Cell free Expression of Cryptic Actinomycetes Gene Clusters for Natural Products Discovery

Project Summary Microbial natural products are known to be rich resources for the search of drug candidates Advances in next generation sequencing have exponentially increased the metagenomic data available from actinomycetes which are known to be prolific producers of bioactive secondary metabolites However much of natural product chemical diversity has not been explored using traditional cultivation dependent strategies Furthermore a significant fraction of gene clusters from cultivated microbial sources remain silent under standard fermentation conditions In theory heterologous expression of natural product gene clusters can provide access to chemical diversity from both uncultivated organisms and cryptic pathways However current cell based heterologous expression approach remains to be the biggest bottleneck for genomic mining as often times it can be very time consuming and expensive and usually limited to a small number of standard hosts In Phase I will this bottleneck by applying our cell free expression technology to heterologously express cryptic actinomycete gene clusters in vitro Cryptic biosynthetic gene clusters of natural products with therapeutic value can be characterized in a high throughput and cost effective fashion We propose to achieve this objective through three specific aims Aim Platform development of diverse actinomycetes cell free systems We anticipate that cell free systems from multiple actinomycete strains will improve the probability of cryptic cluster heterologous expression Aim Identification of cryptic actinomycetes biosynthetic pathways of natural products We will identify never before expressed cryptic actinomycetes biosynthetic pathways and filter based on expression potential in our cell free expression platform Aim High throughput in vitro cell free expression We will run high throughput experiments to then express and characterize unique cryptic gene clusters If successful this valuable platform can be used to activate the pool of bioinformatically available cryptic biosynthetic gene clusters for the discovery of new natural products from actinomycetes with high pharmacological value Phase II efforts will focus on platform scale up and the identification and scaling of potential therapeutic candidates for downstream characterization Project Narrative Natural products remain a valuable resource for novel therapeutics to address human health This proposal uses cell free expression systems as an alternative method to search for novel natural products from actinomycetes By being able to express cryptic gene clusters without the use of cells we hope to exponentially increase the rate of rediscovery of natural products as a reservoir for therapeutics