Development of a Bacterial Host for Natural Product Discovery and Production

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Project Summary Molecules isolated from living systems have consistently served as leads in drug discovery. However, two of the main challenges of drug discovery from natural products are the high rate of rediscovery of known compounds using traditional technologies and low yields. Recent advances in genome sequencing and synthetic biology have spurred a renewed interest in natural product discovery in the private and academic sectors. For instance, genome mining of underexplored taxa increases the chances of novel compound discovery. Moreover, robust host organisms can facilitate discovery efforts by overcoming the common low-yield hurdle. Bacteria belonging to the Burkholderiales order of -Proteobacteria are an emerging source of natural products. We have previously achieved high yield production of autologous polyketide-nonribosomal peptide spliceostatins in a Burkholderia species. We propose to understand, develop, and apply this strain as a host to discover and produce natural products from Burkholderiales and potentially other -Proteobacteria. Although Escherichia coli has long been used as a model bacterial host, the synthetic biology community is moving away from the idea that “one host fits all” to instead have hosts tailored to the source of biosynthetic gene clusters. We hypothesize that the Burkholderia sp. in question can be used as a host to streamline the discovery process by producing heterologous natural products in high yields. Our preliminary data supports this hypothesis as heterologous expression of a model gene cluster encoding the lasso peptide capistruin in this strain led to capistruin production in yields that are at least 65-fold, and up to 580-fold higher than with E. coli. In Aim 1 we propose to test/understand the host by a) testing the breath of the host in terms of source genera within the Burkholderiales and other -Proteobacteria while discovering lasso peptides; and b) investigating the regulation of autologous spliceostatin biosynthesis with the ultimate goal of deriving regulatory parts for heterologous pathway construction. In Aim 2, we will develop the host as a tool for natural product discovery by discovering and characterizing a promoter library and by generating a minimized genome. In Aim 3 we will apply the host by discovering natural products from a newly built Burkholderiales environmental collection. This project is expected to provide tools that will streamline and accelerate natural product discovery from a promising yet underexplored source. The tools and knowledge obtained will be applied to discover novel, bioactive natural products via genome mining of a Burkholderiales strain collection.