Discovery of Novel Natural Products via Synthetic Biology

Microorganisms are a major source of new therapeutic agents. Sequenced genomes and metagenomes provide a tremendously rich source for discovery of novel gene clusters involved in natural product biosynthesis. However, due to the lack of tools to efficiently identify a complete biosynthetic gene cluster, determine the role of each involved gene, and subsequently designate a function to the target gene cluster, only a tiny fraction of those putative natural product biosynthetic gene clusters have been characterized (1). To overcome this limitation, my group has developed a new genomics-driven, synthetic biology enabled method to discover and produce novel natural products from sequenced genomes and metagenomes. By taking advantage of the highly efficient yeast in vivo homologous recombination mechanism, this method refactors the target cryptic pathway together with the genetic elements needed for DNA maintenance and replication in S. cerevisiae, E. coli, and a target heterologous host using a plug-and-play scaffold and a set of heterologous promoters that are functional in the target heterologous host. As proof of concept, we awakened the silent polyketide spectinabilin pathway from Streptomyces spectabilis in Streptomyces lividans (2) and activated a cryptic pathway containing a polyketide synthase-non-ribosomal peptide synthetases from Streptomyces grieseus in Streptomyces lividans, which led to the discovery of two novel tetramic acid natural products that have never been reported in literature (3). Our method bypasses the traditional laborious processes to elicit pathway expression and represents a new platform for discovering novel natural products (4). Currently, in collaboration with several research groups including but are not limited to Adam Arkin, James Allison, Bill Metcalf, and Neil Kelleher that have expertise in bioinformatics, genomics, mass spectrometry, and automation, we are establishing a fully integrated robotic system to automate all the steps in gene cluster refactoring and product detection, which should significantly increase the throughput of our method and discover novel pharmaceutically important natural products.

1.  R. E. Cobb and H. Zhao. “Direct Cloning of Large Genomic Sequences.” Nature Biotechnology, 30, 405-406 (2012).

2.  Z. Shao, G. Rao, C. Li, Z. Abil, Y. Luo, and H. Zhao. “Refactoring the Silent Spectinabilin Biosynthetic Gene Cluster Using a Plug-and-Play Scaffold.” ACS Synthetic Biology, 2, 662-669 (2013).

3.  Y. Luo, H. Huang, J. Liang, M. Wang, L. Lu, Z. Shao, R. Cobb, and H. Zhao. “Activation and Characterization of a Cryptic Polycyclic Tetramate Macrolactam Biosynthetic Gene Cluster.” Nature Communications, 4: 2894 (2013).

4.  R. Cobb, J. Ning, and H. Zhao. “DNA Assembly Techniques for Next Generation Combinatorial Biosynthesis of Natural Products.” Journal of Industrial Microbiology and Biotechnology, 41, 469-477 (2014).