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 1. 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 2. 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 3. High throughput in vitro cell-free expression. We will run high-throughput (384+) experiments to then express and characterize 3 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.

Public Health Relevance

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.?

Agency
National Institute of Health (NIH)
Institute
National Center for Complementary & Alternative Medicine (NCCAM)
Type
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
Project #
1R43AT009522-01
Application #
9348513
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Hopp, Craig
Project Start
2017-04-01
Project End
2018-09-30
Budget Start
2017-04-01
Budget End
2018-09-30
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Synvitrobio, Inc.
Department
Type
DUNS #
079761816
City
San Francisco
State
CA
Country
United States
Zip Code
94107