Genome mining of RiPPs driven by highly efficient pathway reconstruction methods

Ribosomally synthesized and post-translationally modified peptides (RiPPs) constitute an emerging family of natural products, with arising interest in their biosynthetic diversity and therapeutic potentials. Advances in genome sequencing and bioinformatics have significantly accelerated the identification of RiPP biosynthetic gene clusters (BGCs) from genome sequences, however, deciphering the products of these BGCs remains challenging, primarily due to their highly diverse biological origins and elusive genetic regulation machineries. This chapter describes the use of pathway reconstruction approaches for exploring the biosynthetic potential of cryptic RiPP BGCs. Specifically, a plug-and-play pathway refactoring workflow is described, which can effectively rewire the underlying regulatory systems in target BGCs, ensuring their expression in genetically tractable organisms. In addition, the Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE), a method capable of cloning large DNA fragments into selected expression vectors, is provided as an alternative way for investigating BGCs with intricate gene arrangements. Due to their high efficiency and robustness, these methods would be of interest to those working on the genome mining of RiPPs, as well as other families of natural products.