Development and application of synthetic biology tools for natural product research

Natural products, or secondary metabolites, usually refer to bioactive small molecules produced by microorganisms or plants. To date, thousands of natural products have been characterized which reveal diverse structures and bioactivities, making it an ideal resource for discovering drug leads and mining biocatalysts. The advances in bioinformatics and DNA sequencing technology in the 21st century enables the prediction of tremendously large numbers of biosynthetic pathways for natural products directly from genomic database, which indicates Nature’s great potential of making these molecules remains to be explored. However, most of these uncharacterized pathways are naturally under negative regulation or encoded by uncultivable microorganisms and thus turn into “dark matters” for natural product discovery. Pathway refactoring serves as an invaluable synthetic biology tool for natural product discovery, characterization, and engineering. However, the complicated and laborious molecular biology techniques largely hinder its application in natural product research, especially in a high-throughput manner. Therefore, I developed a plug-and-play pathway refactoring workflow for high-throughput, flexible pathway construction, and expression in both Escherichia coli and Saccharomyces cerevisiae. The modular design not only enables the system to accommodate pathways with different number of genes, but also facilitates gene deletion and replacement. As proof of concept, a total of 96 pathways for combinatorial carotenoid biosynthesis were built successfully. The plug-and-play workflow was then used for discovering lanthipeptides and glycocins, both are groups of ribosomally synthesized post-translationally modified peptides (RiPPs) with various antimicrobial activities. A Class IV lanthipeptide with an unusual ring topology was successfully discovered, and its cyclization mechanism was elucidated in vitro. In addition, four novel glycocins were also discovered and one of them was unprecedentedly di-glucosylated on a single serine. Further bioactivity characterization of glycocins revealed that three of them exhibit narrow antimicrobial spectrum and implied the existence of new biological targets of glycocins. In addition, I also characterized the biosynthesis of heat stable antifungal factor (HSAF), a polyketide which belongs to the polycyclic tetramate macrolactam (PTM) series. Previously Zhao group discovered a PTM from Streptomyces griseus and characterized its biosynthesis, indicating a parallel biosynthetic mechanism. To further explore if this is general for the biosynthesis of all PTMs, I made four constructs which are supposed to make different intermediates in the HSAF biosynthesis. After successful isolation and structural characterization of these intermediates, the biosynthetic mechanism of HSAF was elucidated, which clearly supports the assumption that the HSAF pathway also has a parallel biosynthetic mechanism. Finally, to accelerate the engineering of streptomycetes to be ideal chassis for natural product discovery and overproduction, I applied the clustered regularly interspaced short palindromic repeats (CRIPSR)-dCas9 system for gene repression in both Streptomyces lividans 66 and Streptomyces albus J1074. By fusing the RNA polymerase omega factor (RNAP-ω) to dCas9, it was able to activate the silent undecylprodigiosin (RED) gene cluster in S. lividans 66. In addition, to further engineer the CRISPR-dCas9 system to exert titratable repression, an inducible promoter was used for small guide RNA (sgRNA) expression and its performance was analyzed.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste