(9d) Accelerating the Discovery of Novel Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs) through Pathway Refactoring and Robotic Automation

Bioactive natural products derived from microorganisms have proved to be a rich source for drug discovery. Ribosomally synthesized and post-translationally modified peptides (RiPPs), an emerging class of peptide natural products, exhibit diverse biological activities such as antimicrobial and cytolytic activities. To date, hundreds of RiPPs belonging to 41 classes harboring different characteristic modifications have been discovered since the first reported RiPP - nisin in 1928. Recent progress in genome mining has revealed many more RiPPs containing both known and unknown kinds of modification await characterization. However, discovery of new RiPPs is still time-consuming and labor-intensive. To address this limitation, we sought to employ pathway refactoring and robotic automation to study this under-characterized group of natural products. First, we demonstrated the feasibility of using Golden Gate assembly method for rapid RiPPs pathway refactoring by one-pot assembly of a varying number of predesigned expression units with each containing a codon-optimized gene encoding single biosynthetic enzyme driven by a T7 promoter. A total of 114 RiPPs pathways involving 2-9 biosynthetic enzymes were successfully refactored with a 90% success rate. After heterologous expression in E. coli BL21(DE3), 28 of them produced compounds with novel structures as determined by matrix-assisted laser desorption/ionization (MALDI)-TOF mass spectrometry. Of note, a new Class IV lanthipeptide S1022 with an unprecedented ring topology and a Streptolysin-S like linear azol(in)e-containing peptide (LAP) with 9 successive thiazole rings were successfully expressed using this method. In addition, we developed the automated pathway refactoring workflow and successfully implemented it on our robotic system named Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB) (https://www.igb.illinois.edu/iBIOFAB) for fully automated and scalable discovery of RiPPs. The whole pathway refactoring workflow can be divided into three process modules, including DNA assembly, heat-shock transformation, and construct verification. Each process module was converted into a sequence of common unit operations such as liquid handling, centrifugation, and temperature control. Each unit operation and sample transportation routes between unit operations were well-defined and matched with component instruments. Together, we successfully established a high-throughput synthetic biology platform for large-scale RiPPs discovery via Golden Gate assembly-based pathway refactoring method and robotic automation. In the future, more than two thousand uncharacterized RiPP pathways of both known and unknown classes will be investigated using this high-throughput natural product discovery pipeline.