Biosynthesis and exploitation of marine-derived post-translationally modified ribosomal peptides

Compounds from Nature are still a mainstay for drug discovery and in certain therapeutic areas such as cancer, 70% of drugs used in the clinic are of natural origin. Many of these compounds are highly complex and are difficult to re-create in the laboratory. A recent trend has been to try to understand the mechanisms the organisms use to biologically synthesise these molecules. This work is already in a very advanced stage for certain classes of these molecules, which are constructed from small subunits, and are known as polyketides and non-ribosomal peptides. Candidates from each category are already in the clinic, or are being tested on patients to assess their suitability as pharmaceuticals. In some cases the supply from nature is limited, and the organism's processes have been transplanted into easy-to-culture bacteria, which then produces the compound of interest. This is done by taking the DNA which encodes the instructions (the biosynthetic genes) to make the compounds and introducing these into a bacterium. This method has been successfully used to produce prospective drug candidates and to engineer new compounds by changing the instructions. We have recently discovered that potential anti-lymphoma compounds, originally isolated from a marine invertebrate (seasquirt) are in fact produced by its bacterial symbiont, Prochloron. Its instructions for biological synthesis of the compounds are encoded in a much more straightforward way than the classes of compounds mentioned above, and should make it possible to modify them more readily. This class of compounds is relatively unexplored, but evidence from the scientific literature suggests that there may be many more examples of this class of compound in certain types of marine invertebrate. Using the known chemical structure we can predict the way in which it is encoded in the DNA of the producing organism, and thus locate the relevant biosynthetic genes. We will chemically screen a number of target marine invertebrates (seasquirts, sponges) and extract their DNA. After this we will then screen the DNA for the presence of the relevant biosynthetic genes and determine the sequence of their DNA. Doing this on a number of species producing this unusual group of compounds will allow us to understand the rules by which these compounds are synthesised within the organisms. The combined information can then be used to screen marine invertebrates, which are suspected, but not known to, produce similar compounds. Combining this approach with ecological information will enable us to identify organisms, which are likely to produce other compounds of this type with potent biological activity. The outcomes of this work will be the discovery of new biologically active compounds from marine invertebrates together with methods to produce them in a sustainable fashion and modify them to modulate their activity. The method necessitates only a small specimen to be collected for DNA extraction, rather than large scale harvesting. In addition we will gain an understanding of how these unique compounds are biologically synthesised in these primitive organisms.

Modified peptides display a wealth of biological activity and most are constructed via NRPS pathways. An emerging group of metabolites is the post-translationally modified ribosomal peptides. These metabolites are formed from a ribosomally synthesised prepeptide by a complex of monofunctional peptides. Knowledge of the metabolite's chemical structure will therefore enable the prediction of the gene sequence from which it is derived. The biosynthetic rules towards these metabolites are not yet clearly understood. The most complex examples of this class are the patellamides, originally isolated from the seasquirt Lissoclinum patella, but now confirmed to be synthesised by its symbiont, Prochloron didemni. Many seasquirts and sponges produce related peptides that are often attributed to the symbiont. The biosynthesis of many of these bioactive compounds may also be via a ribosomal pathway. The main aim is to use a combination of chemistry, chemoinformatics, molecular biology and bioinformatics to explore these natural products. The discovery of novel biosynthetic pathways towards these bioactive modified peptides would be of interest academically, and will make excellent candidates for biosynthetic engineering. There are two strands to this work; (a) understanding the biosynthetic rules and (b) appreciation of the diversity of these type of compounds in the natural environment and gaining an understanding of how the pathways vary. A large number of molecules have been described from marine sources that are likely to be synthesised by this pathway. We will reisolate these compounds, and isolate the symbiont and its genomic DNA. Following this, the design of degenerate primers for conserved domains in the gene cluster will allow the systematic discovery of novel compounds in this class. Detailed cataloguing of the types of sea squirt and the nature of their symbionts (16S rRNA determination) will enable relationships between these organisms to be understood.