Engineering the Biosynthesis of Cytochalasans for the Development of New Molecular Tools

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Cytochalasans are important fungal natural products which have a very wide range of potent bioactivities. These compounds could be developed into a class of very useful molecular tools, but they are hard to access from nature or by synthetic means. However, enough is now known of their biosynthesis to start to be able to rationally engineer them. In this project we will aim to use new synthetic biology methods in fungi to manipulate biosynthetic genes involved in the production of a number of cytochalasans. We will start by engineering host organisms to allow us to do more genetic manipulations including combined knockout and heterologous expression experiments. We will attempt to systematically re-engineer the polyketide moiety of cytochalasins in terms of chain-length and methylation pattern. We will also change the amino acid partner by rebuilding its biosynthetic proteins. We will engineer late stage tailoring steps by mixing and matching tailoring genes from diverse pathways and fungi. Finally, we will combine mutasynthesis methods with semi-synthesis to expand the range of useful cytochalasan molecular tools which will be used by others in the CytoLabs consortium for exploring cytochalasan activity in vivo and in vitro vs actin; and for the determination of new non-actin targets. We will supply compounds to the CytoLabs compound collection for use by others in bio-assays, and also work with synthetic chemists for the biotransformation of synthetic intermediates, particularly in respect of late-stage tailoring reactions. We will collaborate with scientists in Germany for fungal genome and transcriptome sequencing, and with scientists in France for scale-up of solid fermentations. The project will work towards the rational manipulation of fungal natural products pathways and development effective synthetic biology tools for the manufacture of useful materials. These will play a key role in the CytoLabs consortium with important uses in exploring cell biology, inter-organism interactions, plant pathology and in vitro enzymology.