Linking single cell phenotype to gene expression by single cell transcriptions

Theme: Industrial Biotechnology and Bioenergy

In this project the initial objective will be to purify the antifungal compound)(s) from the bacterial cultures in sufficient quantity for a full structure determination using mainly NMR methods. Once the structure is known work on the biosynthesis will begin. The gene cluster that makes matillamide encodes for both polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) proteins and a partial structure prediction can be made on the basis of the predicted specificities of the acyl transferase and adenylation domains. However much remains unknown, e.g. the starter unit, the role of a unique flavin mono-oxygenase domain in the NRPS, and the role of the other enzymes in the cluster. It is likely that some of the amino acid residues in matillamide will have been modified and we will investigate whether these have be modified pre- or post-NRPS by feeding synthesised precursors to selected gene knock-out strains to see if this restores production of matillamide. Additionally the additional enzymes in the cluster (an aminotransferase, a hydrolase, an oxidoreductase, and a dehydratase) will be over-expressed, to test their enzymic activity on their putative substrates (or synthetic analogues). Genetic engineering, semi-synthesis and metabolic incorporation of unnatural precursors will all be tried as ways to make modified versions of matillamide and the anti-fungal activity of these compounds will be tested to start to build up a structure-activity relationship (SAR) for the antifungal activity. The substrate specificity, structures and mechanisms of these enzymes will be investigated in vitro with a view to engineering modified enzymes to more efficiently make matillamide analogues.