Understanding programming in highly reducing iterative fungal polyketide synthases - a structural and mechanistic approach

Fungal polyketide synthases (PKS) are extremely large multifunctional multi-domain proteins that assemble simple biochemical building blocks in a highly programmed iterative manner to make a huge range of biologically active complex natural products many of which are of importance as drugs or agrochemical agents or as toxins which are detrimental to both human health and agriculture. In simple terms PKSs are like beads on a string where each bead or 'domain' of the PKS represents an individual enzyme function or biological catalyst. The PKS uses simple carbon-carbon bond forming (condensation) reactions followed by a variety of 'tailoring' reactions in which the initial condensation product may be modified by all or some of a series of reactions - reduction (addition of two atoms of hydrogen), dehydration (loss of water) and C-methylation (addition of a single carbon branch), each of which is controlled by specific domains on the PKS. Discovering the molecular factors which control this programming is arguably the greatest remaining problem in our understanding of how nature makes complex natural products, and also represents a great fundamental problem in enzyme catalysis. Understanding of how the programming works, how the number iterative condensation cycle that determines the overall chain length, degree of reduction, and methylation is essentially absent. In this project the individual beads/domains will be isolated so that their individual properties and structures can be studied. Individual beads will then be brought together to study how one influences another, and this process will be repeated to try to build up an overall picture of how the intact PKS works. The project is highly interdisciplinary and will bring together the skills and expertise of natural products chemistry, chemical synthesis, molecular biology, biochemistry, structural biology and bioinformatics. The ultimate aim is to use the understanding gained of how the programming actually works, to manipulate the PKSs to make new products with altered or improved properties of benefit to human and animal health and to provide the pharmaceutical and fine chemical industries with new methods to produce new useful products.

Fungal polyketide synthases (PKS) are large multifunctional multidomain proteins that assemble simple acyl thioester intermediates in an iterative fashion to make a huge range of biologically active complex natural products using the simple reactions of Claisen C-C bond formation followed by varying degrees of reduction and C-methylation each of which is controlled by specific catalytic domains on the PKS. The initially formed structures may then be further modified by a wide range of 'tailoring' enzymes. The genes encoding the PKS and tailoring enzymes are generally clustered on the fungal genome and genome sequencing projects are revealing an overwhelming wealth of these biosynthetic gene clusters. Understanding of the nature of the programming of each iterative cycle which determines the overall chain length, degree of reduction, methylation and cyclisation is essentially absent. Individual domains, di-domains and tri-domains will be expressed and the individual reactions catalysed by the domains will be assayed by synthesis and covalently linking successive assembly intermediates involved in each cycle of chain elongation and reductive/methylation modification to the ACP, followed by monitoring and comparing the efficiency of their conversion by the individual catalytic domains. This will be extended into detailed structural studies of the domains themselves and to domain-domian and domain-substrate interactions that must be responsible for control of programming. This information will be linked with detailed bioinformatics studies to relate the fine differences in gene sequence to the programming steps and ultimately to predict the structures of the metabolite encoded by individual PKS genes. To enable this work and to exploit the many cryptic pathways revealed by genome sequencing, methods for expression of complete and partial fungal biosynthetic gene clusters will be developed.

Natural products are key to the health and well-being of mankind providing us with, amongst others, antibiotics, antifungals and antiparistic compounds for use in medicine and agriculture. By fully understanding how Nature assembles such a diversity of valuable structures at the genetic and protein level, there is enormous potential to exploit this knowledge in the design and synthesis of novel bioactive compounds. Compared to the striking advances made with the bacterial metabolites, the exploitation of fungal systems remains in its infancy. This is particularly ironic given the huge diversity of structural types known and continually be discovered in fungi. This lack of development is brought into sharper focus by rapid advancing discoveries in fungal genomics. Many filamentous fungi produce numerous complex polyketides, and from genomic studies we now know that they also possess the genetic machinery to make many more- some fungi possess at least 16 individual PKS genes. Thus the fungi have the ability to produce more polyketides, and with greater structural diversity, than the current paradigm organisms the actinomycetes. Furthermore, while only a small fraction of the 300,000 known fungi have been chemically investigated, an even smaller fraction (<100) have been genetically investigated for biosynthetic potential. On top of this it is estimated that the known fungal species represent a small fraction of all fungal species in the world (estimates of 3 million). Thus there is a vast potential for future genetic discoveries in fungi that will only be exploited chemically if a full understanding of fungal PKSs is forthcoming. The proposed interdisciplinary programme will shed light on the link between fungal PKS gene sequence and the chemical structures of the products of the encoded synthases. If the programming problem can be solved, it can be exploited for the production of new chemical entities, as has been achieved with the modular, non-iterative bacterial PKS systems. In the longer term this has potentially high impact for the healthcare of society with the generation of new drugs to combat disease. Research into polyketides is of world-wide interest both in academia and industry, and indeed several spin-out companies e.g. Biotica in Cambridge have been established. In the shorter term, the fundamental knowledge gained will be of immediate value to groups both in academic and industry to make advances in our understanding and exploitation of polyketide biosynthesis. Such advances depend upon the availability of well trained personnel. This programme will give the PDRAs valuable experience at the interface between chemistry and biological sciences and arm them with the skills to succeed in a future career in academia or industry. In order that the results can be fully exploited by us and the wider scientific community, communication is vital. The work will be published in internationally leading, peer-reviewed journals. Results will be presented by the investigators and PDRAs at national and international conferences and at meetings with industrial and academic collaborators. Appropriate training will be given to the PDRAs in the preparation of papers, posters and oral presentations to ensure that, alongside their scientific knowledge and skills, they are developing a portfolio of wide transferable skills. The Research and Development Unit at University of Bristol have experience on all aspects of intellectual property and will assist as required. The program is multidisciplinary and as well as frequent meetings between the investigators and PDRAs, further collaborators in Bristol will be involved including Drs Chris Arthur (biological MS), Andy Bailey (fungal gene expression), John Crosby (mass spectrometry, protein chemistry) and Colin Lazaraus (plant and fungal genetics). In addition we have links with industry (e.g. AZ, Biotica, GSK, Vernalis) to facilitate a wider perspective on the research.