Enzymology and engineering of the biosynthesis of polyether antibiotics

This project seeks to combine the efforts of two established research teams in Cambridge to solve a major outstanding problem in chemistry and biochemistry: the way in which a certain large group of natural antibiotics called polyethers are produced in Nature. Polyethers are antibiotics, whose clinical use has been restricted by their toxicity and by the difficulty of synthesising them or modifying them chemically, but which have been recently discovered (for example) to be highly effective against drug-resistant malarial parasites, a major global health threat. There is therefore great interest in developing new biological ways of synthesising libraries of such molecules to test as the starting point for potentially improved drugs of lower toxicity. We already know that to build up such complex small molecules from the simple building blocks inside bacterial cells requires multiple steps, each one catalysed by an enzyme. Some of these are physically tethered together into massive multienzyme complexes, the most complex biological catalysts so far discovered, but all are orchestrated to provide a smooth cascade or chain of reactions so that nothing is wasted and typically a single end-product is made. The opportunity for this research arises because one of us (PFL) has recently (with BBSRC support) succeeded for the first time in cloning and sequencing not one but four giant gene clusters governing the biosynthesis of four different polyethers; and his group has developed genetic methods to harness an industrial bacterial strain which overproduces one of these polyethers called monensin, so that products accumulate at levels up to 1000-fold higher than from the wild type, making the analysis of even minor products possible. Understanding polyether biosynthesis in molecular detail will require diverse experimental approaches including genetic manipulation of the genes inside the bacterial cells to see in what way the altered blueprint directs the synthesis of an altered end-product; and attempts to reconstitute portions of the catalysis in the test tube using purified enzymes and artificial substrates. We aim not only to provide a new appreciation of how the properties of individual enzymes are modulated when combined into such an integrated system, but also to learn the rules of how to alter the genes, or combine the genes from two or more polyether pathways, so as deliberately to divert the biosynthesis to make new target natural products. Our joint project aims to accelerate the further development of this exciting technology, with its broad potential benefits in the pharmaceutical industry.

The biosynthesis of complex antibiotic polyketides by multienzyme pathways in actinomycete soil bacteria represents one of the best studied examples of assembly-line enzymology, in which multiple enzyme activities are orchestrated to produce a specific chemical and stereochemical outcome in the final product. In this project it is aimed to clarify the enzymology, currently obscure, involved in the generation of a large, structurally complex and important class of polyketides, the ionophoric polyethers, which have been widely used commercially in animal husbandry and which also have intriguing activity against drug-resistant malaria, but whose use in human therapy has been inhibited by their toxicity. We then aim to use this knowledge to engineer the production of novel polyethers with potentially useful biological activity. The starting point for this project is our recent genetic analysis of four biosynthetic gene clusters governing the production of different polyethers, and the successful establishment of methods for genetic manipulation of the polyether-producing actinomycete strains. Our preliminary experiments are most advanced for the monensin (mon) genes of Streptomyces cinnamonensis. We intend to establish the mechanism of the stereoselective oxidative cyclisations that lead from putative linear polyketide intermediates to the polyether product, and determine the roles of each enzyme and the timing and stereochemistry of its action. We will also seek to establish the novel mechanism of chain termination on these enzymes. A second distinct method of chain termination is shown by the tetronic acids tetronomycin, tetronasin, and RK-682. We intend to identify the novel genes and enzymes responsible for tetronic acid ring formation in these clusters. Finally, we will use the insights gained to assist the engineered production of novel polyethers, or even compounds of radically different structure, by manipulation of the pathway genes.