DrPatrick Caffrey

Many of the antibiotics used in clinical medicine are derived from natural products synthesised by bacteria and fungi. Micro-organisms also synthesise a number of other important pharmaceuticals. These include anti-cancer drugs such as doxorubicin and epothilone C, immunosuppressants like cyclosporin and rapamycin, and the cholesterol-lowering statins. The need to develop improved treatments for serious diseases has intensified interest in biosynthesis of these compounds. This research group is interested in genetic manipulation of bacterial secondary metabolism to produce useful new compounds. A major focus is the biosynthesis of polyenes; highly effective antifungal antibiotics that disrupt the ergosterol-containing membranes of fungal cells. Polyenes have a broad spectrum of activity and resistance has not emerged as a serious problem after forty years of clinical use. The most serious disadvantages of polyene antibiotics are extreme toxicity and low water-solubility. However, the rising incidence of life-threatening systemic fungal infections is being mirrored by increased resistance to other classes of antifungal antibiotics. Engineered biosynthesis of less toxic polyenes is clearly a worthwhile objective. The most important polyene is amphotericin B, a heptaene produced by Streptomyces nodosus. The macrolactone core of amphotericin B is assembled from acetate and propionate units by a modular polyketide synthase. The late stages involve oxidation of a methyl branch to a carboxyl group, glycosylation with an aminodeoxyhexose sugar, mycosamine, and hydroxylation. We have characterised the amphotericin biosynthetic gene cluster and developed methods for genetic manipulation of S. nodosus. Analysis of the polyketide synthase sequence has given insights into how these enzymes determine the stereochemistry of chiral centres during assembly of polyketide carbon chains. Targeted gene replacements have yielded several amphotericin analogues. To date, the most promising of these is 16-descarboxyl-16-methyl amphotericin B, which is expected to show a reduction in toxicity similar to that of the semi-synthetic derivative amphotericin B methyl ester. Most of the engineered strains produce 10 to 50 mg of novel polyene per litre of culture. Future work aims to maximise these yields to produce large quantities for therapeutic testing. In addition to its antifungal action, amphotericin B also has some activity against prion diseases, enveloped viruses and Leishmania parasites. The new amphotericin analogues will also be tested for improvements in these biological activities. Additional projects include development of methods for genetic manipulation of other antibiotic-producing bacteria and engineering glycosylation of bioactive natural products.