(253d) Rapid Discovery of Ribosomal Natural Products Using Synthetic Biology Approaches

Ribosomally-synthesized and post-translationally modified peptides (RiPPs) constitute a major family of natural products that have been found in all three domains of life. To date, RiPPs have been reported with vast structural diversity and hence a broad range of biological functions, including antimicrobial, anticancer, and antiviral activities, making them an ideal source for drug development. The explosion of genomic data and the advancement of genome mining tools have enabled the identification of RiPP biosynthetic gene clusters (BGCs) in silico, however, attempts to isolate these compounds often fail due to the negative regulation over these BGCs under standard laboratory conditions, a common problem for traditional natural product discovery. To rapidly explore their biosynthetic potential in nature, here I present the genome mining of RiPPs by leveraging synthetic biology approaches. Candidate RiPP BGCs predicted by bioinformatics are categorized into two groups: the high-fidelity group and the class-defining group. The high-fidelity group comprises BGCs that are annotated as characterized yet highly underexplored RiPP classes. Given the high successful rate of refactored BGCs in heterologous expression, an automation friendly plug-and-play method was developed to refactor the high-fidelity BGCs efficiently. While BGCs in the class-defining group encode unknown and usually complicated biosynthetic machinery, a direct cloning strategy named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) is used to manipulate these BGCs for heterologous expression. Afterwards, the initial product screening is performed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Due to the high efficiency and low time consumption of the synthetic biology approaches, the genome mining of RiPP BGCs can be achieved rapidly. The effectiveness of pathway refactoring for high-fidelity BGCs was demonstrated by uncovering a class IV lanthipeptide and four glycocins with interesting structural features and bioactivities, which significantly expanded these two previously underexplored RiPP classes. In addition, two novel classes of RiPPs, named daptides and lipoavitides, were also discovered by the direct cloning approach and characterized with unprecedented biosynthetic machineries. Overall, the synthetic biology based approaches were demonstrated to be highly useful for unveiling novel RiPPs, and have the potential to be developed into a scalable platform for RiPP genome mining.