(228bx) A Plug and Play Pathway Refactoring Strategy for Natural Product Discovery in Escherichia coli and Saccharomyces Cerevisiae

Natural products remain one of the major sources for drug discovery. However, expression of the corresponding biosynthetic gene clusters in heterologous hosts requires a pathway refactoring strategy with high efficiency, fidelity and modularity. Ideally, such a strategy should be automation-compatible in order to increase the throughput by robotic systems. Here, we report a plug and play pathway refactoring strategy for our automation platform, Illinois Biological Foundry for Advanced Biomanufcturing (iBioFAB), based on Golden gate reaction. Briefly, a total of 9 destination sites were predefined in a receiver plasmid and the genes to be refactored can be inserted into any of these sites by using helper plasmids. For destination sites without genes, we also computationally designed 9 linkers, which makes the assembly condition consistent and genes interchangeable. Two sets of helper plasmid were built for pathway expression in Escherichia coli and Saccharomyces cerevisiae, and high fidelity of assembly was observed in both cases. With the usage of counter-selection marker, the whole workflow can be finished in 2 days. As a proof of concept, 96 combinatorial carotenoid pathways were built automatically and a library of products with varying colors were generated successfully.