Chemical Analysis of Hybrid Fungal Megasynthases

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Fungi produce a diverse array of biologically active compounds with roles as pharmaceuticals, agrochemicals and toxins. These include drugs such as the penicillins for treating bacterial infections, anticholesterol compounds such as the statins, and psychoactive drugs such as xenovulene. A major class of these compounds are the polyketides. In fungi polyketides are synthesised by giant multifunctional proteins known as polyketide synthases (PKS) - in turn these giant proteins are encoded by very large PKS genes. We have developed ways of isolating PKS genes from any given fungi which are involved in the biosynthesis of specific chemical compounds. For example we have found PKS gene clusters involved in the biosynthesis of the anticholesterol compound squalestatin, the mycotoxin fusarin A, the pigment tenellin and the psychoactive drug xenovulene from different fungal species. These PKS genes have been transferred to a new fungal host and switched on so that new chemical compounds are made. Despite very similar gene sequences, the four PKS genes encode proteins which can make very different chemical compounds. Thus, a knowledge of the gene sequence for these PKS does not yet help in understanding the processes which occur during the catalysis of polyketide formation in fungi. The PKS proteins evidently carry out a complex series of highly programmed chemical steps. While it is possible to read from the sequence the steps which could be catalysed, the programme controlling the order in which the steps are used, and the number of times they are repeated, is cryptic. The aim of this project is to find out how the giant PKS proteins are programmed. We will take a chemical approach to this problem. BBSRC and EU funded work is currently underway to generate numerous genetic changes in the genes themselves. We expect the genetic changes to lead to the production of modified proteins, which in turn should make new chemical compounds. By detecting, purifying and analysing the chemical structure of these new compounds we will be able to reveal the effect of the genetic change on the programme of the PKS. We expect to generate many tens of genetic experiments and each of these will require the examination of tens of genetic clones in fungi for the production of new compounds. We thus expect to have to examine hundreds to thousands of chemical extracts. We will use a state-of-the-art instrument to automate many of the purification and analysis steps. This instrument will combine High Performance Liquid Chromatography (HPLC) with sensitive, but robust, detectors which will be able to detect new compounds by size (evaporative light scattering detector), mass (mass spectrometer) and ultraviolet light (uv). The instrument will also be able to do automated purification for small samples, and will thus assist the chemist in processing the many hundreds or thousands of samples. We will also use the facilities of the School of Chemistry such as high field NMR and high resolution MS for structural elucidation. The new knowledge chemical structures of the new compounds produced from the mutant PKS will then allow us to elucidate the chemical effect of the genetic changes. We hope to eventually understand the link between gene sequence and chemical compound. This will allow two major advances - the ability to engineer fungal PKS at will to produce new compounds; and the ability to predict what compound will be made by simply reading a gene sequence.