will assemble the predicted complement of genes in an heterologous host to reproduce the backbone of MA and determine if the tetrahydropyran ring is produced when predicted tailoring genes are added to the cloned PKS genes to complete biosynthesis of MA. We will test the need for the nascent MA to be esterified with 9-HN, and whether cassettes with mupW+T and mupOUV+macpE can function in biotransformations to convert model substrates to functionalised tetrahydropyrans. We will establish if 3-hydroxypropionate is the starter for 9-HN, how it is made and the possible role of mupQ, mupS and macpD. Then with isolated MmpB, the most likely candidate for the FAS/ PKS involved in 9-HN synthesis, supplemented with appropriate accessory genes/enzymes we will determine whether biosynthesis can be performed in vivo and in vitro. We will clone/sequence the genes from Alteromonas rava responsible for the biosynthesis of the thiomarinols - secondary metabolites closely related in structure to mupirocin. Genes unique to thiomarinols will be tested for ability to introduce thiomarinol features into mupirocin. Joint with BB/E022367/1 Co-funded by EPSRC.