1. Genome mining
Searching for biosynthetic gene clusters that potentially encode for novel natural products is the most crucial step in the genomics-based approach for new natural products discovery. Although recent advances in sequencing technologies have facilitated rapid sequencing of microbial genomes, it is still impossible to sequence every genome in order to search for natural products biosynthetic gene clusters. A PCR-based screening would be the most viable alternative that allows rapid profiling of biosynthetic gene clusters present in a large pool of un-sequenced genomic DNAs. Core biosynthetic genes that are responsible for biosynthesis of natural product scaffolds and thus likely to have co-evolved with their respective biosynthetic gene clusters can serve as robust phylogenetic markers. Phylogenetic trees built with these genes provide detailed evolutionary pictures of nature’s combinatorial biosynthetic processes. My LAB will develop robust phylogenetic markers for diverse families of natural products that can best represent evolutional histories of their respective biosynthetic gene clusters.
2. Synthetic Biology
Most of the biosynthetic gene clusters recovered from microbial genomes remain transcriptionally silent in common laboratory culture conditions, hampering the chemical and biological characterization of their products. Therefore, the development of a scalable and generally applicable method to activate silent biosynthetic gene clusters will facilitate genomics-based small molecule discovery efforts. The most direct approach toward the activation of silent biosynthetic gene clusters would be promoter engineering in which tightly controlled native promoters are replaced with well-characterized constitutive or inducible promoters.10 However, the use of promoter engineering has so far been limited to simple, mostly single operon biosynthetic gene clusters due to the difficulty of identifying native promoters and also due to the time-consuming promoter replacement process that requires several rounds of homologous recombination. To overcome this limitation, my LAB will develop a generally applicable and easily scalable yeast- and Streptomyces-based multiplex promoter engineering platforms to systematically activate silent biosynthetic gene clusters found in microbial genomes.
3. Retro-biosynthesis for lead optimization
Natural products have served as drug lead compounds in diverse therapeutic areas. The structures of natural product drug leads are often modified to optimize pharmacological activities and reduce toxicities. However, the synthetic organic chemistry approaches such as total synthesis and semi-synthesis are extremely challenging in case of natural products with high structural and stereochemical complexities. Retro-biosynthesis in which natural products biosynthetic gene clusters are genetically modified to generate an array of structural analogs offers a viable alternative. Although there has been an encouraging progress for the past decade, retro-biosynthesis has suffered from two fundamental limitations: 1) a small number of sequenced biosynthetic gene clusters available, 2) the lack of genetic tools for efficient gene cluster refactoring. Recent advances in sequencing technologies have resulted in an infinite amount of sequencing data available in public databases, and continuous emergences of new synthetic biology tools have enabled a rapid, efficient multiplex genetic engineering, opening a new opportunity for retro-biosynthesis. My LAB will harness these resources to create libraries of structural analogs of clinically important natural products.
